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# Introduction
Although traditionally thought to be of minor significance in pharmacology and
toxicology, the clinical importance of CYP2B6 was recently established with the
identification of increasing numbers of CYP2B6 substrates, including clinically
important drugs such as the anticancer agents, cyclophosphamide, ifosfamide and
tamoxifen, the anti-retrovirals efavirenz and nevirapine, the anesthetics
ketamine and propofol, and the central nervous system-active bupropion and
methadone. It was estimated that CYP2B6 is involved in the metabolism of nearly
25% of drugs on the market. Marked intra- and inter-individual variation of
CYP2B6 expression and activity have been described in the literature and were
considered to be attributed mainly to the highly-inducible and polymorphic
nature of this gene. Many drugs and environmental chemicals can alter the
expression of CYP2B6, potentially leading to clinically significant drug-drug
interactions. Given the growing importance of CYP2B6 in drug metabolism, the
need for a better understanding of the molecular mechanisms governing *CYP2B6*
gene expression is evident.
Our understanding of mechanisms underlying transcriptional regulation of CYP2B6
expression has grown substantially during the past two decades, and it has been
well-established that induction of CYP2B6 expression by xenobiotics is mediated
primarily by the constitutive androstane receptor (CAR, NR1i3) and the pregnane
X receptor (PXR, NR1i2) through interactions with phenobarbital responsive
enhancer module (PBREM) and xenobiotic responsive module (XREM) located in the
distal region of CYP2B6 promoter. Nevertheless, large interindividual
variability in the expression of CYP2B6 cannot be entirely explained by this
simplified CAR/PXR-based model. Activation of CAR and PXR is essential but not
sufficient for the optimal regulation of *CYP2B6* gene transcription. Ectopic
expression of CAR or PXR alone failed to fully restore the basal and inductive
expression of CYP2B6 in non-hepatic cells or hepatoma cell lines that express
extremely low levels of liver enriched transcription factors (LETFs). Several
previous reports including our own data demonstrated that expression of *CYP2B6*
gene can be influenced by interactions between CAR/PXR and LETFs such as the
hepatic nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα),
suggesting hepatic factors other than CAR and PXR contribute to the large
individual variations in CYP2B6 expression.
The hepatocyte nuclear factor 3β (HNF3β), also known as forkhead box protein A2
(FOXA2), is a DNA-binding protein that is encoded by the *FOXA2* gene in human,
and plays a pivotal role in the regulation of metabolism-related gene expression
in the liver and pancreas. As a hepatic transcription factor, HNF3β influences
the expression of numerous genes involved in energy metabolism, bile acid
homeostasis, drug metabolism and transport by interacting with other LETFs such
as HNF1α and HNF4α, and nuclear receptors such as the glucocorticoid receptor
and PXR. Disruption of HNF3β binding sites located in the promoter of
glucose-6-phosphatase or CYP3A4 repressed the transcriptional activity of each
respective gene. Recently, Lamba et al., reported that hepatic expression of
CYP3A4 was positively correlated with that of HNF3β. Given the pleotropic roles
of HNF3β in hepatic gene regulation and the known transcriptional cross-talk
between CYP2B6 and CYP3A4, we hypothesized that HNF3β may play a role in the
transcription of *CYP2B6* gene and contribute to the observed large inter- and
intra-individual variations in CYP2B6 expression.
In the current study, we provide experimental evidence to demonstrate that
expression of CYP2B6 is closely associated with the expression of HNF3β in human
primary hepatocytes (HPH). Overexpression of HNF3β enhanced expression and
promoter activity of CYP2B6, while knockdown of HNF3β significantly repressed
CYP2B6 expression in HepG2 cells. Utilizing *in silico* analysis, chromatin
immunoprecipitation (ChIP) and surface plasmon resonance (SPR) binding affinity
assays, characterization of the CYP2B6 promoter revealed two functional enhancer
modules that are responsible for HNF3β-mediated CYP2B6 transcription.
# Material and Methods
## Reagents
Phenobarbital (PB) and 6-(4-Chlorophenyl)
imidazo\[2,1-b\]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime
(CITCO) were purchased from Sigma-Aldrich (St. Louis, MO). Oligonucleotide
primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).
The Dual-Luciferase Reporter Assay System was purchased through Promega
(Madison, WI). Antibodies against CYP2B6 and HNF3β were from Santa Cruz (Dallas,
TX). β-Actin antibody was from Sigma-Aldrich. Matrigel, insulin, and
ITS<sup>+</sup> (insulin/transferrin/selenium) were obtained from BD Biosciences
(Bedford, MA). Other cell culture reagents were purchased from Life Technologies
(Grand Island, NY) or Sigma-Aldrich.
## Plasmid Construction
Luciferase reporter plasmids containing -300bp, -387bp, -600bp, -1kb, -1.4kb and
-2kb fragments of the CYP2B6 promoter region were PCR-amplified by using forward
primers: `5’- GGGGTACCTAGACATACATATACCCAC-3’`, `5’-
GGGGTACCCATACAGGGATGCAAGCAG-3’`, `5’- GGGGTACCGGGATTACAGGTGTGAGC-3’`, `5’-
GGGGTACCTCAGCATCTGCAGGCTTC-3’`, `5’- GGGGTACCACACACCTGGAGCTCAAG-3’`, `5’-
GGGGTACCGGACAATGTAGCCCCAACCC-3’` and the same reverse primer: `5’-
AGTCTACTCGAGCTGCACCCTGCTGCAGCCT-3’`. The PCR products were sub-cloned into the
KpnI and XhoI sites of pGL3-basic vector, resulting in constructs termed
2B6-300bp, 2B6-387bp, 2B6-600bp, 2B6-1kb, 2B6-1.4kb and 2B6-2kb, and the correct
orientation was verified by sequencing. The pCR3-hCAR, pMEX-C/EBPα,
pcDNA3.1/HNF3β expression vectors and the CYP2B6 reporter constructs (2B6-1.6kb
and 2B6-1.8kb) were obtained or generated as described previously. pRL-TK was
used as an internal control.
## Site-directed Mutagenesis
Site-directed mutagenesis was performed by PCR, using the 2B6-2kb construct as
the template, based on the protocol of the QuickChange Multi Site-Directed
Mutagenesis Kit from Stratagene (Santa Clara, CA). Mutated nucleotides in the
HNF3β binding sites are underlined for the HNF3β-a-mut:
`CACTAAGAGTGTACCGCCTGAGTTACTGTGTG`, the HNF3β-b-mut: `CCCCTTTACAT
GTACCAGTCATATAAGCACATAC`, and the HNF3β-c-mut: `CAAAGCTAAGTACCAGAGTGCA
AGCTCACC`. The constructs were sequenced to confirm the presence of the
mutation(s).
## HPH Cultures
Human hepatocytes were isolated as described previously from human liver
specimens obtained from University of Maryland Medical Center with prior
approval by the Institutional Review Board at the University of Maryland at
Baltimore or obtained from Bioreclamation In Vitro Technologies (Baltimore, MD).
Hepatocytes with viability over 90% were seeded at 0.75 × 10<sup>6</sup>
cells/well in 12-well biocoat plates in DMEM supplemented with 5% FBS, 100 U/ml
penicillin, 100 μg/ml streptomycin, 4 μg/ml insulin, and 1 μM dexamethasone.
After attachment at 37°C in a humidified atmosphere of 5% CO2, hepatocytes were
cultured in complete William’s Medium E (WME) and overlaid with Matrigel
(0.25mg/ml). Cell culture medium was replaced on a daily basis.
## Real-Time PCR Analysis
Total RNA from human primary hepatocytes was isolated using the RNeasy Mini Kit
(Qiagen, Valencia, CA) and reverse transcribed using High Capacity cDNA Archive
kit (Applied Biosystems, Foster City, CA) following the manufacturers’
instructions. Real-time PCR assays were performed in 96-well optical plates on
an ABI StepOnePlus Real-Time PCR system (Applied Biosystems) with SYBR Green PCR
Master Mix (Qiagen). Primer sequences for the CYP2B6, CAR, HNF3β, C/EBPα, HNF4α,
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are: CYP2B6,
`5’-AGACGCCTTCAATCCTGACC-3’` and `5’-CCTTCACCAAGACAAATCCGC-3’`; CAR, `5’-
GAGCTGAGGAACTGTGTGGTA-3’` and `5’-CTTTTGCTGACTGTTCTCCTGAA-3’`; HNF3β, `5’-
GGAGCAGCTACTATGCAGAGC-3’` and `5’-CGTGTTCATGCCGTTCATCC-3’`; C/EBPα,
`5’-TCGGTGGACAAGAACAGCAA-3’` and `5’-TTTCAGGAGGCACCGGAATCT-3’`; HNF4α,
`5’-CGAAGGTCAAGCTATGAGGACA-3’` and `5’- ATCTGCGATGCTGGCAATCT-3’`; and
GAPDH,`5’-CCCATCACCATCTTCCAG GAG-3’` and `5’-GTTGTCATGGATGACCTTGGC-3’`. Fold
induction values were calculated according to the equation 2<sup>ΔΔCt</sup>,
where ΔCt represents the differences in cycle threshold numbers between the
target gene and GAPDH, and ΔΔCt represents the relative change in these
differences between control and treatment groups.
## Transient Transfection in HepG2 Cells
HepG2 cells obtained from American Type Culture Collection (Manassas, VA) were
transfected with different CYP2B6 reporter constructs in the presence of HNF3β,
with or without hCAR and C/EBPα expression vectors using X-tremeGENE 9 DNA
Transfection Reagent (Roche Diagnostics Corporation, Indianapolis, IN). Twenty-
four hours after transfection, cells were treated with solvent (0.1% DMSO), PB
(1mM), or CITCO (1μM) for 24 h. Subsequently, cell lysates were assayed for
firefly luciferase activities normalized against the activities of co-
transfected Renilla luciferase using Dual-Luciferase Kit (Promega, WI). Data
were represented as mean ± S.D. of three individual transfections.
## HNF3β overexpression and knockdown in HepG2 cells
Twenty-four hours after seeding, HepG2 cells were infected with negative control
adenovirus or increasing concentrations of adenovirus expressing HNF3β purchased
from Vigene Biosciences (Rockville, MD) for 48 h. For HNF3β-RNAi lentivirus, DNA
oligonucleotides encoding short hairpin HNF3β RNA
(`GATCAAGAACATGTCGTCGTACGTGCTCGAGCACGTACGACGACATGTTCTTTTTTTG`) was inserted into
the BamHI and EcoRI restriction sites of the pGreenPuro™ shRNA expression
lentivector from System Biosciences (Mountain View, CA). Lentivirus was prepared
as described previously. In HNF3β knockdown assay, HepG2 cells were infected
with the lentiviral particles for 96 h. Total RNA and Proteins were prepared for
real-time PCR and western blot analysis.
## Western Blot Analysis
Cell homogenate proteins harvested from Ad-HNF3β overexpressing HepG2 cells were
resolved on SDS—polyacrylamide gels (12%) and electrophoretically transferred
onto blotting membranes. Subsequently, membranes were incubated with antibodies
against HNF3β (diluted 1:200), CYP2B6 (diluted 1:200), or β-actin (diluted
1:50,000). Blots were washed and incubated with horseradish peroxidase secondary
antibodies, and developed using enhanced chemiluminescence Western blotting
detection reagent from GE Healthcare (Pittsburgh, PA).
## CYP2B6 Activity Assay
CYP2B6 metabolic activity assay was performed in adenovirus HNF3β infected HepG2
cells on 24-well plate using P450-Glo ™ CYP2B6 Assay kit (Promega, WI) following
the manufacturer’s instruction. In brief, 48 h after HNF3β adenovirus infection,
culture medium was completely removed and cells were washed twice with Krebs-
Henseleit buffer (BioreclamationIVT, MD), followed by incubation at 37°C for 2 h
in 300 μL Krebs-Henseleit buffer containing 3 μM of Luciferin-2B6 (CYP2B6
specific substrate) and 3 mM of salicylamide. Equal volume of Luciferin
Detection Reagent was added into each well, and mixed at room temperature for 20
min before luminescence detection (Promega, WI). Data were represented as mean ±
S.D. of three individual infections.
## Chromatin Immunoprecipitation Assays
Experiments were performed using a ChIP assay kit according to the
manufacturer’s protocol (Millipore Corporation, Billerica, MA). In brief, 1 ×
10<sup>6</sup> cultured human primary hepatocytes were cross-linked with 1%
formaldehyde for 10 min at 37°C, washed with ice-cold phosphate buffered saline
containing a protease inhibitor cocktail. Nuclear extracts were sonicated.
Immunoprecipitation was performed overnight at 4°C using 5μg of anti-HNF3β
antibody (Santa Cruz Biotechnology) or normal rabbit IgG (Cell Signaling
Technology) followed by precipitation using protein A coupled to agarose beads.
After de-crosslinking and protease digestion, DNA fragments were recovered by
QIAquick PCR purification kit (QIAGEN). CYP2B6 promoters containing HNF3β-a
(distal), HNF3β-c (proximal), and the region between -1.4/-1.6k were amplified
by PCR using primers: `5’-TGGACAATGTAGCCCCAACCC-3’` and
`5’-GATTGGGTGCTCATTGCAGCC-3’`; `5’-CTCATACACATGCAAGGATAC-3’` and
`5’-GAGCAAGTGAATGTGTGGG TG-3’`; and `5’-GTCAGGCGTAGGATGAGACAG-3’`and
`5’-TCTTGAGCTCCAGGTGTGTGC-3’`. A proximal promoter region of *SULT1E1* gene was
used as a negative control as reported previously.
## Plasmon Resonance Binding Assay
Recombinant HNF3β protein purchased from Abcam (Cambridge, MA) was covalently
linked to the surface of a BIAcore CM5 sensor chip (BR-1005-30, Lot#10222950) by
direct immobilization as described previously. Oligonucleotides containing
HNF3β-a (`AGAGTGTAAAGACTGAG`), HNF3β-b (`TACATGTAAAAATCATA`) and HNF3β-c
(`GCTAAGTAAAAAAGTGC`) were used as analytes. The binding assay was performed by
injecting 60 μl each of the oligonucleotides at 10 μM in 10 mM Hepes, pH 7.4,
containing 150 mM NaCl, 3 mM EDTA, and 0.005% P-20 at the flow rate of 30 μl per
minute at 25°C. The association and dissociation between analytes and HNF3β
protein were recorded respectively by SPR with a Biacore 3000 (GE Healthcare,
Piscataway, NJ) following the manufacturer’s instructions. Sensorgrams of the
interaction generated by the instrument were analyzed by the software BIAeval
3.2.
## General data analysis
All data represent at least three independent experiments and were expressed as
mean ± S.D. Statistical significance was determined using one-way analysis of
variance followed by post-hoc Dunnett’s test or Student’s t test where
appropriate. Statistical significance was set at *p* \< 0.05 or *p* \< 0.01.
Linear regression was analyzed using Pearson’s Correlation Coefficient (JMP 7.0;
SAS, NC).
# Results
## Correlation of CYP2B6 and HNF3β expression in HPH
Correlation between the expression of a number of hepatic transcription factors
and CYP2B6 was initially evaluated in a collection of HPH prepared from 35 human
liver donors. Basal mRNA expression of these genes in hepatocytes was determined
by real-time PCR assays. As expected, positive correlations were observed
between the expression of CYP2B6 and that of CAR (R = 0.6844; *p* \< 0.01),
C/EBPα (R = 0.5935; *p* \< 0.01), and HNF4α (R = 0.5479; *p* \< 0.01),
respectively. Interestingly, the abundance of CYP2B6 in these donors was also
significantly correlated with the expression of HNF3β (R = 0.5429; *p* \< 0.01).
These results indicate that besides the known role of CAR, HNF4α and C/EBPα in
CYP2B6 expression, HNF3β may represent another LETF that contributes to the
transcription of *CYP2B6* gene in human liver.
## Ectopic expression of HNF3β alters CYP2B6 expression and activity in HepG2 cells
To further investigate the effects of HNF3β on CYP2B6 expression, adenovirus-
driven HNF3β was used to ectopically over-express HNF3β in HepG2 cells. As shown
in, mRNA expression of HNF3β by virus infection was associated with enhanced
expression of CYP2B6 in HepG2 cells in a concentration-dependent manner. A
similar pattern of increased CYP2B6 protein content and enzyme activity was also
observed. On the other hand, knockdown of HNF3β by lentiviral-shRNA was
associated with decreased expression of CYP2B6 in HepG2 cells. Together, these
results indicate that the intracellular level of HNF3β influences expression of
CYP2B6 in HepG2 cells and support the positive correlation between hepatic
expression of HNF3β and CYP2B6 observed in human liver donors.
## HNF3β activates the transcriptional activity of a CYP2B6 reporter gene
It is well-known that expression of CYP2B6 is predominantly regulated at the
transcriptional level. Thus, we next investigated whether HNF3β could alter the
transcriptional activity of CYP2B6 via a putative luciferase construct
containing the first 2 kb of the CYP2B6 5’-flanking region in a pGL3-Basic
reporter vector. This region contains the PBREM regulatory module that responds
to the nuclear receptor CAR. Consistent with the notion that CAR is
constitutively activated in immortalized cell lines, transfection of CAR
robustly increased the luciferase activity of CYP2B6-2kb, while additional
treatment with PB or CITCO only moderately increased the CYP2B6 luciferase
activity. Notably, transfection of HNF3β dramatically enhanced the activation of
the CYP2B6 reporter independent of chemical treatment. Co-transfection of
hCAR/HNF3β/C/EBPα/CYP2B6-2kb resulted in an additive increase of the luciferase
activity in HepG2 cells. Likewise, co-transfection of hCAR and/or HNF3β/C/EBPα
as aforementioned also increased expression of endogenous CYP2B6 mRNA in HepG2
cells. Together, these findings suggest that the first 2 kb of the CYP2B6
promoter may contain specific sequences that coordinate HNF3β-mediated CYP2B6
transactivation.
## Identification of HNF3β-Response Elements in CYP2B6 promoter
To elucidate the molecular basis underlying HNF3β-mediated CYP2B6 transcription,
we carried out an *in silico* analysis of the first 2 kb upstream sequence of
the CYP2B6 promoter using the MatInspector release professional program. Two
clusters of potential HNF3β binding sites were localized at -1887/-1871bp and
-434/-350bp regions. As shown in, three predicted HNF3β responsive motifs in the
two clusters were designated as HNF3β-a, HNF3β-b, and HNF3β-c. Serial deletion
reporter assays revealed that maximal transactivation of CYP2B6 promoter by
HNF3β was achieved with the CYP2B6-2kb construct that contained all predicted
enhancers. Deletion of HNF3β-a or HNF3β-c significantly repressed the role of
HNF3β in CYP2B6 promoter activation, while elimination of HNF3β-b only exhibited
negligible consequence. In site-directed mutagenesis experiments,
transactivation of CYP2B6-luciferase activity through HNF3β was remarkably
attenuated by the mutation of HNF3β-a or HNF3β-c, while only moderately affected
by HNF3β-b mutation. Interestingly, although our *in silico* analysis failed to
predict a consensus HNF3β binding site between -1.6 kb and -1.4 kb, our
luciferase reporter assay showed that deletion of this 200 bp sequence
significantly reduced HNF3β-mediated activation of CYP2B6 promoter, indicating
the existence of a functional yet unidentified HNF3β binding site in this
region. Together, these data suggest that transcriptional activation of CYP2B6
by HNF3β is mediated through multiple enhancer modules including the newly
identified HNF3β binding sites, particularly the HNF3β-a and HNF3β-c motifs.
## Interaction between HNF3β and enhancers identified in the CYP2B6 promoter
Potential physiological recruitment of HNF3β to the CYP2B6 promoter was assessed
using ChIP assays in cultured HPH from two liver donors (HL#98 and \#107). As
demonstrated in, endogenous HNF3β protein was efficiently recruited to the
distal and proximal promoter regions of CYP2B6 containing HNF3β-a and HNF3β-c,
respectively. Lack of binding to the promoter region of SULT1E1 was used as a
negative control as reported previously. Notably, our results showed that HNF3β
protein was also enriched in the -1.6/-1.4 kb region, further supporting the
presence of a functional HNF3β binding site in this region. The binding kinetics
between HNF3β and CYP2B6 enhancers were further validated using a SPR binding
affinity assay. As shown in, DNA sequences (analytes) containing HNF3β-a or
HNF3β-c bind to HNF3β efficiently, while HNF3β-b exhibits undetectable
association with HNF3β. Detailed kinetics calculation revealed that HNF3β-a had
a higher association rate constant (2.42 × 10<sup>5</sup> 1/Ms) in comparison to
that of HNF3β-c (4.82 × 10<sup>3</sup> 1/Ms) though both shared similar
dissociation rate constants (2.04 ×10<sup>−6</sup> and 3.54 × 10<sup>−6</sup>
1/s, respectively). In consequence, HNF3β-a exhibited a higher binding affinity
(KD = 8.45 ×10<sup>−12</sup> M) than HNF3β-c (KD = 7.34 ×10<sup>−10</sup> M).
Together, these results indicate that HNF3β can efficiently interact with the
CYP2B6 promoter with higher binding affinity to the HNF3β-a than the HNF3β-c
motifs, while there is no detectable binding to the HNF3β-b site.
# Discussion
CYP2B6, an inducible cytochrome P450 isoform predominantly present in the liver,
exhibits large intra- and inter-individual variations in human populations.
Expression of CYP2B6 is controlled by many transcription factors including drug
and hormone responsive nuclear receptors such as CAR, PXR, the vitamin D and
glucocorticoid receptors, and constitutively activated LETFs including HNF4α and
C/EBPα. Our current study reveals that HNF3β represents a novel LETF that can
up-regulate *CYP2B6* gene expression by recognizing and interacting with
multiple enhancer modules including two consensus HNF3β binding sites located at
-1887/-1871bp and -347/-331bp upstream of the CYP2B6 transcriptional start site.
Site-directed mutation of these responsive elements resulted in significant
reduction of the CYP2B6 promoter activity. Moreover, HNF3β protein was
efficiently recruited to the CYP2B6 promoter through direct interaction with
these enhancer modules.
To date, human CAR and PXR have been recognized as the major transcription
factors mediating drug-induced CYP2B6 expression. However, optimal expression of
CYP2B6 was only achieved in HPH that maintain physiologically relevant
expression of most LETFs. Hepatocyte nuclear factors are a heterogeneous group
of evolutionarily conserved transcription factors that are pivotal for the
development and maintenance of liver specific features. Positive correlations
between expression of CYP2B6 and HNF4α or C/EBPα (also named HNF2) were
demonstrated previously in human liver samples. Knockdown of HNF4α expression
decreased the mRNA level of CYP2B6, CAR, and PXR in HPH, while ectopic co-
expression of HNF4α, CAR, and C/EBPα markedly increased *CYP2B6* gene
transcription in HepG2 cells. Using HPH from 35 individuals, we found that
expression of CYP2B6 was positively associated with the expression of HNF3β, in
addition to its known correlation with CAR, HNF4α and C/EBPα. These initial
observations led to a more focused investigation on the role of HNF3β in hepatic
CYP2B6 transcription.
Regarded as one of the master regulators of hepatocyte differentiation and
maturation, HNF3β regulates the expression of numerous hepatic genes by
interacting with respective cis-acting binding elements in the promoters of
these genes, including the gluconeogenic phosphoenolpyruvate carboxykinase,
insulin-like growth factor-binding protein 1, and tyrosine aminotransferase.
Unlike ligand-activated transcription factors, presence of the HNF3β protein
alone in hepatocytes is sufficient to trigger downstream signaling pathways and
the abundance of HNF3β protein often correlates well with the expression of its
target genes. Additionally, cooperation between LETFs and ligand-activated
nuclear receptors can contribute to tissue-specific increase or decrease in
expression of nuclear receptor target genes. For instance, overexpression of
HNF4α facilitates CAR- and PXR-mediated induction of CYP2B6, CYP3A4, and CYP2C9,
while HNF4α-mediated expression of CYP7A1, a key enzyme in cholesterol
metabolism and bile acid synthesis, was down-regulated by competing with CAR for
binding to the direct repeat 1 (DR1) motif in the promoter of CYP7A1. We have
previously shown that a single nucleotide polymorphism introducing a functional
C/EBPα binding site at the proximal region of CYP2B6 promoter synergistically
enhanced PXR-mediated induction of this gene. In the current study, we found
that overexpression of HNF3β increased the activity of CYP2B6 reporter
construct, and the endogenous expression of CYP2B6 mRNA and protein in HepG2
cells. Notably, although overexpression of either CAR or HNF3β increased CYP2B6
promoter activity, their combination only produced additive effects on the
transactivation of CYP2B6 in transfected HepG2 cells, suggesting CAR and HNF3β
may influence CYP2B6 expression independently rather than collaboratively.
Subsequent computer-based analysis revealed the presence of three potential
HNF3β binding sites at -1887/-1871bp, -347/-331bp, and -428/-412bp regions of
the first 2 kb of the CYP2B6 promoter. Luciferase reporter assays demonstrated
that maximal activation of CYP2B6 reporter by HNF3β requires the presence of all
three elements. Of importance, serial deletion and mutation assays further
revealed the importance of the HNF3β-a and HNF3β-c motifs, while HNF3β-b only
exhibited minimal activity. Intriguingly, although no consensus HNF3β binding
site(s) was predicted from our *in silico* analysis in the -1.6k to -1.4k region
of the CYP2B6 promoter, deletion of this region unexpectedly resulted in a
significant reduction of CYP2B6 luciferase activity, indicating the presence of
an unidentified HNF3β binding site which warrants more detailed investigation in
the future.
In characterizing the recruitment of HNF3β to the promoter of CYP2B6 in a more
physiologically relevant system, results from our ChIP assays in HPH confirmed
the interaction between HNF3β protein and chromatin regions harboring the
HNF3β-a and HNF3β-c motifs of *CYP2B6* gene. It is worth noting that HNF3β was
also co-precipitated with the DNA fragment covering the -1.6/-1.4bp region,
where an unidentified HNF3β binding motif was speculated. However, given the
proximity between the -1.6/-1.4bp and the HNF3β-a containing (-1887/-1871bp)
areas, such recruitment may only represent a HNF3β-a dependent phenomenon.
Quantitative kinetics for binding affinities between HNF3β and the three
predicted binding sites were further estimated by a SPR approach. Consistent
with results of the luciferase reporter and ChIP assays, our SPR affinity assays
revealed that the HNF3β-a and HNF3β-c elements were efficiently recruited to the
HNF3β protein, while the HNF3β-b containing region appeared not to be directly
associated with HNF3β.
In conclusion, our data show that HNF3β represents a novel LETF that regulates
the transcription of *CYP2B6* gene, in addition to HNF4α and C/EBPα. Ectopic
expression of HNF3β in HepG2 cells was associated with increased CYP2B6
transactivation and endogenous expression. Such enhancement can be at least
partly explained by the identification and functional characterization of two
consensus HNF3β binding sites located in the 5’-flank of CYP2B6 upstream. These
findings reveal additional mechanistic bases in our understanding of *CYP2B6*
gene transcription, and suggest that expression of CYP2B6 is governed by a
complex regulatory network including genes such as, CAR, PXR, HNF4α, C/EBPα, and
HNF3β. It is worth noting that multiple cis-acting responsive elements for
C/EBPα and HNF4α have also been localized within the first 2 kb of the CYP2B6
promoter. Future studies to elucidate the mechanisms by which these LETFs work
in concert with CAR/PXR to confer the optimal expression of CYP2B6 are
warranted. Given that expression of HNF3β can be disturbed by the fluctuation of
steroids and hormones, altered expression of HNF3β may contribute to the large
interindividual variations in hepatic CYP2B6 expression.
The authors thank Dr. Yinghua Zhang (Department of Physiology, University of
Maryland School of Medicine, Baltimore, MD) for assistance in the Plasmon
Resonance Binding analysis. We are also grateful to members of the Wang
laboratory for discussions and comments on the manuscript.
[^1]: The commercial affiliation of SH with Bioreclamation IVT does not
alter the authors' adherence to PLOS ONE policies on sharing data and
materials.
[^2]: Conceived and designed the experiments: HW. Performed the experiments:
LL DL SH. Analyzed the data: LL DL. Contributed reagents/materials/analysis
tools: SH. Wrote the paper: LL SH HW. |
# Introduction
Neurons are specialized cells responsible for exchanging information with other
neurons or cells through synapses. During development, differentiating neurons
explore the surrounding environment in order to form the correct contacts and
they use highly motile structures called growth cones (GCs) located at the tip
of their neurites. GCs consist of a flat extension, named ‘lamellipodium’ with
varying width from which finger-like submicron diameter structures called
filopodia emerge. The process of polymerization of actin filaments is the main
source of GC protrusion, which is regulated and controlled by several proteins
such as Arp2/3, cofilin, formin and molecular motors, such as myosin, dynein,
controlling different features of cellular motility.
Actin related protein 2/3 complex (Arp2/3) is widely studied for its involvement
in lamellipodia formation and protrusion. Arp2/3 consists of seven subunits and
promotes the formation of branched actin filament networks. Arp2/3 not only
regulates the branching of actin filaments but it is also involved in the
formation and dynamics of filopodia. Inhibition of Arp2/3 causes lamellipodia
retraction and an increase of the actin retrograde flow rate. Arp2/3 is inactive
in its native state and the members of the Wiskott-Aldrich syndrome protein
(WASP) family, downstream of Rac and Cdc42 pathways activate the Arp2/3 complex
to nucleate new filaments. Rac binds the WAVE (WASP family Verprolin Homology
Domain-containing protein) complex to release active WAVE, which promotes actin
polymerization through activation of Arp2/3. WASP and WIP (WASP-interacting
protein), downstream effectors of Cdc42 interact directly with Arp 2/3 complex
to promote filopodia formation. Recently a new protein called Arpin has been
shown to be part of the Rac-Arpin-Arp2/3 inhibitory circuit playing a major role
in steering during cell migration. Rac can both activate and inhibit
Arp2/3-driven actin branching and polymerization to regulate speed,
directionality and persistence of membrane protrusions.
Rho family GTPase has distinct and specific roles in the regulation of growth,
maintenance and retraction of GCs. The mammalian Rho GTPase family currently
consists of three subfamilies, Rho (RhoA, RhoB and RhoC), Rac (Rac1, Rac2 and
Rac3) and Cdc42 (Cell Division Cycle-42) (Cdc42Hs and G25K). RhoA, Rac1 and
Cdc42 are well-studied members of Rho family GTPase controlling distinct
cytoskeletal elements. Activation of Rac1 stimulates actin polymerization to
form lamellipodia, Cdc42 induces the polymerization of actin to form filopodia
or microspikes which are parallel actin bundles within the lamellipodium and Rho
regulates the bundling of actin filaments into stress fibers and the formation
of focal adhesion complexes. The Rho family of GTP-binding proteins are
activated by a variety of growth factors, cytokines, adhesion molecules,
hormones, integrins, G-proteins and other biologically active substances.
Biochemical approaches or analyses of the morphology of fixed cells have shown
that Rho GTPase also involves crosstalk. This may occur through the Rac/Cdc42
effecter PAK, which can negatively regulate Rho GEFs or other mechanisms
including, via reactive oxygen species, phosphorylation and competitive binding
of RhoGDI or binding of GEFs to actomyosin. Depending upon the concentration
and localization of these Rho GTPase, mammalian cells show different morphology,
movement and behavior.
When the rate of actin polymerization overtakes the actin retrograde flow, the
GC protrudes. Retrograde flow refers to the backward flow of the actin filament
network away from the growth cone leading edge into the C-domain. This allows
the addition of actin monomers/oligomers to actin filaments in close contact
with the membrane, pushing the cellular membrane forward, leading to the
protrusion. Mitchison and Kirschner proposed the ‘Molecular Clutch Hypothesis’,
which postulates that an intracellular molecular clutch, formed by interactions
between GC membrane adhesive receptors and the extracellular environment, couple
to the overlying flow of actin filaments to slow down their retrograde rate.
Formation of these ‘clutches’ together with myosin II contractile activity,
provides a traction to pull and move the central region of the GC closer to the
peripheral region, leading to axonal lengthening. Therefore, substrate adhesion
decreases the actin retrograde flow. The decrease in the actin retrograde flow
together with actin polymerization, results in the leading edge protrusion.
The traction force is essential for cell migration, cell shape maintenance,
mechanical signal generation and other cellular functions. There are different
methods to quantify the cellular traction forces. Traction force microscopy
measures the stress of a cell on an elastic gel substrate by detecting the
movement of fluorescent beads embedded at the surface of the gel. With Optical
Tweezers the bead is attached to the cell membrane either to apply the tensile
force or to measure the retrograde flow rate. We have independently developed a
method to estimate the force exerted by the lamellipodia and filopodia by
measuring the displacement of the bead using quadrant photo detector (QPD). In
our case, the bead is not initially attached to the cell membrane and it is kept
in the vicinity of the lamellipodia or filopodia, so that their spontaneous
motion can displace the bead.
We observed that the lamellipodia transiently retract and recover back after 5–8
min of Arp2/3 inhibition. In this study we have investigated the role of Rac1,
in the recovery of lamellipodia in Arp2/3 depleted condition and also in GC
motility, by using Optical Tweezers and specific inhibitors of Arp2/3 (CK-548)
and Rac1 (EHop-016 and F56). Motility of lamellipodia and of filopodia was also
followed and characterized by video imaging. By combining these techniques
together with immunofluorescence we have explored the interaction between Rac1
and Arp2/3 complex and their role in the formation of lamellipodia and filopodia
of Dorsal root Ganglion (DRG) GCs. Here we show that Rac1 acts as a switch and
activates upon inhibition of Arp2/3.
# Results
After 6–8 hours of culture, differentiating DRG neurons have neurites emerging
from their soma. At the tip of the protruding neurites, GCs lamellipodia and
filopodia explore the environment and their motion continues for 1–3 days. The
motility of lamellipodia and filopodia slows down when appropriate connections
are established and the neuronal network is formed; the leading edge of these
lamellipodia can move with a speed 30–100 nm/s exerting a force exceeding 20 pN.
The effect of the inhibitors of specific proteins involved in the regulation of
GC motility was analyzed after 24–48 hours of culture, when the motility of
filopodia and lamellipodia was more pronounced. We focused on the analysis of
inhibitors of small GTPases and of the Arp2/3 complex.
We used the small molecules CK-636, CK-548, CK-666 and CK-869 as inhibitors of
the Arp2/3 complex. All these compounds at a high concentration, i.e. above 100
μM, abolished GC motility completely and in the experiments here described we
used extensively CK548 (CK) as the Arp2/3 inhibitor, since CK decreases the
affinity of rhodamine-N-WASP-VCA for BtArp2/3 complex approximately twofold.
Furthermore, we tested two inhibitors of Rac1 namely, EHop-016 (EH) and F56 and
the Cdc42 inhibitor ZCL-278 (ZCL). In addition to these drugs, CT04 (CT) and GSK
269962 (GSK) were also used as inhibitors of the RhoA and Rock pathways
respectively. In order to check if the effect of the inhibitors was a side
effect of toxicity we also checked their reversibility after washout (WO), as
shown in.
## The effect of partial inhibition of Rac1 and Arp2/3 in lamellipodia motility
The involvement of Rac1 and Arp2/3 in lamellipodia motility of DRG GCs was
studied by analyzing the effect of their inhibitors EHop-016 (EH) and CK-548
(CK) respectively and by quantifying lamellipodia motility using the two
algorithms as described in the Materials and Methods section, based on the
analysis of Z-stack phase contrast video imaging. From the image sequences,
kymographs were obtained by using algorithm I. The ability of lamellipodia to
lift up vertically was quantified by computing the fraction of pixels in focus
at 5 μm above the coverslip obtained by using algorithm II.
When Rac1 activity was inhibited by 20 μM EH lamellipodia still exhibited
protrusion retraction cycles (upper panel) and could lift up in the axial
direction (lower panel). EH effects were reversible and period, persistence
length, retrograde flow rate of lamellipodia returned to control level after
washout. Interestingly, lamellipodia of DRG GCs, treated with 50 μM CK showed a
transient retraction and were not able to lift up vertically in a significant
manner. However, treated lamellipodia recovered their usual motility in 5–8 min
(upper panel) and were able to lift up in the axial direction as in control
conditions (lower panel). Following 50 μM CK treatment, period, persistence
length and retrograde flow rate of lamellipodia were quantified during
lamellipodia recovery period. The average period of protrusion/retraction cycles
of lamellipodia increased significantly, both in the presence of 20 μM EH
(129.6±5.2 s) and of 50 μM CK (115.1±4.2 s) respectively compared to control
conditions (86.5±3.2 s) and after washout of 20 μM EH (90.3±4.1 s). The
persistence length of lamellipodia i.e the maximum extension reached by the
lamellipodia after which they start to retract, increased when Arp 2/3 was
inhibited by 50 μM CK (1.90±0.09 μm) compared to control conditions (1.48±0.07
μm) and after washout of 20 μM EH (1.42±0.1). However, there was no significant
change in the persistence length of lamellipodia when Rac1 was inhibited
(1.56±0.09 μm), but the lamellipodia retrograde flow rate decreased when Rac1
was inhibited (0.05±0.007 μm/s) compared to what observed in control conditions
(0.08±0.005 μm/s), after washout of 20 μM EH (0.07±0.008 μm/s) and in the
presence of Arp2/3 inhibitors (0.09±0.009 μm/s).
## Rac1 activates when Arp2/3 is inhibited
When the activity of Arp2/3 was inhibited by 100 μM of CK lamellipodia shrank
and their motility was completely and permanently suppressed. The growth cone
also lost the adhesion to the substrate and retracted towards the soma (data not
shown). Remarkably, when DRG neurons were treated with 50 μM CK, lamellipodia
showed a transient retraction that continued for 5–8 minutes, but then
lamellipodia recovered their usual motility restoring protrusion and retraction
cycles and were also able to lift up vertically almost as under control
conditions. The results of these experiments suggest that following a partial
inhibition of Arp2/3 another pathway is activated rescuing—to some extent—the
usual GC motility. To test this possibility and to identify the origin of the
recovery of motility in treated lamellipodia, we considered the Rho GTPase
pathways, known to regulate many aspects of intracellular actin dynamics and GC
metabolism. The most extensively studied members of Rho GTPase family are Rho A,
Rac1 and Cdc42. Rac can not only regulate actin polymerization but it can also
increase the availability of free actin-barbed ends by the removal of capping
proteins and it can also increase the availability of actin monomers by
regulating cofilin. These roles of Rac1 could help in the formation and
protrusion of lamellipodia by polymerizing the pre-existing branched actin
filaments at the leading edge of the lamellipodia, in Arp2/3 depleted condition.
In addition, the newly formed actin branches generated by the remaining Arp2/3
can contribute to the lamellipodia protrusion. Therefore, we hypothesized that
Rac1 could mediate the recovery of motility observed in. Lamellipodia that were
first treated with 20 μM EH exhibited an increase in the period of
protrusion/retraction cycles and could move up in the axial direction. Then, the
same lamellipodia were treated also with 50 μM CK: in this case, as expected,
lamellipodia shrank but could not recover their motility even after 10–20
minutes of exposure to these inhibitors. We tested also the simultaneous
application of 20 μM EH and of 50 μM CK, which were mixed and added to the
medium bathing of the neuronal culture at the same time. Lamellipodia exposed
simultaneously to the two inhibitors retracted and did not show any sign of
motility even after 10–20 minutes.
The above results indicate that Rac1 could be behind the recovery of
lamellipodia which were transiently retracted after Arp2/3 inhibition. However,
the Rac1 inhibitor EHop-016 inhibits both Rac3 as well as Cdc42 above the
concentration of 3μM. Therefore, in order to examine the possible role of
the Cdc42 pathway, we used ZCL as a selective inhibitor which is known to target
the binding site of the Cdc42 guanine nucleotide exchange factor, intersectin
(ITSN) and to hinder Cdc42 activation. When 50μM ZCL was added lamellipodia
did not show significant changes in their motility. Subsequent exposure of 50 μM
CK to the same lamellipodia shrank the lamellipodia as usual, but then
lamellipodia did recover after approximately 8 minutes of exposure.
To dispose the possibility of Rac3, in the recovery of lamellipodia, in Arp2/3
inhibited condition, we used F56 as another specific Rac1 inhibitor. It is a
control peptide version of Rac1 Inhibitor W56; comprises residues 45–60 of Rac1
with Trp56 replaced by Phe, which does not affect GEF-Rac1 interaction. When
lamellipodia were treated with 100μM F56, lamellipodia did not show significant
changes in their motility. The same lamellipodia were then exposed to 50 μM CK,
the lamellipodia shrank as usual, but then lamellipodia did not recover even
after 10–20 minutes of exposure. We tested also the simultaneous application of
100 μM F56 and of 50 μM CK, which were mixed and added to the medium bathing of
the neuronal culture at the same time. Lamellipodia exposed simultaneously to
the two inhibitors retracted and did not show any sign of motility after 10–20
minutes (data not shown).
In the Arp2/3 depleted situation, in order to see the role of RhoA and Rock in
the lamellipodia recovery, lamellipodia were exposed to CT (Rho A inhibitor)
and GSK (ROCK inhibitor) (data not shown) independently, before the treatment
with CK. In both situations lamellipodia recovered after 8 minutes of exposure
and, at the end of their retraction, they were also able to reach the same
height as in control conditions. We analyzed in detail the growth cone dynamics
in the presence of the inhibitors of CDC42, RhoA and ROCK signaling pathways.
The period of lamellipodia protrusion/retraction cycles in the presence of ZCL
(101.7±2.6 s), CT (113.6±2.9 s) and GSK (107.8±2.9 s) increased when compared
with the control conditions (86.5±3.2 s). The lamellipodia persistence length
also increased in the presence of CT (1.92±0.13 μm) and GSK (1.8±0.11 μm) but
remained constant in the presence of ZCL (1.35±0.07 μm) when compared to control
conditions (1.48±0.07 μm). The retrograde flow rate decreased in the presence of
ZCL (0.05±0.006 μm/s) but remained constant in CT (0.08±0.01 μm/s) and GSK
(0.09±0.01 μm/s) when compared to control conditions (0.08±0.005 μm/s).
These results discard the involvement of the Rac3, Cdc42, RhoA and Rock pathways
in the recovery of lamellipodia which were transiently retracted after the
inhibition of Arp2/3. It also indicates that Rac1 is crucial for the recovery of
the transient retraction of lamellipodia due to inhibition of Arp2/3.
To confirm the role of Rac1 during the recovery of the transient lamellipodia
retraction due to 50 μM CK, we examined the Rac1-GTP level (the activated form
of Rac1) in cultured DRG neurons with different exposure conditions of CK by
using the G-LISA Rac 1 activation assay kit (Cytoskeleton, Inc., Denver, Colo.).
The Rac1-GTP level, in the presence of 25 and 50 μM CK for 2 minutes did not
show any significant change compared to the control conditions. However, the
Rac1-GTP level significantly increased in the presence of 50 μM CK for 8 minutes
(P\<0.005) i.e. the time during which lamellipodia recovered motility after the
transient retraction. These results confirm that Rac1 activates upon inhibition
of Arp2/3.
## Effect of Arp2/3 and Rac1 inhibitors on the force exerted by lamellipodia
Optical Tweezers was used to investigate the effect of the partial inhibition of
Rac1 and Arp2/3 on the force exerted by lamellipodia. Lamellipodia in control
conditions pushed the trapped beads with a force up to 10–20 pN as previously
described and often beads could be displaced out of the optical trap. The forces
were measured from the same lamellipodia in control conditions and in the
presence of the inhibitors. Exerted forces were analyzed according to four
different stereotyped behaviors previously described, depending on the direction
in which the lamellipodia were exerting the force on the bead: vertical push
(VP), vertical retraction (VR), lateral push (LP) and lateral retraction (LR).
Lamellipodia of DRG treated with a moderate concentration of Rac1 and Arp2/3
inhibitors were able to pull and push a trapped bead, but with a lower force
compared to the force observed in control conditions. In the lateral direction:
in case of LP the lamellipodia force decreased by 30–40% with an increase in the
inhibition of Rac1, however it decreased by 50–65% when Arp2/3 was inhibited
compared to control conditions. The retractile force LR decreased by 40% when
Rac1 was inhibited by 10μM EH, inhibition of Rac1 by 20 μM EH decreased the LR
force more than 70% probably due to a decrease in the lamellipodia retrograde
flow rate. The retractile force LR decreased by 65% when Arp2/3 was inhibited.
In the axial direction: when Rac1 was inhibited by 10 μM EH, the lamellipodia
force in VP and VR decreased more than 60%. Besides, it decreased more than 75%
in all the other VP and VR cases.
These results suggest that lamellipodia were not able to explore the surrounding
environment with an equal force when Rac1 and Arp2/3 were inhibited when
compared to control conditions. In addition, lamellipodia were not able to exert
a larger force in the axial direction than in the lateral direction, when
compared with the control conditions state.
## The effect of Rac1 inhibitors on the rate of lamellipodia protrusion
Lamellipodia in the presence of 10–20 μM EH exerted a lower force but were still
able to extend. In order to measure their rate of protrusion, we used the
Nanopositioner feedback which allows a precise and continuous
measurement of the bead position by employing Optical Tweezers.
All the measurements obtained using the nanopositioner feedback mechanism were
compensated as explained in the Materials and Methods section. In each case, the
total displacement of the bead in the lateral direction was computed. In each
case—control conditions, 10 μM EH and 20 μM EH- 5–6 of such traces were averaged
and plotted against time. Before averaging, traces were aligned so that their
rising phase matched each other. The slopes of these traces were calculated to
determine the lamellipodia protrusion rate. In control conditions, the speed of
protrusion of lamellipodia could reach 100 nm/s (see black trace) and was
reduced to 30–50 nm/s in the presence of 10 μM EH (blue trace in) and to 10–20
nm/s in the presence of 20 μM EH (magenta trace).
These results indicate that inhibition of Rac1 has a similar effect on the
amplitude of the force exerted by lamellipodia and on their protrusion rate.
## Effect of Arp2/3 and Rac1 inhibitors on the force exerted by filopodia and their motility
The filopodia motility and the force exerted by them were quantified by video
imaging, immunocytochemistry and Optical Tweezers. The protruding filopodia tips
were followed in different frames to calculate the filopodia protrusion rate,
and the maximum length of the filopodia was measured as described in the
Materials and Methods section.
In DRG GC the length of the filopodia increased by 60 to 80% when Arp2/3 was
inhibited by 25 and 50 μM CK respectively. When Rac1 was inhibited by 10 μM EH
the length of the filopodia increased by 20%. Remarkably, the filopodia length
increased more than the double when Rac1 was inhibited by 20 μM EH compared to
control conditions. The GCs were then fixed and stained with Alexa 488
phalloidin and imaged to observe the actin localization. The longer filopodia
protruded from the GCs after the inhibition of Rac1 with 20 μM EH and showed an
increase in the total F-actin compared to the controlled filopodia.
The protrusion rate of filopodia did not change when Rac1 and Arp2/3 were
suppressed by their respective inhibitors with a lower concentration. However,
it increased by 30% when Rac1 was inhibited by 20 μM EH. In this case, the
extension of the filopodia length could be the effect of this increase in the
filopodia protrusion rate together with the decrease of the lamellipodia
retrograde flow rate. Surprisingly, the filopodia protrusion rate decreased by
30% when Arp2/3 was inhibited by 50 μM CK.
Inhibition of Rac1 and Arp2/3 significantly decreased the force exerted by
lamellipodia; however, the force exerted by filopodia did not change when Rac1
was inhibited and, with a lower concentration of its inhibitor, Arp2/3 was
suppressed, compared to control conditions. Very rarely filopodia emerged from
lamellipodia exerted a force that is larger than 4 pN in control conditions. The
forces exerted by filopodia were measured in the same neuron before and after
the addition of inhibitors of Rac1 or Arp2/3. In each case collected data from
10 neurons showed that the filopodia force did not changed when Rac1 was
inhibited by 10–20 μM EH and when the Arp2/3 was inhibited by 25 μM CK.
Inhibition of Arp2/3 with 50 μM CK decreased the filopodia force by 20% when
compared to control conditions.
# Discussion
In this study we have characterized the role of Rac1 and Arp2/3 in the motility
and force exerted by lamellipodia and filopodia of DRG GCs. Our results suggest
that in neuronal growth cones, Rac1 acts as a switch that activates following
the inhibition of Arp2/3. Moreover, Arp2/3 and Rac1 not only control the force
exerted by lamellipodia but also the dynamics of filopodia.
## The effect of the inhibition of Rac1 and Arp2/3 on lamellipodia motility
We followed and quantified the protrusion/retraction cycles of DRG lamellipodia
by measuring their period, persistence length and retrograde flow rate using
kymographs (see in the section).
Lamellipodia treated with a small amount of Rac1 and Arp2/3 inhibitors increased
the period of their protrusion/retraction cycles. When Rac1 was inhibited, the
lamellipodia retrograde flow rate decreased, leading to a longer retraction time
and overall cycle period. These effects were then returned to the control level
after the washout of the Rac1 inhibitor, 20 μM EH indicating that the inhibitor
was not toxic to the neuronal growth cone. When Arp2/3 was inhibited, the
lamellipodia retrograde flow rate remained constant but the persistence length
increased. The combination of these two effects increased the period of
protrusion/retraction cycle.
The actin retrograde flow level decreased after the Arp2/3 complex was knocked
down with siRNA in primary cultured hippocampal neurons and neuroblastoma cells
but increased when the Arp2/3 complex was inhibited by CK666 and CK869. We
found that partial inhibition of Arp2/3 with 50 μM CK548, did not affect the
lamellipodia retrograde flow rate after the recovery of lamellipodium motility.
## Recovery of motility following partial inhibition of Arp2/3
When Arp2/3 was inhibited by 100 μM CK548 the growth cone dynamics was
completely abolished presumably because of the loss of adhesion. However, when
Arp2/3 was partially inhibited by 50 μM CK548, lamellipodia transiently shrank
for 5–8 minutes but then recovered their usual motility. The Rho family of
GTPase signaling proteins plays a pivotal role in regulating actin cytoskeleton
and could be involved in the observed recovery of lamellipodia motility. The
best characterized small GTPases of the Rho family are Rac1, Cdc42 and RhoA
which act as molecular switches, cycling between an active GTP-bound state and
an inactive GDP-bound state. To determine the possible role of Rho GTPase
signaling pathways, in the transient retraction and recovery of lamellipodia
when Arp2/3 was inhibited, we used selective inhibitors of Rac1, Cdc42 and RhoA.
Lamellipodia treated with the inhibitor of Rac1, EH showed an increase in their
period of protrusion/retraction cycle and could move in the axial direction. The
lamellipodia regained their motility after the washout of the EH, which show
that the inhibitor effect on the neuronal growth cone was not due by its
toxicity. When the same lamellipodia were later treated also with 50 μM CK548,
lamellipodia showed the usual retraction but did not recover even after 10–20
minutes. Moreover, when treated with both Rac1 and Arp2/3 inhibitors together,
lamellipodia shrank as usual but again they did not recover after 10–20 minutes
of exposure. The higher concentration of EH not only inhibit Rac1 but also Rac3
and Cdc42 in the MDA-MB-435 metastatic cancer cells. To rule out the
possibility that the Rac3 and Cdc42 could be behind the recovery of transiently
retracted lamellipodia in the Arp2/3 inhibited condition we tested the effect of
the specific inhibitor of Cdc42, ZCL and of another specific inhibitor of Rac1,
F56. Transiently retracted lamellipodia following partial Arp2/3 inhibition
condition did recover also in the presence of the Cdc42 inhibitor, ZCL.
Transiently retracted lamellipodia following Arp2/3 inhibition did not recover
when treated also with 100 μM F56.
The level of activated Rac1 following an exposure to 50 μM CK for 8 minutes
significantly increased compared to what observed in control conditions, 25 μM
CK(2 min) and 50 μM CK(2 min). These results indicate the specific role of Rac1
in the observed recovery of motility following partial inhibition of Arp2/3.
A possible mechanism could be mediated by the Integrin pathways. Jacquemet, G.
*et al*. suggested that the engagement of integrin followed by filamin-A, IQGAP1
and RacGAP1 enrollment, deactivates Rac1. Ilić, D. *et* al. and Saunders, R.
M. *et al*. reported that Arp2/3 is recruited to nascent integrin adhesions
through interaction with FAK and vinculin, where it reinforces the link between
integrin and the cytoskeleton. Furthermore, Beckham et al. reported that Arp2/3
inhibition impairs integrin, an extracellular membrane attachment resulting in
either a translocation or treadmilling of mature adhesions. Therefore, it is
possible that inhibition of Arp2/3 could reduce the ligation and clustering of
integrins and further suppress filamin-A, IQGAP1 and RacGAP1 recruitment,
leading to an enhancement of Rac1 activity. Rac1 not only regulates actin
polymerization but also increases the free actin-barbed ends and actin monomers.
Therefore, the enhanced Rac1 activity could promote the formation and protrusion
of lamellipodia, stimulating Arp2/3 by activating the WASP/WAVE family
proteins.To study the role of other Rho GTPase pathways in the transient
retraction and recovery of lamellipodia upon Arp2/3 inactivation, inhibitors of
the respective pathways were used. Detailed quantification showed that the
lamellipodia persistence length significantly increased after CT and GSK
treatment but it remained constant after ZCL treatment, which is also consistent
with what previously reported. The increase in the lamellipodia persistence
length upon CT and GSK treatment is probably due to the crosstalk between RhoA
and Rac1. The lamellipodia retrograde flow rate significantly decreased after
ZCL treatment; however, it remained constant when treated with CT and GSK. As
previously shown, CDC42 promotes retrograde flow rate thus the observed
significant decrease in the lamellipodia retrograde flow rate is the direct
result of CDC42 inhibition. Lamellipodia treated with CDC42 inhibitor increased
the period of their protrusion/retraction cycles. When CDC42 was inhibited, the
lamellipodia retrograde flow rate decreased, leading to a longer retraction time
and overall cycle period. When RhoA and ROCK were inhibited, the lamellipodia
retrograde flow rate remained constant but the persistence length increased. The
combination of these two effects increased the period of protrusion/retraction
cycle. These results show that inhibitors of CDC42, RhoA and ROCK were
functioning appropriately. In addition to that, in all these cases lamellipodia
showed recovery when treated with Cdc42, RhoA and ROCK inhibitors before
treatment with Arp 2/3 inhibitor. Therefore, the involvement of these pathways
in the recovery of lamellipodia motility can be discarded.
## Arp2/3 controls the formation and dynamics of filopodia
In the active states Rac1, Cdc42 and RhoA interact not only with their specific
downstream targets but also cross talk. Specifically, activation of Cdc42
triggers a localized activation of Rac1, initiating the filopodia formation. In
our experiments the presence of actin was confirmed in the filopodia before and
after the inhibition of Rac1 by using immunocytochemistry. Inhibition of Rac1
remarkably increased the protrusion speed as well as the maximum length of the
filopodia. Since Rac1 inhibition reduces the activation of Arp2/3, it is
possible that Rac1 inhibition decreases the formation and protrusion of
lamellipodia, leaving filopodia behind. In addition, a decrease in the
lamellipodia retrograde flow rate and a stable persistence length due to Rac1
inhibition are expected to cause an accumulation of actin at the peripheral
region of the GC, from where the filopodia emerge. A higher concentration of
actin at the base of the filopodia enables the growth of substantially longer
filopodia.
Korobova et al. found that inhibition of Arp2/3 reduced the lamellipodia
protrusion as well as filopodia formation and dynamics. In our case we found
that Arp 2/3 inhibition decreased the protrusion speed of filopodia but it
increased their maximum length. Inhibition of Arp2/3, increased the lamellipodia
persistence length and the retrograde flow rate, which will accumulate less
actin at the periphery of the GC. This may possibly lead to a decrease in the
protrusion speed of filopodia. Moreover, Arp2/3 is required for the formation of
filopodia and inhibition of Arp2/3 could decrease the formation of new
filopodia. The actin accumulated at the periphery of the growth cone upon Arp2/3
inhibition can be utilized by the remaining filopodia to form longer filopodia.
We also found that when Arp2/3 was inhibited, the force exerted by filopodia
decreased compared to control conditions. The above results indicate the direct
involvement of the Arp2/3 in the formation and dynamics of filopodia. On the
other hand, Rac1 inhibition increased the length of filopodia but it did not
change the force they exerted. This indicates that, like Arp2/3, Rac1 may not
directly take part in the formation and dynamics of filopodia.
In conclusion, we show here that Rac1 activates when Arp2/3 is inhibited
possibly through the Integrin pathways acting as a feedback. Besides its role in
lamellipodia formation, Arp2/3 is directly involved in the formation and
dynamics of filopodia, while Rac1 is not involved in the activity of the force
generation of filopodia.
# Materials and Methods
## Neuron preparation
Wistar rats at postnatal days 10–12 (P10-P12) were sacrificed by decapitation
after anesthesia with CO<sub>2</sub> in accordance with the Italian Animal
Welfare Act. The Ethics Committee of the International School for Advanced
Studies (SISSA-ISAS) has approved the protocol (Prot.n. 289-II/7). After
dissection, Dorsal Root Ganglia (DRG) were incubated with trypsin (0.5 mg/ml;
Sigma-Aldrich, Milan, Italy), collagenase (1mg/ml; Sigma-Aldrich) and DNase (0.1
mg/ml; Sigma-Aldrich) in 5 ml Neurobasal medium (Gibco, Invitrogen, Milan,
Italy) in a shaking bath (37°C, 35–40 min). After mechanical dissociation, they
were centrifuged at 300 rpm, resuspended in the culture medium and plated on
poly-L-lysine-coated (0.5 μg/ml; Sigma-Aldrich) coverslips. Neurons were
incubated for 24–48 h and nerve growth factor (50 ng/ml; Alomone Labs,
Jerusalem, Israel) was added before performing the measurements.
## Quantification of lamellipodia and filopodia motility
Neurons were maintained at 37°C in the sample holder of the microscope stage
capable of moving in X and Y directions with nanometer precision and imaged
through 100 X oil immersed, 1.4 NA objective lens mounted on an inverted
microscope (IX80, Olympus). Stacks of phase contrast images of neurons from DRG
ganglia were obtained by Charge couple device (CCD) camera (Olympus Megaview)
and by moving the objective lens vertically. Each stack contains images obtained
in the focal plane of the objective, focused on the coverslip where neurons were
cultured i.e. at height 0 and at 1, 2, 3, 4, 5 and 6 micron above the coverslip.
Stacks of images were acquired with 0.1–1 Hz frequency to quantify the 3D motion
of lamellipodia. Then, for a further analysis, the time lapse image sequence for
each height was extracted by using Xcellence software (Olympus) to create videos
of different height. Two algorithms were developed to quantify the dynamics of
lamellipodia. Algorithm I was designed to quantify in a semi-automatic way the
time course of protrusion/retraction cycles by using an improved version of the
Kymograph. Algorithm II was designed to quantify the vertical motion of
lamellipodia during these cycles.
## Algorithm I
The images at height ‘0’ i.e. the cover slip where neurons were cultured- were
focused and were used to analyze the protrusion/retraction cycles of
lamellipodia. The lamellipodia edges were extracted from each image of the video
by using the difference of Gaussian filter. Lamellipodia edges were tracked and
followed during the entire duration of the video. A profile of the temporal
movement of the lamellipodium edge was obtained. These profiles allowed to
follow and quantify lamellipodia cycles of protrusion and retraction. Then the
regions of interest of each line were cut and lined up with the time course, to
obtain separate kymographs corresponding to each line.
The white dotted line in the kymograph shows the lamellipodia leading edge. The
changes in the grey values show lamellipodia movements. Mainly the ascending
white dotted parts of the dotted line show the protrusion of lamellipodia (white
line showing single protrusion) while the descending white dotted parts of the
line represent the retraction of lamellipodia. The time to complete one
protrusion and retraction by the lamellipodia was considered as a period (T) of
protrusion/retraction cycle of lamellipodia. The maximum protrusion length after
which lamellipodia starts retracting (white line, dl; micrometers) was defined
as the persistence length of lamellipodia. The dark appearances in the kymograph
during each retraction of lamellipodia represent the retrogradely moving
lamellipodia features (black line showing single lamellipodium retrograde flow).
The slope of the lines drawn on these dark appearances was calculated to find
out the lamellipodium retrograde flow rate (dx/dt; micrometer per second). Each
parameter, the lamellipodia period of the protrusion/retraction cycles, the
persistence length and the retrograde flow rate, were calculated by extracting
these features from many kymographs and averaged over for statistical
significance.
## Algorithm II
Lamellipodia not only show periodic motion of protrusion and retraction but,
during retraction, they also lift up and ruffle. To study the axial motion of GC
lamellipodia, image sequences taken at different heights i.e. 0, 1, 2…6 were
acquired and analysed. Algorithm II was based on the theory of defocusing, in
which a pixel is assumed to be in focus at a specific height when its intensity
equalises with the background intensity of the image of that height. The
background intensity of the image for each height was computed as the median of
pixel intensities of the image for that height. The number of pixels in focus at
a specific height was obtained and normalized by dividing it by the total number
of pixels in focus at all the given heights. In this way, the fraction of pixels
of the lamellipodium in focus at different heights, was extracted and plotted
against time. In this manner it was possible to study the maximal height reached
by the lamellipodia edge during retraction in different conditions. Usually
lamellipodia lift up high around the maximal retraction, so, in our experiments,
their cyclic motility could be characterized both by the kymograph and by the
fractional height that was reached (Figs).
In order to quantify the motility of filopodia, phase contrast time lapse image
sequences acquired at height ‘0’ were analyzed. An Imagej (Image processing and
analysis in Java) software was used to measure the maximum length of the
filopodia and plug-in, ‘manual tracking’ was used to identify the protrusion
rate of the filopodia.
## Force Measurements
The force exerted by lamellipodia and filopodia was calculated by measuring the
displacement of the optically trapped bead and the known trap stiffness.
Unlike traction force microscopy or other similar measurement methods,
initially, the bead was not in contact with the cell membrane but was kept in
the vicinity of the motile lamellipodia and filopodia. In this way the
lamellipodia and filopodia can displace the bead in a spontaneous manner.
The Optical Tweezers (OT) set-up used for force measurements was as previously
described. The optical tweezers set-up was built as described in Ref. 31.
## Nanopositioner feedback
In the OT setup, the detection of the position of the bead was based on the
interference signal in the back focal plane, monitored with Quadrant Photo
Detector (QPD). Often lamellipodia were able to push the bead out of the linear
range–typically 200 nm—in which the QPD could provide a reliable measurement. To
overcome this situation, a feedback mechanism, based on a nanopositioner stage-
Nanodrive (Mad City Labs, USA) was used.
To summarize, from the detected x and y coordinates of the bead the displacement
‘r’ of the bead position from the centre of the trap was computed as sqrt
(x^2+y^2). The nanodrive stage brings back the bead into the centre of the
optical trap when r is larger than the threshold (which is usually set to be
equal to 200 nm). By using the information of the displacement of the nanodrive
stage (lower panel) and the bead position determined by the QPD (X, Y axis
original) we recovered the x-y axis of the compensated displacement.
## Immunostaining
Cells were fixed in 4% paraformaldehyde containing 0.15% picric acid in
phosphate-buffered saline (PBS), saturated with 0.1 M glycine, permeabilized
with 0.1% Triton X-100, saturated with 0.5% BSA in PBS (all from Sigma-Aldrich,
St.Louis, MO) and then incubated for 1h with primary antibodies. The secondary
antibodies were goat anti-rabbit 594 Alexa (Invitrogen, Life Technologies,
Gaithersburg, MD, USA) and anti-mouse IgG<sub>2a</sub> biotynilated (Santa Cruz
Biotechnology, Santa Cruz, CA) and the incubation time was 30 min. F-actin was
marked with Alexa Fluor 488 phalloidin, whereas biotin was identified by Marina
Blue-Streptavidin (Invitrogen, Life Technologies, Gaithersburg, MD, USA) and
incubated for 30 min. All the incubations were performed at room temperature
(20–22°C). Cells were examined using a Leica DMIRE2 confocal microscope (Leica
Microsystems GmbH, Germany) equipped with DIC and fluorescence optics, diode
laser 405nm, Ar/ArKr 488nm and He/Ne 543/594nm lasers. The fluorescence images
(1024x1024 pixels) were collected with a 63X magnification and 1.3 NA oil-
immersion objective. Leica LCS Lite and Image J by W. Rasband (developed at the
U.S. National Institutes of Health and available at <http://rsbweb.nih.gov/ij/>)
were used for image processing.
## Rac1 activity assay
The Rac1-GTP level (the activated form of Rac1) was determined in DRG neurons in
control conditions, 25 μM CK (2 min), 50 μM CK (2 min) and 50 μM CK (8 min)
using the G-LISA Rac 1 activation assay kit (Cytoskeleton, Inc., Denver, CO,
catalog number BK128) according to the manufacturer’s instructions.
After experimental treatment, neurons were washed with ice-cold (4°C) PBS and
then lysed in ice-cold lysis buffer. The lysate was clarified at 10000 x g at
4°C for 1 min, a 20 μl aliquot was taken for a protein assay, and the remaining
lysate was separated into at least two aliquots, snap frozen in liquid nitrogen,
and stored at −70°C until the start of the ELISA portion of the assay. Protein
concentrations were determined using the Precision Red Advanced Protein Assay
that came with the kit. Absorption of the ELISA wells was determined with a
Multiskan™ GO Microplate Spectrophotometer (Thermoscientific, USA).
We acknowledge the financial support of the following projects within the
Seventh Framework Programme for Research of the European Commission: the FOCUS
Project n. FP7-ICT-270483, the NEUROSCAFFOLDS Project n. 604263. The author,
Wasim A. Sayyad undertook this work with the support of ‘ICTP TRIL Programme’,
Trieste, Italy. We thank M. Lough for editing the paper, Andrea Raffin for
developing the LabView based algorithm and Priyadharishini V. for helping in
protein estimation.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: VT. Performed the experiments:
WAS. Analyzed the data: WAS. Contributed reagents/materials/analysis tools:
WAS PF. Wrote the paper: VT WAS PF. Designed the software used in analysis:
PF.
[^3]: Current address: Department of Molecular, Cellular and Developmental
Biology, Yale University, New Haven, Connecticut, United States of America |
# Introduction
Fibromyalgia (FM) is a syndrome primarily characterised by chronic, widespread
musculoskeletal pain. Its prevalence is estimated to be 3–6% of the world
population, and it occurs predominantly in women, with a female to male ratio of
10:1. FM symptoms are not restricted to pain, but often include a heterogeneous
group of other conditions, such as hyperalgesia and/or allodynia, physical and
mental fatigue, disrupted or non-restorative sleep, headache, irritable bowel,
psychiatric disorders, cognitive impairment, and other functional complaints.
The aetiology of this syndrome is not completely understood, but a crucial role
seems to be played by complex interactions among biological, genetic,
psychological, and socio-cultural factors, such as medical illness,
neuroendocrine disturbances, stress, and psychiatric disorders. In particular,
high levels of stress and psychiatric symptoms may negatively influence the
perception of disease severity, functional ability, and the threshold and
tolerance for pain. Some authors have indeed suggested that the development of
FM might stem from stress-induced disruption of the hypothalamic-pituitary-
adrenal (HPA) axis. Exposure to prolonged stressful conditions can alter the
function of the HPA axis, with a consequent increase in the production of
corticotropin-releasing factor and potentially amplified pain perception. For
this reason, FM is often defined as a *central sensitisation syndrome*, caused
by increased sensitivity of the central nervous system to pain signals.
Among psychological factors, the high prevalence of depression (20–80%) and
anxiety disorders (13–64%) has been widely reported. Only recently, researchers
have also started to explore emotional functioning in FM syndrome, with their
attention focused on alexithymia, a multifaceted personality construct that
affects the regulation of a person’s own emotions. Alexithymia is characterised
by difficulty in identifying and describing subjective feelings, difficulty in
distinguishing between feelings and bodily sensations of emotional arousal,
restricted imagination processes, and a stimulus-bound, externally oriented
cognitive style. Most of these studies have reported high levels of alexithymia
in patients with FM, suggesting the presence of a deficit in emotional self-
awareness.
On the other hand, the detection and experience of emotional signals from other
people have only been sparsely investigated in FM syndrome. A link between
alexithymic traits and deficits in the processing of other people’s emotions has
been highlighted in both healthy individuals and specific clinical populations,
e.g. affective disorders, eating disorders, borderline and psychopathic
personality disorders, schizophrenia, somatoform disorders. To the best of our
knowledge, only one study to date has examined the ability to identify other
people’s emotions in FM syndrome. The results of this study highlighted the fact
that patients with FM had reduced performance in a face-recognition task, with a
higher percentage of misclassifications of emotional expressions compared with
healthy controls. In addition, pain intensity, alexithymia, depression, and
anxiety were inversely related to recognition performance, while psychiatric co-
morbidity and medication had no impact on performance.
The ability to decipher information about the intentions and affective states of
social partners is crucial for the implementation of appropriate behaviour
during social interactions. This complex process is part of the so-called
*social cognition domain*, defined as the ability to construct mental
representations of the relations that exist between oneself and others and to
flexibly use these representations to function effectively in one’s social
environment. Examples of social cognition abilities are the capacity to
represent other people’s intentions and beliefs (i.e. Theory of Mind, ToM), and
the ability to share and recognise the emotions and sensations of others.
From a neurological point of view, ToM and emotional processing abilities are
associated with overlapping but distinct brain networks. Common areas of
activation are the prefrontal cortex, the superior temporal sulcus, and the
temporo-parietal junctions. These areas form the basis for making inferences
about mental states. However, these areas are not sufficient for the evaluation
of one’s own and other’s emotions, and it is necessary to recruit the additional
involvement of emotional networks, in particular those of the amygdala. In fact,
while the amygdala is not involved in ToM processes per se, its role is crucial
in processing basic and social emotions, related both to the self and to others.
The social cognition domain includes a series of different abilities, which
gradually evolve throughout the lifetime. An open issue concerns the
relationship between these capacities, particularly ToM processes, and the
higher-level cognitive skills known as executive functions (EF). Growing numbers
of studies are trying to address this relationship in patients with different
psychiatric and neurological conditions.
EF refers to all of the skills that people use to control and coordinate their
cognitive abilities and behavior. These are essential for independent everyday
functioning in life, and for the establishment of adaptive social relations. In
the last decade, evidence for the multifaceted nature of EF has replaced the
idea of a unitary function. Among the several classifications that have been
proposed to distinguish EF subcomponents, the model of Miyake et al. identified
three separate types of operations: Updating, Shifting, and Inhibition.
*Updating* is related to working memory and requires monitoring and coding
information as well as replacing old non-relevant information with new relevant
information. *Shifting* implies the ability to shift attention between different
sub-tasks or different elements of the same task. *Inhibition* is concerned with
the individual’s ability to withhold dominant, automatic or prepotent responses
when they are inappropriate, and is considered to be a key component in planning
abilities. Fisk and Sharp later added a fourth subcomponent, and revised the
model of Miyake and colleagues. The factor, called *Access*, refers to the
processes involved in verbal fluency tasks, which are believed to mediate access
to representations in long-term memory.
Currently, there are two opposing views about the relationship between ToM
abilities and EF. Some authors believe that ToM is a circumscribed cognitive
process, independent of general intellectual functioning and other cognitive
domains, included EF. Basing their ideas on theoretical and experimental data,
others have suggested instead that lower-level perceptual abilities (e.g.
detection of gaze direction and voice recognition) that are required for an
appropriate implementation of ToM skills, may be related to specific and
circumscribed cognitive domains, while higher-order ToM processes, involving
interpreting and associating information as well as hypothesising, would be the
result of a more general ability regarding metarepresentation and EF.
The present study is based on both clinical evidence, which has highlighted high
levels of emotional distress (depression, anxiety, and in particular,
alexithymia) in patients with FM, and neuroimaging and neuropsychological data
that has reported functional and structural alterations in brain areas crucial
for ToM and emotional processing abilities (i.e. the prefrontal cortex and
amygdala) in these patients, as well as cognitive deficits in the EF domain. On
these bases, the present study aimed to address two main objectives. The first
goal was to evaluate the social-cognitive profile of patients with FM, and
analyse ToM and emotional processing abilities. In particular, four different
areas of the social cognition domain were examined: (1) regulation of one’s own
emotions; (2) empathic capacities; (3) recognition of other’s emotions; (4)
representation of other people’s affective mental states (i.e., affective ToM).
The second goal was to explore the possible relationships between the
performance on executive-function tasks and performance on social-cognition
tasks in patients with FM. Furthermore, relationships between social-cognitive
abilities and demographic, clinical and psychological variables were also
investigated for explorative purposes.
# Materials and Methods
## Ethics Statement
The study was approved by the San Giovanni Battista University Hospital’s ethics
committee and was conducted in accordance with the Declaration of Helsinki. All
the participants gave their written informed consent to participate in the
study.
## Participants and procedure
Forty female participants with FM (51.75 ± 7.76 years of age) were consecutively
recruited from the Fibromyalgia Integrated Outpatient Unit (FIOU), a
multidisciplinary unit based on the collaboration between rheumatologists,
psychologists, and psychiatrists at the San Giovanni Battista University
Hospital of Turin. All patients had a main diagnosis of fibromyalgia, made by
rheumatologists who are experts in the field. In addition, a psychiatric
interview based on DSM IV-TR axis II criteria (Diagnostic and Statistical Manual
of Mental Disorders, 4th edition, Text Revision) was performed by an expert
psychiatrist, in order to exclude FM patients with personality disorders.
Exclusion criteria were as follows: less than 18 years old, low education level
(\<5 years), and the presence or history of a neurological or a severe
psychiatric disorder. Forty-one healthy women (51.83 ± 7.78 years of age) were
recruited to the HC group. Exclusion criteria for the HC group were the presence
of rheumatic diseases or chronic pain, as well as the presence or history of a
neurological or psychiatric disorder.
## Pain evaluation
As an index of pain intensity, the item “Pain” of the Italian version of the
Fibromyalgia Impact Questionnaire (FIQ) was used to assess the average intensity
of pain in the previous week on a scale ranging between 0 and 10.
## Psychological assessment
The presence of symptoms of depression and anxiety was assessed using the
Italian version of the Hospital Anxiety and Depression Scale (HADS). It consists
of 14 items on a 0 to 3 range, and is divided into two subscales, one for
depression (HADS-D) and one for anxiety (HADS-A). Each subscale score ranges
from 0 to 21 and a score of 8 (cut-off) or more suggests a level of
depression/anxiety symptoms that is clinically relevant.
## Neuropsychological assessment
Neuropsychological assessment was performed, using tests for short-term memory
(Digit Span-Forward—DS F), learning (Rey auditory-verbal learning test—AVLT),
and attention (Trail-making test—TMT—A-B). For the investigation of executive
functions, four different tests were used, specific for each one of the four
subcomponents into which EF has been divided according to the models of Miyake
et al. and Fisk and Sharp. Specifically, the Digit Span-Backward (DS B) was
employed for evaluating the Updating component, the TMT B for Shifting, the
Tower of London (ToL) for Inhibition, and the verbal fluency (FAS) for Access.
## Social cognition assessment
**Twenty-Item Toronto Alexithymia Scale (TAS-20)**. Alexithymia was assessed
using the Italian version of the Toronto Alexithymia Scale (TAS-20). Subjects
were asked to indicate the extent to which they agreed or disagreed with each
statement on a five-point Likert scale. The results provide a TAS-20 total
score, and three subscale scores that measure different aspects of alexithymia:
*difficulty identifying feelings* (Factor 1), which measures the inability to
distinguish specific emotions and between emotions and the bodily sensations of
emotional arousal; *difficulty describing feelings* (Factor 2), which assesses
the inability to verbalise one’s emotions to other people; and *externally-
oriented thinking* (Factor 3), which evaluates the tendency of individuals to
focus their attention externally and not on the inner emotional experience. The
TAS-20 cut-off scores are as follows: ≤51 no alexithymia, 52–60 borderline
alexithymia, ≥61 alexithymia. This scale has shown good internal consistency and
test-retest reliability, as well as convergent, discriminant and concurrent
validity, and it is currently one of the most utilised instruments in studies of
alexithymia and emotion.
**Empathy Quotient (EQ)**. The EQ is a validated self-report questionnaire,
employed to assess the capacity to empathise with another, i.e. to recognise
another’s affective state and to respond to this with an appropriate emotion.
The EQ comprises 60 items, broken down into two types: 40 items assessing
empathy and 20 filler/control items, included to distract the participant from a
relentless focus on empathy. For each empathy item, a person can score 2, 1, or
0, so the EQ has a maximum score of 80 (higher scores indicate greater empathy).
The EQ is able to detect considerable individual, gender, and group differences,
in both general-population and clinical samples.
**Ekman 60 Faces**. The Italian version of this test was used to assess the
recognition of facial expressions pertaining to basic emotions. The Ekman 60
Faces Test uses photographs of the faces of 10 actors (six female and four male)
selected from the Ekman and Friesen series. Each actor displays one of the six
basic emotions investigated (happiness, sadness, disgust, fear, surprise, and
anger). The subject is required to respond verbally, deciding which of the six
labels for basic emotions that are placed below each photograph can best
describe the facial expression shown. The maximum test score (indicating best
performance) is 60 for all six emotions and 10 for each basic emotion.
**Reading the Mind in the Eyes Test (RME)**. The RME was employed to assess the
ability to represent other people’s affective mental states. In the test, the
experimenter presents a set of 36 photographs of the eye region of various human
faces. Participants are required to choose among four words that are printed on
the page that the picture appears on, using the criterion of which word best
describes the mental state of the person depicted in the photograph.
Participants have unlimited time to decide, and a glossary is provided.
Participants have to put themselves into the mind of another person and
recognise his or her complex mental state. In the gender-recognition control
task, participants are asked to judge the gender of the person in each of the 36
photographs. For both the experimental (mental state attribution) and control
(gender attribution) conditions, the maximum score indicating the best
performance is 36.
## Statistical analyses
All the statistical analyses were conducted using IBM SPSS Statistics, version
20.0. Normal distribution was assessed using indices of asymmetry and kurtosis.
Non-parametric equivalent tests were performed on data that violated this
assumption. For normally distributed variables, independent *t*-tests were used.
In order to evaluate the possible relationships between variables, Spearman or
Pearson correlations were computed, as appropriate.
# Results
## Demographic, clinical and psychological data
Data on the demographic and psychological variables are presented in. The two
groups were matched for age and education.
For psychological assessment we used data from the HADS total score and the
scores for subscales HADS-A and HADS-D. The results showed significantly higher
scores in patients with FM both for the total score (*p*\< 0.001) and for each
of the two subscales evaluating anxiety (*p*\< 0.001) and depression (*p*\<
0.001). According to the cut-off scores of the HADS, 67.5% (27/40) of the
patients with FM showed a clinically relevant level of both anxiety and
depression, compared with 34% (14/41) for anxiety (*Χ*<sup>2</sup>(1) = 9.011,
*p*\< 0.001) and 19.5% (8/41) for depression (*Χ*<sup>2</sup>(1) = 19.000, *p*\<
0.001) in the HC group.
Concerning the clinical characteristics of the FM group, patients reported 6.47
(± 5.81) years of duration of illness and a high rate of pain intensity (7 ±
2.55 to the item “Pain” of the FIQ).
## Neuropsychological assessment
The comparisons between the neuropsychological scores of the two groups are
shown in. Patients with FM performed worse than the HC group on all the four
tasks evaluating EF (*p* values ranging from \< 0.001–0.011). Furthermore, a
poorer performance in the FM group also emerged in the DS F (*p* = 0.005), in
the AVLT-Delayed recall (*p* = 0.006) and in the TMT B-A (*p* = 0.004). No
statistically significant differences were found on the other neuropsychological
measures.
In order to bring out the individual differences that could be flattened by
group analyses, the individual scores were analysed comparing for each test the
number of subjects with impaired or borderline performance according to the age-
and education-corrected scores (equivalent score ≤1). The results showed that a
significantly higher number of patients with FM compared with HC had a deficient
performance in the DS F (short-term memory) and B (working memory), and in the
Delayed recall task of AVLT (episodic memory).
## Social cognition tasks
Data from social cognition tasks are reported in.
**Regulation of own emotions**. Concerning alexithymia, statistical analyses
revealed the presence of significant differences between FM and HC on the TAS-20
total score (*p*\< 0.001), and on the F1 (*p*\< 0.001) and F2 subscales (*p* =
0.011); in all these comparisons patients with FM scored higher than HC.
According to the TAS-20 cut-off scores, 27.5% (11/40) of the patients with FM
were alexithymic and 45% (18/40) were borderline, compared with 7% (3/41) and
19.5% (8/41), respectively, in the HC group.
**Empathic capacity**. No significant difference between the two groups was
found in the EQ score.
**Recognition of others emotions**. Concerning the Ekman 60 Faces Test,
independent *t*-tests revealed the presence of significant differences between
the two groups on total score (*p* = 0.010), and on two of the six emotions
investigated by means of the test, i.e. anger (*p* = 0.049) and disgust (*p* =
0.016). Once again, patients with FM showed significantly lower scores,
indicating a reduced ability to recognise other people’s emotions, especially
anger and disgust.
**Representation of other people’s affective mental states**. No significant
difference between FM and HC was found in the control task for RME. In the
experimental condition, patients with FM evidenced a significantly lower
performance on the mental states attribution task (*p* = 0.007).
## Correlations
The second aim of this study was to investigate the possible relationships
between social cognition tasks and EF measures in patients with FM. Moreover, we
also evaluated the correlations between social cognition tasks and demographic,
clinical and psychological data. To do that, we only considered the variables
that showed a significant difference in the comparison between FM and HC, i.e.
TAS-20 F1, F2, and total score; Ekman anger, disgust and total score; and RME
experimental task.
Correlations between social cognition measures and EF tasks are listed in. As
shown, no significant correlation was found, with the only exception of a low
positive correlation between the Ekman total score and the DS B. In addition, we
verified the possible relationships between social cognition measures, EF tasks
and duration of illness (DI) in FM group and no significant correlation emerged
(DI and FAS: *r* = -0.276, *p*: ns; DI and DS B: *r* = -0.132, *p*: ns; DI and
ToL: *r* = 0.196, *p*: ns; DI and TMT B: *r<sub>s</sub>* = 0.185, *p*: ns; DI
and TAS-20 F1: *r* = 0.319, *p*: ns; DI and TAS-20 F2: *r* = 0.333, *p*: ns; DI
and TAS-20 Total: *r* = 0.342, *p*: ns; DI and Ekman Anger: *r* = 0.087, *p*:
ns; DI and Ekman Disgust: *r<sub>s</sub>* = -0.175, *p*: ns; DI and Ekman Total:
*r* = -0.036, *p*: ns; DI and RME Experimental: *r* = -0.053, *p*: ns).
Regarding the relationship between social cognition measures and demographic,
clinical and psychological variables, no significant correlations were detected
between age, HADS-A, HADS-D, FIQ-pain, and the RME experimental or the Ekman
anger, disgust and total score; a positive correlation was only found between
the RME experimental and the level of education (*r* = 0.359, *p* = 0.023).
However, significant correlations were detected between demographic, clinical
and psychological variables and the TAS-20 scores. In particular, positive
correlations were found between the TAS-20 total score and the HADS-A (*r* =
0.334, *p* = 0.035), the HADS-D (*r* = 0.630, *p* \< 0.001), and the FIQ-pain
(*r* = 0.518, *p* = 0.001). Likewise, positive correlations were found between
the TAS-20 F1 and the HADS-A (*r* = 0.462, *p* = 0.003), the HADS-D (*r* =
0.476, *p* = 0.002) and the FIQ-pain (*r* = 0.442, *p* = 0.004). Finally, the
TAS-20 F2 was positively correlated only with the HADS-D (*r* = 0.537, *p* \<
0.001) and the FIQ-Pain (*r* = 0.344, *p* = 0.030).
# Discussion
The present study aimed to address two main objectives. Firstly, we evaluated
the social-cognitive profile of patients with FM, investigating ToM and
emotional processing abilities. Secondly, we analysed the relationship between
EF deficits and social cognition tasks in patients with FM. Correlations between
demographic, clinical and psychological variables, and measures of social
cognition were also evaluated.
The results highlighted a significant difference between patients with FM and
the HC group in most of the social cognition tasks employed. In particular, the
FM group showed significantly higher levels of alexithymia, especially in the
subscales “Difficulty in identifying feelings” and “Difficulty describing
feelings” of TAS-20, compared to the control sample. These data are in line with
most of the studies that have evaluated the prevalence of alexithymia in
patients with FM.
A similar significant difference was found for the experimental task of the RME,
while no significant difference was observed for the control task. Patients with
FM experienced specific difficulties in representing other people’s affective
mental states that cannot be attributed to a basic sensory deficit. The Ekman 60
Faces Test results also showed the presence of significant differences (lower
performance of patients with FM) between the two groups both for the total score
and for two of the six emotions investigated by means of the test, i.e. anger
and disgust; no significant differences were found for other emotions. These
data are in line with the only study that has investigated the ability to
recognise another’s facial emotions in patients with FM. As mentioned above,
these authors showed that patients with FM had reduced performance in the facial
affect recognition task, with a higher percentage of misclassifications of
emotional expressions compared with the HC group.
The only social cognition task, in which no differences between the two groups
were found, was the EQ. In this case, the FM group didn’t report a lower
capacity for empathy compared to the control group.
Concerning the general cognitive profile, neuropsychological assessment revealed
the presence of significant differences in most of the measures. In particular,
the FM group displayed significantly lower performance on the verbal fluency
(FAS), the DS B and F, the AVLT-Delayed recall, and the TMT B and B-A, compared
to HC. These data are consistent with previous studies that have reported
cognitive deficits in attention, memory, and EF domains in patients with FM. In
particular, Park et al. found that patients with FM demonstrated lower
performance on measures of working memory, free recall, verbal fluency, and
verbal knowledge, but showed intact speed of processing, compared with age- and
education-matched controls. Significantly, patients with FM in that study
performed no differently from controls who were 20 years older on most cognitive
tasks, with the exception of speed of processing and vocabulary. Only self-
reported pain on the Arthritis Impact Measurement Scales predicted poor
cognitive performance in the FM group. Measures of depression, anxiety, and the
McGill Pain Questionnaire scores were all unrelated to poor cognitive
performance. Verdejo-Garcia et al. observed that in the Wisconsin Card Sorting
Test, women with FM showed poorer performance than healthy women on the number
of categories and non-perseverative errors, but not on perseverative errors.
Patients with FM also exhibited an altered learning curve in the original Iowa
Gambling Task (IGT) (where reward is immediate and punishment is delayed),
suggesting compromised emotion-based decision-making. This was not the case in
the variant IGT (where punishment is immediate but reward is delayed),
suggesting hypersensitivity to reward. Self-reported pain intensity and pain
interference were significantly associated with task performance. In contrast,
cognitive performance was not associated with measures of negative mood (i.e.
affective distress) or duration of pharmacological treatment, and was very
mildly associated with personality characteristics.
Finally, from a psychological standpoint, our group of patients with FM
presented with significantly higher levels of depressive and anxiety symptoms
(67.5% in both cases) compared with the HC group. These results corroborate,
once again, the high prevalence of psychological distress reported in previous
studies of patients with FM.
To the best of our knowledge, this is the first study to investigate social-
cognitive abilities in a homogenous sample of patients with FM. The results show
that patients with FM have impairments both in the regulation of their own
affect and in the recognition of other’s emotions, as well as in representing
other people’s affective mental states. There is evidence that appropriate
behaviour in social interactions is determined by the ability to decipher
information about the intentions and affective states of social partners. Thus,
impairments in facial affect recognition and difficulties in accurately
inferring other people’s affective mental states may lead to substantial
difficulties in interpersonal contacts (e.g. interaction problems with family
and friends, or social isolation), which have been already reported in patients
with FM. Furthermore, poor psychosocial functioning and unsatisfactory
relationships might contribute to the genesis and maintenance of chronic pain,
intensifying the symptomatology in individuals with FM.
From a neurological standpoint, the brain networks relevant for pain and
emotional processing partially overlap. The amygdala plays a crucial role in
sharing emotional experiences and in recognising emotions in oneself and others.
In particular, this structure is involved in the decoding of emotional
expressions, and modulates the activity of the fusiform gyrus, which constitutes
the most prominent face-selective area of the brain. Neuroimaging studies have
indeed shown sensitivity of the amygdala to the kind and valence of facially
expressed emotions. The insular cortex may also be relevant in this context. It
has been reported, for instance, that impaired disgust recognition is associated
with reduced insula activity. In addition to their prominent role in emotional
functioning, the amygdala and the insula are integral parts of the neural
network underlying pain. Specifically, both structures are involved in
transmitting the affective dimension of pain perception and are altered in
patients with FM. The hyperactivity of the pain network due to central nervous
system sensitisation, may lead to an increased demand on structures such as the
amygdala and insula, reducing the available resources for other functions such
as emotional processing.
As far as the second goal of this study is concerned, we investigated whether EF
measures in the FM group were related to the different social cognition tasks
that were used. In addition, we also analysed the possible relationships between
demographic, clinical and psychological variables, and measures of social
cognition. Concerning EF tasks, no correlations were found between social
cognition tasks and each of the four subcomponents of the EF domain that were
analysed. The only exception was represented by a low positive correlation
between the Ekman total score and the DS B. Regarding the demographic, clinical
and psychological variables, correlation analyses showed no relationship between
Ekman total score, anger and disgust, RME experimental, on the one hand, and
anxiety, depression and pain intensity, on the other hand. Positive correlations
were only detected between the latter measures (anxiety, depression, and pain)
and the total score and scores for the F1 and F2 subscales of the TAS-20. This
result is consistent with previous studies that have investigated the presence
of alexithymia in FM patients. In particular, Steinweg et al. found higher
levels of alexithymia in FM patients compared with either general medical or
rheumatoid arthritis patients. However, they also revealed that alexithymia was
strongly associated with moderate-to-severe depression, but no group differences
were detected when mood disturbance was controlled for.
Taken together, as far as the first aim of this study is concerned, the results
show the presence of several impairments in social cognition skills in patients
with FM. As for the second aim, i.e. to explore the possible relationships
between the performance on executive-function tasks and the performance on
social-cognition tasks in patients with FM, we found that the latter are largely
independent of both EF deficits and symptoms of psychological distress. The only
exception seems to be represented by alexithymia; in fact, psychological
disorders, but not EF deficits, seem to play a role in explaining the high
levels of alexithymia found in the FM sample. Concerning other measures of
social cognition, no relationship was found with EF deficits or symptoms of
psychological distress. In our sample, impairments in ToM and emotional
processing ability appeared to be independent of the EF domain.
This study also has some limitations. Firstly, even though we enrolled an
adequate number of patients with FM, our study is still limited by a relatively
small sample size. Secondly, the self-reported measures we used might have
elicited a bias towards social desirability, masking the real profile of some
individuals. Thirdly, although in patients with FM there is evidence of
structural and functional alterations in brain areas crucial for ToM and
emotional processing abilities (i.e. the prefrontal cortex and the amygdala), we
didn’t directly measure the activity in these brain structures. Future studies
should include neuroimaging evaluations and use performance-based instruments
for the analysis of both empathic capacity and alexithymia, in addition to
traditional self-reported tests.
In spite of these limitations, the findings reported in the present study
represent the first contribution towards understanding the complex social-
cognitive profile of patients with FM. The impairments reported in tasks that
evaluate ToM and emotional processing abilities highlight the importance of
adequately assessing these abilities in clinical practice. In this way, it could
be possible for clinicians to plan better pharmacological and/or psychological
treatment based on each patient’s needs.
The authors would like to thank Valentina Tesio, Stefania Brighenti and
Francesca Monoli for their help in collecting data.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: MDT LC MA. Performed the
experiments: MDT FC EF RT. Analyzed the data: MDT LC RBA MA. Contributed
reagents/materials/analysis tools: LC RBA MA. Wrote the paper: MDT LC RBA
MA. |
# Introduction
Recently, genome editing by the CRISPR/Cas9 system has became a widely used
strategy to study gene functions in cells and *in vivo*. In the widely-used
CRISPR/Cas9 system derived from *Streptococcus pyogenes*, the Cas9 nuclease
creates a double strand break in the genome at a site complementary to the guide
RNA, which is then repaired by various repair machineries of the cell. This
often leads to the introduction of indels, resulting in an efficient loss of
function of gene. However, even with the most efficient delivery strategies,
gene disruption does not reach 100% efficiency in cell culture. For this reason,
researchers have often performed isolation of mutant clones before doing their
assay of interest. Therefore, a strategy to identify mutant clones quickly and
accurately is very important. The indels caused by CRISPR/Cas9 editing are often
small, thus standard PCR with genomic DNA (gDNA) is not useful to detect the
mutants since the amplicon sizes are not different enough. The usage of Cas9
nuclease with two guide RNAs targeting relatively distant sites enables an easy
discrimination by inducing a bigger deletion detectable by PCR (not to be
confounded with the double-nickase strategy where the offset should be small
enough to get a double strand break). However, with this strategy only clones
having the large deletion in all alleles are discriminated, and the potentially
useful mutant clones having only small indels are ignored, reducing the success
rate of obtaining mutants. Also, this strategy doubles the effort of plasmid
preparation as well as the chance of having off-target effects.
Multiple methods to identify CRISPR/Cas9-induced indels have been published. A
strategy that is often used is the mismatch cleavage assay. This method uses
enzymes that cleave DNA heteroduplexes at mismatch sites. Although being easy to
perform, this method does not discriminate wild type clones and mutant clones
harbouring identical mutations in both alleles. Also, heterozygous mutants are
not discriminated from homozygous mutants with different mutations between
alleles. These issues can be avoided by mixing wild type PCR amplicon with the
amplicons of the analysed samples. However, this adds another step for the
experiment, and DNA concentrations of PCR products have to be quantified in
order to have a correct ratio of mixed amplicons, since this affects the
cleavage efficiency. An ideal method should discriminate completely mutated
cells from the others without the need of too many steps, while being cost
effective, and not requiring special laboratory equipment. In case of difficult-
to-transfect cells or when doing multiplex gene disruption, hundreds of clones
might have to be analysed. Therefore, it is also desirable to reduce any steps
like template DNA quantification and PCR product purification.
None of the existing methods fulfil all the above requirements. For example,
high-resolution melting analysis is easy and quick, but requires specific
instruments. Also, since different type of indels will affect the melting
behaviour of PCR products, unambiguous identification of homozygous mutants
might be difficult. Capillary electrophoresis of PCR products enables accurate
detection of indels, but requires specific instruments. Quantitative PCR
efficiently identifies mutations, but the highly quantitative nature of this
method gives rise to the need to quantify and normalize template DNA amount.
Heteroduplex mobility assay is simple to perform, but has similar drawbacks to
mismatch cleavage assays when applied to clone analysis. Restriction fragment
length polymorphism and RNA-guided engineered nuclease restriction fragment
length polymorphism are almost ideal. However, the former requires a convenient
restriction site at the cleavage site, and for the latter one needs to generate
*in vitro* the nuclease complex used for restriction analysis, which might be
laborious. In addition, enzyme restriction is another step added to PCR, which
one might want to avoid if possible. Most of the methods stated above have the
benefit of being able to estimate the mutation efficiency (with more or less
accuracy) in bulk cells. This degree of quantitation is not required when the
only requirement is to detect fully mutated clones. Thus, we wanted to establish
a method that is easy to perform without the need to normalize template DNA
amount, and requiring only minimal laboratory instruments, even if it might be
less quantitative than the ones stated above. Here, we show that when primers
are appropriately designed, a simple PCR using a set of primers flanking the
target site of interest, together with an additional one overlapping with the
site cleaved by Cas9, enables the identification of homozygous mutant clones.
# Materials and methods
## Cell culture
McA-RH7777 cells and HeLa MZ cells were cultured in Dulbecco’s Modified Eagle
Medium (DMEM high glucose, GlutaMAX, pyruvate), supplemented with 10% fetal
bovine serum and 100 U/mL penicillin-streptomycin (all from Thermo Fisher
Scientific, Waltham, MA, USA).
## Plasmid construction for genome editing
Plasmid backbones for CRISPR/Cas9 experiments were either pX330, which contains
expression cassettes for both Cas9 and single guide RNAs (sgRNAs), or an in-
house constructed plasmid (pUC-U6-sg) for expressing only sgRNAs. The latter was
constructed by incorporating the sgRNA expression cassette from pX330 into a
pUC19 backbone. The insert and the pUC19 backbone were amplified by PCR using
PrimeSTAR GXL DNA Polymerase (TAKARA Clonthech, Otsu, Shiga, Japan) with the
primers listed in, and assembled using the Gibson assembly system (New England
Biolabs, Ipswich, MA, USA). Target sites for genome editing were selected using
either the Zhang laboratory algorithm (<http://crispr.mit.edu>) or CRISPOR
(<http://crispor.tefor.net>), choosing those having low off-target scores and
high on-target scores. The pairs of oligo DNA were synthesized at Microsynth AG
(Balgach, St. Gallen, Switzerland). Plasmids were constructed by Golden Gate
assembly, based on the Yamamoto laboratory protocol with slight modifications.
Restriction and ligation was performed in a single tube using FastDigest Bpi I
(Thermo Fisher Scientific) and quick ligase (New England Biolabs, Ipswich, MA,
USA) in quick ligase buffer. Tubes were incubated at 37°C and 25°C for 5 minutes
each, and this cycle was repeated three times. Then, an additional Bpi I
digestion was done for 1 hour. Products were transformed into STBL3 chemical
competent cells (Thermo Fisher Scientific), plasmids were sequence-verified by
Sanger sequencing by Fasteris SA (Plan-les-Ouates, Geneva, Switzerland), and
obtained in large scale using either QIAFilter plasmid midiprep kit (QIAGEN,
Hilden, Germany) or PureLink HiPure midiprep kit (Thermo Fisher Scientific).
## Genome editing in cultured cells
Genome editing in McA-RH7777 cells was performed based on a previously described
Hprt co-targeting strategy, which was optimized for our cell lines. In this
method, mutant cells are strongly enriched by selecting cells having a mutation
in the co-targeted Hprt1 gene, which confers resistance to 6-thioguanine (6-TG,
Sigma-Aldrich, St. Louis, MO, USA). Transfection of plasmids (450 ng of
pX330-based plasmid for the target gene, and 50 ng of pUC-U6-sg-based plasmid
for rat Hprt1 gene) was done using lipofectamine 3000 (Thermo Fisher
Scientific), by reverse transfection; the mixed transfection reagents were put
in an empty 24 well plate, and 100,000 cells were seeded on them in a volume of
1 mL. Cells were maintained in culture with occasional passages. Nine days post-
transfection, cells were detached and diluted in a medium containing 4 μg/mL
6-TG. Selection was done for one week, with a medium change at day 4. To isolate
triple knockout clones, cells were transfected with equal amounts of the three
pX330-based plasmids to target Sgpl1, Sgpp1, and Sgpp2, and subjected to
limiting dilution 5 days post-transfection on a type I collagen (Sigma-
Aldrich)-coated 96 well plate. Conditioned medium was added at 20% to promote
the growth of clonal populations. Genome editing in HeLa MZ cells was done
without co-targeting (empty pX330 plus pUC-U6-sg-based plasmid for human HPRT1
gene), using the same transfection method. Clones of HeLa MZ cells were obtained
by limiting dilution into 96 well plates. To analyse polyclonal HPRT1-mutated
HeLa MZ cells, the transfectants were selected with 4 μg/mL 6-TG 5 days post-
transfection.
## Primer design
Primer sequences for the amplification of genomic DNA were designed using
primer-BLAST (<http://www.ncbi.nlm.nih.gov/tools/primer-blast/>). Melting
temperature was calculated using the same program. The primers were typically
designed to amplify 700 to 900 bp, with the Cas9-cleaved site located between
150 to 300 bp from one end. For competition-based PCR, the inner primers were
designed only after amplification of the outer amplicon was confirmed. Except
for the initial experiments during optimization, the inner primers were designed
to have a melting temperature not exceeding 60°C, with the 3’ end extending 3
bases beyond the cleavage site. If this design resulted a primer shorter than a
16-mer, the primer was designed as a 16-mer regardless of its melting
temperature. Then, the outer primer having the same orientation with the inner
one was redesigned (when required) to have a lower melting temperature than it.
Details are provided in.
## Isolation of genome DNA and confirmation of mutation rates
Genomic DNA was isolated as following. Cells were lysed in lysis buffer (20 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 0.67% (w/v) SDS, and 124 μg/mL proteinase K (Roche
Life Science, Rotkreuz, Zug, Switzerland)) at 55°C for at least 4 hours. DNA was
precipitated with isopropanol, pellets were washed with 70% ethanol, and were
dissolved in TE buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA). TIDE (Tracking of
Indels by DEcomposition) analysis was performed to confirm that samples used in
the optimization steps are mutants. For this, target regions were amplified by
PCR (primers are listed), amplicons were treated with Exonuclease I and FastAP
alkaline phosphatase (both from Thermo Fisher Scientific), followed by heat
denaturation, and directly used for Sanger sequencing. Competition-based PCR was
performed using a mixture of 3 primers at 0.2 μM each. PCR reactions were
carried out using ExTaq polymerase (Takara Clontech) or PrimeSTAR GXL
polymerase. For ExTaq reactions, 5% (v/v) DMSO was added. PCR reactions with the
following temperature cycles were performed using a T Professional TRIO
Thermocycler (Biometra, Göttingen, Germany): 30 seconds at 94°C, 30 seconds at
60°C, and 50 seconds at 72°C (total 35 cycles). For PrimeSTAR GXL reactions, the
conditions were: 10 seconds at 98°C, 15 seconds at 60°C, and 50 seconds at 68°C
(35 cycles). PCR products were analysed by agarose gel electrophoresis,
typically using 1.5% (w/v) gels in TBE buffer (89 mM Tris-borate, 2 mM EDTA).
Bands were visualized using EZ-VISION DNA dye (amresco, Solon, OH, USA) or
ethidium bromide (Sigma-Aldrich). Primers are listed in.
## Image processing and quantification of band signals
For clarity on printed pages, signals of some images were adjusted using
photoshop (Adobe, San Jose, CA, USA), while maintaining the linearity of
signals. For quantification of signals, images without adjustment were used. The
digital gel images were analysed using ImageJ. We obtained images using
different exposure times, and did quantification with those that did not have
saturating signals.
## Analysis of 6-TG sensitivity
Individual HeLa MZ clones were placed in different wells of a 96 well plate with
approximately 20% confluency, and incubated with 6 μg/mL 6-TG (day 0). Medium
change (still containing 6-TG) was done at day 4, and cells were fixed with
methanol at day 6. Cells were stained with 0.5% crystal violet (in 20% methanol)
for visualization.
## Analysis of mutant clones by sequencing
To analyse genome sequencing results of clones that were selected by
competition-based PCR, TIDE analysis was done for each locus. When TIDE analysis
gave sufficient explanation for the mixed sequencing chromatograms, it was
concluded that the clone contained the indels proposed by the algorithm. When an
indel larger than 50 bases was present, this allele could not be calculated by
TIDE, so chromatogram deconvolution was done manually. Control and mutant
chromatograms were compared by eye, and the aberrant sequence was estimated from
their difference, which was matched with downstream sequence in case of large
deletions. If a large stretch of aberrant sequence was found to be not
corresponding to any sequence of the locus, this was interpreted as an insertion
of a relatively random sequence.
When different alleles of mutant clones had to be analysed, target regions were
amplified by PCR using PrimeSTAR GXL (primers at), amplicons were cloned using
Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific), and transformed into
STBL3 chemical competent cells. Bacterial colonies containing plasmids with
inserts were selected by colony-direct PCR, and these PCR products were used for
direct Sanger sequencing after Exonuclease I and FastAP alkaline phosphatase
treatment as written above.
# Results
## Competition-based PCR detects mutant cells
A previous study showed that quantitative PCR with one of the primers
overlapping with the Cas9 cleavage site enables the detection of mutations in
zebrafish. In the same study, endpoint PCR was used only to detect mutant
fragments after isolation into plasmids. This motivated us to develop an
endpoint PCR-based method to detect mutant clones of cultured cells, without the
need to isolate fragments into plasmids. To optimize such a method, we used gDNA
from mutant cells that were established for another unpublished study. Since
this manuscript focuses on the detection strategy, details about the generation
and characterization of these mutant cells will be described elsewhere. In this
study, the PCR strategy is designed to detect mutations generated by wild type
*Streptococcus pyogenes* Cas9 nuclease (hereafter simply described as Cas9). The
potential use of this strategy to detect mutations induced by other genome
editing methods will be addressed in the Discussion.
We used McA-RH7777 cells that have mutations in Sgpl1 gene for initial
optimization. First, we performed a standard endpoint PCR with one primer
overlapping with the Cas9 cleavage site, placing the cleavage site between the
third and fourth bases from the 3' end of the primer. As can be expected, the
amount of PCR products did not differ when using gDNA from wild type or mutant
cells, nor did it when using different template DNA amounts. Thus, as is well
known, amplification reaches a plateau in conventional end point PCR, and even
if the affinity of the primer with template DNA from mutant cells is reduced,
the amount of final products is unchanged. This shows that mutants cannot be
discriminated using this design of primers, unless using quantitative PCR as in
the previous study. Next, we designed a PCR strategy where one pair of primers
is designed to flank the Cas9 cleavage site (the outer primers), and another
primer (the inner primer) overlaps with the cleavage site. Among the outer
primers, the one proximal to the Cas9 cleavage site will be called the “F-out”
primer, and the other one the “R-out” primer. The inner primer will be called
“F-in” when it directs to the same direction as F-out, and “R-in” in the other
case (see also for the R-in orientation). The amplicon of outer primers will be
called “out-amplicon”, and the one from the inner primer and the corresponding
outer primer will be called “in-amplicon”. Under such a PCR condition,
competition between amplicons should occur. First, since the amount of
substrates (dNTPs and primers) is not infinite, the amplification of one
amplicon reduces the amount of substrates that can be used for the other.
Second, if the inner primer is present on a template DNA, the elongation from
the outer primer that is located upstream will be blocked (unless using a
polymerase with strand-displacing activity). Thus, when an amplicon is strongly
amplified, the other one is reduced. The fact that out-amplicons can be used as
a template for PCR of in-amplicon, but not the inverse, makes this competition
even more complex (not depicted in the Figure). When gDNA from a mutant is
utilized, the efficiency of inner primer binding should be lower, shifting the
balance of amplification toward the out-amplicon. We thought that this design
would lead to a different ratio between out- and in-amplicons when using mutant
gDNA, enabling their discrimination from wild type. This design was inspired by
the fact that before the appearance of real-time PCR, quantitative PCR was done
based on competition of primers caused by the addition of a competitor DNA. The
difference of the current design is that it does not require the preparation of
a competitor DNA.
Based on this design, we tested whether competition between individual amplicons
indeed occurs. We did PCR of the Sgpl1 locus from wild type gDNA using
individual primer pairs (F-out and R-out or F-in and R-out), or with the three
primers mixed (upper). The signal of the out-amplicon was strongly inhibited
when the inner primer was present (lanes 1 versus 3). On the other hand, the
signal of the in-amplicon was unaffected by the presence of F-out primer (lanes
2 versus 3). We did the same experiment using genomic DNA from Sgpl1 mutant
cells. This time, the signals of both of the amplicons were weaker when the
three primers were used than when doing PCR individually (bottom). Thus, as
assumed, each amplicon has a negative effect on the other. In wild type cells,
the out-amplicon was so strongly inhibited that it could not affect the amount
of the in-amplicon. The balance of inhibition was different in mutant cells due
to the lower affinity of the inner primer. Therefore, wild type and mutant DNA
could be discriminated by calculating the ratio of out- and in-amplicon when
doing PCR with the three primers. We will call this PCR method “competition-
based PCR (cbPCR)”.
## Optimization of cbPCR
Having shown that cbPCR can discriminate mutant DNA from wild type, we next
investigated the factors that affect the efficiency of this method in order to
reduce the chance of potential identification errors. This was important since
CRISPR/Cas9 leads to various indel sizes, thus some might be more or less easily
discriminated (due to differences in inner primer affinity), and we thought that
optimization was required to enable discrimination of all of them from wild
type. We first tested whether there is an optimal orientation of the inner
primers. Based on the nomenclature defined above, the F-in primer provides a
larger in-amplicon, which utilizes more dNTPs. We did cbPCR using F-in or R-in
as the inner primer. The use of F-in led to a stronger inhibition of out-
amplicons than when R-in was used. Both primers enabled the discrimination of
mutant and wild type DNA using the ratio of out- and in-amplicons, but the
difference was bigger when F-in was used. This enabled a better genotype
discrimination due to a stronger inhibition of out-amplicon when using wild type
DNA. The same pattern was seen when another gene, Sphk1, was tested. In this
case, in-amplicons were absent when cbPCR was done with mutant DNA regardless of
inner primer orientation, but the use of F-in led to a stronger inhibition of
out-amplicon when using wild type DNA. Thus, when primer design allows it, the
inner primer should be designed in the direction of F-in.
We next tested the effect of F-in primer length. A longer F-in primer might
increase its affinity with template DNA, leading to a stronger inhibition of
out-amplicon when using wild type DNA, thus improving mutant discrimination.
However, a longer primer might also increase the tolerance to mutation in the
template, reducing mutant discrimination. Therefore, it was difficult to predict
whether the F-in primer should be long or short. We did cbPCR using F-in primers
of varying lengths. As expected, a longer F-in primer leads to a smaller out- to
in-amplicon ratio in wild type DNA, but at the same time increased the tolerance
for mutations (see the appearance of in-amplicons in PCR products of mutant
gDNA). Therefore, the best discrimination was achieved when the F-in primer was
short, as long as it enabled efficient detection of in-amplicon in wild type DNA
(21 mer). In our PCR conditions, we usually achieved this condition when the
F-in primer was designed to be the longest while not exceeding a melting
temperature of 60°C. We also routinely designed the F-out primer with a melting
temperature lower than the F-in primer, in order to increase the inhibition of
out-amplicon when doing cbPCR with wild type DNA. We used this primer design as
a default. We next tested whether by using this default setting, we could
discriminate mutants of different genes by cbPCR. For this, we used gDNA from 8
mutant cell lines that were generated a priori. Without further optimization of
primer design, we could clearly discriminate 7 out of 8 mutant lines from wild
type using cbPCR, by the absence of in-amplicons. The exception was the Plpp2
gene, where the in-amplicon was also absent in cbPCR product of wild type gDNA.
Except for this, using the default primer settings, most of mutants could be
discriminated by cbPCR.
## Guidelines for primer refinement
If we could overcome the problem of Plpp2 mutant detection, we should be able to
determine how a failed primer design could be improved. We first did
conventional PCR to test whether the F-in primer could bind to the template or
not. When used without competition, the F-in and R-out primer set enabled the
amplification of a detectable in-amplicon. However, in cbPCR settings, the out-
amplicon outcompeted the in-amplicon even in wild type DNA. This result shows
that by doing PCR with individual primers, we can define whether the
amplification of in-amplicon just failed or whether there was a too strong
competition. If the inner primer does not work, we should either use the one
with the other direction (R-in primer), or change PCR conditions (annealing
temperature and/or enzyme) to make it work. If the inner primer does work, then
adjustment of the balance of PCR amplification efficiency could be done. Since
the inner primer did work for Plpp2, we did the latter. We tested F-out primers
of different lengths, and found that slightly shorter ones (17 or 18 bp) enabled
the detection of in-amplicons in cbPCR using wild type, but not mutant DNA.
Therefore, by refining primer design in failed experiments, we could design
primer sets that discriminate mutants. As far as we tested, whenever an in-
amplicon is efficiently detected by cbPCR using wild type gDNA, mutants are
discriminated (when using the default primer design).
Since heterozygous mutant cells should also be present in screening experiments,
we tested whether completely mutant cells can be correctly discriminated from
them. For this experiment, we selected targets for which we had weak signals of
in-amplicons when doing cbPCR (Figs), because we thought that these targets
might be more difficult to discriminate. We mixed 1:1 wild type and mutant DNA
to obtain artificial heterozygous mutant DNA, and did cbPCR. Since we pre-
selected targets with weak in-amplicon signals, some bands were difficult to
visualize by eye, but a complete absence of in-amplicon was seen only when
completely mutant DNA was used. The difference was clearer when we analysed the
signals from gel images by Image J. The latter analysis revealed that the
signals of in-amplicons tend to be reduced in heterozygous mutants, but less
obviously than in completely mutant clones. Thus, the calculated ratios between
out- and in-amplicons for heterozygotes were higher than for wild type, but the
differences were not as pronounced as when using knockouts. This shows that the
increase of amplicon ratios is not linear according to wild type copy number,
and that a change in wild type copy number from one to zero causes a bigger
shift than a change from two to one. Therefore, the results suggest that this
strategy can be used for an easy screening to discriminate completely mutant
clones from wild type or partially mutant clones.
## Testing the efficiency of mutant detection
Finally, we evaluated the error rate of cbPCR for mutant clone screening. For
this, we mutated the HPRT1 gene in HeLa MZ cells using CRISPR/Cas9 and isolated
clones. HPRT1-deficient cells are resistant to the toxicity of a nucleotide
analog, 6-thioguanine (6-TG). Thus, by comparing 6-TG resistance and cbPCR
results, we were able to evaluate how efficiently this method discriminates
mutants from the others. This experiment was done using two different guide RNAs
targeting different sites for HPRT1, and samples from one experiment served as
negative controls for the other experiment (since the mutation sites were
distant enough). In one experiment, all 6-TG resistant clones had a complete
absence of in-amplicons when analysed with cbPCR. When out- to in-amplicon
ratios were calculated and ranked, only 6-TG resistant clones had infinite
values due to absent in-amplicons. One 6-TG sensitive clone (clone 19) had an
out- to in-amplicon ratio which was intermediate between controls and 6-TG
resistant clones. Sequencing analysis showed that this clone had a 2 bp deletion
and a wild type allele. In addition, we selected six 6-TG sensitive clones that
had ratios comparable to controls for sequencing, and found that all were
completely wild type. Thus, for this target, the accuracy of mutant
discrimination was 100%, and sequencing also suggested that partial mutants
could be detected to some degree.
When we did the same experiment with another target site of HPRT1, some 6-TG
resistant mutants had detectable in-amplicons, but calculation of the out- to
in-amplicon ratio enabled us to correctly discriminate them from the 6-TG
sensitive clones. This shows that mutants are not always detected as a complete
lack of in-amplicon, but can be discriminated using the ratios between out- and
in-amplicons. One exception was a 6-TG sensitive clone (#10) that was ranked as
a mutant based on quantification of amplicon ratios. By doing cbPCR with a
shorter F-in primer, we could obtain a complete absence of in-amplicon in all
the mutants. However, the 6-TG sensitive clone \#10 was still classified as a
mutant even with this optimized primer design. From this result, we speculated
that clone \#10 was indeed a mutant, but retained an active HPRT enzyme. We did
sequencing to detect the pattern of mutations in clone \#10, and found that one
allele contained a point mutation without indels. This mutation was silent,
leading to a protein with wild type primary sequence. Therefore, clone \#10 was
indeed a mutant, but was 6-TG sensitive due to a wild type HPRT protein. We
further performed sequencing of all the clones having intermediate out- to in-
amplicon ratios (between zero and infinite), as well as four clones each from
samples having either zero or infinite ratios. All the analysed clones with zero
values were complete wild type. Heterozygous clones (8 and 23) had slightly
increased ratios but at a lower degree than for homozygous mutant clones. As a
tendency, mutants with bigger indels had bigger changes in ratios, and all
mutants with infinite ratios had only large (\>4) indels. Importantly,
homozygous mutants with the smallest indels had bigger changes in ratios than
the heterozygous mutant clone 23 that had a large deletion, meaning that the
size of indels affect less the results than the wild type copy number. As a
conclusion, cbPCR had also 100% accuracy (at least for the 6-TG resistant clones
and the sequenced clones) for discriminating mutants of this locus, even without
the further optimization that we did to obtain a complete disappearance of in-
amplicons when doing cbPCR with mutant gDNA. This also shows that cbPCR is
sensitive enough to detect even a point mutant. The sequencing results suggested
that although the size of indels affects the quantification results, the
presence or absence of wild type alleles have a bigger impact, thus enabling the
discrimination of complete mutants.
# Discussion
In this manuscript, we described competition-based PCR (cbPCR) as a method to
detect mutant clones after genome editing experiments. The competitive factor in
this experiment is very important. If the discrimination was based only on the
lack of binding between a primer and its target, the primer (and the annealing
temperature) should be designed to give a marginal binding to the target, which
is lost even by the smallest indel. When competition is present, this strict
optimum is no longer needed, since a decreased affinity is sufficient to give a
different outcome. The advantage of this method is that it requires only
equipment for PCR and agarose gel electrophoresis (or any other alternatives),
thus can be performed in almost all laboratories, and at low cost. The method is
so simple that we speculate that similar methods might already be used, even
without recognizing that the detection is based on competition between
amplicons, and not solely based on loss of inner primer binding. However,
existing reviews or online experimental guides
(<http://blog.addgene.org/crispr-101-validating-your-genome-edit>) did not
describe such a method for clone identification, thus we thought that it would
be useful to share the theory and design optimization steps for cbPCR. We
provide a protocol to explain the steps for establishing cbPCR for different
targets. Although optimization of additional factors such as extension time or
cycling numbers (to test whether we need to reach an amplification plateau or
not for efficient mutant detection) might be done, these issues were not
investigated in this manuscript since the detection efficacy was already
satisfactory with the current design.
The present method can discriminate homozygous mutants from the other undesired
clones, even when the mutation is the same in both alleles. This is extremely
important, since mutations induced by CRISPR/Cas9 experiments are not completely
random. Indeed, we have found cases where \~60% of the mutants had the same
indel at the target site. In such biased cases, many of the mutant clones will
have the same mutation in all alleles, and screening of mutant clones with
mismatch cleavage assays or heteroduplex mobility assay will result in the
misidentification of many mutant clones, unless wild type PCR amplicon is added
to all samples. Therefore, cbPCR is easier and might have a higher detection
power than these other methods.
The success of mutant detection relies on the design of primers that can
identify efficiently the mutants. The default setting in our laboratory for
primer design was good enough to detect mutants of 7 out of 8 genes.
Furthermore, we could easily optimize primer design for the remaining one.
Therefore, it is not difficult to obtain primer sets that work efficiently. One
constraint for primer design is that the inner primer should overlap with the
Cas9 cleavage site. It can be imagined that for some genes, this constraint will
lead to the design of primers that are non-specific or that do not bind to the
target, due to secondary structure or other reasons. We speculate that the non-
specificity issue is not a problem, since we usually design guide RNAs that
avoid off-target effects, thus the target should be unique in the genome. For
the second issue, as far as we have tested, we did not find an inner primer that
could not bind to its target. We routinely use two polymerases (ExTaq or
PrimeSTAR GXL) for PCR, and all of the targets have been amplified by at least
one of them. The freedom to design two orientations of the inner primer
(although F-in orientation is preferable than the other) also reduces the
probability to fail in the design of inner primers. In this manuscript, we often
used mutant DNA that was generated a priori. From these experiments, we found
that whenever the primers are designed based on the default setting and two
bands are seen in cbPCR using wild type DNA, mutants can be discriminated.
Therefore, even if one does not have mutant DNA a priori (as should be in most
cases), a successful primer set could be designed based on cbPCR results using
wild type DNA. If the in-amplicon is seen in wild type (and preferably also the
out-amplicon, which should demonstrate that it is not overcompeted by the in-
amplicon), then the primers should work. It should be noted that even if the
primer is not fully optimized, the power of mutant detection by cbPCR is still
very high (compare).
In this manuscript, only random mutations induced by the widely-used wild type
*Streptococcus pyogenes* Cas9 nuclease were analysed. However other genome
editing methods exist to induce random mutations, such as Transcription
Activator-Like Effector Nucleases, double-nickase using single-mutant Cas9, or
CRISPR systems from other species. We believe that cbPCR can detect mutations
induced by other methods, as far as the mutation site is predictable. Since some
of these genome editing strategies can induce staggered (instead of blunt)
double strand breaks, it is possible that in such cases the prediction of where
indels happen is more difficult, thus the placement of the inner primer might be
more complicated. Indeed, indels generated by a double-nickase strategy could
happen in two different sites separated from each other by more than 20 bp,
making the design of an inner primer that can overlap with all the mutations
impossible. Therefore, the type of double strand break should be considered
before designing a cbPCR approach to analyse clones. Another application of
genome editing is precise editing based on homology-directed repair using donor
DNA. We speculate that for such experiments, we can detect mutant clones with
the desired mutation by designing an inner primer having a perfect match with
the desired mutant site (and not the wild type site). By doing so, correctly
mutated DNA should be detected by an increase in the ratio between in- and out-
amplicons, since only correct mutants have efficient amplification of the in-
amplicon, which is the inverse case of what is done in this manuscript. This
hypothesis still has to be examined.
It should be emphasized that the experiment was specifically designed for
detection of fully mutated clones. We do not expect this method to be
quantitative enough for estimation of mutation rate in polyclonal cells, or for
the detection of heterozygous mutant cells, although the results still suggest
some degree of quantitation. Indeed, heterozygous mutants could be discriminated
by cbPCR at least in all the experiments done in this manuscript, but only a
limited number of targets were analysed until now. It should also be noted that
some variability in signal ratios is seen between experiments (for example,
compare), so it is appropriate to include at least one wild type sample in all
experiments as a control, while avoiding comparison of ratio values between
experiments done in different days. In addition, we recommend doing sequencing
after the screening by cbPCR. Although the experiment with HPRT1 mutants
suggests a very low misidentification rate, we have not yet evaluated the
misidentification rate in large numbers of target genes. The ease of cbPCR is
especially useful when large numbers of clones have to be analysed. Such a
requirement might arise when multiplex genome editing is done. In such
experiments, one can do cbPCR for all targets first, rank clones based on the
ratio of out- and in-amplicons of each target, and sequence only genomic DNA of
those cells that are expected to have mutations in all the targets. This will
reduce drastically the time and cost needed to obtain the correct clones needed.
We could obtain triple knockout cells easily using this strategy. We do not
recommend to directly sequence cbPCR products, since the in-amplicon might serve
as a megaprimer for the out-amplicon, leading to sequencing reads that are
partially wild type, even in completely mutated clones.
To conclude, we established a method to discriminate fully mutated clones from
the other undesired ones, based on a simple PCR experiment. The method is cost-
and time effective, can be performed with standard equipment while being at
least equally sensitive to existing methods.
# Supporting information
McA-RH7777 cells and HeLa MZ cells were kindly provided by Christian Toso
(University of Geneva) and Marino Zerial (Max Planck Institute), respectively.
The plasmid pX330 was deposited to Addgene (plasmid \#42230) by Feng Zhang
(Broad Institute). We thank all the members of the Riezman laboratory for
valuable comments.
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** TH. **Data curation:** TH. **Formal analysis:**
TH. **Funding acquisition:** TH HR. **Investigation:** TH. **Methodology:**
TH. **Project administration:** TH HR. **Resources:** TH HR.
**Supervision:** HR. **Validation:** TH HR. **Visualization:** TH HR.
**Writing – original draft:** TH. **Writing – review & editing:** TH HR. |
# Introduction
The riding posture is very important because an awkward riding posture may cause
discomfort to the riders and increase the risk of developing musculoskeletal
disorders (MSD) in long-term exposure. De Cássia et al. stated that body
discomfort is an indicator of many MSD due to prolonged discomfort. A sustained
flexion of lumbar lordosis (low back) during driving usually is the main factor
in the generation of low back pain. According to Makhsous et al., sitting
decreases lumbar lordosis compared to the standing posture, resulting in
increased disc pressure and low back muscle activity. This is because while
sitting, the ischial tuberosity mainly supports the majority of the upper body
weight. Increased pressure in this area is significantly associated with
increased spinal load. Riding a motorcycle exposes the officers to excessive
physical demand, especially for prolonged periods. They will tend to feel
discomfort and possibly fatigue while sitting in the same position with
restricted movements. Thus, to improve spinal posture and comfort in
transportation settings, seat design has a significant role and efficiency.
A well-designed seat would decrease pressure within the spinal discs, spinal
ligaments, and gluteal muscle. Lumbar support is the solution for the problem,
whereby the weight and pressure of the trunk are taken by the back support and
increased lumbar lordosis. The use of a vehicle seat (lumbar support) has been
significantly associated with the reduction of muscle discomfort and low back
pain during prolonged riding and driving journey. Adjustable lumbar support with
extra cushioning, which provided a massage-like comfort, has been proved to
reduce transmission of vibration, which indirectly increases muscle comfort by
improving oxygen and blood flow to the tissue. According to Mansfield et al.,
the seat shape, user suitability, seat material, duration of sitting in the same
position, vibration, and posture changes are among the factors that influence
seat comfort.
Traffic police is a police officer who serves to enforce the rules related to
traffic. They have to perform various work tasks, such as escorting important
person to any events, patrol selected locations, and finding any road offenders.
All this work task requires them to use a motorcycle as their vehicle. At
present, the officer riding a motorcycle takes at least five hours per day
during duties. Therefore, traffic police riders have to endure prolonged riding
duration. Rashid et al. believed that continuous riding of a standard motorcycle
for an extended time would result in a high level of postural fatigue and health
problems. From a previous research study on the CBX 750P21 motorcycle, it was
found that 88.3% of traffic police riders suffered from MSD with 34.3% of them
suffering from lower back pain due to static posture and prolonged sitting while
riding the motorcycle. Another previous study also revealed that more than half
(54.7%) of the traffic police riders rode high-powered motorcycles for an
average of 5.64 hours per day with a fixed posture leading to increased
discomfort from prolonged sitting that enhanced muscle fatigue. This showed that
most of their working time involves riding motorcycles. However, to the best of
the authors’ knowledge, no research has been done on the spinal riding posture
during a motorcycle ride, especially among police officers in Malaysia who use a
motorcycle to perform their duties daily.
To solve this problem, ergonomics is the best solution for scientific research
in man-machine interaction at the workplace since this field involves fitting
machine to workers comfortability to improve their working performance, reduce
fatigue, and stress. The ergonomic application is very significant in areas
involving prolonged and static riding activities that directly affect the
riders’ healthy spinal posture and reduce muscle fatigue. Lumbar support with a
built-in massager system is an intervention in this study to solve the ergonomic
issue among motorcycle riders. The use of a back (lumbar) support has been
proved to help maintain the natural spine lordotic lumbar curvature of the
person while sitting compared to those without back support. In a clinical and
laboratory setting, automobile seat massage is utilised widely, and it has been
proved efficacious in recovering from postural fatigue. However, research and
data on the effectiveness of this intervention seat, lumbar support with a
built-in massager system, in an in-field setting (on-the-road) are insufficient.
Thus, the present study integrates a massager system and lumbar support with the
existing seat which an on-road test was conducted to maintain and support the
spinal posture of the riders more efficiently as the test involved prolonged and
static riding. In this study, the spinal angle profile was measured and assessed
to determine the effectiveness of theprototype seat in supporting the spinal
body posture of the riders throughout 20 minutes motorcycle ride. Thus, the
present study seeks to evaluate the effect of lumbar support with a built-in
massager system (prototype seat) among traffic police riders.
# Materials and methods
## Study design
A randomised controlled trial, pretest-posttest control group design was
conducted among 24 traffic police riders who ride a high-powered motorcycle
(Honda CBX 750). A simple random sampling was used in this study which the
subjects were randomly assigned to the control group (12 riders) and
experimental group (12 groups). Data collection commenced in March 2020 and
finished in July 2020.
## Eligibility criteria
Eligible subjects were male riders only because almost 90% of traffic police
riders are male. BMI was between 18.5 to 29.9 kg/m<sup>2</sup>. The age group
recruitment was between 20 and 39 years old because more than 40 years old are
usually prone to experiencing low back pain due to the age factor and changes in
spinal posture. At least one year of experience in high-powered motorcycle
riding. Exclusion criteria were the presence of any injury (under treatment or
taking any medication related to muscle pain), especially lower back pain, in
the 12 months preceding this survey.
## Sample size
To calculate the appropriate sample size, we use the formula of group comparison
guidelines in a study done by Donnelly et al. as a reference. The estimated SD
(30.2), estimated larger mean (57.2) and estimated lower mean (19.0) were used
in this formula. In this study, the desired power is 80% and the significant
level is 95%. Thus, each experimental and control group would have 10 subjects.
However, an additional 20% dropout rate was added in the sample size
calculation. Thus, each experimental and control group will have 12 subjects.
Therefore, a total of 24 subjects was selected in this study.
## Participant recruitment
The recruitment strategy involved taking the name list of all officers working
at the Kuala Lumpur Traffic Police Station. The name lists were obtained from
the Human Resources Department. However, 97 officers from the escort department
only were chosen to ensure the consistency of data in which different department
have different task or job and the duration of riding police’s motorcycle. Only
24 respondent who fulfilled the criteria were selected for the study. For
qualification criteria, respondents were asked to fill up the pre-survey form
and the BMI were measured by the main researcher. After the eligible subjects
were selected, then the main researcher randomly assigned 24 of them into
control and experimental group. This study’s sampling method was simple random
sampling, in which all the subjects’ names were numbered into pieces of paper
and put in a container. Then, the subjects were chosen randomly into the
experimental or control group by Fishbowl Technique. Each subject in the
experimental group received lumbar support with a built-in massager system
during post-test. The pre-test and post-test were conducted at a one-week
interval. Detail about study flow was illustrated in the CONSORT diagram.
Subjects (n = 12) allocated into the control group had undergone post-test
session without the lumbar support with a built-in massager system (existing
seat) during 20 minutes riding session. On the other hand, the experimental
group (n = 12) had undergone a post-test session with the lumbar support with a
built-in massager system (prototype seat) for 20 minutes riding session.
Before the experimental session began, subjects were allowed to choose the
Truposture smart shirt that fits their body. This was done to ensure the point
of each sensor would be placed in the correct position of the spine. There were
no cases of unable to follow up or incomplete experimental testing as planned in
this study. All subjects completed the follow-up stage (n = 24) in both
intervention groups. This study had no excluded analysis cases as all the data
were successfully recorded without any missing data.
## A prototype
The lumbar support on the prototype seat can be adjusted either upward and
downward according to the lumbar height position for the comfortability of the
riders. There is also a massage fitted inside the lumbar support with two
rotating balls. The lumbar massage was set at a ratio of 1 minute on and 4
minutes off. It was used for 20 minutes in the experimental riding session. It
can support up to 150 kg body weight.
## TruPosture
A spinal posture angle was obtained using the TruPosture Smart Shirt connected
with the TruPosture mobile app among the police riders while riding a motorcycle
with an existing seat for both groups; control and experimental during the pre-
test. Then, during the post-test, the control group used the same motorcycle
seat, and the experimental group used a prototype seat containing adjustable
lumbar support with a build-in massager system. The TruPosture Smart Shirt
covered different regions of the spinal alignment, with five nano-sensor
technology attached undershirt. The five sensors cover the thoracic vertebrae,
lumbar, and the pelvic regions (T1, T8, L1, L3, and Pelvis).
The technology helped the researcher monitor the entire spinal’s alignment and
curvature in several modes, including standing and sitting. The TruPosture
mobile apps interface and Windows software were utilised as these tools help the
researcher track the posture and record the movement of posture in real-time.
The sensors detect the real-time changes of spinal posture angle based on the
spinal movement.
Based on, the blue curve represents the spinal reference posture (ideal posture)
at 0 minutes, and the orange curve indicates the actual spinal riding posture
throughout 20 minutes. The number on the left and right side of the interface
represents the angle reading for each spinal point sensor. The positive and
negative values of angle indicate the spinal position, which is forward and
backward respectively.
The validity of the equipment had been tested and approved by the previous study
which was suitable for monitoring the posture of the spine. The reason for
choosing this method is that the spinal sitting pattern studies were limited to
laboratory and clinical settings only. According to Ma’arof et al., the research
field provides vital outcomes and real-world motorcycling information. Thus,
TruPosture Smart Shirt is the most suitable method to monitor the spinal change
pattern among motorcyclists on-road with the application of real working
conditions.
## Data collection
In this study, the spinal reference posture at the 0 minutes riding session was
set first. The subjects wore the TruPosture Smart Shirt during the monitoring,
according to the ideal riding posture. An upright riding posture is the best
posture in riding this type of motorcycle based on the standard operating
procedure (SOP) of Honda CBX750P motorcycle riding. According to Ma’arof et al.,
a good riding posture for the upright posture is that the arms should bend
slightly when gripping the throttle and sitting in the upright position. The
shoulders and elbows are held easily on the holds without exceeding or over-
expanding the elbows. Elbows are flexed, and lower arms are parallel to the
ground. Furthermore, the legs are held near the fuel tank while relaxing the hip
and pelvis. This posture was set as a spinal reference posture for riding, and
the angle for each sensor was recorded. However, this is the real problem for
the riders, as they could not maintain the ideal posture in medium- and long-
distance trips. Thus, the subjects were asked to ride a motorcycle for only 20
minutes. This is because the average commute time of traffic police riders for
one task is less than 30 minutes. Moreover, According to Deros et al., 82.5% of
the discomfort variance divergence is accounted for after being seated for 20
minutes and it takes less than five minutes in postural changes without back
support. The riding posture was assessed by the same person (an ergonomist) in
the pretest-posttest session.
This study was conducted at the MEX highway route (the route between the
commercial centre of Kuala Lumpur to the Federal Administrative capital of
Putrajaya and Kuala Lumpur International Airport), with good road condition and
maintenance. The route was chosen as it was commonly used in their work task as
escort riders. The pre-post test results were then compared between the two
groups.
## Quality control
A pre-test was conducted approximately 10% of the sample size. Two respondents
who had undergone pre-test were not included in the real experimental testing as
it was not to prove the superiority of the treatment but to test the procedures
and processes and estimate parameters for the main trial sample size
calculation. Other than that, the pre-test could ensure that all the instruments
were working properly and in good order. The other purpose of this study was to
familiarise the researcher with the placement of sensors of Truposture smart
shirt to avoid any mistakes during the data collection process.
To ensure a good quality control, the supplier had demonstrated the handling of
Truposture smart shirt to the researcher and the possible errors that might
occur. The measurement procedure used was obtained from the Truposture smart
shirt Manual Book. The respondents must ensure that the size of the shirt was
fitted with their body to make sure the sensor placed aligned their spinal
correctly. Then, the respondents need to ride a motorcycle for 20 minutes. Each
test was taken around 10:00 am to 10:20 am, to ensure consistency of traffic
conditions. The measurement was taken for 20 minutes because the average time
taken by traffic police riders in riding motorcycle was approximately 20 minute
per task.
## Statistical analysis
The collected data from the questionnaire and measurement were analysed using
the IBM SPSS Software (Version 26). The Shapiro-wilk test was used in this study
to determine the normality of data distribution for variables. The normality of
data distribution was assumed when the p-value was more than 0.05. The present
study found that the data variables were not normally distributed. Thus, a non-
parametric test was used in this study. The data collected were analysed using
the Mann-Whitney test and Wilcoxon signed-ranked test to verify a statistically
significant difference between and within the control and experimental groups.
The dependent variable of this study was the spinal angle (0<sup>th</sup> and
20<sup>th</sup> minutes). The measurement of the spinal angle was done by using
Truposture smart shirt. The study was conducted using a 95% confidence level,
80% of power, and the results of p≤0.05 were considered significant.
## Ethics approval
This study was submitted and approved by the Ethics Committee, Universiti Putra
Malaysia (reference number: UPM/TNCPI/RMC/JKEUPM/1.4.18.2 (JKEUPM)). Permission
from the subjects of this study was obtained with their written consents before
the study was conducted. Privacy of the information and confidentiality of the
subjects were and are always protected.
# Results
## Subjects
In total, 24 traffic police riders were included in the study, and there were no
drops out. All subjects enrolled completed the study protocol as planned. The
subjects’ demographic data from both the control group and the experimental
group are tabulated in ; no significant differences were observed between the
two groups.
## Distribution of five different sensors for spinal posture angle
The mean spinal posture angle in each sensor for the control and experimental
groups is presented in. The graph showed that the experimental group (post-test)
values are successfully maintained with minimal changes to the spinal posture
angle compared to the other groups. The positive and negative values of angle
indicate the position of the spinal, which is forward and backwards,
respectively.
Meanwhile, the line graph in show the trend comparison of spinal posture angle
deviation throughout the 20 minutes riding session. The line graph compares the
level of spinal angle deviation in four groups (control pre-test, experimental
pre-test, control post-test, and experimental post-test). Based on the graph
line comparison, a deviation of spinal angle increased in each sensor, and
Sensor 1 experienced the greatest deviation from the other sensors. From the
graph, a deviation of spinal angle on each group shows an upward trend. However,
the spinal angle deviation in the experimental (post-test) group is always at a
lower level than the other groups. It showed that the experimental group
intervention has a huge improvement in maintaining posture, which only involves
slight spinal angle deviation changes from the spinal reference angle throughout
the 20 minutes ride. The details of the spinal posture angle profile throughout
20 minutes are available as supporting information;.
## Comparison of five different sensors
compares the spinal angle (0<sup>th</sup> and 20<sup>th</sup> minutes) between
pre-test and post-test study for five different sensors within two groups. As
depicted in this table, a Wilcoxon signed-rank test revealed a statistically
significant difference between pre-test and post-test session in all sensors at
the 0<sup>th</sup> and 20<sup>th</sup> minutes in the experimental group
(p\<0.05). Meanwhile, no statistically significant difference is observed among
the control group in all five sensors (p\>0.05). The results obtained from a
Mann-Whitney test showed that there was no statistically significant difference
in spinal angle between the control group and experimental group in the pre-test
session as shown in. However, there were statistically significant differences
in the spinal angle between control and experimental groups for all sensor
(p\<0.05) throughout 20 minutes of a motorcycle ride.
# Discussion
The present study found that the existing seat contributes to the huge changes
in Sensor 1 (Thoracic 1) throughout 20 minutes of riding for both groups. This
could be explained when the riders applied a slumped posture at the end of the
20 minutes of riding due to muscle fatigue while adopting an upright posture
without back support. Kwon et al. explained that the thoracic angle is usually
related to the outcome of a slumped posture. These findings were supported by
Ma’arof et al., who reported that an upright riding posture involved higher
muscular activity compared to the forward-lean posture, which makes riders
muscle less comfortable, leading to fatigue. Shoulder stiffness is frequently
encountered by motorcyclists, including young and healthy riders. Therefore,
officers using existing seat proved that they could not maintain an ideal and
good posture while riding a motorcycle, indicating the leading cause of concern
for this problem.
The application of lumbar support with a built-in massager system showed a lower
deviation angle in the trend compared to the group without intervention. This
happened because the upright posture without back support (exiting seat) leads
to the rider’s unnatural spinal curve. This can be explained by the fact that
this prototype helps correct the spinal posture (upright) of motorcyclists
better than the absence of lumbar support. These findings are parallel with the
previous research that reported the lumbar support on a motorcycle seat was
capable of providing good posture and reduce muscle discomfort of riders during
the riding process. This is supported by Ceunen et al., who stated that the
upright sitting posture with lumbar support is efficient to change pelvis and
spinal structure to their natural curve during sitting, which reduces the load
on the ischial tuberosity and lower spinal, reduce muscular activity, maintained
lumbar lordosis; thus, potentially reduce the risk of developing low back pain.
Alyami and Albarrati also found that the workstation would be safer when the
posture’s biomechanical risk could be reduced by applying ergonomic rules in
design that would support healthy (ideal) body posture.
A statistically significant difference was noted throughout this research in all
sensors at the 0<sup>th</sup>, and 20<sup>th</sup> minutes (p\<0.05) between
pre-test and post-test measurement within the experimental group. However, there
was no statistically significant difference in spinal angle within the control
group. This proved that the intervention of the prototype seat in the current
study could effectively maintain spinal posture in an upright riding position
throughout 20 minutes riding session. The value of spinal angle deviation
between the pre-test and post-test in the experimental group throughout 20
minutes of riding also showed that the slumped spinal posture could be prevented
with lumbar support and a built-in massager system.
The analyses between-group differences also found that there was a significant
difference in all spinal angle between control and experimental groups
throughout 20 minutes of a motorcycle ride. The findings in the current study
proved that the prototype seat provides a positive effect on the spinal posture
of motorcyclists in adjusted and maintaining their spinal posture which can
serve as a channel for the distribution of the force as well as static loading
by the body. In this case, the intradiscal pressure could be reduced and the
supporting back muscle could be enhanced as shown in.
Another crucial finding in this study involved applying the massager system (4
minutes off to 1 minute on), whereby the results of the experimental group
showed relatively not much deviation compared to the other groups. The value of
spinal angle deviation in the experimental group was reduced after every four
minutes. This could be possibly due to the massager in the lumbar system
improves spinal posture from an awkward posture (slump) over time. These results
proved the theory that the massager in the lumbar area could reduce muscle
fatigue, increase blood flow and oxygenation, and maintain body posture
effectively.
Contrarily, Tanaka et al. reported differences that suggested the lumbar massage
failed to exert significant changes in any electromyographic (EMG) measurement.
Besides that, Van Poppel et al. reported that lumbar support was not useful in
preventing low back pain. The outcome of this study contradicted the study by
Kolich et al., where they pointed out that one minute of lumbar massage in every
five minutes of the driving session has a beneficial effect on the lower back
muscle activity. A study conducted by Franz et al. suggested that lumbar support
usage with lightweight massager in automobile seats has been reported to
decrease muscle discomfort during the driving process and improve lumbar
lordosis.
There were several limitations in the present study. Only male traffic police
riders were recruited. Thus, it cannot be used to generalise the whole
population. A future study might include female traffic police riders and other
occupations such as food delivery. Even though we found a positive outcome of
this prototype in maintaining spinal posture, since this study was conducted in
an on-the-road setting, there was a limited objective measurement that can be
done such as the measurement of muscle fatigue using EMG electrode (clinical
testing) due to environmental factors such as road condition and vibration. This
study was unable to blind the respondents to the condition that they were
exposed to, which might impact the posture riding in this study. This is because
traffic police riders were aware of which seat they were using, and as such,
there was no way to prevent a bias toward the prototype seat. However, it is
assumed that any bias towards the motorcycle seat prototype would disappear
through the riding duration if the seat did not truly adjust and maintain riding
posture throughout the riding process. Due to time constraints (pandemic
outbreak) and limited resources such as budget, it was not possible to include
all aspects of the problem with a bigger sample size in this research. Besides
that, there was only one prototype available for this research.
# Conclusion
In summary, the present study proved that lumbar support with a built-in
massager system successfully maintains and improves the spinal posture angle
ergonomically throughout 20 minutes of riding. Although there is a positive
outcome in this research, there is still a lack of evidence (i.e. muscle
fatigue, discomfort, muscle activity) for this study. Hence, further study in
the artificial laboratory and clinical settings as well as virtual testing is
needed to support these findings.
# Supporting information
The author would like to thank all of the people who were involved in this study
especially the Royal Malaysian Police (RMP).
10.1371/journal.pone.0258796.r001
Decision Letter 0
Mosa
Ahmed Mancy
Academic Editor
2021
Ahmed Mancy Mosa
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
27 May 2021
PONE-D-21-13337
Assessment of spinal angle profiles among high-powered traffic police motorcycle
riders
PLOS ONE
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Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#1: Partly
Reviewer \#2: No
Reviewer \#3: No
Reviewer \#4: No
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
2\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#1: No
Reviewer \#2: No
Reviewer \#3: No
Reviewer \#4: No
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
3\. Have the authors made all data underlying the findings in their manuscript
fully available?
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Reviewer \#1: Yes
Reviewer \#2: Yes
Reviewer \#3: Yes
Reviewer \#4: No
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: No
Reviewer \#2: Yes
Reviewer \#3: No
Reviewer \#4: No
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
5\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: Dear Authors,
This is an interesting research area. While the study has some merits to
consider for publication several clarifications are needed to assist in the
decision. My comments and questions are as follow:
1\. What is the new contribution of this study? What was previously known about
the ergonomic function and effect of the prototype seat, and how will this study
add to the existing knowledge? Why is it important to measure the spinal angle
profile?
2\. The problem statement highlighted in the introduction does not match the aim
of the study which is an experimental study comparing between an intervention
and control group.
3\. Please clarify whether there is any sample size calculation undertaken prior
to the study, and provide the relevant references for the sample size
calculation. What was the power of the study based on the sample size
calculation?
4\. Please provide adequate references for the prototype and the TruPosture app.
5\. Please provide a detailed explanation on the recruitment process, and the
allocation to control and intervention group.
6\. What was the dependent variable in the statistical analysis. please provide
the operational definition of the dependent variable.
7\. Please provide the general description of your study participants in the
results section, and describe the characteristics between the experimental and
control group to show that they are comparable.
8\. Do you expect any confounding factors in your final analysis to look at the
difference in outcome between the intervention and control group? What are the
appropriate statistical tests to be used to control for the confounders, if
there is any?
9\. please explain clearly the within and between groups comparison in the final
analysis of the study outcome.
10\. Please align the discussion with the aim of the manuscript. What is it that
you plan to highlight? is it about the
usability of the TruPosture smart Shirt in measuring posture, or it is about
showing the effect of the prototype seat on posture of the police riders?
11\. What is the study limitation?
12\. Please revise the manuscript title to reflect on the aim and experimental
design.
Reviewer \#2: 1. SUMMARY OF THE RESEARCH AND OVERALL IMPRESSION –
The manuscript is well written with concise language, but I have major concerns
on the methodology that prevent me to endorse acceptance at current stage. I see
that sound methodology is lacking and that compromise the data analyses and
could have led to potentially wrong results and misleading conclusion.
Recommended course of action is to re-look at the methods and try to conform to
standard reporting guidelines such as CONSORT guideline. The authors can expand
the study sample size, with proper calculation and reference to previous study
to ensure its adequate to draw to solid conclusion. Current study can serve as
preliminary or pilot study to come up with better technical standard of
experimental methodology and sufficient description on the details.
2\. DISCUSSION OF SPECIFIC AREAS FOR IMPROVEMENT –
a\) Methodology (major issues)
There are several issues with the methodology and analysis that need to be
clarified, address and described in great detail. The methodology section should
be expanded and clarified to support the conclusion and validate the findings.
As the methods section is lacking, it might be premature to draw sound and valid
conclusion.
• There is no mention on the specific study design and how the randomisation is
done (authors only mentioned randomisation once in Figure 2). Is this a
randomised controlled trial? Why is it not mentioned in the title? Has it been
registered in any trial’s registry?
• There is no mention of how the sample size is calculated. How is the sample
size being determined? Is the sample size adequate to represent the study
population? Is the sample size adequate to meet the assumption of statistical
analyses and come to conclusion?
• Eligibility criteria is not clear (authors only mention no history of MSD and
low back pain, but what about baseline age, height, weight, BMI, fat percentage,
gender, ethnicity, years of service, and other factors that might influence the
spinal angle profile?).
• There is no mention on how the recruitment is done. Is it convenience? Can it
be representative of the study population? Who enrolled the participants?
• How is the allocation to the control and intervention group being done? Is
there any blinding involved? What are the methods used to do randomisation and
type of randomisation (when allocating participants to control or intervention
group)? The authors did not mention who did the randomisation and who assigned
the participants interventions.
• There is no mention of the time period of data collection defining
recruitment, pre and post-test. How long is the gap between pre and post-test?
What specific time of the day the experiment being conducted? (is the timing and
duration of work on that particular experiment day influence the spinal angle
profiles?) What is the justification for 20 minutes duration of the experimental
riding session? (authors mention riders spend at least 5 hours per day on
motorbikes).
• The author did not mention anything on the instrument validation and
calibration. How are the sensors being placed? Is it done by the same person for
both control and intervention groups? Has the apps being tested and validated
before? How accurate is the reading?
• The author did not mention whether both groups are being assessed by the same
person (e.g. posture for riding). If it’s not done by the same person, do the
researchers take into account inter-rater reliability?
b\) Discussion
There is no mention or any discussion on the experiment’s limitations in the
discussion part. The authors did not discuss potential source bias and
confounder, threat to validity that might compromise the findings.
c\) Results
• The authors did not report the sociodemographic and baseline characteristics
of both control and experimental group. How does the researcher ensure that both
groups are similar at baseline? How can the researcher then conclude that the
outcome is due to their intervention, rather than existing differences? Are
there any additional methods of analyses such as subgroup analyses to account
for confounder or differences in the baseline?
• There is no justification on why non-parametric statistical analyses was
chosen. What are the assumptions and limitation of the analysis? Have all the
criteria being met?
• Too much data being presented that can be summarised in a sentence or in
simpler table. For example, Table 1 presents a range of minimum and maximum
value for each reading throughout 20 minutes – can be summarised by providing
the mean/median value. Easier to interpret and understand, rather than the
reader have to go thru each range.
d\) Others
• Figure 1 – no legend to help reader understand the figure. Which line belongs
to control and intervention group?
• Figure 2 – suggest authors to follow CONSORT flow diagram format (more
details)
3\. OTHER POINTS
Authors have provided adequate literature review to justify the problem
statement, significance of the study and burden of the disease. The authors also
adequately described previous research and gap of the study.
The data presented has potential to be published if its extended and properly
developed. Current study can serve as preliminary findings or pilot to come up
with better protocols and larger sample size to test the same hypotheses and
draw more concrete results and conclusion.
Reviewer \#3: Thank you for the opportunity to review your manuscript entitled
“Assessment of spinal angle profiles among high-powered traffic police
motorcycle riders”. The topic is interesting; however, I believe the study was
not appropriately designed to address the research question. The analysis and
reporting should not lead to the conclusion made by the authors. The
introduction needs to be strengthened. I have highlighted some points that I
believe would improve the quality of the manuscript.
Abstract
Line 30: the purpose of the study described in the abstract does not match with
what was done in the methods. This was an intervention study and I suggest
reformatting the purpose of the study.
Line 40: Are we really interested in a pre-test VS post-test analysis (within
group analysis)? Reporting the between group comparison is more insightful…
Introduction
Provide some statistics of MSD/low back in the population
Line 51: provide a full meaning of MSD
Line 59: I believe it was meant to be “been” instead of “seen”
Line 61: has instead of have
Line 67: delete “the”
Line 81: The purpose of the study needs to be reformulated
The introduction lacks content. The consequences of increased lumbar lordosis
are not fully described. There are some grammatical errors that need to be
corrected.
Materials and Methods
More information on the eligibility criteria (inclusion and exclusion criteria)
is needed
Any reference for the TruePosture mobile app? Has this app been used before in
any study? What are the validity and reliability properties of the app?
How were the groups defined? Were the participants randomly assigned to the
groups? What are the baseline characteristics of the control and experimental
groups?
Why did the authors not perform a between-group analysis? That comparison is
more interesting than all those pre-test vs post-tests performed.
Results
Data on the participants missing
The data reported does not indicate whether the spinal change pattern in the
intervention group is superior to the control group.
Discussion and conclusion
Not sustained by the analysis and results presented
Reviewer \#4: Abstract
Background
Context and research gap were not indicated
Materials and Methods
Type of study, sampling method and data collection method were not specified
Major statistical analysis was not stated
Main body
Materials and Methods
It has major methodological defect.
Type of study, sampling method and data collection method were not specified
Gold standard of experimental study was not succinctly stated.
Outcome was not assessed
Major statistical analysis was not indicated.
Why Wilcoxon signed rank test and median was used?
Reviewer \#5: Line 51: Please indicate the full meaning of MSD when been used
for the first time in write up
Line 178: Consider replacing "respondents" with "subjects" which best suits the
study and its concept
Please give reasons for the sample size choice and indicate the exclusion and
inclusion criteria for participating in the study
Line 243: "The value of spinal angle deviation between the pre-test and post-
test....." Indicate the angle of deviation to make your point clear
There are some typographical errors indicated in the attached document. Please
revise them accordingly
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: No
Reviewer \#2: No
Reviewer \#3: No
Reviewer \#4: No
Reviewer \#5: No
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10.1371/journal.pone.0258796.r002
Author response to Decision Letter 0
4 Jun 2021
Dear Dr Ahmed Mancy Mosa (Academic Editor),
Thank you for giving me the opportunity to submit a revised draft of my
manuscript titled Assessment of spinal angle profiles among high-powered traffic
police motorcycle riders. We appreciate the time and effort that you and the
reviewers have dedicated to providing your valuable feedback on my manuscript.
We are grateful to the reviewers for their insightful comments on our paper. We
have been able to incorporate changes to reflect most of the suggestions
provided by the reviewers. We have highlighted the revisions within the
manuscript. The line mention in the author response.
Here is a point-by-point response to the reviewers’ comments and concerns.
Reviewer 1
1\. What is the new contribution of this study?
Thank you for pointing this out. The new contribution of this study is:
• The application of the method (Truposture smart shirt) in spinal profile
measurement was evaluated in an occupational setting (on-the-road) which riding
in a real working condition was applied.
• In motorcycling industry, new invention for an ergonomic motorcycle seat had
been developed and tested.
2\. What was previously known about the ergonomic function and effect of the
prototype seat, and how will this study add to the existing knowledge? Why is it
important to measure the spinal angle profile?
We agree with this comment. Therefore, the relevant justification has been made
in the introduction section (line 92- 99).
2\. The problem statement highlighted in the introduction does not match the aim
of the study which is an experimental study comparing between an intervention
and control group.
We agree with this comment and has made changes accordingly in introduction
section (line 94-96).
3\. Please clarify whether there is any sample size calculation undertaken prior
to the study, and provide the relevant references for the sample size
calculation. What was the power of the study based on the sample size
calculation? You have raised an important point here.
We agree with this comment and the sample size calculation has been added in
sample size section (line 122-129).
4\. Please provide adequate references for the prototype and the TruPosture app.
Adequate references for the prototype and Truposture app have made (line
186-189).
5\. Please provide a detailed explanation on the recruitment process, and the
allocation to control and intervention group.
Thank you for pointing this out. The recruitment process has been explained
accordingly (line 131-158).
6\. What was the dependent variable in the statistical analysis. please provide
the operational definition of the dependent variable.
The dependent variable and the operational definition have been added in the
statistical analysis as suggested (line 223-225).
7\. Please provide the general description of your study participants in the
results section, and describe the characteristics between the experimental and
control group to show that they are comparable.
Thank you for pointing this out. The general description of the participants has
been discussed and tabulated in Table 1 (line 234-242).
8\. Do you expect any confounding factors in your final analysis to look at the
difference in outcome between the intervention and control group? What are the
appropriate statistical tests to be used to control for the confounders, if
there is any?
The confounding factors in this study was expected with the difference age,
gender, BMI and present of low back pain. However, these criteria were
controlled based on inclusion and exclusion criteria during sampling (line
144-121).
9\. please explain clearly the within and between groups comparison in the final
analysis of the study outcome.
Thank you for pointing this out. We agree with this comment and has made changes
accordingly in results and discussion section (line 316-330).
10\. Please align the discussion with the aim of the manuscript. What is it that
you plan to highlight? is it about the usability of the TruPosture smart Shirt
in measuring posture, or it is about showing the effect of the prototype seat on
posture of the police riders?
Agree. We have, accordingly, revised the discussion part and removed the
unrelated part.
11\. What is the study limitation?
Thank you for pointing this out. We have added the study limitation accordingly
in the discussion section (line 350-359).
12\. Please revise the manuscript title to reflect on the aim and experimental
design.
We agree with this comment. Therefore, we have revised and changed the
manuscript title into “Effectiveness of lumbar support with built-in massager
system on spinal angle profiles among high-powered traffic police motorcycle
riders: A randomised controlled trial”
Reviewer 2
1\. • There is no mention on the specific study design and how the randomisation
is done (authors only mentioned randomisation once in Figure 2). Is this a
randomised controlled trial? Why is it not mentioned in the title? Has it been
registered in any trial’s registry?
Thank you for the input. Yes, it is randomised controlled trial. However, we
apologise for overlooking this matter. Thus, the title and study design have
been revised as suggested (line 108-112).
It has not been registered in any trial’s registry. We are sorry for this and
hope for your understanding. Nevertheless, the sampling method is approved and
supported by the Ethical Committee of Universiti Putra Malaysia after consulting
their expertise.
2\. • There is no mention of how the sample size is calculated. How is the
sample size being determined? Is the sample size adequate to represent the study
population? Is the sample size adequate to meet the assumption of statistical
analyses and come to conclusion?
You have raised an important point here. We agree with this comment and the
sample size calculation has been added (line 122-129).
3\. • Eligibility criteria is not clear (authors only mention no history of MSD
and low back pain, but what about baseline age, height, weight, BMI, fat
percentage, gender, ethnicity, years of service, and other factors that might
influence the spinal angle profile?).
You have raised an important point here. We agree with this comment and has
included relevant information and explanation accordingly (line 114-121).
However, due to time (pandemic outbreak) and budget constraints (e.g: fat
percentage analyser), we are unable to include all aspects of the problem and
other factors.
Thank you for pointing this out. For future studies, this will be a great
insight and we will be sure to use it. Also, we will highlight this matter as
the limitation in this study.
4\. • There is no mention on how the recruitment is done. Is it convenience? Can
it be representative of the study population? Who enrolled the participants?
Thank you for pointing this out. The participant recruitment has been included
and explained accordingly (131-158).
5\. • How is the allocation to the control and intervention group being done? Is
there any blinding involved? What are the methods used to do randomisation and
type of randomisation (when allocating participants to control or intervention
group)? The authors did not mention who did the randomisation and who assigned
the participants interventions.
Thank you for pointing this out. The participant recruitment has been included
and explained accordingly (131-158).
6\. There is no mention of the time period of data collection defining
recruitment, pre and post-test. How long is the gap between pre and post-test?
Thank you for pointing this out. The participant recruitment has been included
and explained accordingly (line 144).
7\. What specific time of the day the experiment being conducted? (is the timing
and duration of work on that particular experiment day influence the spinal
angle profiles?) What is the justification for 20 minutes duration of the
experimental riding session? (authors mention riders spend at least 5 hours per
day on motorbikes). You have raised an important point here.
We agree with this comment and have included the relevant information and
justification in data collection section (line 205-209).
8\. • The author did not mention anything on the instrument validation and
calibration. How are the sensors being placed? Is it done by the same person for
both control and intervention groups? Has the apps being tested and validated
before? How accurate is the reading?
We agree with this comment. Therefore, we have discussed this information in
TruPosture section (line 186-192).
9\. • The author did not mention whether both groups are being assessed by the
same person (e.g. posture for riding). If it’s not done by the same person, do
the researchers take into account inter-rater reliability?
You have raised an important point here. Thus, the relevant information has been
added accordingly (line 209-210).
10\. b) Discussion
There is no mention or any discussion on the experiment’s limitations in the
discussion part. The authors did not discuss potential source bias and
confounder, threat to validity that might compromise the findings.
Thank you for pointing this out. We have added the study limitation accordingly
(line 350-364).
11\. c) Results
• The authors did not report the sociodemographic and baseline characteristics
of both control and experimental group. How does the researcher ensure that both
groups are similar at baseline? How can the researcher then conclude that the
outcome is due to their intervention, rather than existing differences? Are
there any additional methods of analyses such as subgroup analyses to account
for confounder or differences in the baseline?
Thank you for pointing this out. The general description of the participants has
been discussed and tabulated in Table 1 (line 234-242).
12\. • There is no justification on why non-parametric statistical analyses was
chosen. What are the assumptions and limitation of the analysis? Have all the
criteria being met?
We agree with this comment and has made changes accordingly in statistical
analysis section (line 218-223).
13\. • Too much data being presented that can be summarised in a sentence or in
simpler table. For example, Table 1 presents a range of minimum and maximum
value for each reading throughout 20 minutes – can be summarised by providing
the mean/median value. Easier to interpret and understand, rather than the
reader have to go thru each range.
Agree. We have removed this table accordingly and the mean value of spinal
posture angle changes throughout 20 minutes riding for both groups had already
presented in Figure 3 (line 250).
15\. d) Others
• Figure 1 – no legend to help reader understand the figure. Which line belongs
to control and intervention group?
Actually Figure 1 is an example of Truposture mobile apps interface and not
related with the control and intervention group results.
• Figure 2 – suggest authors to follow CONSORT flow diagram format (more
details)
Thank you for your suggestion. Thus, Figure 2 has been changed and followed
CONSORT format (Fig1: Line 146).
Reviewer 3
1\. Abstract
Line 30: the purpose of the study described in the abstract does not match with
what was done in the methods. This was an intervention study and I suggest
reformatting the purpose of the study.
The purpose of the study has been reformulated accordingly (line 30-31).
2\. Line 40: Are we really interested in a pre-test VS post-test analysis
(within group analysis)? Reporting the between group comparison is more
insightful…
Thank you for pointing this out. Thus, results and discussion related to
comparison between groups have been added accordingly
3\. Introduction
Provide some statistics of MSD/low back in the population The statistics of MSD
and low back pain among traffic police riders have been provided as suggested.
(line 78-85)
4\. Line 51: provide a full meaning of MSD
The full meaning of MSD has been provided as suggested (line 50).
5\. Line 59: I believe it was meant to be “been” instead of “seen”
Thank you for the comment. The relevant word has been changed accordingly (line
104-106).
6\.
Line 61: has instead of have
Thank you for the comment. The relevant word has been changed accordingly.
7\. Line 67: delete “the”
Thank you for the comment. The relevant word has been deleted accordingly.
8\. Line 81: The purpose of the study needs to be reformulated
The purpose of the study has been reformulated accordingly.
9\. The introduction lacks content. The consequences of increased lumbar
lordosis are not fully described. There are some grammatical errors that need to
be corrected.
Thank you for pointing this out. The consequence of decreased lumbar lordosis
has been explained accordingly (line 52-60).
10\. Materials and Methods
More information on the eligibility criteria (inclusion and exclusion criteria)
is needed
You have raised an important point here. We agree with this comment and has
included relevant information and explanation accordingly (114-121).
10\. Any reference for the TruePosture mobile app? Has this app been used before
in any study? What are the validity and reliability properties of the app?
Adequate references for the Truposture app have been added and discussed (line
186-189).
11\. How were the groups defined? Were the participants randomly assigned to the
groups? What are the baseline characteristics of the control and experimental
groups?
We agree with this comment. Thus, the relevant information has been added and
explained accordingly.
12\. Why did the authors not perform a between-group analysis? That comparison
is more interesting than all those pre-test vs post-tests performed.
Thank you for pointing this out. Thus, results and discussion related to the
comparison between groups have been added accordingly.
13 Results
The data reported does not indicate whether the spinal change pattern in the
intervention group is superior to the control group.
Thank you for pointing this out. Thus, results and discussion related to the
spinal change pattern between groups have been added accordingly.
14\. Discussion and conclusion
Not sustained by the analysis and results presented
The analysis and results presented have been revised.
Reviewer 4
1\. Context and research gap were not indicated.
The research gap was explained in line 85.
2\. Type of study, sampling method and data collection method were not
specified.
It has major methodological defect.
Type of study, sampling method and data collection method were not specified
Gold standard of experimental study was not succinctly stated.
Thank you for pointing this out. We agree with this and have incorporated your
suggestion throughout the manuscript accordingly.
3\. Major statistical analysis was not indicated.
Why Wilcoxon signed rank test and median was used?
The reason the analysis was chosen has been discussed in details (line 218-223).
Reviewer 5
1\. Reviewer \#5: Line 51: Please indicate the full meaning of MSD when been
used for the first time in write up.
Thank you for pointing this out. The change has been made accordingly (line 50).
2\. Line 178: Consider replacing "respondents" with "subjects" which best suits
the study and its concept
Thank you for pointing this out. The change has been made throughout the
manuscript accordingly.
3\. Please give reasons for the sample size choice and indicate the exclusion
and inclusion criteria for participating in the study
You have raised an important point here. We agree with this comment and has
included relevant information and explanation accordingly (line 114-129).
4\. Line 243: "The value of spinal angle deviation between the pre-test and
post-test....." Indicate the angle of deviation to make your point clear
Thank you for the comment. Actually, we want to highlight the effect in the
experimental group. However, we have made some changes based on your suggestion
“The value of spinal angle deviation in the experimental group….”
5\. There are some typographical errors indicated in the attached document.
Please revise them accordingly Thank you for pointing this out. The typological
error has been revised accordingly.
10.1371/journal.pone.0258796.r003
Decision Letter 1
Mosa
Ahmed Mancy
Academic Editor
2021
Ahmed Mancy Mosa
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
12 Jul 2021
PONE-D-21-13337R1
Effectiveness of lumbar support with built-in massager system on spinal angle
profiles among high-powered traffic police motorcycle riders: A randomised
controlled trial
PLOS ONE
Dear Dr. Karuppiah,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
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review process.
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Kind regards,
Ahmed Mancy Mosa, Ph.D.
Academic Editor
PLOS ONE
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Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
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Reviewer \#3: All comments have been addressed
Reviewer \#4: (No Response)
Reviewer \#5: All comments have been addressed
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#3: Partly
Reviewer \#4: Yes
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#3: No
Reviewer \#4: Yes
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
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fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
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be available. If there are restrictions on publicly sharing data—e.g.
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Reviewer \#3: Yes
Reviewer \#4: Yes
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
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Reviewer \#4: Yes
Reviewer \#5: Yes
\*\*\*\*\*\*\*\*\*\*
6\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#3: The authors appropriately revised the manuscript and made the
corresponding adjustments. The amount of time devoted to this paper is evident.
I have some more comments.
Additional comments:
How was the randomization performed? Any use of software or what was used for
the randomization?
Table 3 is not understandable and needs to be reformatted. The formatting of
this Table is fine for a within group difference analysis like the authors did
for Table 2 but not for a between group difference analysis. A Table should be
self-explanatory and this one is not. What does the p-value in the control
column indicate? Difference between baseline data (Precont VS Preexp)?
Similarly, what does p-value in the experimental column indicate? Difference
between after intervention data (Postcont VS Postexp)?
I would even suggest comparing the median of changes between control and
experimental groups.
Line 277: The sentence about no significant difference at baseline should be
presented before the results of after intervention. This is a good sign for a
between group comparison after intervention.
Reviewer \#4: Abstract
Background
Context and research gap were not stated.
methods
major statistical analysis was not succinctly stated
Main body
Methods
Sampling technique was not clear
data quality control was not indicated
Major statistical analysis was not indicated
Reviewer \#5: The manuscript is well written and technically sound. All the
issues have been addressed by the author.
However, there are a couple of technical and grammatical errors that have been
noticed:
Line 132: “ the recruitment strategy used by taken the name list of all
officers”
It should rather read “ the recruitment strategy involved taking the name list
of all the officers”
Line 139: “….then a main researcher was randomly assigned them …….”
It should rather read “………, then the main researcher randomly assigned them into
control and experimental
Line 189: “this equipment had been tested validity….”
It should rather read “the validity of the equipment had been tested and
approved……”
Other comments have been made in the attached document
\*\*\*\*\*\*\*\*\*\*
7\. PLOS authors have the option to publish the peer review history of their
article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-
and-peer-review-process#loc-peer-review-history)). If published, this will
include your full peer review and any attached files.
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Reviewer \#3: **Yes: **Libak Abou
Reviewer \#4: No
Reviewer \#5: No
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Please note that Supporting Information files do not need this step.
10.1371/journal.pone.0258796.r004
Author response to Decision Letter 1
15 Jul 2021
Dear Dr Ahmed Mancy Mosa (Academic Editor),
Thank you for giving me the opportunity to submit a second revised draft of my
manuscript titled effectiveness of lumbar support with built-in massager system
on spinal angle profiles among high-powered traffic police motorcycle riders: A
randomised controlled trial. We appreciate the time and effort that you and the
reviewers have dedicated to providing your valuable feedback on my manuscript.
We are grateful to the reviewers for their insightful comments on our paper. We
have been able to incorporate changes to reflect most of the suggestions
provided by the reviewers. We have highlighted the revisions within the
manuscript.
Here is a point-by-point response to the reviewers’ comments and concerns.
Reviewer 3
1\. How was the randomization performed? Any use of software or what was used
for the randomization?
The randomization of this study was explained using the list name and Fishbowl
technique (line 140-142).
2\. Table 3 is not understandable and needs to be reformatted. The formatting of
this Table is fine for a within group difference analysis like the authors did
for Table 2 but not for a between group difference analysis. A Table should be
self-explanatory and this one is not. What does the p-value in the control
column indicate? Difference between baseline data (Precont VS Preexp)?
Similarly, what does p-value in the experimental column indicate? Difference
between after intervention data (Postcont VS Postexp)? I would even suggest
comparing the median of changes between control and experimental groups.
We are sorry for the confusion, and for our understanding we did this as
mentioned in the earlier comments (i. Line 40: Are we really interested in a
pre-test VS post-test analysis (within group analysis)? Reporting the between
group comparison is more insightful…ii. Please explain within and between groups
comparison in the analysis of the study outcome.).
Thus, we created Table 2 for within-group analysis and Table 3 reporting
between-group comparisons as suggested. The p-value in Table 2 represents a
value for a significant/not significant between pre-test and post-test studies
within two groups and p-value in Table 3 explain a value for a significant/not
significant between control and experimental groups.
3\. Line 277: The sentence about no significant difference at baseline should be
presented before the results of after intervention. This is a good sign for a
between group comparison after intervention.
Thank you for your suggestion. We agree with this comment. The revision has been
made on lines 293-297.
Reviewer 4
1\. Background:Context and research gap were not stated.
i\. The context and research gap of traffic police riders suffered low back
pain, and MSD which no research has been done on the spinal riding posture
during a motorcycle ride was explained in line 72-87.
ii\. The context and research gap in which there is a lack of data on the
effectiveness of this intervention seat, lumbar support with a built-in massager
system, in an in-field setting (on-the-road) was explained in line 96-99.
2\. methods: major statistical analysis was not succinctly stated.
The major statistical analysis used in this study was the Mann-Whitney test and
Wilcoxon signed-ranked test. The details of the statistical analysis were
explained in line 233-243.
3\. Methods: Sampling technique was not clear.
The sampling technique was explained in Participant Recruitment (line 132-158).
4\. data quality control was not indicated.
Thank you for pointing this out. We agree with this comment. The data quality
control had been added in line 216-231.
Reviewer 5
1\. Line 132: “ the recruitment strategy used by taken the name list of all
officers”
It should rather read “ the recruitment strategy involved taking the name list
of all the officers”
Thank you for pointing this out. The change has been made accordingly (line
132).
2\. Line 139: “….then a main researcher was randomly assigned them …….”
It should rather read “………, then the main researcher randomly assigned them into
control and experimental Thank you for pointing this out. The change has been
made accordingly (line 139).
3\. Line 189: “this equipment had been tested validity….”
It should rather read “the validity of the equipment had been tested and
approved……”
Thank you for pointing this out. The change has been made accordingly (line
189).
4\. Other comments have been made in the attached document
Thank you for the comments. We agree with this and have incorporated your
suggestion throughout the manuscript accordingly.
10.1371/journal.pone.0258796.r005
Decision Letter 2
Mosa
Ahmed Mancy
Academic Editor
2021
Ahmed Mancy Mosa
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
6 Sep 2021
PONE-D-21-13337R2Effectiveness of lumbar support with built-in massager system
on spinal angle profiles among high-powered traffic police motorcycle riders: A
randomised controlled trialPLOS ONE
Dear Dr. Karuppiah,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
a revised version of the manuscript that addresses the points raised during the
review process.
Please, consider all the comments
Please submit your revised manuscript by 16 September 2021. If you will need
more time than this to complete your revisions, please reply to this message or
contact the journal office at <plosone@plos.org>. When you're ready to submit
your revision, log on to <https://www.editorialmanager.com/pone/> and select the
'Submissions Needing Revision' folder to locate your manuscript file.
Please include the following items when submitting your revised manuscript:A
rebuttal letter that responds to each point raised by the academic editor and
reviewer(s). You should upload this letter as a separate file labeled 'Response
to Reviewers'.A marked-up copy of your manuscript that highlights changes made
to the original version. You should upload this as a separate file labeled
'Revised Manuscript with Track Changes'.An unmarked version of your revised
paper without tracked changes. You should upload this as a separate file labeled
'Manuscript'.
If you would like to make changes to your financial disclosure, please include
your updated statement in your cover letter. Guidelines for resubmitting your
figure files are available below the reviewer comments at the end of this
letter.
If applicable, we recommend that you deposit your laboratory protocols in
protocols.io to enhance the reproducibility of your results. Protocols.io
assigns your protocol its own identifier (DOI) so that it can be cited
independently in the future. For instructions see:
<https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-
protocols>. Additionally, PLOS ONE offers an option for publishing peer-reviewed
Lab Protocol articles, which describe protocols hosted on protocols.io. Read
more information on sharing protocols at
<https://plos.org/protocols?utm_medium=editorial-
email&utm_source=authorletters&utm_campaign=protocols>.
We look forward to receiving your revised manuscript.
Kind regards,
Ahmed Mancy Mosa, Ph.D.
Academic Editor
PLOS ONE
Journal Requirements:
Additional Editor Comments (if provided):
\[Note: HTML markup is below. Please do not edit.\]
Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. If the authors have adequately addressed your comments raised in a previous
round of review and you feel that this manuscript is now acceptable for
publication, you may indicate that here to bypass the “Comments to the Author”
section, enter your conflict of interest statement in the “Confidential to
Editor” section, and submit your "Accept" recommendation.
Reviewer \#3: (No Response)
Reviewer \#4: All comments have been addressed
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#3: Yes
Reviewer \#4: Yes
\*\*\*\*\*\*\*\*\*\*
3\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#3: No
Reviewer \#4: No
\*\*\*\*\*\*\*\*\*\*
4\. Have the authors made all data underlying the findings in their manuscript
fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
manuscript fully available without restriction, with rare exception (please
refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
participant privacy or use of data from a third party—those must be specified.
Reviewer \#3: Yes
Reviewer \#4: No
\*\*\*\*\*\*\*\*\*\*
5\. Is the manuscript presented in an intelligible fashion and written in
standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
articles must be clear, correct, and unambiguous. Any typographical or
grammatical errors should be corrected at revision, so please note any specific
errors here.
Reviewer \#3: Yes
Reviewer \#4: No
\*\*\*\*\*\*\*\*\*\*
6\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#3: Thank you for responding to my comments. I believe the authors
dedicated a lot of time going through all the reviews. Congrats on that! I still
believe the authors did not explain the randomization method. Mentioning that
randomization was performed is not enough, how was it done? They are several
randomization methods? Which one did you use?
Also, the authors compared pre and post in exp and control group (fine);
compared pre (cont) vs pre (int) and post (cont) vs (pos int), which is also
fine but the most important comparison was not performed. The mean (median)
difference between group is the one that is really informative and should be
compared to the MCID.
Reviewer \#4: Abstract
Background
Research gap was not stated
method
major statistical analysis was not specified
Main body
major statistical analysis was not specified
results
The major statistical model output were not presented
\*\*\*\*\*\*\*\*\*\*
7\. PLOS authors have the option to publish the peer review history of their
article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-
and-peer-review-process#loc-peer-review-history)). If published, this will
include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may
still be made public.
**Do you want your identity to be public for this peer review?** For information
about this choice, including consent withdrawal, please see our [Privacy
Policy](https://www.plos.org/privacy-policy).
Reviewer \#3: No
Reviewer \#4: No
\[NOTE: If reviewer comments were submitted as an attachment file, they will be
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While revising your submission, please upload your figure files to the Preflight
Analysis and Conversion Engine (PACE) digital diagnostic
tool, <https://pacev2.apexcovantage.com/>. PACE helps ensure that figures meet
PLOS requirements. To use PACE, you must first register as a user. Registration
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have any questions when using PACE, please email PLOS at <figures@plos.org>.
Please note that Supporting Information files do not need this step.
10.1371/journal.pone.0258796.r006
Author response to Decision Letter 2
13 Sep 2021
Here is a point-by-point response to the reviewers’ comments and concerns.
No. Comments from Reviewer 3
1\. Thank you for responding to my comments. I believe the authors dedicated a
lot of time going through all the reviews. Congrats on that! I still believe the
authors did not explain the randomization method. Mentioning that randomization
was performed is not enough, how was it done? They are several randomization
methods? Which one did you use?
Firstly, we want to apologize because we are not clear on the type of
randomization intended by the reviewer. However, this is the best explanation
that we can give for this remark.
A randomised controlled trial, pretest-posttest control group design was
conducted among 24 traffic police riders who ride a high-powered motorcycle
(Honda CBX 750). A simple random sampling was used in this study which the
subjects were randomly assigned to the control group (12 riders) and
experimental group (12 groups). Data collection commenced in March 2020 and
finished in July 2020.
Further explanation had been explained in line 138-165.
2\. Also, the authors compared pre and post in exp and control group (fine);
compared pre (cont) vs pre (int) and post (cont) vs (pos int), which is also
fine but the most important comparison was not performed. The mean (median)
difference between group is the one that is really informative and should be
compared to the MCID. Thank you for pointing this out. However, this MCID
actually will be discussed in details in our future paper. The main objective
for this work is to evaluate the effect of lumbar support with a built-in
massager system on spinal angle profiles among traffic police riders which focus
on comparison of pretest-posttesst and between control-experimental groups.
Nevertheless, for future studies, this will be a great insight and we will be
sure to use it.
No. Comments from Reviewer 4
1\. Reviewer \#4: Abstract
Background
Research gap was not stated
method
major statistical analysis was not specified
Main body
major statistical analysis was not specified
results
The major statistical model output were not presented
We really appreciate your valuable comments here. We find it really useful to
improve our manuscript. After discussion with our co-authors, we have improved
our manuscript (in the 2nd revision) based on the given comments as below: -
i\. The context and research gap of traffic police riders suffered low back
pain, and MSD which no research has been done on the spinal riding posture
during a motorcycle ride was explained in line 72-87.
ii\. The context and research gap in which there is a lack of data on the
effectiveness of this intervention seat, lumbar support with a built-in massager
system, in an in-field setting (on-the-road) was explained in line 96-99.
The major statistical analysis used in this study was the Mann-Whitney test and
Wilcoxon signed-ranked test. The details of the statistical analysis were
explained in line 233-243.
The sampling technique was explained in Participant Recruitment (line 132-158).
However, the similar comments are asked again in this 3rd revision. Thus, we
decided to explained this in the abstract since it has a word abstract in the
beginning of the comments.
Traffic police riders are exposed to prolonged static postures causing
significant angular deviation of the musculoskeletal, including the lumbar angle
(L1-L5). This postural alteration contributes to awkward posture,
musculoskeletal disorders and spinal injury, especially in the lower back area,
as it is one of the most severe modern diseases nowadays. Thus, the study aimed
to evaluate the effect of lumbar support with a built-in massager system on
spinal angle profiles among traffic police riders. A randomised controlled trial
(pre-testpost-test control design) was used to assess spinal angle pattern while
riding the high-powered motorcycle for 20 minutes. Twenty-four traffic police
riders were randomly selected to participate and 12 riders were assigned to the
control group and 12 riders to the experimental group. The pre-test and post-
test were conducted at a one-week interval. Each participant was required to
wear a TruPosture Smart Shirt (to monitor spinal posture). The TruPosture Apps
recorded the spinal angle pattern. The data indicated that the police riders
using motorcycle seat with lumbar support and built-in massager system showed a
huge improvement in maintaining posture which only involves slight spinal angle
deviation changes from the spinal reference angle throughout the 20 minutes
ride. The data collected then were analysed using the Mann-Whitney test and
Wilcoxon signed-ranked test to verify a statistically significant difference
between and within the control and experimental groups. There were significant
differences in all sensors between the control group and experimental groups
(p\<0.05) and within the experimental group. According to the findings, it can
be said that the ergonomic intervention prototype (lumbar support with built-in
massager system) successfully helps to maintain and improve the natural curve of
the spinal posture. This indirectly would reduce the risk of developing
musculoskeletal disorders and spinal injury among traffic police riders.
We do provide the justification as requested as based on our understanding.
Please do advise us in details if we need further revision as we feel your
valuable comments will improve further our manuscript. Thank you.
10.1371/journal.pone.0258796.r007
Decision Letter 3
Mosa
Ahmed Mancy
Academic Editor
2021
Ahmed Mancy Mosa
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
6 Oct 2021
Effectiveness of lumbar support with built-in massager system on spinal angle
profiles among high-powered traffic police motorcycle riders: A randomised
controlled trial
PONE-D-21-13337R3
Dear Dr. Karuppiah,
We’re pleased to inform you that your manuscript has been judged scientifically
suitable for publication and will be formally accepted for publication once it
meets all outstanding technical requirements.
Within one week, you’ll receive an e-mail detailing the required amendments.
When these have been addressed, you’ll receive a formal acceptance letter and
your manuscript will be scheduled for publication.
An invoice for payment will follow shortly after the formal acceptance. To
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For more information, please contact <onepress@plos.org>.
Kind regards,
Ahmed Mancy Mosa, Ph.D.
Academic Editor
PLOS ONE
Additional Editor Comments (optional):
Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. If the authors have adequately addressed your comments raised in a previous
round of review and you feel that this manuscript is now acceptable for
publication, you may indicate that here to bypass the “Comments to the Author”
section, enter your conflict of interest statement in the “Confidential to
Editor” section, and submit your "Accept" recommendation.
Reviewer \#3: (No Response)
Reviewer \#4: All comments have been addressed
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#3: (No Response)
Reviewer \#4: Yes
\*\*\*\*\*\*\*\*\*\*
3\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#3: (No Response)
Reviewer \#4: Yes
\*\*\*\*\*\*\*\*\*\*
4\. Have the authors made all data underlying the findings in their manuscript
fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
manuscript fully available without restriction, with rare exception (please
refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
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10.1371/journal.pone.0258796.r008
Acceptance letter
Mosa
Ahmed Mancy
Academic Editor
2021
Ahmed Mancy Mosa
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
11 Oct 2021
PONE-D-21-13337R3
Effectiveness of lumbar support with built-in massager system on spinal angle
profiles among high-powered traffic police motorcycle riders: A randomised
controlled trial
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# Introduction
The enterohemorrhagic *Escherichia coli* (EHEC) O157:H7 causes acute
gastroenteritis, hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in
humans. The latter is a severe infection sequelae characterized by thrombotic
microangiopathy, hemolytic anaemia and acute renal failure which can lead to
long-term kidney damage or fatal outcome. *E.coli* O157:H7 was first recognized
as an important human pathogen in 1982 during an investigation of a food-borne
disease outbreak in the United States and has caused many outbreaks in the past
three decades, with a wide range of clinical illness. In 2006, an outbreak
associated with spinach caused high rates of bloody diarrhea (22/23),
hospitalization (13/23) and HUS (7/23), suggesting that the outbreak strain,
TW14359, has evolved to higher virulence. Further studies have shown that
TW14359 expresses higher levels of many virulence genes and a range of other
chromosomal and pO157-encoded genes. It has also been shown that TW14359 has
better adherence to epithelial cells than *E.coli* O157:H7 strain Sakai.
An outbreak of *E. coli* O157:H7 occurred in Xuzhou, China in 1999, causing
about 20,326 infections, 195 hospitalized HUS patients and 177 deaths. Our group
has recently sequenced the outbreak strain Xuzhou21. We also discovered that a
novel conjugative plasmid pO157_Sal was present in Xuzhou21 and was found nearly
unique in the outbreak isolates in China. The pO157_Sal contains 52 ORFs and has
a full set of genes for the type IV secretion system (T4SS), but no known
virulence-related genes were identified. Several genes found on pO157_Sal are
homologous to transcriptional regulatory genes such as *stpA* and *hha* ¸ both
of which have been reported to be implicated in virulence or environmental
adaptation. In addition, the pO157_Sal *mpr* gene is homologous to *stcE* on
pO157 which encodes a zinc metalloproteinase and may play a role in adherence.
We further showed that Xuzhou21 has the capacity to provoke elevated
proinflammatory responses with levels of IL-6 and IL-8 induction being
significantly higher than that induced by EDL933. Xuzhou21 also carries a highly
inducible Stx2 prophage.
RNA-Seq (whole transcriptome shotgun sequencing) is a next generation sequencing
platform based assay of genome-wide bacterial gene expression. RNA-Seq can
reveal the entire transcriptional landscape and has less systematic bias
compared to microarray technology. To further enhance our understanding of the
Xuzhou outbreak strain and to investigate the global effects of pO157_Sal on the
gene expression, virulence and adaptation of Xuzhou21, we cured the pO157_Sal
plasmid from Xuzhou21 and compared the transcriptomic differences by RNA-Seq
between the wild-type strain Xuzhou21 and the pO157_Sal cured strain Xuzhou21m.
# Results
## Plasmid curing and complementation
Xuzhou21 was treated with SDS and high temperature to cure the pO157_Sal plasmid
and curing was confirmed by PCR using primer pairs, ehxA-F/ehxA-R and
p247-F/p247-R, targeting *ehxA* and *traL* specific for amplification of pO157
and pO157_Sal respectively. Xuzhou21 was positive for these two genes while the
pO157_Sal cured strain Xuzhou21m was only positive for *ehxA*. We found that the
efficiency of curing pO157_Sal plasmid was 1%∼3%. We further sequenced Xuzhou21m
using Illumina sequencing to confirm that no other changes occurred in the
genome during plasmid curing. Reads were mapped to Xuzhou21 genome with an
average 114-fold coverage. No insertion or deletion was found and plasmid pO157
was intact in Xuzhou21m. No DNA fragment from plasmid pO157_Sal was detected in
Xuzhou21m ****. Fifty-eight tentative SNPs were identified. However, using RNA-
Seq data, only two non-synonymous SNPs, one each from two genes of unknown
function (CDCO157_2530 \[C to A\] and CDCO157_2534 \[G to T\]), were confirmed
to be genuine.
We further created a pO157_Sal complemented strain, Xuzhou21c. The plasmid
pO157_Sal was first marked with the kanamycin resistance gene using one-step
gene inactivation and was transformed into Xuzhou21m successfully. The presence
of the pO157_Sal in this complemented strain Xuzhou21c was confirmed by PCR
using pO157_Sal specific primer pair, p247-F and p247-R.
Q-PCR was used to determine the relative copy number of pO157 and pO157_Sal in
Xuzhou21, Xuzhou21m using 2<sup>−ΔΔCT</sup> method. The target genes on pO157
and pO157_Sal were *espP* and *traL*. The reference chromosomal gene was g*apA*.
Q-PCR showed that the two strains had 2.35±0.49 and 2.21±0.24 copies of pO157
respectively. The copy number of pO157_Sal in Xuzhou21 was 2.04±0.18. These
results suggested that the two plasmids have similar copy numbers and the curing
of pO157_Sal has no effect on the copy number of pO157.
## Transcriptional profiling of Xuzhou21 and Xuzhou21m
To determine the effect of pO157_Sal at transcriptomic level, the transcriptomes
of Xuzhou21 and Xuzhou21m were compared using RNA-Seq. Cells were cultured in LB
broth to exponential phase and mRNA were sequenced using Illumina sequencing
****. About 6.5 million reads were obtained for both Xuzhou21 and Xuzhou21m,
giving a 212 fold coverage respectively. About 1.9 and 1.4 million “clean reads”
were obtained for Xuzhou21 and Xuzhou21m respectively after filtering out any
reads with more than 10% uncalled bases (Ns) or with more than 50% of bases with
a base quality less than 5.0. The reads were mapped to Xuzhou21 reference
genome. Allowing for up to 2 mismatches for a read, 67.9% of Xuzhou21 and 63.35%
Xuzhou21m reads were mapped to the Xuzhou21 genome. The reads covered 70.13% and
65.6% gene regions for Xuzhou21 and Xuzhou21m respectively. Sequence reads
located in intergenic regions account for 28.28% and 29.89% of all sequence
reads obtained for Xuzhou21 and Xuzhou21m respectively. Although expression of
intergenic regions may have important information, these reads were not analyzed
further.
As a result, a high-resolution transcriptomic map of the Xuzhou21 genome was
generated. The level of gene expression was calculated using reads per kilobase
per million reads (RPKM) method. Since the reproducibility was relatively low in
genes with low RPKM value, only genes with RPKM≥10 were considered expressed. We
found that 3991 of 5214 genes from Xuzhou21 had a RPKM greater than ten, 963
genes have a RPKM greater than zero but less than ten, and 260 genes had no
reads (RPKM = 0). Ninety-five percent of the genes of Xuzhou21 genome had an
RPKM\>0 and were thus transcribed. One hundred and sixteen genes (2.22%) had a
RPKM greater than 1,000 representing highly expressed protein coding genes,
among which, 42 and 24 encode ribosomal proteins and hypothetical proteins
respectively. Other highly transcribed genes including genes encoding elongation
factor Tu, MokW, cold shock protein CspE and flagellin. MokW is only 51 amino
acids long and little is known of its function. Of the 1223 genes with RPKM less
than ten, 432 (35.32%) were genes of unknown function and a further 369 (30.17%)
had putative functions.
Of the 5070 chromosomal genes and 92 pO157 plasmid genes, 4814 (94.95%) and 88
(95.65%) were transcribed respectively while all pO157_Sal genes were
transcribed in Xuzhou21. In contrast, 94.62% (4749/5070) and 95.65% (88/92) of
the chromosomal and pO157 genes were expressed In Xuzhou21m respectively.
Interestingly, pO157_Sal genes showed much higher level of expression on average
than genes on the chromosome and pO157 ****.
## Validation of RNA-Seq results using RT-qPCR
To validate RNA-Seq results, 167 genes were selected to validate their level of
expression using RT-qPCR. We selected 51 highly (RPKM\>500), 72 moderately
(500\>RPKM\>100) and 44 poorly (100\>RPKM\>10) expressed genes to encompass the
wide spectrum of variation in expression levels. As shown in, RT-qPCR data is
consistent with the RNA-Seq data (R<sup>2</sup> = 0.5505, *P*\<0.01). The slope
of the trend line as shown in is less than 45 degrees, indicating that RNA-Seq
is generally less sensitive than RT-qPCR for quantification of gene expression.
Thus RNA-Seq gave a conservative estimate of the number of differentially
expressed genes.
## Differentially expressed genes between Xuzhou21 and Xuzhou21m
There were 3733 genes with RPKM ≥10 in both strains as candidates for
differential expression analysis. To provide a reliable comparison, genes with
RPKM less than10 in both strains were excluded. However genes that had an
RPKM\<10 in one strain but were highly expressed (RPKM ≥10) in the other (231 in
Xuzhou21 and 265 in Xuzhou21m) were included as candidates. Consequently, 168
genes with 2-fold change or greater and *P*\<0.05 were identified as
significantly differentially expressed genes. These genes are listed in along
with their functions and their levels of expression. Excluding the 52 genes
located on pO157_Sal, 67 genes were up-regulated and 101 genes were down-
regulated in Xuzhou21. In the 67 up-regulated genes, the expression level of 24
genes in Xuzhou21m was zero; and in the 101 down-regulated genes, the expression
level of 12 genes in Xuzhou21 was zero. The vast majority of the differentially
expressed genes were chromosomal. Five genes were from pO157 including three
down-regulated and two up-regulated genes. Sixteen of the up-regulated genes
were hypothetical proteins with unknown functions, while 28 of the down-
regulated genes were hypothetical proteins.
The up-regulated genes in Xuzhou21 were in 15 gene function classes based on
cluster of orthologous genes (COG) classification ****. Eleven up-regulated
genes belong to the COG classes of transport and metabolism of amino acids,
carbohydrates, inorganic ions, lipids and nucleotides and six in the class of
energy production and conversion. Two genes encoding pilus/fimbrial assembly
proteins, FimA and PilN, and a gene involved in curli production (CsgG) were up-
regulated in Xuzhou21. Curli is associated with biofilm formation, adhesion and
invasion. It is interesting to note that several genes are related to stress
response including heat shock chaperone gene *ibpB*, cold shock protein gene
*cspE*, hyperosmotically inducible periplasmic protein gene *osmY*, peripheral
inner membrane phage-shock protein gene *pspD*, multidrug efflux system subunit
gene *mdtA*, adenine-specific DNA-methyltransferase gene *dam* and oxidation-
reduction related genes, suggesting that pO157_Sal enhances expression of these
genes which play a role in stress response.
The down-regulated genes also encompass a range of COG gene function classes.
Twenty three down-regulated genes belong to the COG classes of transport and
metabolism of amino acids, carbohydrates, inorganic ions, lipids and
nucleotides. Five genes are related to virulence including *ler*, *ehxD*, 3 LEE
effector genes and 1 non-LEE effector gene. Five genes were in the class of cell
motility and are involved in pilus biogenesis and assembly, which may also
affect virulence. Thus, the pO157_Sal has a repression effect on the expression
of these metabolic and virulence associated genes.
## pO157_Sal enhances hyperosmosis and bile salt resistance
Since the RNA-Seq data showed that several genes related to stress responses
were more highly expressed in Xuzhou21, which suggests that pO157_Sal plays a
role in stress response, we tested resistance to bile salt and osmotic pressure.
The growth rates of Xuzhou21, Xuzhou21m and Xuzhou21c in the M9 basal medium
with 0.2% glucose were the same. In contrast, the growth rates of these three
strains in the M9 medium containing an additional 0.37% NaCl or 0.67% NaDC were
different. The generation time of Xuzhou21 and Xuzhou21m when cultured in M9
containing the additional NaCl, were 48.8±1.25 minutes and 56.3±3.21 minutes
(*t* test, *P* = 0.048) respectively, while the generation time of Xuzhou21 and
Xuzhou21m when cultured in M9 containing NaDC were 86.3±3.40 minutes and
100.0±7.50 minutes respectively (*t* test, *P* = 0.016) respectively. The
generation times of Xuzhou21c when cultured in M9 containing NaCl and NaDC were
51.3±5.51 minutes and 88.3±1.44 minutes respectively and were similar to those
of Xuzhou21 ****. Therefore, pO157_Sal promoted the growth of Xuzhou21
significantly under both conditions.
## The effect of pO157_Sal encoded global gene regulator Hha
Analysis of the genes on pO157_Sal found that only two known homologs of global
gene regulators *stpA* and *hha* that may play the role in hyperosmosis and bile
resistance in Xuzhou21. We attempted to test this by constructing a plasmid
carrying a pO157_Sal *stpA* or *hha*. We initially used a constitute expression
vector pMD®20 T (TAKARA). However, the empty T vector has a high background
effect on Xuzhou21m. We then used an arabinose inducible vector pBAD30, which
was found to has no effect on Xuzhou21m, to create pBAD30 constructs carrying
the pO157_Sal *stpA* and *hha* separately and transformed to Xuzhou21m as
Xuzhou21m+*stpA* and Xuzhou21m+*hha* respectively. However, Xuzhou21m+*stpA* did
not grow under arabinose induction. We then tested resistance to osmotic
pressure and bile salt of Xuzhou21m+*hha*. As shown in, Xuzhou21, Xuzhou21c and
Xuzhou21+*hha* grew similarly while Xuzhou21m grew significantly slower than the
other three strains (t test, *P*\<0.05).
We further tested whether *hha* affects the expression of the LEE associated
T3SS genes, six of which *ler*, *ehxD*, 3 LEE effector genes (*espH, espF,
espG*) and 1 non-LEE effector gene (*espY3*) were differentially expressed based
on RNA-Seq data. The expression of *espF* and *espH* in Xuzhou21m+*hha* grown in
LB to late exponential phase (OD<sub>600</sub> = 3.0) and under induction of
0.05% arabinose were measured by qPCR. The transcription levels of *espF* and
*espH* in Xuzhou21m were 3.2±0.44 (*t* test, *P* = 0.001) and 1.9±0.27 times
(*t* test, *P* = 0.003) higher than Xuzhou21. The transcription levels of *espF*
and *espH* in Xuzhou21m+*hha* were reduced to 0.5±0.06 (*t* test, *P* = 0.003)
and 0.3±0.04 times (*t* test, *P* = 0.001) of those of Xuzhou21.
# Discussion
To understand further the role of the novel plasmid pO157_Sal, we created a
pO157_Sal cured Xuzhou21m strain for comparative studies. Using RNA-Seq, a high-
resolution transcriptomic map of Xuzhou21 was constructed which showed that 95%
of the genes were transcribed (RPKM\>0). The expression of over 168 genes
including 163 genes from the chromosome and 5 genes from the pO157 plasmid were
affected by plasmid pO157_Sal. Thus the presence of pO157_Sal has a far reaching
effect on the biology of Xuzhou21. pO157_Sal specific genes in Xuzhou21 were all
transcribed. Notably, pO157_Sal genes showed much higher expression level on
average than other genes in the chromosome and pO157.
pO157_Sal affects bile salt resistance since Xuzhou21m grew 16% slower than
Xuzhou21 in the presence of bile salt. The growth rate of the complemented
strain Xuzhou21c was restored to the same level as the wild type. Seven
genes/systems that were up-regulated by pO157_Sal can be associated with this
role including multidrug efflux system subunit gene *mdtA*, the adenine-specific
DNA-methyltransferase gene *dam*, hyperosmotically inducible periplasmic protein
gene *osmY* and oxidation-reduction related genes ****. The multidrug efflux
system encoded by *mdtABCD* locus could expel bile salt from the cytoplasm after
they breach the cell membrane and thus increase resistance to bile salt. The
adenine-specific DNA-methyltransferase coded by *dam* is important for bile
resistance by controlling integrity of the envelope as shown in *Salmonella
enterica*. *dam* mutants are sensitive to bile salt mostly in exponential-growth
phase. Four genes related to oxidation-reduction may also affect bile salt
resistance since proteins involved in oxidation-reduction reactions are
differentially expressed after exposure to bile salt.
pO157_Sal also affects resistance to osmotic pressure as shown by growth rate
difference. Differentially expression of two genes (*osmY* and *pspD*) related
to osmotic resistance was observed. *osmY* encodes a periplasmic protein OsmY
which is commonly involved in osmotic stress and resistance to bile salt. *pspD*
and the other 4 genes in the *psp* operon was up-regulated by pO157_Sal although
only *pspD* reached the cutoff. The major function of the *psp* regulon is to
stabilize and maintain proton motive force within a stressed cell and thus may
also promote resistance to osmotic pressure.
In addition, heat shock chaperone gene *lpbB* and cold shock protein gene *cspE*
related to heat and cold resistance respectively were up-regulated in Xuzhou21.
*E. coli* cold shock protein CspA family consists of nine proteins (CspA to
CspI). CspE and CspC are constitutively produced at 37°C and up-regulate the
expression of the gene encoding global stress response regulator RpoS through
*rpoS* message stabilization. In addition, CspE functions as a negative
regulator for *cspA*. CspA alters the secondary structure of RNA, making it more
susceptible to degradation.
Very few virulence-associated genes were found to be differentially expressed
between Xuzhou21 and Xuzhou21m ****, which may be related to the exponential
growth phase and the medium we used. Previous studies show that LEE1-3 genes
usually express much higher than exponential phase in the transition from the
late exponential phase to the stationary phase. In the stationary phase, the
expression of LEE-encoded genes are down-regulated. For examples, the *ler*
promoter activity increased greatly from the mid-exponential phase to the
stationary phase. Ler directly regulates genes within the LEE PAI as well as
genes elsewhere in the genome. Therefore, genes located on LEE were up-regulated
in late exponential phase via *ler*. Interestingly pO157_Sal represses *ler* and
four LEE and non-LEE encoded effectors as described above, consistent with
repression of virulence genes during late exponential growth reported
previously. It would be interesting to examine the effect of pO157_Sal on these
genes in the stationary phase. Previous studies have shown that virulence genes
are up-regulated when O157:H7 is grown in minimal medium. The expression of
virulence genes of O157:H7 have also used DMEM and LB medium previously. Future
studies will be conducted using other media to compare with expression in LB to
shed further light on the effects of pO157_Sal on the expression of virulence
genes.
Additionally, there are 7 genes related to bacteria motility including 5 down-
regulated (*hofC, yadN, papD, fimL* and major tail protein V gene) and 2 up-
regulated (*pilN* and *fimA*) genes by pO157_Sal. But no difference in motility
between Xuzhou21 and Xuzhou21m was found when conventional soft agar and U-shape
tube motility assay was performed (data not shown).
pO157_Sal clearly has a major effect on the expression of chromosomal and pO157
genes, in particular genes associated with cell metabolism. Since a large number
of genes of different functions were differentially expressed and these genes
are distributed across the chromosome, the effects of pO157_Sal on these genes
must be exerted through regulation by global regulatory genes present on
pO157_Sal. There are two genes homologous to global regulatory genes, *stpA* and
*hha* on the pO157_Sal plasmid.
Previous studies showed that the chromosomal homolog of *stpA* plays a similar
but minor role to H-NS, both of which bind DNA nonspecifically as global gene
regulators with certain differential effect. The differential effect may be
associated with their intracellular concentration of which H-NS is higher than
StpA. There are 2 *stpA* homologs on the chromosome (CDCO157_3291 and
CDCO157_1668). Both homologs had no transcriptional difference between Xuzhou21
and Xuzhou21m with RPKM values of 1035.06 and 898.659 for CDCO157_1668 and
283.562 and 275.738 for CDCO157_3291 respectively. The pO157_Sal plasmid copy of
*stpA* is homologous to the two chromosomal copies (the pO157_Sal *stpA* shares
46% and 43% DNA identity with CDCO157_3291 and CDCO157_1668 respectively). The
RPKM value of pO157_Sal *stpA* is much higher (2818.8) and may play a
functionally similar but larger role. Studies have also shown that the
expression of the chromosomal *stpA* in *E. coli* K-12 is up-regulated by high
osmolarity during cell growth, further pointing to a regulatory role of StpA in
the resistance to osmotic pressure. Unfortunately, Xuzhou21m+*stpA* did not grow
in M9 when *stpA* expression was induced using arabinose and thus the function
role of *stpA* could not be confirmed. The failure to grow may be associated
with overexpression of *stpA*which can orgnize DNA into compact higher order
structure via the magnesium in M9 medium.
Hha could form heteromeric complexes with H-NS and StpA and have multiple
effects on several virulence or adaptation associated genes,. Hha enhances the
ability of H-NS to repress the *ehx* operon on the pO157, and mediates
repression of *ler* leading to reduced expression of the *esp* operon. Hha also
controls biofilm formation by repressing the transcription of rare codon tRNAs
and repressing transcription of fimbrial genes. RNA-Seq showed that both *ler*
and *ehxD* were repressed by pO157_Sal. The transcription of *espF* and *espH*
was repressed by Hha in Xuzhou21m+*hha*. Therefore we conclude that the *hha*
located on pO157_Sal represses the transcription of the LEE encoded T3SS and its
effectors. Both the whole pO157_Sal plasmid and single gene (*hha*)
complementations of Xuzhou21m showed that *hha* plays a role in the resistance
to NaCl and bile salt.
It is interesting to note that there is also a *hha* homolog present in pO157 as
well as on the chromosome. The pO157 *hha* homolog (pO157_0071) shares 37% amino
acid identity with the pO157_Sal *hha*, although the two homologs have no
significant similarity at DNA sequence level. The RPKM values of the chromosomal
copy, CDCO157_0501, were 845.206 and 1022.92 in Xuzhou21 and Xuzhou21m
respectively, indicating no transcriptional difference. In contrast, the RPKM
values of the pO157 copy, CDCpO157_0071, were 117.155 and 0 in Xuzhou21 and
Xuzhou21m respectively, and it seems that pO157_Sal turns on the expression of
pO157_0071. Therefore, even if pO157_0071 plays a similar role to the pO157_Sal
*hha* it is conditional upon the activation by pO157_Sal.
Previous study have shown that *hha* regulates the expression of *csgD* which is
a positive regulator of curli genes encoded by *csgDEFG* and *csgBAC* operons.
However, the RNA-Seq data showed some opposing effects of the pO157_Sal *hha* on
the curli genes, *csgG* of the *csgDEFG* operon was up-regulated in Xuzhou21
while *csgA* of the *csgBAC* operon was down-regulated in Xuzhou21. The RPKM
values of *csgD* had no difference between Xuzhou21 and Xuzhou21m, suggesting
that the Hha regulation is independent of CsgD. Overall the expression of both
operons was low with RPKM values on average of 8.42 and 8.87 in Xuzhou21 and
Xuzhou21m respectively. This may be due to the growth phase of the cells we used
for RNA-Seq as curli fimbriae are usually expressed higher during the stationary
phase.
The pO157_Sal *mpr* gene encodes a putative zinc metalloproteinase, which is
homologous to *stcE* on pO157. StcE plays a role in intimate adherence of
*E.coli* O157:H7 to HEp-2 cells by cleavage of glycoproteins from the cell
surface. Thus, the Mpr homolog encoded on pO157_Sal may also be involved in
adherence. Future studies will assess the role of the *mpr* gene in virulence.
In conclusion, plasmid pO157_Sal affected the expression of 168 genes involved
in a range of functions under the conditions tested and may contribute to the
resistance to NaCl and NaDC. The wide-ranging effect we observed is likely to be
a result of gene regulation by global regulators encoded on the plasmid. The
*stpA* and *hha* homologs on pO157_Sal are the most likely regulators playing
this role and we have confirmed the effect of *hha* on resistance to NaCl and
NaDC. Further studies will be needed to determine the effects by these
regulators specifically and their effects on virulence.
# Materials and Methods
## Bacterial strains, plasmids and growth conditions
The *E. coli* O157:H7 strain Xuzhou21 was isolated from feces of a HUS patient
from an outbreak in China in 1999.The nonpathogenic *E.coli* K12 MG1655 was used
as negative control. The bacteria were routinely grown in Luria-Bertani (LB)
broth or on LB agar plates (pH 7.2). Chloramphenicol (35 µg/ml), kanamycin (40
µg/ml), ampicillin (100 µg/ml) and arabinose (0.05%) were added as required.
For growth assays, the bacteria were incubated overnight in 5 ml LB broth at
37°C, and were collected and washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na<sub>2</sub>HPO<sub>4</sub> and 2 mM KH<sub>2</sub>PO<sub>4</sub>, pH 7.2–7.4)
for three times. The bacteria were adjusted to about OD<sub>600</sub> = 0.90
with PBS and 40 µl were inoculated into 4 ml M9 minimal medium (12.8 g/L
Na<sub>2</sub>HPO<sub>4</sub>, 3.0 g/L KH<sub>2</sub>PO<sub>4</sub>, 0.5 g/L
NaCl, 1.0 g/L NH<sub>4</sub>Cl, 2 mM MgSO<sub>4</sub>, 0.1 mM CaCl<sub>2</sub>
and 0.001% thiamine), with the addition of 0.2% glucose or 0.2% arabinose (for
gene complemented strains and pBAD30 negative control). Additional differential
compositions were NaDC (0.67%) to test bile salt resistance or NaCl (0.37%) to
test osmotic stress resistance. We tested a range of NaDC and NaCl
concentrations initially. The concentration gave the best difference and thus
were selected for the final growth assays. The growth was measured as optical
density at 600 nm at hourly intervals. All assays were performed in triplicate,
and the results were expressed as mean± standard deviation (SD).
## Curing of plasmid pO157_Sal
The plasmid pO157_Sal was removed from Xuzhou21 using sodium dodecyl sulfate
(SDS) and high temperature treatment. Briefly, steps were (i) Xuzhou21 was
inoculated in 5 ml LB broth at 37°C with shaking for 16 h. (ii) 50 µl of the
above culture was inoculated in 5 ml fresh LB broth with 0.05% SDS at 37°C with
shaking for 16 h. (iii) 50 µl of the above culture was inoculated in 5 ml fresh
LB broth at 42°C with shaking for 16 h. Repeat steps (ii) and (iii), and then 50
µl of the culture was inoculated in 5 ml fresh LB broth at 37°C with shaking for
16 h. The culture was then 10-fold serial diluted and spread on LB plates in
order to obtain single colonies. The PCR primers used to identify the plasmid
pO157_Sal were p247-F (5′-AGCGCATCGCTACAAGCACA-3′) and p247-R
(5′-ACGACAACCCCACCGAGGCT-3′). The PCR primers used to identify the pO157 were
ehxA-F (5′-AGCTGCAAGTGCGGGTCTG-3′) and ehxA-R (5′-TACGGGTTATGCCTGCAAGTTCAC-3′).
The plasmid and chromosomal DNA integrity were confirmed by genome sequencing.
Xuzhou21 cured of the pO157_Sal plasmid was named as Xuzhou21m.
## Plasmid and single gene complementation of Xuzhou21m
Vector pRS551 was used as PCR template for kanamycin resistant (Km<sup>r</sup>)
gene.The Km<sup>r</sup> gene was inserted between pO157_Sal_36 and *nikB* gene
without disrupting any open reading frame (ORF) using a one-step gene
inactivation method as described by Datsenko and Wanner. The km<sup>r</sup>
marked pO157_Sal was extracted using SDS alkaline lysis method and introduced
into Xuzhou21m by electroporation using gene pulse II apparatus (Bio-Rad, USA).
The complemented strain was named as Xuzhou21c.
The single gene complementation (*stpA* and *hha*) strains were constructed
using pBAD30 (Ap<sup>r</sup>) due to its lower copy number in this study. The
*stpA* and *hha* genes were amplified from purified Xuzhou21 DNA template by
PCR. The primers used were listed in. The *stpA* and *hha* products were cloned
into the *Eco*RI-*Hin*dIII site of pBAD30 and transformed by electroporation
into Xuzhou21m, creating Xuzhou21m+*stpA* and Xuzhou21m+*hha* respectively.
Empty pBAD30 was transferred into Xuzhou21m as negative control.
## DNA and RNA isolation
Genomic DNA were extracted from both Xuzhou21 and Xuzhou21m using Wizard Genomic
DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer's
protocols.
For total RNA isolation, the bacteria were inoculated in 5 ml LB broth at 37°C
with shaking for 16 h. 50 µl of the above culture was inoculated in 5 ml fresh
LB broth and the culture was shaken at 37°C for about 2.5 h until the
OD<sub>600</sub> reached 0.6. 500 µl of the culture were mixed with 1 ml RNA
protect bacterial reagent (Qiagen, Hilden, Germany) to stabilize RNA according
to the manufacturer's instructions and was centrifuged at 5000×g for 10 minutes
to pellet the cells, which were then resuspended in 100 µl TE buffer (30
mMTris·HCl,1 mM EDTA, pH 8.0) containing 15 mg/ml lysozyme and 1 mg/ml
proteinase K. Total RNA were then isolated according to the standard protocol
using an RNeasy mini kit (Qiagen). The RNA quantity and integrity were analyzed
using Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was stored at −80°C
for the use of transcriptome analysis.
We extracted RNA from 3 separate biological experiments and these RNA samples
were pooled for RNA-seq. We did not perform RNA-Seq for each biological
replicate separately partly due to the cost of RNAseq at that time. Further
Illumina RNA-seq data are highly replicable with relatively low technical
variation.
## Plasmid copy number and major virulence genes transcriptional level
The copy number of pO157 was determined using real-time PCR with the Rotor-gene
Q machine (Qiagen). The primer sequences and annealing temperatures are listed
in. Expression level of major virulence genes was measured by reverse-
transcription quantitative PCR (RT-qPCR) using One Step SYBR PrimeScript RT-PCR
Kit II (Takara, Dalian, China) in the Rotor-Gene Q machine (Qiagen). Primer
sequences and annealing temperatures are listed in. The relative expression
level of target genes and major virulence genes, genes copy numbers were
calculated as 2<sup>−ΔΔCT</sup>. The mRNA expression level of each target gene
was normalized to the expression level of *gapA*. Each assay was performed in
triplicate.
## Genome sequencing, RNA sequencing and mapping
The Xuzhou21m genome was sequenced using Illumina paired end sequencing. All
reads were mapped to the complete genome in previous study (Genbank accession
no. CP001925, CP001926 and CP001927) by SOAP pipeline.
The total RNA extracted from Xuzhou21 and Xuzhou21m were first treated with
Ribo-Zero™ rRNA Removal kit to remove rRNA. The mRNA was fragmented and produced
cDNA libraries primed with random hexamers. cDNA was selected by size,
amplificated using PCR and then sent to sequencing using Illumina Hiseq™ 2000
commercially.
The RNA-Seq data have been submitted to GEO database and the GEO accession
number is GSE44846
(<http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44846>).
## Bioinformatics analysis and transcriptome analysis
Images generated by sequencers were converted by base calling into nucleotide
sequences, which are called raw data or raw reads and are stored in FASTQ
format. Reads were discarded if containing only adaptors, unknown bases more
than 10% of a read, or more than half the bases of a read with quality less than
5. Reads after filtering were called clean reads, on which all following
analyses were based.
Xuzhou21 genome sequence was used as the reference for RNA sequencing mapping
and functional analysis. Clean reads were mapped to reference genome and genes
sequences respectively using SOAP2. Mismatches no more than 2 were allowed in
the alignment. The distribution of reads was plotted by its location in the
reference genome, and then divided into gene region and intergenic region.
Genome and gene coverage was calculated by counting the number of reads mapped
to the genome and individual genes respectively. The gene expression was
calculated using the RPKM method. RPKM ratio and p value were used to evaluate
the difference between two samples. Differential expression genes (DEGs) were
chosen with a P\<0.05 and a ratio of 2 or greater.
## Validation of the transcriptome results by RT-qPCR
To confirm the results of the gene expression data from RNA-Seq, the expression
levels of 167 selected genes that maintain higher, moderate and lower expression
levels in RNA-Seq were measured using RT-qPCR. Primer sequences and annealing
temperatures for these genes examined are in. The *gapA* gene was used for
within sample normalization.
## Statistical analysis
The results were analyzed using the statistical software package SPSS 15.0 for
Windows (IBM SPSS). Statistical analysis was performed using the *t* test.
Values of *P* ≤0.05 were considered statistically significant.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: HZ CC YX CY RL JX. Performed
the experiments: HZ YX XZ HS QC YC QM XNB XL. Analyzed the data: CC HW XY HZ
YX XX. Contributed reagents/materials/analysis tools: AZ XMB. Wrote the
paper: HZ YX CC XX RL JX. |
# 1 Introduction
Experimental hardware for quantum computing has been steadily improving in the
past twenty years, indicating that a useful quantum computer that outperforms a
classical computer may eventually be built. However, until a large-scale and
viable quantum computer has been realized, numerically simulating quantum
circuits on a classical computer will be necessary for predicting the behavior
of quantum computers.
Such simulations can play an important role in the development of quantum
computing by (1) numerically verifying the correctness and characterizing the
performance of quantum algorithms, (2) simulating error and decoherence due to
the interaction between the quantum computer and its environment, and (3)
improving our understanding of the boundary between classical and quantum
computing in terms of computational power, for which recent efforts for
characterizing the advantage of quantum computers over classical computers serve
as an example of this direction.
In this work, we consider the problem of quantum circuit simulation as one where
we are given a quantum circuit and an initial state, with the goal of
determining the probability of a given output state. Several approaches are
possible for such simulation tasks. The most general method is to represent the
state vector of an *N*-qubit state by a complex unit vector of dimension
2<sup>*N*</sup> and apply the quantum gates by performing matrix-vector
multiplications. This is essentially the approach adopted in, for example. This
method has the advantage that full information of the quantum state is
represented at any point during the circuit propagation. However, the
exponential cost of storing and updating the state vector renders it prohibitive
for simulating circuits larger than ∼45 qubits. On the other hand, for a wide
class of circuits with restricted gate sets and input states, efficient
classical simulation algorithms are available. For example, the numerical
package Quipu has been developed for taking advantage of prior results on the
stabilizer formalism to speed up general quantum circuit simulation. Finally,
path integral-based methods have also been proposed—though they do not improve
the simulation cost, they lead to reduced memory storage requirements.
Other than considering the gate sets involved, an alternative perspective of
viewing a quantum circuit is through its geometry or topology. Under this view,
a quantum circuit is simulated via tensor network contractions. An advantage of
viewing quantum circuits as tensor networks is that one can afford to ignore the
particular kinds of quantum gates used in a circuit, and instead only focus on
the graph theoretic properties. While general quantum circuits involving
universal sets of elementary gates are likely hard to simulate on a classical
computer, this geometric perspective sometimes allows for the efficient
simulation of a quantum circuit with a universal gate set, provided that it
satisfies certain graph theoretic properties. We note that at least one open
source implementation of tensor network simulation for quantum circuits already
exists, called TNQVM, which can simulate tensor networks but also focuses on
integrating algorithms with real quantum hardware. Aside from the field of
quantum computation, tensor networks and related methods are an important and
active area of research in the simulation of quantum mechanical problems in
theoretical physics.
Among others, *treewidth* is an important graph theoretic parameter that
determines the efficiency of contracting a tensor network of quantum gates. A
property of graphs that is actively studied in the graph theory literature, the
treewidth provides important structural information about a quantum circuit.
Namely, if the circuit’s underlying tensor network has treewidth *T*, it is
shown in that the cost of simulating the circuit is *O*(exp(*T*)). In treewidth
is also used for estimating the classical resource needed for simulating certain
quantum circuits.
Motivated by the importance of tensor networks in quantum circuit simulation in
general (and for example quantum computational supremacy tests in particular),
it is useful to have a circuit simulation platform singularly dedicated to
tensor network contraction. One immediate challenge in contracting tensor
networks is to find an efficient contraction ordering, which relies on
explicitly or implicitly finding a reasonable *tree decomposition* of the
underlying graph (definitions are further discussed in Section 2). However,
finding the optimal contraction ordering (or equivalently finding the minimum-
size tree decomposition, or finding the treewidth of a graph) is NP-complete.
Therefore one must typically resort to heuristic methods when finding this
decomposition.
For this study, we implemented a plug-and-play tensor network (TN) contraction
code with two contraction schemes. Other schemes were attempted, but were
significantly inferior to those that became part of the software package.
However, there are likely other heuristic schemes that outperform our stochastic
algorithm, and this is an avenue worth pursuing. For a large set of quantum
circuits, our tensor network based methods are shown to be less costly than
simulation of the full Hilbert space, by comparing to simulations using the
LIQ*Ui*\|\> software package. We emphasize again that the tests in this report
give timing data for finding the expectation value of a measurement performed
after implementing a quantum circuit, not for fully characterizing a circuit’s
final state.
The remainder of the paper is organized as the follows. Section 2 sets up the
definitions and notations used in the paper. Section 3 describes the heuristic
methods used for contracting the quantum circuit tensor networks, along with
other relevant details of the code implementation. Section 4 presents the
example quantum circuits used as benchmarks for demonstrating the performance of
our contraction algorithms. Section 5 gives results of comparisons between the
qTorch contraction methods, and between qTorch simulations and LIQ*Ui*\|\>’s
Hilbert space simulations.
# 2 Preliminaries
In this section, we provide an overview of relevant concepts and definitions.
All graphs that we consider in this paper are undirected. We denote a graph as
*G*(*V*, *E*), consisting of the set of nodes *V* = {*v*<sub>1</sub>,
*v*<sub>2</sub>, ⋯, *v*<sub>*n*</sub>} and edges *E* ⊆ *V* × *V*.
Two relevant concepts are a graph’s tree decomposition and treewidth. A
*tree decomposition* of a graph *G*(*V*, *E*) is a pair (*S*, *T*(*I*, *F*)),
where *S* = {*X*<sub>*i*</sub>\|*i* ∈ *I*} is a collection of subsets
*X*<sub>*i*</sub> ⊆ *V* and *T* is a tree (with edge set *F* and node set *I*),
such that ∪<sub>*i*∈*I*</sub>*X*<sub>*i*</sub> = *V*. Two nodes
*X*<sub>*i*</sub> and *X*<sub>*j*</sub> are connected by an edge only if the
intersection between *X*<sub>*i*</sub> and *X*<sub>*j*</sub> is not null. The
*width* of a tree decomposition (*S*, *T*) is
max<sub>*i*∈*I*</sub>\|*X*<sub>*i*</sub>\| − 1. The *treewidth* of a graph *G*
is the minimum width among all tree decompositions of *G*.
Another important concept in tensor network methods is the *linegraph* of graph
*G*, denoted by *L*(*G*). *L*(*G*) is itself an undirected graph, with each edge
in *G* corresponding to a node in *L*(*G*). Two nodes in *L*(*G*) are connected
if and only if these two nodes’ corresponding edges in *G* are connected to the
same node in *G*. There exists an optimal tree decomposition of *L*(*G*) that
provides the optimal contraction ordering of *G*.
In the context of this work, a *tensor* is defined as a data structure with rank
*k* and dimension *m*. More specifically, each tensor is a multidimensional
array with *m*<sup>*k*</sup> complex entries. Each index may have a different
dimension, though in this work each index has the same dimension *m* = 4. A
tensor $A_{i_{1},i_{2},i_{3}...i_{k}}$ has *k* indices, which take values from 0
to *m* − 1.
A *tensor contraction* is a generalized tensor-tensor multiplication. Here a
rank (*x* + *y*) dimension *m* tensor *A* and a rank (*y* + *z*) dimension *m*
tensor *B* are contracted into *C*, a rank (*x* + *z*) dimension *m* tensor.
$$\begin{array}{r} {C_{i_{1},i_{2}...,i_{x},k_{1},k_{2}...,k_{z}} =
\sum\limits_{j_{1},j_{2}...,j_{y} \in {\{ 0..,m - 1\}}}{A_{i_{1},i_{2}...,i_{x},
j_{1}...,j_{y}}B_{j_{1},j_{2}...,j_{y},k_{1}...,k_{z}}}} \\ \end{array}$$
Note that the number of floating point operations performed is
*m*<sup>*x*+*y*+*z*</sup>, exponential in the number of indices contracted on
*y* and the rank of the resulting tensor (*x* + *z*). It is important to point
out that pairwise contractions are always optimal. In other words, a function
that contracts three or more nodes at a time will not achieve an improvement in
scaling.
A *tensor network* is a graph *G* = (*V*, *E*) with tensors as vertices, and
edges labeled by a set of indices. The rank of each tensor is given by the
number of edges connected to it. An edge from one tensor to another indicates a
contraction between the two tensors, and multiple connected edges indicate a
contraction on multiple indices. shows an example of a tensor network. Note that
a tensor may have open edges, *i.e*. edges that do not connect to any other
tensor, though this possibility is not allowed in the current version of qTorch.
A contraction ordering or contraction scheme determines the order in which the
tensor network is contracted. The ordering chosen for the contraction will
greatly affect the computation and memory requirements, because some contraction
orderings can result in much larger intermediate tensors than others. Although
in this work we focus on contracting the tensor network to a scalar that equals
the expectation value of the quantum circuit’s measurement, the goal of a tensor
network algorithm is often *not* to contract it to a scalar. An example of this
is the infinite tensor networks used to study periodic systems in condensed
matter physics.
An important goal is to avoid tensors of large intermediate rank when
contracting the network, as floating point operations grow exponentially with
tensor rank. However, it is often the case that increasing the tensor rank is
unavoidable. A simple example of this issue is a tetrahedral graph of rank 3
tensors, which cannot be contracted without forming intermediate tensors of rank
greater than 3. The larger the treewidth of *L*(*G*) is, the more one will be
forced to create new tensors of higher rank as the network is contracted,
greatly increasing the computational cost.
We note that tensor network methods are commonly used to efficiently find
approximate solutions—indeed this is often the main source of a TN’s utility. In
approximate TN methods, the space of the smaller values of the eigenspectrum are
removed, after e.g. performing a singular value decomposition on the tensor.
This allows one to contract to form a larger tensor, then renormalize its size
before continuing to contract the network. Though this strategy is useful in the
context of quantum circuits as well, the current version of qTorch is limited to
numerically exact contractions of the tensor network.
Before contracting, the tensor network graph must first be created from a
quantum circuit, a procedure that has been summarized in previous work. Each
node on the graph represents one of the following: An initial state of the qubit
(usually \|0〉〈0\|), a gate operation, or a measurement. The initial density
matrix is represented as a rank 1 dimension 4 tensor (i.e. a vector), \[*ρ*<sub>
\|0〉〈0\|</sub>;*ρ*<sub>\|0〉〈1\|</sub>;*ρ*<sub>\|1〉〈0\|</sub>;*ρ*<sub>\|1〉〈1\|</s
ub>\]. Measurement nodes are rank 1 as well. All indices in the graph are
dimension 4, regardless of rank. Nodes corresponding to quantum gates are
represented in superoperator form. Hence a gate *U* which acts on the quantum
state as *ρ* → *UρU*<sup>†</sup> is represented by the superoperator
$\widetilde{U}$. The same operation can be expressed as $\left.
\widetilde{\rho}\rightarrow\widetilde{U}\widetilde{\rho} \right.$, where
$\widetilde{\rho}$ is the Lindblad representation of the density operator.
Single qubit gates correspond to rank 2 tensors and two-qubit gates correspond
to rank 4 tensors. The graph’s connectivity is identical to the connectivity of
the original quantum circuit.
We end this section with explicit examples of tensors for standard quantum
circuit components. Tensors for other circuit components can be viewed in the
source code for qTorch.
The initial state \|0〉〈0\| corresponds to the tensor $$\begin{array}{r}
{{\widetilde{\rho}}_{0} = \lbrack 1,0,0,0\rbrack.} \\ \end{array}$$
Superoperator tensors for the Pauli matrices are $$\begin{array}{r}
{\widetilde{X} = \begin{bmatrix} 0 & 0 & 0 & 1 \\ 0 & 0 & 1 & 0 \\ 0 & 1 & 0 & 0
\\ 1 & 0 & 0 & 0 \\ \end{bmatrix};\widetilde{Y} = \begin{bmatrix} 0 & 0 & 0 & 1
\\ 0 & 0 & {- 1} & 0 \\ 0 & {- 1} & 0 & 0 \\ 1 & 0 & 0 & 0 \\
\end{bmatrix};\widetilde{Z} = \begin{bmatrix} 1 & 0 & 0 & 0 \\ 0 & 1 & 0 & 0 \\
0 & 0 & 1 & 0 \\ 0 & 0 & 0 & 1 \\ \end{bmatrix}.} \\ \end{array}$$
The CNOT gate is represented as a sparse rank 4 tensor for which only the
following entries are nonzero: $$\begin{array}{r}
{{\widetilde{U}}_{CNOT}:{\widetilde{U}}_{0000} = {\widetilde{U}}_{0101} =
{\widetilde{U}}_{0202} = {\widetilde{U}}_{0303} = {\widetilde{U}}_{1011} =
{\widetilde{U}}_{1110} = {\widetilde{U}}_{1213} = {\widetilde{U}}_{1312} =} \\
{{\widetilde{U}}_{2022} = {\widetilde{U}}_{2123} = {\widetilde{U}}_{2220} =
{\widetilde{U}}_{2321} = {\widetilde{U}}_{3033} = {\widetilde{U}}_{3132} =
{\widetilde{U}}_{3231} = {\widetilde{U}}_{3330} = 1} \\ \end{array}$$
Finally, the nodes for measurement are rank 1 tensors. ${\widetilde{M}}_{X}$,
${\widetilde{M}}_{Y}$, and ${\widetilde{M}}_{Z}$ correspond respectively to
determining expectation values for measurements in the *X*, *Y*, and *Z* bases.
Note that using ${\widetilde{M}}_{X}$, ${\widetilde{M}}_{Y}$, or
${\widetilde{M}}_{Z}$ is equivalent to inserting a Pauli gate at the end of the
circuit before implementing ${\widetilde{M}}_{Trace}$. $$\begin{array}{r}
{{\widetilde{M}}_{Trace} = \begin{bmatrix} 1 \\ 0 \\ 0 \\ 1 \\
\end{bmatrix};{\widetilde{M}}_{X} = \begin{bmatrix} 0 \\ 1 \\ 1 \\ 0 \\
\end{bmatrix};{\widetilde{M}}_{Y} = \begin{bmatrix} 0 \\ i \\ {- i} \\ 0 \\
\end{bmatrix};{\widetilde{M}}_{Z} = \begin{bmatrix} 1 \\ 0 \\ 0 \\ {- 1} \\
\end{bmatrix}} \\ \end{array}$$
# 3 Contraction schemes and implementation details
For many problems in quantum physics to which matrix product states (MPS) or
other tensor network methods have been applied, an efficient contraction scheme
is often obvious from the underlying structure of the Hamiltonian. However,
efficient contraction schemes are not available for arbitrary tensor networks. A
general heuristic contraction scheme is important for the simulation of general
quantum circuits, when one does not know a priori the topological properties of
the underlying tensor network problem.
## 3.1 Contraction schemes
qTorch implements two algorithms for determining the contraction ordering. For
what we call the line graph (*LG*) method, outlined in Algorithm 1, we first
create the line graph of the quantum circuit’s graph. Then, the software package
QuickBB is used to determine an approximately optimal tree decomposition of this
linegraph. The resulting tree decomposition is used to define the order of
contraction. This linegraph-based approach was first described by Markov and
Shi.
QuickBB uses a so-called anytime algorithm, meaning that it can be run for an
arbitrary amount of time, such that when the program is stopped it provides the
best solution found thus far. The algorithm is based on the branch and bound
(B&B) method, though the authors use several techniques based on modern graph
theory to improve efficiency in the pruning and propagation steps, making
QuickBB faster at finding low-width tree decompositions than vanilla B&B.
The second contraction scheme is a simple stochastic procedure we refer to as
*Stoch* (Algorithm 2). First, a wire is randomly chosen. If the rank of the
contracted tensor is higher than the highest rank of the two nodes, plus a given
threshold, the contraction is rejected. After a fixed number of rejected
contraction attempts, the threshold is relaxed.
**Algorithm 1** Contraction via TD of L(G)
1: Create line graph L(G) of graph G
2: *π* ← (Calculate approx. optimal elimination ordering of L(G))
3: Eliminate wires of G in order *π*
**Algorithm 2** Simple stochastic contraction
1: Define *G* ← The tensor network Graph
2: *Threshold* ← −1
3: Define *MaxRejections* ← Maximum Number of Rejections
4: **repeat**
5: Choose a random wire *w*
6: (*A*, *B*) ← (Nodes of *w*)
7: Cost ← rank(*C*) − max(rank(*A*), rank(*B*))
8: **if** Cost ≤ Threshold **then**
9: Contract *w* to form node *C*
10: *Rejections* ← 0
11: *Threshold* ← -1
12: Update *G*
13: **else**
14: *Rejections* ← *Rejections* + 1
15: **if** *Rejections* \> *MaxRejections* **then**
16: *Threshold* ← *Threshold* + 1
17: *Rejections* ← 0
18: Continue
19: **until** Graph completely contracted
## 3.2 Threading
The tensor contractions are parallelized using the C++ standard library’s
`std::thread` class. A particular tensor-tensor contraction is parallelized if
the cost of contracting a pair is higher than a provided threshold. We implement
other parallelization schemes at the network level, i.e. splitting up the nodes
into separate groups to compute on different threads, but the vast majority of
the parallelization speedup comes from threading the tensor–tensor contractions.
Currently, qTorch does not support parallelization across multiple compute nodes
within a cluster, but it allows the user to specify the number of threads
(default of 8).
## 3.3 Estimating the answer string
qTorch computes expectation values of the form 〈*ψ*\|*M*\|*ψ*〉, where *M* is a
measurement operator such as a Pauli string, and \|*ψ*〉 is the quantum state
produced by the circuit. If instead one wishes to capture all the information of
this final state of *n* qubits, it generally requires *O*(2<sup>*n*</sup>)
repetitions of the algorithm. However, many quantities of interest may be
calculated efficiently. For instance, the probability that one measurement
operator (e.g. a Pauli string) will provide a particular outcome can be
estimated in just one contraction of the tensor network, a result essential to
simulating the variational quantum eigensolver (VQE).
qTorch provides a heuristic scheme to output a high-probability answer string
from the circuit, which we summarize here. Though this scheme is not used for
the results presented in Section 5, it may be useful in the future for
simulating algorithms (like QAOA) where the goal is to estimate a most likely
bit string.
The scheme is implemented as follows. First we run one simulation, and measure
in the computational basis to project the first qubit into 0 or 1. Based on the
resulting expectation value from the simulation, we choose the value for the
first qubit that has the greater probability. If the 0 and 1 are equally likely,
one is chosen randomly. In the next full contraction, we set the resulting
binary value as the measurement outcome for the first qubit in the next
simulation, and repeat with a projective measurement on the second qubit. We
continue this process for the rest of the qubits. As we show below, this method
often gives a good approximation of the most likely final computational basis
state. In original tests on 3-regular graphs of 30 vertices, the scheme (used on
Quantum Approximate Optimization Algorithm \[QAOA\] circuits) gave bit strings
that provided good estimates to the solution of the Max-Cut problem (average
approximation ratio of 94% compared to the exact brute force solution).
As a way to test the general applicability of this scheme, we performed some
tests on more general circuits than the QAOA problem. These tests are meant to
provide some early insight into how useful this heuristic would be for
estimating the most likely bit string of a quantum algorithm, for the users who
are interested in running this string estimation subroutine. We note that it is
abundantly clear that in many cases this scheme does not produce a string
closest to the most likely string—indeed, if it was a generally accurate scheme
then we would have no need for a quantum computer.
In the remainder of this section, we consider the most likely bit string of the
final state \|*ψ*〉 = ∑<sub>*i*</sub> *ψ*<sub>*i*</sub>\|*i*〉, which we define as
argmax<sub>*i*</sub>\|*ψ*<sub>*i*</sub>\|<sup>2</sup>, where the vectors
{\|*i*〉} are in the computational basis. We apply a unitary of the form
$$\begin{array}{r} {U_{p}\left( \mathbf{\beta},\mathbf{\gamma} \right) =
\prod\limits_{j = 1}^{p}\text{exp}\left( i\beta_{j}\sum\limits_{i = 1}^{n}X_{i}
\right)\text{exp}\left( i\gamma_{j}D_{j} \right)} \\ \end{array}$$ where the
matrix *D*<sub>*j*</sub> is a diagonal matrix with entries chosen randomly from
the integers {1, ⋯, *nm*}. Here *m* is a parameter that could be interpreted as
the number of clauses, if this were a QAOA problem. The elements of the
*p*-dimensional vectors ***β*** and ***γ*** are drawn uniformly from \[0, *π*\]
and \[0, 2*π*\] respectively. We use the construction of
*U*<sub>*p*</sub>(***β***, ***γ***) to emulate the form of parametrized unitary
operations used in QAOA with the same *p*. Starting from the uniform
superposition over all 2<sup>*n*</sup> bit strings \|*s*〉, we apply
*U*<sub>*p*</sub> to compute the final state $\left| \Psi \right\rangle =
U_{p}\left| s \right\rangle = \sum_{i = 0}^{2^{n} - 1}\psi_{i}\left| i
\right\rangle$. Let *p*<sub>*i*</sub> = \|*ψ*<sub>*i*</sub>\|<sup>2</sup> denote
the probability distribution associated to the QAOA-like output state \|Ψ〉. We
ran 10,000 trials (with n = 6, m = 10, and p = 2) using, and ranked the result
by how many bit strings in the true state were more likely than our outputted
bit string.
Conceptually, our likely string estimation algorithm can be thought of as
falsely assuming that the output state is a product state. Suppose we apply our
algorithm to the state \|Ψ〉. The product state then reads $$\begin{array}{r}
{|\Psi^{\prime}{\rangle =}\left( \alpha_{1} \middle| 0 \right\rangle +
\beta_{1}{\left| 1 \right\rangle)} \otimes \left( \alpha_{2} \middle| 0
\right\rangle + \beta_{2}{\left| 1 \right\rangle)} \otimes \cdots\left(
\alpha_{n} \middle| 0 \right\rangle + \beta_{n}{\left| 1 \right\rangle)}} \\
\end{array}$$ where \|*α*<sub>*k*</sub>\|<sup>2</sup> is the probability of \|0〉
that the algorithm obtains at the *k*<sup>th</sup> step, with an analogous
definition for *β*<sub>*k*</sub>. With this conceptual framing, we also
numerically study the 1-norm distance ∥**p**′ − **p**∥<sub>1</sub> between the
approximate distribution **p**′ which the algorithm effectively assumes and the
actual distribution **p**.
The results are shown in. Here we use the number of qubits *n* = 6, with
parameters *m* = 10 and *p* = 2. shows that most of the time our algorithm
produces a high ranking bit string—roughly 90% of the time the output of the
algorithm is among the top 10% most likely bit strings. shows that the 1-norm
distance between the approximate and exact distributions is less than 0.1 for
nearly all of the data points. These results suggest that our heuristic for
estimating an output bit string will produce acceptable estimates for some
circumstances—in QAOA for instance, where one might be interested in a good
approximate (as opposed to exact) solution to the constraint satisfaction
problem.
## 3.4 A note on noise
It is possible to simulate noise within the quantum circuit model, by mapping a
noise model onto a set of one-qubit or multi-qubit operators.
Note that any quantum operation can be expressed in terms of Kraus operators
{*E*<sub>*j*</sub>} $$\begin{array}{r} \left.
\rho\rightarrow\sum\limits_{j}E_{j}\rho E_{j}^{\dagger} \right. \\ \end{array}$$
where {*E*<sub>*j*</sub>} are called Kraus operators, and
$\sum_{j}E_{j}E_{j}^{\dagger} = 1$ because for our purposes the noise process is
assumed to be trace-preserving. A noise model can be expressed in terms of such
Kraus operators, which can in turn be expressed as superoperators for insertion
into the quantum circuit’s tensor network.
The most commonly used approximations assume that noise on different qubits is
uncorrelated, which allows for single-qubit “noise gates” to be used. In this
case, because rank 2 tensors can always be contracted without increasing the
rank of the resulting tensors, the cost of simulating the resulting “noisy”
quantum circuit would not substantially increase. One common and easily
implementable approximation is the Pauli twirl approximation, which approximates
a noise process purely in terms of Pauli rotations, and therefore can be
implemented with the built-in quantum gates of qTorch.
A more physically realistic noise model would assume correlated noise, which
necessitates the insertion of noise gates that operate on at least two qubits.
In this case, the tree width of the circuit’s underlying line graph, and hence
the complexity of the problem, would increase in all but the most trivial cases.
qTorch does not incorporate built-in noise gates. Instead, we include
functionality that allows for user-defined gates.
# 4 Circuit simulations
Here we describe the classes of quantum circuits that were simulated for this
work.
## 4.1 QAOA / Max-Cut
The quantum approximate optimization algorithm (QAOA) was recently developed,
for the purpose of demonstrating quantum speedup for combinatorial problems on
low-depth quantum circuits. Given a constraint satisfaction problem (CSP), a
QAOA quantum circuit produces an output that provides approximate solutions.
Several aspects of QAOA have been studied since its introduction, including its
application to different classes of CSP, implementations of different classical
optimization routines, and numerical and analytical comparisons to classical
algorithms.
We use qTorch to simulate QAOA for the Max-Cut problem, a combinatorial problem
that has been a focus of QAOA. Given an arbitrary undirected graph, the goal of
Max-Cut is to assign one of two colors to each node, so as to maximize the
number of cuts. A cut is any edge that connects two nodes of different color. A
more detailed explanation of QAOA is given in the Appendix, though we summarize
important aspects of the algorithm here.
In QAOA, a set of constraints is mapped to a an objective function represented
by a set of operators. Specifically for the Max-Cut problem, the object function
is $$\begin{array}{r} {C = \sum\limits_{\langle ij\rangle}C_{\langle
ij\rangle},} \\ \end{array}$$ with $$C_{\langle ij\rangle} = \frac{1}{2}(1 -
\sigma_{i}^{z}\sigma_{j}^{z}),$$ where 〈*ij*〉 represents the edge between nodes
*i* and *j*, $\sigma_{k}^{z}$ is the Pauli-Z operator on qubit *k*, and each
node in the underlying Max-Cut graph (which is related to but not the same as
the quantum circuit’s graph) corresponds to one qubit in the quantum circuit.
Define two operators *U*(*C*, *γ*) and *U*(*B*, *β*) as $$\begin{array}{r}
{U(C,\gamma) = e^{- i\gamma C} = \prod\limits_{m = 1}^{n}e^{- i\gamma C_{m}}} \\
\end{array}$$ and $$\begin{array}{r} {U(B,\beta) = e^{- i\beta B} =
\prod\limits_{k = 1}^{q}e^{- i\beta\sigma_{k}^{x}}.} \\ \end{array}$$ where $B =
\sum_{k = 1}^{q}\sigma_{k}^{x}$, *σ*<sub>*x*</sub> is the Pauli-X operator, *q*
is the number of qubits, and *n* is the number of clauses (for Max-Cut this is
the number of edges). These two operators are applied *p* times (with different
*γ* and *β* allowed for each step), with a larger *p* providing a better
approximation. The *γ* and *β* parameters are modified with a classical
optimization routine to maximize the cost function. The cost function is
evaluated after each measurement, with the bit string that resulted from the
measurement.
To generate the graphs for the underlying Max-Cut problem, we made random
*k*-regular graphs by placing edges randomly throughout a given vertex set to
satisfy a given regularity, before ensuring that disconnected graphs are
rejected. QAOA/Max-Cut Quantum circuits based on these graphs are then
constructed.
In the numerical results of this paper, we report only the timing for a single
contraction of each quantum circuit. A full analysis of QAOA is beyond the scope
of this work. However, we note that once the graphs have been created, qTorch
currently has the functionality to optimize the QAOA angles using the classical
optimization library NLopt. Finally, one can use qTorch to estimate a Max-Cut
for the randomly-generated graph, using the most-likely bit string estimation
method described above.
## 4.2 Hubbard model
Quantum simulation of fermionic systems is one of the most relevant applications
of quantum computers, with direct impact on chemistry and materials science,
including for the design of new drugs and materials. Among all the algorithms
proposed for quantum simulation of fermions, the quantum variational algorithm
(VQE) and related approaches are arguably the most promising for near-term
hardware because they have lower circuit depth requirements. We note that many
types of chemistry-related circuits can be prepared with the software package
OpenFermion.
In the VQE algorithm, a quantum computer is employed to prepare and measure the
energy of quantum states associated with a parameterized quantum circuit. The
approximate ground state of a Hamiltonian is obtained by variationally
minimizing the cost function (corresponding to e.g. the molecular energy) with
respect to the circuit parameters using a classical optimization routine. This
hybrid quantum-classical approach offers a good compromise between classical and
quantum resources. Classical simulations of the VQE algorithm for tens of qubits
could provide insights into the complexity of the circuits used for state
preparation and help design better ansatzes for the quantum simulation of
fermions.
As an example of a VQE simulation, we used qTorch to classically simulate
variational circuits employed for the quantum simulation of 1D Hubbard lattices.
We consider half-filled Hubbard models on *N* sites, with periodic boundary
conditions.
To construct variational circuits for these systems, we considered the
variational ansatz introduced by Wecker et al. In this case, the Hubbard
Hamiltonian is divided as *H* = *h*<sub>*h*</sub> + *h*<sub>*U*</sub>, where
*h*<sub>*h*</sub> is the sum of hopping terms in the horizontal dimension and
*h*<sub>*U*</sub> is the repulsion term. The variational circuit is constructed
as a sequence of unitary rotations by terms in the Hamiltonian with different
variational parameters, with the sequence being repeated *S* times. In each
step, there are two variational parameters, $\theta_{U}^{b}$ and
$\theta_{h}^{b}$, where *b* = 1, ⋯, *N* such that $$\begin{array}{r} {U =
\prod\limits_{b = 1}^{S}\left\lbrack U_{U}\left( \frac{\sigma_{U}^{b}}{2}
\right)U_{h}\left( \theta_{h}^{b} \right)U_{U}\left( \frac{\theta_{U}^{b}}{2}
\right) \right\rbrack} \\ \end{array}$$ where
*U*<sub>*X*</sub>(*θ*<sub>*X*</sub>) denotes a Trotter approximation to
exp(*iθ*<sub>*X*</sub> *h*<sub>*X*</sub>), and *X* can be *U* or *h*. For our
numerical simulations, we employed the variational circuit of with *S* = 1 using
a 1-step Trotter formula for all the *U*<sub>*X*</sub>(*θ*<sub>*X*</sub>) terms.
Notice that this is approximate for the *h*<sub>*h*</sub> term, which comprises
a sum of non-commuting terms. We also assigned the value of 1 to all variational
amplitudes. The corresponding unitary was mapped to a quantum circuit using the
Jordan-Wigner transformation and the circuit was generated using a decomposition
into CNOT gates and single-qubit rotations.
# 5 Results
Simulations were performed on the NERSC Cori supercomputer, using one “Knights
Landing” (KNL) node per simulation, each of which contains 68 cores and 96 GB of
memory. Each LIQ*Ui*\|\> simulation was run on a full node as well, using Docker
(computational details in the Appendix). The free version of LIQ*Ui*\|\> allows
for the simulation of 24 qubits. Because full Hilbert space simulation scales
exponentially regardless of the quantum algorithm’s complexity, we would not
have been able to simulate more than ∼31 qubits on one of these compute nodes.
For each set of parameters (regularity and number of vertices/qubits) 50
instances of Max-Cut/QAOA circuit were created. For higher qubit counts and
higher regularities, only a subset of these circuits were completed, since many
simulations exceeded memory capacity. In this section, *LG* or *qTorch-LG* refer
to the use of qTorch with the linegraph-based contraction, *Stoch* or *qTorch-
Stoch* refer to qTorch with stochastic contraction. To determine a qTorch-LG
contraction ordering, QuickBB simulations were run for an arbitrary time of 3000
seconds for each quantum circuit. The plotted qTorch results do not include the
QuickBB run time.
We note that LIQ*Ui*\|\> implements a thorough set of important optimizations,
which makes it a fair benchmark against which to compare qTorch. For example,
LIQ*Ui*\|\> fuses many gates together before acting on the state vector, and
uses sparse operations. qTorch, on the other hand, does not yet use sparsity at
all (even when the circuit consists primarily of sparse CNOT gates), which is
one of several optimizations that we expect would further improve performance.
LIQ*Ui*\|\> is the fastest simulation method to use for the Hubbard simulations,
as shown in. This is because the treewidth of the circuit’s graph increases
substantially with the number of qubits, even for these short-depth circuits.
The result is not surprising—if the algorithm were easy to simulate with a
tensor network on a classical computer, then it would not have been worth
proposing as a candidate for a quantum computer.
Simulation timing results for 3-, 4-, and 5-regular Max-Cut/QAOA circuits are
shown using Tukey boxplots in Figs and. Stoch and LG simulation times are of
similar order of magnitude for these circuits, though LG is generally faster.
The exception is the 3-regular graph problems, where Stoch often appears to find
a more efficient contraction than the 3000-second run of QuickBB does. We note
that if the QuickBB algorithm were run for infinite time before beginning the
contraction, then qTorch-LG should always (except in very simple graphs)
contract the circuit faster than qTorch-Stoch. This is because, while the Stoch
search is purely local (considering only individual wires), the tree
decomposition approach of QuickBB implicitly considers the effects of multiple
contraction steps. Note that actual search time of Stoch is negligible compared
to the tensor contraction time. Note that LIQ*Ui*\|\> begins to outperform
tensor contraction methods once the algorithm is run on 5-regular graphs,
because the increased circuit complexity leads to larger intermediate tensors in
qTorch.
Note that in principle, Hilbert space simulation can be considered a subset of
TN contraction, where the state vector is simply a large tensor. Hence one might
expect that there would not be a crossover point at all, *i.e*. that in the
worst case TN contraction would not ever be slower than Hilbert space
simulation. However, because our implementation considers density matrices
instead of state vectors, one would in fact expect this crossover point to
exist. The largest tensor in qTorch would have 4<sup>*N*</sup> entries, while
the state vector has just 2<sup>*N*</sup> entries. The various choices made in
software implementations for qTorch and LIQ*Ui*\|\> would also affect the
position of this crossover point.
Using a single Cori NERSC node, we were able to contract quantum circuits of 90
qubits for a very small subset of the simulated graphs, though not on enough
graphs to report statistics. Full Hilbert space methods would be limited to ∼30
qubits on these nodes, and indeed previous simulation packages have not yet
surpassed 46 qubits, using thousands of nodes.
Interesting trends appear when the simulation time is plotted against regularity
of the Max-Cut problem’s graph. It is notable that the LG method runs out of
memory before the Stoch method does. As previously mentioned, the LG method
contracts more efficiently the longer QuickBB has been run, and we chose 3000
seconds as an arbitrary QuickBB limit for all circuits. There is a trade-off
between running a longer QuickBB simulation and instead immediately using the
Stoch method. Even with few qubits, at higher regularities the full Hilbert
space simulation (using LIQ*Ui*\|\>) performs better. This is expected, since as
the complexity of the quantum circuit increases, higher-rank intermediate
tensors appear.
shows simulation time as the estimated upper bound for the treewidth increases,
for Max-Cut/QAOA circuits of 18 qubits. These include 3- through 7-regular
graphs. This treewidth upper bound is simply the treewidth of the tree
decomposition that defines the contraction ordering. The plot shows the expected
general trend of an increase simulation time with increased treewidth,
regardless of contraction scheme.
Finally, we note that we were easily able to perform simulations of 100 qubits
for less complex graphs. To report one such example, we produced a random
3-regular graph with a slightly different procedure from that given in of
Section 4.1. Beginning with a 2-regular graph (i.e. a ring) of 100 vertices, we
added edges between random pairs of vertices until all vertices were of 3
degrees. Contracting this graph’s Max-Cut/QAOA circuit took ∼150 seconds.
# 6 Conclusions
We have implemented a tensor contraction code for the efficient simulation of
quantum circuits. We compared a stochastic contraction scheme to one based on
the line graph of the quantum circuit’s graph, showing that the latter is more
efficient in most circuits simulated herein. However, there is a subset of cases
for which calculating an efficient approximate optimal tree decomposition of the
line graph takes longer than contracting the circuit stochastically, in which
case the stochastic scheme is superior. For the circuits studied in this work,
our simulations suggest that the point at which qTorch is no longer faster than
LIQ*Ui*\| \> occurs in QAOA/Max-Cut approximately when the Max-Cut graph has a
regularity of five. In the future, qTorch may be used to estimate these points
of equivalent computational cost in other classes of circuits, which may help to
determine which simulation method to use in simulations.
Several immediate algorithmic improvements are possible for this software. The
use of sparse tensors would reduce the number of floating point operations for
some relevant circuits. Tensor decompositions (such as the singular value
decomposition) along with trimming can be added as intermediate steps, as has
been done in tensor network based simulations of physical systems. Additionally,
more advanced parallelization methods would allow for faster calculation of a
tree decomposition as well as faster contractions.
# Supporting information
We are grateful to Edward Farhi and Aram Harrow for discussions about QAOA, to
Salvatore Mandrà for general discussions, and to Dave Wecker for helpful advice
on using LIQ*Ui*\| \>. N.S. acknowledges the use of resources of the National
Energy Research Scientific Computing Center, a DOE Office of Science User
Facility supported by the Office of Science of the U.S. Department of Energy
under Contract No. DE-AC02-05CH11231. A.A.-G. and E.F. were supported by the
Office of Naval Research under grant N00014-16-1-2008 (Vannevar Bush Faculty
Fellowship). A. A.-G. and Y.C. were supported by NSF grant CHE-1655187. |
# Introduction
Initiation of inflammation following infection requires recognition of the
invading microbe by innate immune pattern recognition receptors (PRRs) that
signal in response to pathogen-associated molecular patterns (PAMPs). PRRs
recognize self- and microbe-associated molecules. Members of the Toll-like
receptor (TLR) family of PRRs are transmembrane receptors that are expressed
either on the cell surface or within the endosomal compartment and respond to a
variety of PAMPs. Murine TLR3, TLR7 and TLR9 are expressed in the endolysosome
and are implicated in recognition of viral dsRNA, ssRNA and dsDNA,
respectively,. Ligation of the nucleic acid-sensing TLRs results in
transcription of antiviral genes including type I IFNs (IFN-α/β) and
proinflammatory cytokines. TLR3 responses require signaling through the adaptor
molecule Toll/IL-1R domain-containing adapter-inducing interferon-β (TRIF),
while TLR7 and TLR9 are dependent on the adaptor molecule myeloid
differentiation primary response gene 88 (MyD88) to activate transcription
factors and induce gene transcription,.
Murine cytomegalovirus (MCMV) is a betaherpesvirus that can establish acute
infection in multiple organs including the liver. Acute MCMV infection induces
an early systemic proinflammatory cytokine response including high levels of
type I IFNs, IFN-γ, IL-12 and TNF-α. Infection in the liver induces early
production of IFN-α, predominantly by plasmacytoid dendritic cells (pDCs), by 40
h post-infection. Type I IFN production mediates downstream responses including
chemokine and cytokine production as well as monocyte/macrophage, natural killer
(NK) cell and T cell recruitment. Early type I IFN signaling is necessary for NK
cell recruitment to the liver, where they deliver the antiviral cytokine IFN-γ
within the first 48 h post-MCMV infection. The NK cell IFN-γ response is an
important early step in the control of liver infection. This response induces
IFN-γ-dependent chemokines, which contribute to the recruitment of CD8+ T cells
to the liver. Liver CD8+ T cell responses occur by days 5 and 7 post-MCMV
infection and are an important source of cytokines late in acute infection that
contribute to resistance against MCMV.
While early responses to MCMV infection in the liver are well understood, it
remains unclear how the virus is sensed in this compartment. This is in contrast
with other sites, namely the spleen, in which studies by our group and others
have definitively shown a role for TLR9 and MyD88 signaling in IFN-α,
proinflammatory cytokine and cellular responses in addition to restriction of
virus replication. Although TLR7 alone does not appear to have a strong role in
MCMV recognition, TLR7 and TLR9 combined deficiency was shown to severely impair
pDC responses against MCMV in the spleen. A significant but minor role for TLR3
signaling in the spleen has also been suggested in response to MCMV infection.
In the liver, however, studies by our group have demonstrated that early innate
responses are TLR9-independent but MyD88-dependent. Liver pDCs from mice
genetically deficient in TLR9 produce wild-type (WT) levels of IFN-α at 40 h
post-MCMV infection, with intact downstream cellular and proinflammatory
cytokine responses. Further, TLR9-deficient mice do not exhibit elevated liver
viral titers. Conversely, MyD88-deficient mice have severely impaired liver
cytokine and cellular responses, and are unable to control virus replication in
this compartment. MyD88 is a common adaptor molecule for TLR9 and TLR7
signaling; however, evaluation of TLR7-deficient mice also demonstrated that
TLR7 signals alone were not required to initiate liver antiviral defense.
These TLR-independent but MyD88-dependent antiviral responses suggested possible
redundancies among TLR signals in the liver compartment in response to MCMV
infection. To investigate this possibility, we utilized mice containing an H412R
missense mutation in the endoplasmic reticulum protein UNC93B1 to address the
combined function of nucleic acid-sensing TLRs in the liver during acute MCMV
infection. The UNC93B1 mutation (known as ‘triple d’ or ‘3d’) impairs signaling
through TLR3, TLR7 and TLR9 due to improper trafficking of these receptors to
the endosomal compartment, and has been shown to affect exogenous antigen
presentation. Our studies show that proinflammatory cytokine production after
early infection with MCMV is dependent on UNC93B1. Further, UNC93B1 deficiency
exacerbates liver disease and increases viral burden, although MCMV-specific
CD8+ T cell responses are not impaired. Collectively, these results suggest a
level of redundancy within the liver to promote viral recognition by
demonstrating that a combination of nucleic acid-sensing TLRs contributes to
innate inflammatory responses during MCMV infection.
# Results
## Systemic cytokine production is impaired in 3d mice during MCMV infection
Considering the potential of endosomal TLR signals to induce proinflammatory
cytokine expression, UNC93B1 deficient 3d mice were first assessed for systemic
IFN-α, IFN-γ, IL-12 and TNF-α production during early infection with a moderate
(5×10<sup>4</sup> PFU) dose of MCMV. C57BL/6 (WT) and 3d mice were uninfected or
MCMV-infected for 40 h or 48 h. Serum was collected at indicated time points and
IFN-α, IFN-γ, IL-12p70 and TNF-α were measured by enzyme-linked immunosorbent
assay (ELISA). In WT mice, maximal production of IFN-α, IFN-γ and IL-12p70 was
detected at 40 h post-MCMV infection before declining by 48 h post-infection
(A–C). In contrast, 3d mice exhibited lower serum levels of these cytokines in
response to MCMV infection. Specifically, while serum IFN-α reached 1300±420
pg/mL at 40 h post-MCMV infection in WT mice, IFN-α production was reduced by
three-fold at this infection time point in 3d mice (450±300 pg/mL), with
comparable levels maintained at 48 h post-infection. Likewise, while average
IFN-γ concentrations in WT mice reached maximal levels of 530±245 pg/mL at 40 h
post-MCMV infection, 3d mice failed to induce detectable levels of this cytokine
. IL-12p70 production similarly peaked at 40 h post-MCMV infection in WT mice,
with levels reaching 1000±600 pg/mL. 3d mice, however, produced 12-fold less
IL-12p70 at this infection time point. Serum TNF-α levels were elevated in
response to MCMV infection at both 40 h and 48 h post-infection in WT mice
(69±10 and 63±17 pg/mL, respectively). In 3d mice, average concentrations of
TNF-α were 6-fold lower than WT at 40 h and 2.5-fold lower than WT at 48 h after
infection. These results indicate a requirement for endosomal TLR signals for
early systemic proinflammatory cytokine production, and concur with previous
reports.
## Liver cytokine production is impaired in MCMV-infected 3d mice
Having observed a reduction in the level of proinflammatory cytokines in the
serum of 3d mice, cytokine responses in liver cells from 3d mice infected with
MCMV were evaluated to address the impact of endosomal TLR signaling in a
localized tissue site of infection. The best characterized liver cytokine
responses are IFN-α and IFN-γ. Therefore, to determine whether combined
endosomal TLR signaling contributes to the production of these cytokines during
MCMV infection, IFN-α and IFN-γ in liver homogenates and in individual cell
populations were measured in WT and 3d mice uninfected or infected with MCMV for
40 h and 48 h. As shown in, WT mice displayed four-fold higher levels of IFN-α
than 3d mice at 40 h post-infection, with increased levels still evident at 48 h
following infection. Since pDCs expressing the marker PDCA-1 have been shown to
produce the majority of liver IFN-α at 40 h post-MCMV infection, this cell type
was examined in WT and 3d livers during early infection. There was evidence of
pDC accumulation in the livers of both WT and 3d mice. However, liver pDCs from
3d mice were impaired in their ability to express IFN-α. There were 4-fold fewer
PDCA-1+ pDCs expressing intracellular IFN-α at 40 h, and 3-fold fewer at 48 h
post-infection, in 3d mice as compared to WT. This trend was also reflected in
the proportion of PDCA-1+ IFN-α+ pDCs at 40 h and 48 h after infection in 3d
mice (0.8%±0.4% and 2%±1%) compared with WT (2%±1% and 6%±1%).
3d mice similarly demonstrated a defect in liver IFN-γ production in response to
MCMV infection. In WT mice, IFN-γ reached maximal levels of 3500±1200 pg/g liver
at 40 h before contracting by approximately half at 48 h post-infection. In
contrast, at 40 h post-infection, 3d mice induced 5-fold less IFN-γ than WT. NK
cells are an important early source of IFN-γ in the liver during MCMV infection,
and accumulated at this site in both WT and 3d mice. Using intracellular
cytokine staining, results shown in demonstrate a 7-fold reduction in the
absolute numbers of NK1.1+ TCRβ- liver cells expressing IFN-γ in 3d mice at 40 h
post-infection as compared to WT. There were also fewer IFN-γ-expressing liver
NK cells in 3d mice by proportion (0.8%±0.3% and 1%±0.4%) when compared to WT
(6%±2% and 4%±0.4%) at 40 h and 48 h, respectively. Together, these results
demonstrate that, in addition to an effect on systemic cytokine production,
combined endosomal TLR signals can affect the expression of critical
proinflammatory cytokines in the liver during MCMV infection.
## Liver T cell responses are not impaired in 3d mice during MCMV infection
The innate immune response is important both in establishing early control of
virus replication and in coordinating downstream adaptive responses. Following
MCMV infection, virus-specific CD8+ T cells are recruited to the liver within 5
days and control viral replication at this site through release of cytotoxic
molecules and production of cytokines such as IFN-γ and TNF-α. Given the abated
liver cytokine responses observed in 3d mice, the effect of endosomal TLR
signaling on liver CD8+ T cell responses was examined at late time points during
acute MCMV infection. The results shown in demonstrate comparable absolute
numbers of CD8+ T cells in WT and 3d mice at days 5 and 7 post-MCMV infection, a
trend that was also reflected in proportion (data not shown). To determine
whether CD8+ T cells in 3d mice were properly activated against MCMV infection,
intracellular expression of IFN-γ and TNF-α in CD8+ T cells was examined
following *ex vivo* restimulation with H-2D<sup>b</sup> M45 viral peptide, an
immunodominant epitope of MCMV. CD8+ T cells from 3d mice expressed these two
cytokines at day 5 and day 7 post-MCMV infection by proportion and absolute
numbers at levels that were comparable or slightly increased over WT (B–E). As
an indication of degranulation, surface expression of CD107a was also examined
on liver CD8+ T cells from mice infected with MCMV; however, no differences in
CD107a expression were detected between 3d and WT mice (data not shown). These
results suggest that MCMV-specific CD8+ T cell responses in the liver are not
compromised in the absence of endosomal TLR signals.
## Increased sensitivity to virus-induced liver disease in 3d mice during MCMV infection
Previous studies have demonstrated resolution of virus-induced liver disease
after 5 days of MCMV infection in WT mice. Therefore, given the impaired
inflammatory responses observed in the absence of endosomal TLR signaling, liver
sections prepared from 3d and WT mice that were uninfected or infected for 3, 5,
or 7 days were hematoxylin and eosin (H&E) stained to evaluate pathology. The
histological appearance of liver sections from uninfected 3d and WT mice
appeared comparable (A and B). By day 3 post-MCMV infection, clusters of
infiltrating cells or inflammatory foci, which have been shown to coincide with
sites of MCMV antigen expression, were present in WT mice and persisted through
day 5 before inflammation was resolved by day 7 post-infection (C, E and G).
While the inflammatory foci per area of liver were equally apparent in liver
sections from 3d mice infected for 3 days, there was an increased presence of
cytomegalic inclusion bodies characteristic of MCMV-infected cells that were not
readily apparent in WT mice. By day 5 post-infection, livers from 3d mice were
characterized by widespread areas of inflammation compared to the more punctate
foci in WT livers (E and F). Moreover, the inflammatory foci per area of liver
in 3d mice at day 5 post-infection were significantly more numerous and
contained a greater number of nucleated cells compared to WT. However, by day 7
post-infection, inflammation in 3d mice showed signs of resolution that were
similar to WT (G and H).
To further evaluate the effects of endosomal TLR responses on overall liver
function, the liver enzyme alanine aminotransferase (ALT) was measured in serum
samples from WT and 3d mice that were uninfected or infected with MCMV for 3, 5
or 7 days. Uninfected mice had comparable baseline levels of systemic ALT. By
day 3 of infection, similar elevations in ALT levels were detected in both
groups. In contrast, by day 5 post-infection, the levels of systemic ALT in 3d
mice reached values of 900±400 U/L and were three-fold higher than the ALT
levels measured in WT mice (270±100 U/L;), indicating augmented liver disease.
In contrast, by day 7 post-infection, 3d mice demonstrated a sharp decline in
systemic ALT levels to values of 117±63 U/L, which were comparable to ALT levels
exhibited in WT mice (108±85 U/L). Thus, at this time point of acute infection,
both groups of mice exhibited ALT levels near baseline levels, consistent with
the resolution of virus-induced liver pathology observed in 3d and WT mice (G
and H;). Taken together, these results suggest that in the absence of endosomal
TLR signals, there is pronounced liver inflammation accompanied by a transient
increase in liver damage during acute MCMV infection.
## Impairment of viral clearance in 3d mice
Given the diminished early cytokine responses and enhanced liver disease
observed in 3d mice, the contribution of endosomal TLR responses to control of
virus replication in the liver was assessed in WT and 3d mice infected with
MCMV. Compared with those in WT mice, viral titers were elevated by 1 log on day
3 of infection in 3d mice, and remained significantly higher at day 5 post-
infection when compared to WT. Ultimately, while WT livers showed evidence of
viral clearance at days 7 and 9 post-infection, 3d liver virus titers remained
approximately 2 logs higher at these times of infection as compared to WT mice.
Thus, endosomal TLR signaling contributes to the control of MCMV replication in
the liver during acute infection.
# Discussion
The aim of these studies was to identify the TLR signaling pathways required for
the innate recognition of virus infection in the liver, a common target organ of
many viruses that significantly contributes to innate immune defenses. Moreover,
the liver contains various innate immune cells that express TLRs, ; however, the
role of TLRs in host defense against infection at this site remains largely
unclear. Because responses in the liver do not appear dependent on individual
TLRs, we utilized 3d mice, which lack endosomal TLR3, TLR7 and TLR9 signaling
due to a mutation in the endoplasmic reticulum-resident protein UNC93B1 , to
address the contribution of endosomal TLRs in liver antiviral defenses against
acute MCMV infection. The results demonstrated impaired production of
proinflammatory cytokines by NK cells and pDCs in livers from MCMV-infected 3d
mice. Additionally, 3d mice had elevated viral titers in the liver that
coincided with transient but exacerbated liver disease, although virus-specific
CD8+ T cell responses were not affected. Interestingly, a previous study
demonstrated that TLR3 was not required for the generation of adaptive antiviral
responses to MCMV, although there is evidence that TLR3 signaling contributes in
part to the early control of MCMV infection by the systemic induction of type I
IFN. Other studies have implicated a synergistic role for TLR7 and TLR9 in
promoting MCMV recognition and immune defense in the spleen. The impaired liver
responses observed in 3d mice that were not apparent in TLR9 or TLR7-deficient
mice suggests that a level of redundancy unique to innate immunity is in place
within infected tissue sites to rapidly respond to viral infection. Overall,
these studies advance our understanding of the process of viral recognition in
the complex liver environment and suggest that UNC93B1 is a critical
intermediate factor in innate virus sensing activated by MCMV.
Previous studies have demonstrated impaired serum cytokine production and
increased susceptibility to infection in 3d mice infected with a high lethal
dose of MCMV ; however, our study is the first report to document the
contribution of the 3d mutation to impaired MCMV defense in the liver. In
agreement with the study by Tabeta *et al.*, our results, using a moderate dose
of MCMV, demonstrated a diminished serum cytokine response, reduced splenic
IFN-α production and elevated viral titers in the spleen (data not shown). These
results were not unexpected given the known role for the nucleic acid-sensing
TLRs in MCMV recognition and the subsequent production of proinflammatory
cytokines and type I IFN by splenic pDCs. Production of type I IFN is a critical
early step in antiviral defense, and we further demonstrated diminished levels
of liver IFN-α in MCMV-infected 3d mice. This defect was in part due to an
impaired ability of liver pDCs to express this cytokine and is consistent with
previous reports identifying pDCs as an important early source of IFN-α in
response to TLR7 and TLR9 ligands,. Altogether, these results concur with
previous studies indicating that pDCs are the predominant leukocyte producer of
type I IFN in the liver during early MCMV infection, and demonstrate that
production of these cytokines by pDCs is influenced by the 3d mutation.
Despite impairments in liver IFN-α production in 3d mice, it is notable that
this response was not totally abrogated. This suggests the presence of
alternative pathways to type I IFN production in the liver, and may be the
reason that IFNα/βR-deficient mice die by day 5 in response to infection with a
moderate dose of MCMV, as reported previously, while 3d mice do not. Studies
have also demonstrated production of type I interferon from cells other than
pDCs at 44–48 hours following MCMV infection. In addition, the liver contains a
variety of parenchymal and non-parenchymal cells that express TLRs and are
capable of type I IFN production,. There may also be TLR-independent pathways
leading to the production of these cytokines in response to MCMV, including
cytoplasmic RNA- and DNA-sensing receptors, as have been demonstrated with other
virus models but have yet to be examined in the context of MCMV infection,.
It has been established that NK cell inflammatory responses and production of
IFN-γ are essential to defense against MCMV in the liver –. In 3d mice, NK cell
production of IFN-γ in the liver was severely impaired during early MCMV
infection and likely contributed to increased viral burden and liver pathology.
The reduced levels of IFN-γ in NK cells may reflect the deficiency of serum
IL-12 seen in 3d mice. These results concur with previous reports that type I
IFN regulates IL-12 production by conventional DCs and consequently the
production of IFN-γ by NK cells. The defect in liver cytokine production in 3d
mice is reminiscent of that reported for MyD88-deficient mice. Notably, however,
MyD88-deficient mice exhibited more severe liver pathology and greater
elevations in viral titers when compared to WT than we observed in 3d mice.
Interestingly, mice deficient in TLR7 and TLR9 exhibited decreased levels of
systemic IFN-α/β and increased susceptibility to MCMV infection. Taken together,
these observations further support the notion that the liver possesses
compensatory mechanisms to combat viral infection in the combined absence of
endosomal TLR signals.
Accordingly, despite the early effects of endosomal TLR deficiency, 3d mice were
able to mount robust CD8+ T cell cytokine responses. It should be noted that the
3d defect has previously been shown to impair exogenous antigen presentation,
including cross presentation, which has a reported role in priming CD8+ T cell
responses during MCMV infection. However, we detected no overt defect in CD8+ T
cell responses within the first seven days of MCMV infection in the liver.
Further, examination of activation markers suggested that a similar proportion
of CD8+ T cells from 3d mice were more highly activated when compared to WT
(data not shown). Several studies have demonstrated the contribution of
activated virus-specific CD8+ T cells to effective hepatic immunity against MCMV
infection. In addition, the normal development of adaptive responses despite
impaired innate responses is well documented during MCMV infection. Studies have
shown that reduced levels of type I IFN do not affect the accumulation or
activation of antigen-specific CD8+ T cells in response to low or moderate MCMV
inoculums. Likewise, while IL-12 is critical in inducing NK cell IFN-γ
expression, T cell responses can occur in an IL-12-independent manner,. NK cells
have the potential to negatively regulate CD8+ T cell responses during MCMV
infection –; thus, it is probable that impaired NK cell function in the absence
of endosomal TLR signals contributes to inflated T cell responses. The presence
of increased virus in the liver may also contribute to the robust T cell
recruitment and cytokine production observed at late infection time points in 3d
mice. Interestingly, although the severity of viral liver pathology was more
pronounced in 3d mice, inflammation and liver injury subsided late in infection.
These observations suggest that CD8+ T cells in the liver can respond to limited
amounts of type I interferon for activation in the presence of compromised
innate responses, emphasizing the prevalence of compensatory mechanisms in place
within the liver to deal with infection and promote adaptive immunity. In
contrast, IFN-α/βR deficiency negatively impacts innate inflammatory responses
and resistance to MCMV infection in the liver,.
In conclusion, this study indicates that UNC93B1, which is essential for
combined endosomal TLR signaling, contributes to development of effective innate
immune responses to an acute virus infection in the liver. Our results show that
this contribution involves modulation of early innate proinflammatory cytokine
production from liver pDCs and NK cells and subsequent control of MCMV
replication and pathology before activation of an adaptive immune response.
Altogether, these results highlight a process of virus recognition with multiple
pathways in place to promote host resistance to infection in the liver
microenvironment.
# Methods
## Mice
Pathogen-free C57BL/6J mice were obtained from the Jackson Laboratory (Bar
Harbor, ME). C57BL/6 *Unc93b1<sup>3d/3d</sup>* mice were a kind gift from Dr.
Bruce Beutler (The Scripps Research Institute, La Jolla, CA) and were generated
as described. C57BL/6J mice were housed and *UNC93b1<sup>3d/3d</sup>* mice were
bred in pathogen-free mouse facilities at Brown University. Male and female mice
aged 8–10 weeks were used in experiments. This study was carried out in strict
accordance with the recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All animal work was
approved by the Brown University Institutional Animal Care and Use Committee
(Protocol Number: 0909082 and 0903035).
## Virus Infection and viral titer determination
MCMV Smith strain was used in all experiments. This strain was prepared as a
salivary gland-passaged stock from CD1 mice. Moderate dose infection
(5×10<sup>4</sup> PFU per mouse) was initiated on day 0 by intraperitoneal
injection. In vivo responses were measured at indicated time points. For
infectious viral titer quantification, organs were weighed, homogenized in cold
supplemented DMEM (Invitrogen Life Technologies) and supernatants were collected
following centrifugation. Serially diluted samples were used to inoculate
confluent monolayers of bone marrow stromal cells (ATCC M2-10B4) in 24-well
tissue culture plates and incubated for one hour at 37°C, 5% CO<sub>2</sub>.
Following incubation, inoculums were removed and monolayers were overlaid with a
1×DMEM/0.5% low-melt agarose solution. Cells were incubated for 7 days at 37°C,
5% CO<sub>2</sub>, then fixed in 10% buffered formalin and stained with crystal
violet. Plaques were counted to determine viral titer as previously described.
## Sample preparation
Liver leukocytes were prepared as previously described. Briefly, following
mechanical dissociation of tissue, red blood cells were removed by lysis with
ammonium chloride and leukocytes separated by Percoll density gradient. To
generate homogenates for cytokine analysis, the liver caudate lobe was
homogenized in RPMI 1640 (Invitrogen Life Technologies) and supernatants
collected following centrifugation. Serum was isolated from whole blood by
centrifugation in the presence of heparin and stored at −80°C until further use
in cytokine analyses or ALT assays.
## Cytokine analysis
Liver homogenates and serum were tested for cytokines by standard sandwich
ELISA. IFN-γ, IL-12p70 and TNF-α were assayed by DuoSet (R&D Systems). IFN-α was
measured by VeriKine mouse ELISA kit (R&D Systems). Limits of detection were 15
pg/mL for DuoSets and 12.5 pg/mL for VeriKine ELISA kits.
## Flow cytometric analysis
The following fluorochrome-conjugated mAbs were used in flow cytometric
analyses: NK1.1-PE and TCRβ-APC to distinguish NK cells; CD8α-PECy7 and TCRβ-
FITC to distinguish CD8+ T cells; and PDCA-1-APC (Miltenyi Biotec) to identify
pDCs. Prior to surface staining, cells were incubated with anti-CD16/CD32 mAb to
block nonspecific binding of Abs to Fcγ III/II receptors (clone 2.4G2). Unless
otherwise noted, antibodies were obtained from BD Biosciences or eBioscience.
For intracellular staining of cytokines, cells were treated with Brefeldin A
(eBioscience) for 4 hours at 37°C, 5% CO<sub>2</sub> and permeabilized prior to
labeling with IFN-α-FITC (R&D Systems), IFN-γ-PE, or TNF-α-APC (BD Biosciences).
When indicated, leukocytes were stimulated for 5 hours with 100 ng/mL MCMV M45
peptide in addition to Brefeldin A treatment. Samples were acquired using a
FACSCalibur and analyzed with BD Cell Quest software. For analysis, viable cells
were gated by FSC and SSC. Isotype controls were used to set positive analysis
gates.
## Liver histology and serum ALT analysis
Portions of the median liver lobes were isolated, fixed in 10% neutral buffered
formalin, and paraffin embedded. Tissue sections (5 µm) were stained with H&E
and analyzed microscopically. Images shown were photographed at the indicated
magnification with a DP70 digital camera and software (Optical Analysis
Corporation). Inflammatory foci, defined as discrete clusters containing between
6–60 nucleated cells, were quantitated as described previously. In brief,
inflammatory foci were identified, at a magnification of 200, as clusters of
cells in totals of 8×50 µm<sup>2</sup> areas per representative tissue. In some
cases, liver sections had larger areas of inflammation with \>60 cells with less
defined foci, and are indicated as such. Numbers of nucleated cells per
inflammatory foci were counted in 20 individual foci per representative tissue
at a magnification of 400. Liver alanine aminotransferase (ALT) was measured in
serum samples by Marshfield Labs (Marshfield, WI).
## Statistical analysis
Student's *t* test was used to determine statistical significance of
experimental results when indicated (p≤0.05).
We acknowledge Paula Weston from the Molecular Pathology Core Facility at Brown
University for assistance with histology sample preparations. We would also like
to thank Dr. Bruce Beutler of the Scripps Research Institute, La Jolla, CA, for
generously providing UNC93B1*<sup>3d/3d</sup>* mice for these studies.
[^1]: Conceived and designed the experiments: MJC TSM. Performed the
experiments: MJC PJG. Analyzed the data: MJC TSM. Wrote the paper: MJC TSM.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
Since the 1980s, lithic researchers have worked to develop a series of methods
to measure reduction intensity, particularly as a tool for interpreting curation
or re-sharpening of tools and morphological variability, and they have applied
them to both experimental and archaeological assemblages. Among them, the
quantitative reduction indices proposed by Dibble (the ratio of the remaining
surface area to platform area), and Kuhn (the ratio of flake thickness at the
point where retouch scars terminate to maximum medial thickness, also well-known
as a Geometric Index of Reduction) are the two most influential indices. These
two indices and their modified versions are still frequently applied in current
research, which confirms their usefulness. Generally speaking, these methods for
measuring reduction mainly focus on morphological attributes to estimate
reduction intensity. In other words, as reduction continues, the corresponding
size, shape and other morphological properties change as well. Shott and Weedman
have summarized them as three specific methods, namely, a simple size
measurements method, a geometric measurements method, and an allometric method
that relates shape and other attributes to size.
Allometry has also influenced the reduction study of handaxes. Based on the
reduction hypothesis, McPherron reanalyzed 38 British handaxe assemblages
classified by Roe into either pointed or ovate groups. He argued that these
shapes actually reflect different reduction intensities, with pointed handaxes
in an initial stage of reduction and ovate handaxes in a later stage. For
quantitatively measuring the reduction of handaxes, McPherron employed linear
measurements, namely the tip length, overall length and width in his study and
assumed that pointed handaxes would have both a long tip length and a long
overall length. As reduction continued, tip length and length decreased quickly,
but width decreased at a lower rate, finally leading to the formation of ovate
shaped handaxes with lower elongation values (Length/Width). Thus, through
comparing the tip length, length and elongation ratio, McPherron argued that it
is possible to estimate the reduction intensity of different handaxe
assemblages. This allometric method provided a new perspective in interpretation
of the morphological variability of handaxes, and more importantly, it shifted
attention from the final form of handaxes to their reduction process and flaking
strategies. Consistent with this allometric method, most current reduction
intensity analyses of handaxes are now integrated into studies of morphological
variability.
In contrast to McPherron’s analysis of the size and shape of handaxes in
relation to re-sharpening, McNabb et al. proposed a method for the technological
study of ‘shaping’, without reference to re-sharpening. By recording the extent
of secondary flaking (flake scars \> 1.5cm in length) and the degree of edge
trimming (flake scars \< 1.5cm in length), the authors identified five
subcategories of secondary flaking for each face of a handaxe and five ordinal
scales of edge trimming for each section of a handaxe (see McNabb et al., Figs).
The frequencies of these attributes are then classed by the extent of reduction
as light, moderate or extensive. This is a detailed and useful approach to
reduction analysis of handaxes involving technological attributes. However, a
potential problem may lie in the subjective divisions of different types of
secondary flaking and different scales of edge trimming, which can vary
according to the observer.
Another easily applied approach used by some researchers is the direct counting
of the number of scars on a handaxe. Scar numbers are relatively easier to
quantify at different stages of reduction for Early Stone Age (Lower
Palaeolithic) handaxes than for the generally small-sized tools in the Middle
Stone Age (Middle Palaeolithic) and Later Stone Age (Upper Palaeolithic). The
assumption here is that extensively retouched handaxes would have more flake
scars than less reduced handaxes. Using this concept, Hou et al. compared the
number of flake scars on the Bose Large Cutting Tools (LCTs; \~0.803Ma) with the
number of scars on LCTs in two western Acheulean assemblages of similar age
(0.99Ma-0.7Ma; Olorgesailie Members 1 through 7 in Kenya and Bed IV of Olduvai
in Tanzania). They concluded that the Bose LCTs have a similar number of scars
as the western Acheulean, and therefore they support the proposal that there is
no technological difference between handaxes in the East and West. In addition,
in Sharon’s comparative study of handaxes from Africa, West Asia and India, the
number of scars was also regarded as an important attribute for the analysis of
reduction extent.
Although the counting of flake scars is a useful and easily applied approach, it
also has one limitation. As mentioned by Sharon, the visible number of flake
scars on discarded handaxes is likely lower than the flake scars generated
during manufacture, as a portion of the piece is lost in the process. For
example, a handaxe with 20 flake scars but of a smaller size is not definitely
less retouched than a handaxe of larger size with 30 flake scars. Considering
the number of scars in conjunction with the size of handaxes would make this
attribute size-independent and improve its value. Coincidentally, in the
analyses of core reduction intensity of some East African Oldowan sites, Braun
et al. also suggested that flake scar number divided by mass of the piece is a
more appropriate measure of reduction intensity.
For exploring the use-life and implied human behaviors of handaxes (e.g., raw
material transport), Shipton proposed a flake scar density index in his analysis
of Indian and East African material. Specifically, the scar number on a handaxe
is divided by the product of the handaxe length and width as an indication of
the surface area. The principle here is that a handaxe will start off with a low
flake scar density, and as the reduction progresses, the value of flake scar
density will steadily increase. This is a size-independent method which
addresses the limitation of the scar number approach discussed above. Due to the
imprecision in measuring surface area, Shipton et al. then applied a 3D
technique to capture the area more accurately, producing a 3D surface area.
Simultaneously, Clarkson used a similar method to measure the reduction
intensity of different types of cores (with bifaces included as one core type),
and he introduced the Scar Density Index (SDI, or the ratio of flake scar number
to 3D surface area). Moreover, Clarkson, Clarkson et al. and Shipton and
Clarkson have used both experimental and archaeological materials to reinforce
the reliability of this index.
The purpose of this paper is to present a 3D quantitative analysis of reduction
intensity of a handaxe assemblage from the Danjiangkou Reservoir Region (DRR),
central China. Here, reduction of the DRR handaxes includes both shaping and the
probable re-sharpening process, as these two aspects cannot be objectively
distinguished, especially in cases where the reduction intensity is generally
low, as in DRR. In addition to the 3D Scar Density Index (SDI), a Flaked Area
Index (FAI) which can quantify the reduction extent in different parts (i.e.
tip, medial and base) of a handaxe, will also be used.
# Materials and Methods
## Materials
The handaxes analysed in this paper are from both surface collections and
excavations on the third terrace (T3) of the Danjiangkou Reservoir Region (DRR),
central China. Systematic investigations and excavations over the last two
decades in this area have revealed it as another important handaxe-bearing
region in China, along with the well-known regions of Dingcun, Bose and Luonan.
To ensure accurate measurements of surface area and volume for handaxes, only
complete specimens are employed in our study, namely 92 handaxes in total (see
for raw data of each specimen). Of these, 76 are surface collected and 16 are
excavated from the third terrace of the Han River, the longest tributary of the
Yangtze River and the main feeder of the Danjiangkou Reservoir. The surface-
collected material has been statistically confirmed to be consistent with
excavated specimens in both morphology and technology. In terms of the type of
shaping of the DRR T3 handaxes, with the exception of one indeterminate
specimen, 37 (40.2%) were bifacially shaped, 38 (41.3%) were partly bifacially
shaped and only 16 (17.4%) were shaped unifacially (See). The ESR, OSL and
palaeomagnetic dating of the third terrace at the Shuangshu and Maling 2A sites
indicate that these two handaxe-bearing sites formed in the Middle Pleistocene.
In addition, 25 surface collected handaxes from the second terrace (T2) of the
Han River were also used in a comparative study of differences in reduction
intensity through time in the DRR. The T2 Dishuiyan site is dated to ca 100–50
ka by the OSL and TT-OSL methods. Here more than 20 handaxes comparable to the
25 surface-collected specimens used in this study were excavated.
## Ethics statement
The surface-collected handaxes (N = 101) were retrieved during field
investigations carried out by one of the co-authors, Chaorong Li, and permission
to study these materials was issued by the Institute of Vertebrate Paleontology
and Paleoanthropology (Beijing), Chinese Academy of Sciences, in which these
specimens are stored. The excavated handaxes (N = 16) are stored in the
Danjiangkou Museum in Danjiangkou City, Hubei Province, and study of these
materials was permitted by this museum. We ensure that the fieldwork did not
involve endangered or protected species.
## 3D scanning and measuring
For capturing the 3D image of each handaxe, we used two types of laser scanners.
The NextEngine 3D Laser Scanner was used in the field, as it is light and
portable. To scan the whole surface of a handaxe, we conducted two separate
scans in vertical and horizontal views which were subsequently merged into one
complete 3D image. In the laboratory, the Range 7 3D Laser Scanner was used, as
it gives excellent resolution but is difficult to carry in the field. Handaxes
were rotated manually to obtain a complete 3D image. The mean value of triangles
representing the degree of resolution is two times higher with the Range 7
scanner than with the NextEngine scanner. Holes on images were filled using the
Geomagic Studio software, regardless of the scanner used.
After attaining the 3D images, we then imported them into the Avizo Fire 3D
Imaging Software (version 8.0) to accurately calculate the surface area and
volume of the handaxes. The segmentation function of this software was also
applied to divide the handaxes into three portions with the piece aligned along
the long axis, using the distal end as the guide. The scar coverage of each
portion was then accurately extracted using this software. An example is given
in, which shows the proportional division of the surface into distal, medial and
proximal parts based on the length, for which the area of flake scar coverage is
then calculated in each sector.
## Indices of SDI and FAI
Flake scar density (equal to the Scar Density Index, SDI) has been used as an
effective indicator of reduction intensity of Indian and East African handaxes.
In an experimental test of the reliability of SDI, the regression analysis of
transformed log SDI and log % Mass Remaining of bifaces produced a very strong
relationship (*R*<sup>*2*</sup> = 0.916). In addition, a recent study of the
experimentally reduced handaxes also indicates a strong relationship
(*R*<sup>*2*</sup> = 0.803) between increasing SDI and decreasing % original
mass. For this reason, the 3D SDI was adopted in our analysis of reduction
extent of the DRR handaxes. All visible flake scars on a handaxe were counted in
this study, regardless of their interpretation as shaping vs. refining scars.
Because of the fresh condition of the DRR handaxes, it is easy to count the scar
numbers. And because most of these handaxes were made on primary flakes or
cobble opening flakes, dorsal scars on flake handaxes were mainly formed by the
subsequent flaking. Therefore, all dorsal scars were counted as flaked area.
However, the flaked area does not include the ventral surface of handaxes made
on flakes if no scars are present.
As a result of the use of 3D technology, a new index, Flaked Area Index (FAI,
flaked area divided by the total surface area), is now possible. A reasonable
assumption for this index is that the flake scars area on handaxes increases
with reduction. The unretouched blank for a handaxe would have a FAI value of 0,
while a completely retouched handaxe would reach a FAI value of 1. Through the
accurate measuring of the flaked area in different sectors of the handaxes, we
can estimate not only the overall reduction intensity, but also the reduction
intensity of the different parts, which is a benefit of FAI. We need to bear in
mind that the flaked area does not necessarily relate to the number of flake
scars. This is particularly applicable to hard hammer percussed handaxes, in
which a small number of large scars can produce a large area of scar coverage,
and conversely, a large number of small scars can produce a small area of scar
coverage. Despite this, the FAI index can reflect the general pattern of the
reduction extent of handaxes.
In addition, it should be noted that both the SDI and FAI indices will reveal
the relative extent of reduction, but not the actual mass lost during the
reduction. In order to investigate quantitatively how much mass has been lost in
the reduction process, it is necessary to conduct knapping experiments in future
research.
# Results
## The application of SDI to reduction intensity
Correlation analysis between volume and SDI shows that the relationship between
volume and SDI is significant (*r* = 0.523, *p* \< 0.001), although there is
considerable variation. In addition, to test the effect of outliers, we exclude
specimens (N = 9) whose SDI values are larger than 0.1. Results show that the
correlations between volume and SDI with and without outliers are very close to
each other (*r* = 0.523 vs. *r* = 0.520). Therefore, the DRR handaxes can be
confirmed to be made from similar size cobbles, and we suggest that the size-
independent SDI used in this study is appropriate for measuring the reduction
intensity of handaxes. shows three handaxes (left side of the figure) that are
low in volume and weight but high in SDI, and three handaxes (right side of the
figure) that are high in volume and weight but low in SDI. Detailed information
for each specimen is provided in.
Although the indices of SDI used here cannot show how much mass is lost during
reduction, they do provide us with information about the relative intensity of
handaxe reduction. Adapting statistical models used in demography, Shott and
colleagues suggest that different distributional patterns of reduction could
correspond to different cumulative-survivorship curves. In other words, the fit
between the length of handaxe use and SDI has the ability to reveal the
underlying use-lives of tools and the related human behaviours. In, we can see
that most of the DRR handaxes possess lower SDI values and locate on the left
side of the histogram, with 83.5% (N = 76) of them lower than 0.09 on the SDI
value. Only a small number of handaxes has relatively higher SDI values and
locate on the right side of this diagram. Therefore, it is reasonable to infer
from that most of the DRR handaxes were less extensively reduced and generally
had short use-life before discard. This situation may relate to the use of
locally available raw materials close to the site and brief occupation periods.
For visualizing the reduction intensity of the DRR handaxes, six handaxes with
the values of SDI from 0.08 to 0.09 are presented in (see for information on
individual specimens).
## The application of FAI to reduction intensity
Correlation analysis between SDI and FAI shows that these two indices are
significantly related (*r* = 0.424, *p* \< 0.001; see), although there is
considerable variation. This indicates the validity of FAI in estimating the
reduction intensity of DRR handaxes. Based on the 3D segmentation of handaxes
into three proportional parts, namely, tip, middle and base, we can examine the
pattern of reduction intensity in each sector. and show that the values of FAI
for tips are high, with 34.8% of them scoring in the range 0.75–0.99 and 41.3%
of them fully covered by flake scars. The mean value of FAI for tips is 0.87.
For the middle sections, 59.7% are concentrated in the range 0.26–0.75 and 30.4%
in the range 0.76–0.99, and the mean value for middle sections is 0.69. The FAI
values for the bases are generally low, with 32.6% scoring in the range
0.01–0.25, 34.8% in the range 0.26–0.50 and 23.9% in the range 0.51–0.75, and a
mean value of 0.37. Therefore, we can conclude that most of the shaping was
invested in the tips of the DRR handaxes, while the bases usually have the least
reduction, with an intermediate degree of reduction in the middle sectors. The
reason for this pattern is likely related to the functional differences for each
part: the tip is the most utilised part, while the middle may be related either
to use or to shaping of the tip; and the smooth cobble surface is often left on
the base for holding comfort. This conclusion is supported by using the sector
method which records shaping status and calculates the proportion of shaping or
cortex covered in each sector in the whole handaxe assemblage. Analysis of 36
handaxes found from 2004 onwards shows that shaping was mainly concentrated in
the distal ends of DRR handaxes (86.8%), while the proximal ends were mainly
occupied by cortex (50.0%), with a smaller percentage of shaping (38.9%; see Li
et al.).
The overall extent of reduction for the whole body of handaxes ranges from
0.26–0.99, with over 45.7% in the range of 0.51–0.75, and the mean value of FAI
is 0.60. If a cut-off point of 0.75 is used to represent the boundary between
extensive (\> 0.75) and moderate reduction (≤ 0.75), we can see that more than
three quarters (78.3%) of handaxes in DRR were only moderately reduced. This
result is consistent with our analyses of the SDI, where results show that the
DRR handaxes generally show a low extent of reduction.
## Comparing reduction intensity for different types of raw materials, blanks and shaping
Quartz phyllite (N = 67) and trachyte (N = 15) were the most frequently used raw
materials for DRR handaxes, although the number of trachyte pieces is much lower
than the number of quartz phyllite in the current study sample. Both raw
materials are abundant and locally available in the nearby gravel layers of the
Han River. The comparisons of SDI and FAI by raw material show substantial
overlap (left side of Figs). This observation is supported by the *t*-test (*t*
= -0.305, *p* = 0.761 for SDI; *t* = 0.478, *p* = 0.634 for FAI), which shows
there is no statistically significant difference in the levels of reduction
between the two raw materials. We can, therefore, further infer that a
consistent reduction strategy was employed despite raw material differences.
In contrast, there is a clear trend of lower SDI and FAI with cobble blanks (N =
32) compared with flake blanks (N = 45). This is visible on the middle part of
Figs and. The *t*-test also shows significant differences in the levels of
reduction between the two blank groups (*t* = 2.438, *p* \< 0.05 for SDI; *t* =
2.708, *p* \< 0.01 for FAI). During the experimental test of the effects of
blank type on handaxe reduction, Shipton and Clarkson also noticed that cobble
blanks tend to have lower SDI compared to flake blanks for a given percentage of
mass lost. This difference in DRR T3 handaxes probably relates to the large flat
ventral surface provided by flake blanks and their relative thinness compared to
cobbles, both of which facilitate reduction.
In terms of the effect of types of shaping on the reduction intensity, the
Kruskall-Wallis test shows that there are considerable differences for both SDI
(ChiSquare = 25.6, *p* \< 0.0001; see) and FAI (ChiSquare = 39.0, *p* \<
0.0001; see) among the three shaping groups, namely, bifacial, partly bifacial
and unifacial. Bifacially shaped handaxes have the greatest mean values of SDI
(0.087) and FAI (0.743), while the unifacially shaped handaxes have the least
mean values of SDI (0.042) and FAI (0.458), with partly bifacially shaped
handaxes being intermediate (mean SDI = 0.058; mean FAI = 0.532). Therefore, it
is clear that different types of shaping have a strong influence on the degree
of reduction for the DRR T3 handaxes.
## Comparing the reduction intensity of handaxes from T2 and T3 of the DRR
The 3D quantitative method provides an objective way to compare reduction
intensity through time. Here, the 25 handaxes from the second terrace of the DRR
are compared with the 92 handaxes from the third terrace analysed in the
foregoing sections. The preliminary age of handaxes from the second terrace is
the early Late Pleistocene (100–50 ka), while the handaxes on the third terrace
are dated to the Middle Pleistocene. Therefore, handaxes in the DRR provide an
opportunity to examine the regional change in reduction intensity from the
Middle to the early Late Pleistocene. Because of the relatively small sample
size from T2, we do not conduct a statistical analysis according to different
types of raw materials, blanks and shaping, as was done for the sample from T3.
Attributes used in this analysis include the number of scars, surface area,
volume, total flaked area, and the indices of SDI and FAI. The *t*-test shows
that there are no statistically significant differences (*p* \> 0.05) between
handaxes from T2 (the second terrace) and T3 (the third terrace) in any of these
attributes and thus no technological change in reduction intensity through time
in the DRR is presented.
# Summary and Conclusion
The extensive application of reduction intensity indices in the past three
decades has remarkably improved the ability of lithic analysts to interpret
human behaviour. With the aid of 3D scanning technology, in this paper we
applied two quantitative reduction indices, the Scar Density Index (SDI) and the
Flaked Area Index (FAI), to the analysis of reduction intensity of the T3 DRR
handaxes. The SDI in this study shows that most of the handaxes in DRR have a
relatively low intensity of reduction, which also indicates a generally short
use-life as argued by Shott and Sillitoe’s reduction distribution model. In
addition, the short use-lives of these handaxes may suggest that open-air sites
along the river terrace were not occupied by hominids for a long time. The
analysis of FAI also shows that the overall reduction intensity of the DRR
handaxes represents a least-effort reduction strategy, with 78.3% of handaxes
only moderately reduced (FAI ≤ 0.75). The detailed FAI analysis of the different
parts of the DRR handaxes shows that tips generally show the most reduction,
while the bases show the least, a pattern which is relatively common in some
Chinese handaxe assemblages. It is not surprising that the handaxe tip would
receive most attention in shaping. The middle section could be functional if the
edge were used, but it relates also to shaping of the piece overall. The base of
a handaxe was the holding unit, with only limited or no shaping present.
The two raw materials used in DRR, both locally available, did not have an
influence on the reduction intensity, suggesting that the behavioural
interpretation of short-term use is correct. In addition and because of the
abundance of raw materials, the DRR handaxe knappers seem to have employed an
expedient exploitation strategy. However, the type of blanks and the shaping
types did play a role in the reduction extent of the DRR handaxes. Those made on
flake blanks generally show a higher level of reduction than those made on
cobble blanks, presumably because flake blanks were thinner than cobbles, and
they provided a large flat surface which made reduction easier. In terms of
shaping, bifacially shaped handaxes show a greater degree of reduction than
partly bifacial handaxes, with the unifacial handaxes showing the least
reduction. The preliminary comparison of handaxes from T2 and T3 of the DRR
suggests that there is no technological change in the reduction intensity from
the Middle Pleistocene to the early Late Pleistocene in this region, although
more specimens from terrace two need to be analysed.
The results presented here demonstrate that the application of quantitative
technological indices is necessary and useful in estimating the reduction
intensity of handaxes. According to this estimation, we can further investigate
the behaviour of handaxe makers in the DRR, such as their adaptation to the
local raw materials, their energy investment in making handaxes, and the use-
life of handaxes. The potential of the indices used in this paper has been
confirmed; however, as we have mentioned already, these indices can only
indicate the relative extent of handaxe reduction and they still need to be
further tested. In future research, experiments will be conducted to further
evaluate the mass lost at different levels of the index values, and to support
the validity of the current indices. Additionally, owing to the long lasting and
widespread use of handaxe technology in the Pleistocene, the reduction intensity
of handaxes at different developmental stages and in different regions will be
further examined to address the technological evolution and adaptive behaviour
of Acheulean hominids. Finally, this study has provided detailed information on
the nature of handaxes in the DRR, which will serve as a comparative sample for
a better overall understanding of these industries in China, in comparison with
the western Acheulean.
# Supporting Information
We would like to thank Kristian J. Carlson, Tea Jashashvili, Matt Caruana, and
Raymond Couzens for their help and suggestions on 3D scanning and the statistics
in this research. And we thank Shannon McPherron and one anonymous reviewer for
their invaluable and insightful suggestions for improving this manuscript.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: HL KK CRL. Performed the
experiments: HL. Analyzed the data: HL. Contributed
reagents/materials/analysis tools: KK. Wrote the paper: HL KK CRL. |
# Introduction
DNA double-strand breaks (DSBs) are among the most serious types of DNA damage
in cells and can lead to genetic instability and tumorigenesis. DSB may be
induced by exogenous genotoxic insults, such as ionizing radiation, but also
occur spontaneously in various cellular processes including DNA replication and
V(D)J recombination. There are two major pathways for DSB repair: non-homologous
end joining (NHEJ) and homologous recombination (HR). NHEJ directly ligates the
two broken ends of a DSB and is accessible throughout the cell cycle. In
contrast, HR is an error-free repair mechanism that primarily uses the intact
sister chromatid as a template for repair and predominates in S-phase cells.
The physiological importance of HR is underscored by the association of cancer
predisposition and developmental defects with mutations in HR genes. The tumor
suppressors BRCA1 and BRCA2, key players at different stages of HR, are
frequently mutated in familial breast and ovarian cancers. Other HR proteins,
including PALB2 and RAD51 paralogs, have also been identified as tumor
suppressors. On the other hand, tumor cells with defective HR repair show
increased sensitivity to chemotherapeutic reagents that act via the induction of
DNA damage, including platinum-containing agents and PARP inhibitors. These
observations suggest that HR-proficient tumor cells might be sensitized to
chemotherapeutics if HR repair could be therapeutically inactivated.
In genetic engineering, HR represents a powerful tool to precisely manipulate
the genome for experimental uses. Its use revolutionized the genetic approach to
study biological processes in mice by the late 1980s with the generation of the
first gene targeted knockout mice. Since then, thousands of genes have been
modified in mouse embryonic stem cells by HR with gene-targeting vectors.
However, the low efficiency of HR in vertebrate somatic cells limits the utility
of this approach. Following the discovery that induction of a DSB increases the
frequency of homology directed repair (HDR) by several orders of magnitude,
targeted nucleases have emerged as the method of choice for improving the
efficiency of HDR-mediated genetic alterations. ZFNs (zinc finger nucleases),
TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9
(clustered regularly interspaced short palindromic repeats/CRISPR-associated
system 9) are all engineered endonucleases that can introduce DSBs at desired
locations in the genome. Once a targeted DSB has been made, HDR may reconstruct
the cleaved DNA using an exogenous DNA template.
Various cell-based assays have been developed for the accurate measurement of HR
events. The use of fluorescence has proved to be an effective approach for
detecting HR and the most commonly used assay is the direct repeat green
fluorescent protein (DR-GFP) assay. The DR-GFP reporter consists of two tandem,
inactive copies of the GFP gene, which can be integrated into the cellular
genome or expressed transiently. The first GFP copy (SceGFP) has a promoter and
contains the I-SceI restriction site with an in-frame stop codon and the second
copy (iGFP) harbours truncations at both ends. After cleavage within SceGFP by
I-SceI or a Cas9 nuclease, HR uses iGFP as a template to restore the GFP gene to
its functional form. Then, GFP fluorescence can be measured using flow
cytometry.
Here, we report the development of a novel fluorescence-based assay for
evaluating cellular HR activity. As opposed to the DR-GFP assay, in our system
HR reconstitutes the full-length coding sequence of the enhanced green
fluorescent protein (eGFP), which fluoresces much more intensely than GFP, thus
facilitating the detection of HR events. Our assay also differs from the DR-GFP
assay in that our two non-functional copies of eGFP are not in tandem. Rather, a
single copy of the HR substrate is integrated as a single copy into the AAVS1
safe harbor locus and the template donor is delivered exogenously via AAV
particles, mimicking the scenario of the HR-mediated correction of a monogenic
disorder. Following this procedure, we have detected changes in HR rates
resulting from the altered expression of DNA repair genes, similar to those
previously reported. We provide a well-characterized tool that will help to
further investigate the mechanisms that control HR and analyze the effects of
exogenous manipulations, with i.e., drugs, miRNAs, and genes on HR mediated
correction.
# Materials and methods
## Cells
Human colon cancer cell line HCT116 was obtained from the American Type Culture
Collection (Manassas, VA). Human embryonic kidney cell line HEK-293 optimized
for the packaging of AAV virions (AAV-293) was obtained from Agilent
Technologies (Santa Clara, CA). Both cell lines were handled according to each
manufacturer’s recommendations.
## Plasmids construction
The sequences of the primers used in this study are shown in. The pAAV-MCS-
eGFPΔ3’ plasmid (also named as peGFPΔ3’) used to generate the HR reporter cell
line was obtained through the following steps. Firstly, a fragment containing
the 5’AAVS1 region homology arm (832 bp) followed by a puromycin cassette (1014
bp) was amplified from pAAV-CAGGS-eGFP plasmid with primers 1F and 1R. pAAV-
CAGGS-eGFP plasmid was a gift from Rudolf Jaenisch (Addgene plasmid \# 22212;
<http://n2t.net/addgene:22212>; RRID:Addgene_22212). The puromycin cassette
(SA-T2A-Puro-pA) is promoterless and contains a splice acceptor site followed by
the coding sequence of the T2A peptide, the puromycin resistance gene and a
polyadenylation signal. Secondly, a 1290 bp fragment containing human
cytomegalovirus (CMV) immediate early (IE) promoter and eGFP lacking the last 73
bp was amplified from peGFP-C1 plasmid (Clontech) with primers 2F and 2R.
Thirdly, the 3’AAVS1 region homology arm (1300 bp) was amplified from HCT116
genomic DNA using 3F and 3R. The three fragments were digested and cloned into
the NotI site of pAAV-MCS (Stratagene). With respect to the designation of the
primers used in this work, F stands for forward, and R for reverse.
The HR donor plasmid, pAAV-MCS-eGFPΔ5’ (also named as peGFPΔ5’), was obtained
through the following steps. Firstly, a 929 bp fragment containing eGFP lacking
the first 38 bp at 5’ end was amplified from peGFP-N1 plasmid (Clontech) with
primers 4F and 4R. Secondly, an EcoRI-SalI fragment (1582 bp) containing a
blasticidin resistance cassette flanked by loxP sequences was obtained from a
pBluescript II based plasmid previously generated in our lab. Thirdly, the
3’AAVS1 region homology arm (1300 bp) was obtained as described for pAAV-MCS-
eGFPΔ3’ plasmid. The three fragments were digested and cloned into the NotI site
of pAAV-MCS (Stratagene). AAV2 particles were produced by co-transfecting 293
c18 cells (ATCC CRL-10852) with the pAAV-MCS- eGFPΔ5’ donor plasmid, pHelper and
pAAV-RC (at a 1:1:1 ratio). Three days later cells were harvested and AAV were
released by 4 freeze-thaw cycles. Viral titers were determined by SybrGreen
based real time qPCR using ITR_F and ITR_R primers as previously described.
pHelper and pAAV-RC plasmids were obtained from Stratagene.
The RAD52 gene of *Saccharomyces cerevisiae* (ScRAD52) was cloned into the
expression vector pET15b (Novagen) for His-tagged production of TAT-NLS-RAD52
where TAT peptide (GRKKRRQRRR) promotes cell permeability and NLS peptide
(KKKRKV) is a nuclear localization signal. Wild-type RAD52 sequence was
amplified by PCR from the genomic DNA of *Saccharomyces cerevisiae* with primers
ScRAD52_F1 and ScRAD52_R1 and cloned into HindIII/XhoI digested pTriEx-HTNC
vector immediately downstream the His-TAT-NLS sequence. pTriEx-HTNC was a gift
from Klaus Rajewsky (Addgene plasmid \# 13763). The resultant construct was
digested with NcoI and XhoI and the His-TAT-NLS-ScRAD52 fragment was cloned into
NcoI/XhoI digested pET15b, thus obtaining pET15b-TAT-NLS-ScRAD52. pET15b-TAT-
NLS-ScRAD52 was transformed into BL21 (DE3) and the selected bacteria were
grown. His-TAT-NLS-ScRAD52 expression was induced with 1 mM IPTG for 3 h and the
recombinant protein was purified using Nickel-Sepharose beads from the soluble
fraction of the bacterial extracts. Recombinant protein was stored in a solution
containing 50% (v/v) glycerol, 20 mM HEPES (pH = 7.4) and 500 mM NaCl. Several
concentrations of TAT-NLS-ScRAD52 ranging from 0.02 to 2 μM were tested for
their capacity to increase the HR frequency. The maximum frequencies were
obtained with concentrations equal to or greater than 0.2 μM, and a significant
level of cytotoxicity was observed only at concentrations higher than 1.8 μM.
The TAT-NLS-ScRAD52 experiments shown in this work were performed using the
fusion protein at a concentration of 0.2 μM.
DNA fragments encoding ScRAD52, RAD51, and RAD52 Flag-tagged at the N-termini
were generated by PCR and cloned into mammalian expression vector pcDNA3
(Invitrogen). The Flag sequence was added to the forward primers. The
restriction sites used in the cloning step are shown in. ScRAD52 was amplified
with the primer pair ScRAD52_F2/ ScRAD52_R2 using pET15b-TAT-NLS-ScRAD52 plasmid
as template. Human RAD51 was amplified from the plasmid CMV-hRad51 using primers
hRAD51_F/hRAD51_R. CMV-hRad51 was a gift from David Liu (Addgene plasmid \#
125570; <http://n2t.net/addgene:125570>; RRID:Addgene_125570). Human RAD52 was
amplified from the plasmid pMM1413-SUMO-RAD52 using primers hRAD52_F/hRAD52_R.
pMM1413-SUMO-RAD52 was a gift from Mauro Modesti (Cancer Research Center of
Marseille). The resultant constructs were named pScRAD52, phRAD51, and phRAD52.
The plasmid encoding for Flag-PALB2 was identified as phPALB2 in this work and
corresponds to the expression plasmid pDEST-FRT/T0-Flag-PALB2. pDEST-
FRT/T0-Flag-PALB2 was a gift from Daniel Durocher (Addgene plasmid \# 71114;
<http://n2t.net/addgene:71114>; RRID:Addgene_71114). The constructs were
transfected into the reporter cell line when indicated, and the expression of
the Flag-tagged HR promoters was analyzed by Western Blot using mouse monoclonal
antibodies against Flag peptide (clone M2, Sigma-Aldrich) and β-actin (AC-40;
Sigma-Aldrich) as the loading control.
## Generation of the HCT116-eGFPΔ3’ reporter cell line and HR-mediated rescue of eGFP expression
HCT116 cells were nucleofected with pAAV-MCS-eGFPΔ3’ plasmid and AAVS1 ZFN mRNA
(Sigma-Aldrich). AAVS1 ZFN mRNA encodes a pair of ZFNs that target the genomic
integration site of AAVS1. Targeted integration of pAAV-MCS-eGFPΔ3’ in
puromycin-resistant individual clones was verified by PCR using the following
primers: P1F and P1R for analysis of 5’-arm recombination; P2F and P2R for
analysis of 3’-arm recombination. Homo- and heterozygosity of the eGFPΔ3’
transgene at the AVVS1 locus was explored by PCR using primers P1F and P2R
located outside the homology arms. The single copy integration of eGFPΔ3’ into
the AAVS1 locus was verified by Multiplex Ligation-Dependent Probe Amplification
(MLPA) and droplet digital PCR (ddPCR) (see below). The resultant cell line was
named HCT116-eGFPΔ3’. HCT116-eGFPΔ3’ cells were transduced with AAV particles
containing pAAV-MCS-eGFPΔ5’ donor plasmid (MOI of 10<sup>3</sup>). HR leads to
reconstitution of full-length eGFP coding sequence and the appearance of green
fluorescent cells 48 hours post-transduction. Individual clones were obtained by
limiting dilution in the presence of blasticidin (5 μg/ml) and were analyzed by
PCR with primers P3F and P3R. The following primers against human SDHA were used
for the genomic DNA loading control PCR: SDHA_F and SDHA_R. The restored full
length eGFP cassette was also sequenced and its expression analyzed by Western
Blot using mouse monoclonal antibodies against GFP (clone B34, Biolegend) and
β-actin (AC-40; Sigma-Aldrich) as the loading control.
## Multiplex ligation-dependent probe amplification (MLPA)
MLPA reactions were performed according to the manufacturer’s general
recommendations (MRC-Holland) with the use of the probes designed and generated
according to the strategy developed and described in detail before. Briefly, 100
ng DNA in 5 μl from parental HCT116 and reporter cell line were denaturated for
5 min at 98°C, cooled to room temperature and mixed with 1.5 μl of probes mix
(containing 1.5 fmol of each probe), and 1.5 μl of SALSA hybridization buffer.
The reaction was then denatured at 95°C for 1 min and probes were hybridized to
their respective targets at 60°C for 16–20 h. After the addition of 32 μl of
ligation mixture, the hybridized probes were ligated together at 54°C for 15
min. After heat inactivation, ligation reaction was cooled to room temperature,
mixed with 10 μl of PCR mixture (polymerase, dNTPs, and universal primers, one
of which was labeled with fluorescein) and subjected to PCR amplification for 35
cycles. The MLPA products were subsequently diluted 20x in HiDi formamide
containing GS Liz600, which was used as a DNA size standard and separated by
capillary electrophoresis (POP7 polymer) using an ABI Prism 3130XL apparatus
(Applied Biosystems). The electropherograms were visualized and analyzed using
GeneMarker software v1.91 (2.4.0). The eGFP-specific MLPA probes and 7 control
MLPA probes are listed in. For each probe, the sequences of the 5’ and 3’ half-
probes, universal sequences to which PCR primers are targeted, the “stuffer”
sequence, and the target specific sequence are provided. Details on gene loci
and chromosome locations for control probes are also provided.
## Droplet digital PCR (ddPCR)
The ddPCR was performed using the QX200 system and EvaGreen Supermix (BIO-RAD),
according to the manufacturer’s general recommendations, as generally described
and used before. To determine the exact copy number of the eGFP transgene in the
reporter cell line, we designed two test-amplicons entirely located in the
transgene (eGFP1 and eGFP2) and two control-amplicons \[C1 (up) and C2 (down)\],
located upstream (2 Kb) and downstream (overlapping exon 2 of *PPP1R12C*) of the
transgene site (AAVS1), respectively. The following sets of primers were used:
(i) T1F and T1R for test amplicon eGFP1 (187 bp in length); (ii) T2F and T2R for
test-amplicon eFGP2 (163 bp in length); (iii) C1F and C1R for control-amplicon
C1 (up) (170 bp in length); and (iv) C2F and C2R for control-amplicon C2 (down)
(238 bp in length). Of note, genomic DNA from the reporter cell line was
digested with the *CviQ*I restriction enzyme (New England Biolabs) prior to
ddPCR. *CviQ*I was chosen because it cuts the transgene between the sequences
corresponding to amplicons eGFP1 and eGFP2, which prevents tandemly repeated
transgenes from being incorrectly identified as a single copy. Briefly, ddPCR
procedure was as follow: the PCR mixture containing 10 μl of EvaGreen Supermix
(Bio-Rad), 1 μl of 4 μM forward primer, 1 μl of 4 μM reverse primer, 4 μl (80
ng) of DNA from the tested cell line and 6 μl of water was partitioned into
20,000 droplets with the use of a QX200 ddPCR droplet generator (Bio-Rad). The
generated droplets were transferred to a 96-well plate and amplified in a T100
Thermal Cycler (BioRad) under the following conditions: 5 min at 95°C, followed
by 40 cycles of 30 s at 95°C, 30 s at 60°C, and 45 s at 72°C, followed by 2 min
at 72°C, 5 min at 4°C, 5 min at 90°C for enzyme inactivation and holding at
12°C. The amplified products were analyzed using a QX200 droplet reader (Bio-
Rad), and the number of positive and negative droplets were counted using the
QuantaSoft version 1.7.4.019 software (Bio-Rad). The copy number of eGFP was
calculated based on the number of positive droplets in eGFP-specific PCR
reactions (either eGFP1 and eGFP2) normalized against the average number of
positive droplets in control reactions \[C1 (up) and C2 (down)\]. Control
amplicons were selected in diploid regions and their copy numbers set to 2.
## HR assay and screening protocol
In all the assays, HCT116-eGFPΔ3’ cells were sequentially treated with: (i) the
selected siRNA oligoes, or expression plasmids encoding HR promoters, (ii) a
TALEN pair designed to facilitate HR, and (iii) AAV particles containing the
donor plasmid. In brief, HCT116-eGFPΔ3’ were plated on 12 well plates at 125,000
cells/well. Forty-eight hours later, cells were transfected with siRNAs using
TransIT-X2 (Mirus Bio). The siRNAs used in this work are listed in. Silencer
Select highly potent and chemically modified siRNAs were used at a final
concentration of 5 nM, whereas classical siRNAs were used at a final
concentration of 40 nM. Alternatively, cells were transfected with 400 ng of the
expression plasmids encoding HR promoters using TransIT-X2. Twenty-four hours
after the first treatment, cells were transfected with a TALEN pair designed to
facilitate HR. The TALEN pair (named eGFP) targeted a region within the AAVS1
locus adjacent to the 3’end of eGFPΔ3’ in the HCT116-eGFPΔ3’ cell line. We used
the fuzznuc software provided by the EMBOSS package to identify the targeting
sequences: `5’-TGCCAGAACCTCTAAGGTTT-3’` (sense component) and
`5’-TCCCTCCCAGGATCCTCTCT-3’` (antisense component). TALEN expression vectors
were constructed using the LIC (ligation-independent cloning) TAL Effector
Assembly Kit (Addgene \#1000000023). The kit provides a TALE repeat unit library
of 2-mer fragments that were assembled into an expression ready TALEN construct
in 2 hierarchical assembly steps. Six hours after the transfection of
HCT116-eGFPΔ3’ cells with 1 μg of each of the TALEN expression plasmids, cells
were transduced with AAV particles containing pAAV-MCS-eGFPΔ5’ donor plasmid
(MOI of 10<sup>3</sup>). When indicated, reporter cells were treated with TAT-
NLS-ScRAD52 (at a final concentration of 0.2 μM) 5 hours after the transfection
of the TALEN plasmids and 1 hour before the infection with the AAV particles.
After 24 hours, medium was replaced with fresh medium, and after the next 24
hours (48 hours post-transduction), the recombination frequency was determined
as the percentage of cells expressing eGFP protein following flow cytometry
analysis using a Beckman Coulter Gallios (Beckman Coulter). Data were analyzed
using Kaluza software (Beckman Coulter) and were subsequently corrected for
transfection and transduction efficiencies ****. Dead cells and debris were
excluded based on scatter signals and propidium iodide fluorescence. When
indicated, cells were fixed, stained with Hoechst 33342 (5 μg/ml) and imaged
using a Nikon Eclipse 90i fluorescent microscope coupled to a Nikon DS-Ri1 CCD
camera.
The transfection efficiency of the eGFP-TALEN plasmids and siRNAs using TransIT-
was \>85% in all the HR experiments shown in this work. Transfection efficiency
was measured in parallel to HR experiments by cotransfecting HCT116-eGFPΔ3’ with
either eGFP-TALEN plasmids, expression plasmids or siRNAs under the same
conditions as in the HR experiments but in combination with 1 μg of peGFP-C1.
Twenty-four hours later, cells were analyzed for eGFP by flow cytometry analysis
and the percentage of eGFP positive (eGFP+) cells was determined to estimate the
transfection efficiency. The efficacy of the siRNAs in gene silencing was
validated by a SYBR Green based real-time PCR assay using the primers listed in.
HPRT1 and β-actin were used as housekeeping genes. All siRNAs tested showed over
70% target knockdown at 48 hours post-transfection.
Transduction efficiency of AAV2 was tested using particles carrying the entire
eGFP gene. AAV2 particles were prepared in 293 c18 cells using the plasmids
pAAV-MCS-eGFP, pHelper and pAAV-RC (at a 1:1:1 ratio), as described above.
HCT116 were transduced with different doses (from MOI 10 to 10<sup>4</sup>). The
highest transduction efficiency (measured as frequency of eGFP+ cells) achieved
with AAV2 in HCT116 was 10±1.5% (mean ± SEM) at MOI 10<sup>3</sup>, and
corroborate previous findings.
## Statistics
All analyses were performed using Prism software (GraphPad). Data are expressed
as mean ± SD and were analyzed by using the unpaired Student’s t-test. In all
graphs, \* p \< 0.05, \*\* p \< 0.01, \*\*\* p \< 0.001.
# Results
## Development of a novel eGFP-based HR reporter system
In our assay, HR leads to the restoration of the full-length eGFP coding
sequence from two different truncated eGFP copies. eGFP is a variant of the
wild-type GFP with higher-intensity emission than GFP, thus facilitating the
detection of HR events. In order to generate the two truncated eGFP copies, DNA
sequences for amino acid residues shown to be essential for eGFP fluorescence
were deleted, such that the coding sequence lacked 72 bp at the 3’ end in the
eGFPΔ3’ copy, and 42 bp at the 5’ end in eGFPΔ5’ (including the start codon).
The assay was designed for the fragment eGFPΔ3’ (the recombination substrate) to
be integrated in the genome, while eGFPΔ5’ (the donor template) is delivered
exogenously via AAV particles.
We optimized the assay system in the human colon cancer cell line HCT116, which
is highly proficient in gene targeting and has been widely used to study the
molecular mechanisms of HR. To avoid position effects, we aimed to integrate
eGFPΔ3’ into the adeno-associated virus integration site 1 (AAVS1) within the
intron 1 of PPP1R12C on human chromosome 19. We chose the AAVS1 locus as the
target because it is considered a safe harbor locus for integrating transgenes,
since it is constitutively expressed across a variety of cell types, including
HCT116 cells, and biallelic disruption of PPP1R12C results in no discernible
phenotype.
The targeting construct (peGFPΔ3’) contained a left and right homology arm to
the AAVS1 genomic integration site flanking the eGFPΔ3’ transgene and a
puromycin cassette. The targeting construct design strategy is shown in. The
eGFPΔ3’ transgene was under the control of the CMV promoter. The puromycin
selection cassette was preceded by a splice acceptor site. Therefore, its
expression relied on splicing and was driven by the endogenous PPP1R12C
promoter. Finally, HCT116 were nucleofected with the targeting construct
peGFPΔ3’ and a well-validated pair of ZFNs engineered to target the AAVS1 locus
and increase the targeting rate.
Puromycin-resistant clones were screened for successful integration of the
transgene by PCR analysis of genomic DNA using different combinations of primers
located in the transgene and outside the 5’ and 3’ homology arms as illustrated
in shows representative results of PCR analysis of 4 correctly targeted clones
which were named HCT116-eGFPΔ3’ followed by a serial number. PCR using primers
outside the arms showed all clones were heterozygotes for the eGFPΔ3’ transgene.
We randomly selected the cell clone HCT116-eGFPΔ3’ \#1 for further experiments.
The presence of the transgene eGFPΔ3’ in the reporter cell line was confirmed by
MLPA using two pairs of probes specific for eGFP (eGFP1 and eGFP2) and seven
probes specific for diploid control regions (C1 to C7). shows electropherograms
of MLPA reactions performed on genomic DNA extracted from parental HCT116 and
HCT116-eGFPΔ3’ \#1 cell lines. eGFP-specific signals were present in the
HCT116-eGFPΔ3’ \#1 electropherogram (red arrowheads), but not in the parental
HCT116 electropherogram, which confirmed the presence of the transgene in the
HCT116-eGFPΔ3’ \#1 cells. The peak heights for eGFP were approximately half
those of the control probes suggesting that HCT116-eGFPΔ3’ \#1 cells were
heterozygous for the transgene. Next, ddPCR was performed to precisely determine
the eGFPΔ3’ gene copy number and rule out integration at other off-target sites.
Pairs of primers were designed to amplify two test-amplicons (eGFP1 and eGFP2)
located entirely within the eGFPΔ3’ transgene and two control-amplicons (C1 and
C2) specific for the diploid flanking regions. Of note, genomic DNA from the
reporter cell line HCT116-eGFPΔ3’ \#1 was digested with the *CviQ*I restriction
enzyme prior to ddPCR. *CviQ*I was chosen because it cuts the transgene between
the sequences corresponding to amplicons eGFP1 and eGFP2, thus preventing
tandemly repeated transgenes from being misidentified as a single copy. As shown
in, the fluorescence intensity of the HCT116-eGFPΔ3’ \#1 DNA droplets when eGFP1
and eGFP2 assays were used was half of that obtained with the control assays,
indicating the HCT116-eGFPΔ3’ \#1 reporter cell line contains a single copy of
the eGFPΔ3’ transgene.
Next, we produced AAV particles harboring the HR donor template that contained a
truncated eGFP lacking the first 42 bp, a blasticidin cassette and the 3’AAVS1
homology arm (pAAV-MCS-eGFPΔ5’). AAV particles were added onto HCT116-eGFPΔ3’
\#1 cells at a MOI of 10<sup>3</sup> viral particles/cell. Two days after
transduction, eGFP+ cells (also named HCT116-rec-eGFP) were detected (**)**. The
frequency of recombination was 4.3x10<sup>-3</sup> ± 1.6x10<sup>-3</sup>. To
formally determine whether or not fluorescence cells had undergone HR, we
isolated fluorescent cells to assess if they harbored full-length eGFP coding
sequences. Single HCT116-rec-eGFP cell clones were obtained by limiting dilution
and then expanded to clonal cell lines in the presence of blasticidin. HR-
induced restoration of an intact eGFP gene in fluorescent cells was assessed by
PCR and sequencing. Western blot analysis confirmed the rescue of eGFP
expression in HCT116-rec-eGFP cells. The absence of a truncated form in
HCT116-eGFPΔ3’ \#1 cells is commented in the discussion section.
## Validation of the HR system
Next, we evaluated the ability of our assay to identify factors affecting HR. To
measure HR activity, eGFP+ cells were counted by flow cytometry and the raw data
was subsequently corrected for transfection and transduction efficiencies as
described in Materials and Methods and in.
In all the experiments that will be described below, HCT116-eGFPΔ3’ \#1 cells
were pre-treated with a pair of TALENs designed to induce DSB in the region
adjacent to the 3’end of eGFPΔ3’ (the eGFP TALEN pair). The design and
generation of the TALEN expression vectors are described in Materials and
Methods. The eGFP TALEN pair was added 6 hours prior the transduction with AAV
particles containing the donor template. As expected, adding the eGFP TALEN pair
led to a significant increase of the basal frequency of specific HR events (from
3.6x10<sup>-3</sup> ± 1.6x10<sup>-3</sup> to 28x10<sup>-3</sup> ±
6.5x10<sup>-3</sup>).
First, we tested how adding TAT-NLS-ScRAD52 impacted the frequency of HR. RAD52
is an important HR protein that has the strongest effect on *Saccharomyces
cerevisiae*. In this regard, the yeast protein, ScRAD52, has been demonstrated
to be more effective than its human homologue in promoting HR. The TAT-NLS-
ScRAD52 fusion protein was generated as described in the Materials and Methods
section. The TAT peptide made ScRAD52 cell permeable and the NLS targeted the
protein to the nucleus. TAT-NLS-ScRAD52 was added to the HCT116-eGFPΔ3’ \#1
reporter cell line 5 hours after the transfection of the eGFP TALEN vectors and
1 hour before the infection with the AAV particles containing the donor plasmid.
As expected, the addition of TAT-NLS-ScRAD52 resulted in an approximate 3-fold
increase in HR frequency ****. We also explored the effect of the overexpression
of the following human genes known to promote HR: RAD51, RAD52, and PALB2. The
HCT116-eGFPΔ3’ \#1 reporter cells were transfected with expression plasmids
encoding the different Flag-tagged HR promoters and 24 hours later they were
transfected with the eGFP TALEN vectors. Six hours after the transfection of the
TALEN vectors, the reporter cells were transduced with AAV particles containing
the donor plasmid. As expected, the overexpression of PALB2 and ScRAD52 led to
an increase in HR frequency ranging from approximately 2.5-fold increase in the
case of ScRAD52 to 2.8-fold increase when PALB2 was overexpressed ****. It is of
note that the plasmid encoded ScRAD52 increased HR frequency approximately as
much as TAT-NLS-ScRAD52. In contrast, the overexpression of RAD51 and RAD52 had
no significant effect on HR frequency. We comment on this result in the
discussion section. The expression of the Flag-tagged proteins was confirmed by
Western Blot ****.
Second, we explored the effect of various siRNAs targeting well-characterized
DNA repair-related genes on the frequency of HR. In these assays, the
HCT116-eGFPΔ3’ \#1 reporter cells were transfected with different siRNAs and
then treated with the eGFP TALEN and donor plasmid at the same times as those of
the overexpression experiments described above. We used two siRNAs for each of
the following genes: RAD51, RAD52, PALB2, CTDP1, XRCC6, LIG4, CBP and SMCHD1. As
indicated above, RAD51, RAD52, and PALB2 play a major role in HR-mediated DNA
repair, as does CTDP1. The efficacy of the siRNAs in gene silencing was
validated by real-time PCR assay ****. As expected, the silencing of RAD51,
PALB2 and CTDP1 led to a significant decrease in the HR rate ****. The silencing
of RAD52 had no effect, which we comment on in the discussion section. The XRCC6
and LIG4 genes encode protein Ku70 and human ATP-dependent DNA ligase
respectively, which are the components of the NHEJ repair pathway. It has been
reported that a decrease in NHEJ leads to a compensatory increase in HR.
Accordingly, XRCC6- and LIG4-silenced reporter cells exhibited increased
frequency of HR. Similarly, reporter cells silenced for CBP and SMCHD1
expression exhibited increased frequency of HR. It is important to note that
both CBP and SMCHD1 facilitate the recruitment of NHEJ factors at DSB sites, so
their depletion was also expected to lead to an increase in HR rates. Of note,
the silencing of XRCC6 and CBP led to the greatest increase in HR. Similar
results were obtained between different siRNAs against the same gene in all
cases.
# Discussion
In this study, we present the development and validation of a novel assay in
which HR reconstitutes the full length eGFP from a 5’ and a 3’ truncated copies
of eGFP.
The eGFPΔ3’ fragment, the HR substrate, consisted of a eGFP copy lacking the
nucleotides encoding the last 24 amino acids, which are required for the
protein’s fluorescence. This transgene was integrated into the AAVS1 locus of
HCT116 cells, which are commonly used for studying HR mechanisms. Although AAVS1
is considered a well validated safe harbor in the human genome, it is important
to recall that transgene integration at this locus disrupts the gene phosphatase
1 regulatory subunit 12C (PPP1R12C). As described above, the transgene contained
a puromycin cassette preceded by a splice acceptor sequence in addition to the
CMV promoter-driven eGFPΔ3’. As reported for other transgenes containing splice
acceptor sites, the insertion of the puromycin cassette into the AAVS1 locus led
to a downregulation of PPP1R12C mRNA levels ****. In this regard, it is
important to mention that although the haploinsufficiency or complete
inactivation of PPP1R12C does not lead to a discernible phenotype, the
consequences of this disruption should be further investigated. Another point we
want to highlight is that the eGFPΔ3’ transgene was integrated as a single copy,
and this is relevant when calculating the frequency of recombination because the
appearance of a fluorescent signal in a cell is the result of a single HR event.
Finally, it should be noted that the expression of the truncated eGFPΔ3’ copy
could not be detected by western blot using an antibody against full length
protein, suggesting the degradation of aberrant transcript and protein.
Interestingly, the transgene has persisted over several passages (over 40) in
our reporter cell line, which is likely due to the chromosomal stability of
HCT116.
The donor template consisted of a copy of eGFP lacking 42 bp at the 5’ end
(including the start codon) and was delivered into the reporter cells via AAV
particles. AAVs are on the rise as a powerful tool in gene therapy thanks to
their lack of pathogenicity, wide range of cell tropism and long-term gene
expression among other properties. It is also well known that recombinant AAV
vectors facilitate homologous recombination in mammalian cells at high
efficiencies (even up to 10x10<sup>-3</sup>) and this is thought to be due, at
least in part, to its single stranded nature. The recombination frequency in our
system was 3.6x10<sup>-3</sup> ± 1.6x10<sup>-3</sup>, which falls within the
range of values obtained by others using recombinant AAVs.
Finally, our study also validates the ability of our assay to identify factors
affecting HR. Of note, all validation experiments were performed in the presence
of a pair of TALENs designed to induce DSB in the region adjacent to the 3’ end
of eGFPΔ3’. As expected, the addition of the eGFP TALEN pair led to a
significant increase of the basal frequency of specific HR events (by
approximately 8-fold), which helped improve the system.
ScRAD52 is more effective than its human homologue in promoting HR both in vitro
and in vivo. We observed that overexpression of ScRAD52 resulted in an
approximate 3-fold increase in HR frequency when either the recombinant protein
or the plasmid encoded protein were used. Similarly, the overexpression of PALB2
led to an increase in the HR frequency as previously reported by others using
the DR-GFP reporter system. In our assay neither RAD51 nor RAD52 had any
significant effect on HR frequency. In this regard, it is important to mention
that it has been previously reported that the overexpression of RAD51 does not
have a significant effect on HR rates using DR-GFP system, while the
overexpression of its dominant negative forms which are ATPase mutants results
in a 8-fold decrease in HR frequency. On the other hand, Kim et al. have shown
that the overexpression of RAD51 and/or RAD52 reduces DSB-induced HR while
enhances spontaneous HR. The distinct results obtained by us and others may
reflect differences in recombination substrate structures or different levels of
overexpression.
The impact of siRNA-mediated silencing of RAD51, RAD52, PALB2, CTDP1, XRCC6,
LIG4, CBP and SMCHD1 on the HR frequency that we report here is similar to that
observed in previous genome wide HR screens and single gene approaches by
others. Thus, as expected, RAD51, PALB2, and CTDP1 depletion resulted in a
marked reduction of eGFP+ cells whereas depletion of NHEJ-related genes led to
an increase in their number. This last result highlights the tight regulation of
the balance between NHEJ and HR. In the case of RAD52, its silencing had a
negligible effect on HR frequency. This apparently unexpected result has been
reported by others, indicating that RAD52 is not essential to HR.
In conclusion, we have established a sensitive assay for measuring the
efficiency of HR in the context of the DNA repair of an integrated transgene
using an exogenous DNA template. This novel tool will be useful to accurately
test the impact of continually emerging tools for HR-mediated genome editing,
drugs, miRNAs and genes on HR and DNA repair. We anticipate that refining the
precision of repair during in vitro culture will contribute to developing
innovative gene therapy products.
# Supporting information
We thank Roberto Cantalapiedra and Raquel Carretero for technical support.
[^1]: The authors have declared that no competing interests exist.
[^2]: Current address: Institute of Pharmacological Research (ININFA),
University of Buenos Aires–National Scientific and Technical Research
Council (CONICET), Buenos Aires, Argentina
[^3]: ‡ These senior authors contributed equally to this work. |
# Introduction
The renin-angiotensin system (RAS) plays a central role in the development and
regulation of blood pressure response. Angiotensin II (Ang II), the effector
peptide of the pro-hypertensive axis of the RAS that also includes angiotensin
converting enzyme (ACE) and the Angiotensin -type 1 receptor (AT<sub>1</sub>R),
exerts diverse physiological actions in both the peripheral and central neural
systems. The anti-hypertensive counterbalance to these mediators includes ACE2,
Ang-(1–7) and the Mas receptor. Importantly, all these essential components of
the RAS, including renin and angiotensinogen, as well as various cardiovascular-
modulatory aminopeptidases, are synthesized within the brain, suggesting the
existence of a comprehensive intrinsic brain RAS. Recent evidence suggests that
dysregulation of the individual brain RAS axes may play a critical role in the
development and maintenance of hypertension. Ang II, acting through the
AT<sub>1</sub>R, plays a prominent role in the central regulation of blood
pressure by activating the sympathetic nervous system, regulating fluid and salt
balance and the secretion of aldosterone, amongst other actions. Previous
studies suggest that systemically delivered Ang II likely acts upon the
circumventricular organs, where the blood brain barrier is weak or absent, and
subsequently activates hypothalamic and brain stem sites such as the
paraventricular nucleus (PVN) and ventrolateral medulla, contributing to
sympathoexcitation and hypertensive response. Experimental evidence indicates
that the hypothalamic PVN is an important center for integrating Ang II-induced
neural outflow signals for the pressor response and sympathetic vasomotor tone.
Recent findings from our lab and others suggest that the RAS, in addition to
inducing neurohumoral excitation, also increases the production of
proinflammatory cytokines (PICs), such as tumor necrosis factor-alpha (TNF), in
brain cardiovascular regulatory centers, and has been shown to contribute to the
neurogenic component of hypertension, both through direct actions and through
modulating reactive oxygen species (ROS) signaling pathways. A chronic increase
in peripheral Ang II levels is proposed to initiate a cascade of signaling
events involving PICs and ROS in brain cardioregulatory sites raising
sympathetic activity, hypertension and end organ damage. A study by Marvar et
al. showed that Ang II-mediated hypertension is caused by central mechanisms and
described a feed-forward process in which the central pressor effects of Ang II
lead to activation of T cells, which in turn, promote vascular inflammation and
further raise blood pressure, leading to severe hypertension. In addition, PICs
can be produced locally in the brain by glia and neurons, thereby contributing
to the neuroinflammatory response implicated in the pathogenesis of
hypertension. These observations, coupled with the emerging role of PICs and the
little known role of the anti-hypertensive axis of the RAS in hypertension, led
to hypothesize that the central effects of Ang II are, at least in part,
mediated by the activation of PICs, especially TNF. The resultant of these
actions are the differentially dysregulated RAS axes within cardiovascular
relevant brain regions, including the PVN, ultimately enhancing the neurogenic
hypertensive response. In the current study, we investigate this hypothesis by
examining central TNF inhibition via intracerebroventricular (ICV) etanercept
infusion, a soluble TNF receptor fusion protein, on pro- and anti-hypertensive
RAS components in the PVN in Ang II-induced hypertension.
# Materials and Methods
## Ethics Statement
All animal and experimental procedures in this study were reviewed and approved
by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State
University in compliance with National Institutes of Health Guide for the Care
and Use of Laboratory Animals.
## Experimental Design
Male Sprague-Dawley rats (10–12 weeks old) were used in this study. Animals were
housed in a temperature-controlled room (25±1°C) and maintained on a 12∶12 hour
light:dark cycle with free access to food and water. The rats were implanted
with radio-telemetry transmitters to measure blood pressure, and subjected to
ICV infusion of etanercept (ETN; 10 µg/kg/day) or artificial cerebrospinal fluid
(aCSF) (Alzet, model 1004; 0.11 µl/hr), with and without subcutaneous infusion
of Ang II (200 ng/kg/min) for 4 weeks. Osmotic minipumps (Alzet, model 2004;
0.25 µl/hr) were filled with Ang II dissolved in 0.9% saline or saline alone,
and were implanted subcutaneously in the retroscapular area. The rats were
divided into 4 groups: 1) Control group: saline minipumps+ICV aCSF, 2) ETN
group: saline minipumps+ICV etanercept, 3) Ang II group: Ang II minipump+ICV
aCSF, and 4) Ang II+ETN group: Ang II minipump+ICV etanercept. At the end of the
study, rats were euthanized; the hearts and brains were collected and stored at
−80°C until further analysis.
## Blood Pressure Measurement
Blood pressure was measured continuously in conscious rats implanted with radio-
telemetry transmitters (Model TA11PA-C40, Data Sciences International, St. Paul,
MN). Rats were anesthetized with a ketamine (90 mg/kg) and xylazine (10 mg/kg)
mixture (i.p.) and placed dorsally on a heated surgical table. The adequacy of
anesthesia was monitored by limb withdrawal response to toe pinching. An
incision was made on the ventral surface of the left leg, and the femoral artery
and vein were exposed and dissected apart. The femoral artery was ligated
distally, and a small clamp was used to temporarily interrupt the blood flow.
The catheter tip was introduced through a small incision in the femoral artery,
advanced into the abdominal aorta such that the catheter tip was distal to the
origin of the renal arteries, and secured into place. The body of the
transmitter was placed into the abdominal cavity and secured to the abdominal
wall. The abdominal musculature was sutured and the skin layer was closed
following implantation. Rats received benzathine penicillin (30000 U, i.m.) and
buprenorphine (0.1 mg/kg, s.c.) immediately following surgery and 12 h
postoperatively and allowed to recover for seven days.
## ICV Cannula Implantation
Following the transmitter recovery period, the rats were implanted with ICV
cannula for infusion of etanercept or aCSF. The rats were anesthetized with
ketamine (90 mg/kg) and xylazine (10 mg/kg) mixture (i.p.) and the head was
positioned in a Kopf stereotaxic apparatus. An ICV cannula was implanted into
the right lateral cerebroventricle (1.3 mm caudal to bregma, 1.5 mm lateral to
the midline, and 3.5 mm ventral to the dura) according to Paxinos and Watson,
and fixed to the cranium using small screws and dental cement. A 4-week osmotic
minipump was implanted subcutaneously and connected to the infusion cannula via
the catheter tube to deliver etanercept or aCSF into the brain.
## Measurement of Plasma IL-10
At the end of the study, blood was collected in chilled EDTA tubes; plasma was
separated and stored at −80°C until assayed. Circulating levels of IL-10 were
quantified in the plasma using a commercially available rat IL-10 ELISA kit
(Invitrogen) according to manufacturer’s instructions.
## Real Time RT-PCR
The PVN punches were made from frozen brain sections using a Stoelting brain
punch (Stoelting). Total RNA was isolated from PVN tissue using RNeasy plus
micro kit (Qiagen) and cDNA was synthesized using iScript cDNA synthesis kit
(Bio-Rad). Real Time PCR amplification reactions were performed with iQ SYBR
Green Super mix with ROX (Bio-Rad) using the ABI Prism 7900 Real time PCR
machine (Applied Biosystems). Data were normalized to GAPDH expression by the
ΔΔC<sub>T</sub> comparative method.
## Western Blot Analysis
Western blot analysis was performed according to standard protocols. The PVN
tissue was homogenized with RIPA lysis buffer. The protein concentration was
measured using a bicinchioninic acid protein assay kit (Pierce). Equal amounts
of protein (5 µg) were separated by SDS-PAGE on 10% or 12% gels, transferred on
to PVDF membrane (Immobilon-P, Millipore), and blocked with 1% BSA in TBS-T at
room temperature for 60 min. The membranes were subjected to immunoblot analyses
with anti-ACE (Santa Cruz, 1∶500), anti-AT<sub>1</sub>R (Santa Cruz, 1∶500),
anti-ACE2 (Santa Cruz, 1∶500), anti-AT<sub>2</sub>R (Santa Cruz, 1∶200), anti-
Mas (Alomone Labs, 1∶500), anti-NOX-2 (BD Biosciences, 1∶500), anti-NOX-4 (Santa
Cruz, 1∶1000), anti-iNOS (Santa Cruz, 1∶500), anti-nNOS (Santa Cruz, 1∶500), and
anti-GAPDH (Santa Cruz, 1∶1000) antibodies. The membranes were washed and
incubated with anti-rabbit or anti-goat secondary antibodies (Santa Cruz,
1∶5000) for 1 hour at room temperature. Specific bands were detected using an
enhanced chemiluminescence kit (Amersham). The bands were quantified by
densitometry using Chemidoc XRS system and Quantity-One software (Bio-Rad) and
were normalized to GAPDH expression.
## Determination of NAD(P)H Oxidase Activity in the PVN
Homogenates were prepared from PVN samples and total protein concentration was
determined using a bicinchoninic acid protein assay kit (Pierce). The NAD(P)H
oxidase activity was measured using lucigenin enhanced chemiluminiscence
detection of superoxide as previously described. In brief, the homogenates were
diluted in modified HEPES buffer containing 140 mM NaCl, 5 mM KCl, 0.8 mM
MgCl<sub>2</sub>, 1.8 mM CaCl<sub>2</sub>, 1 mM Na<sub>2</sub> HPO<sub>4</sub>,
25 mM HEPES, and 1% glucose (pH 7.0). The reaction was started by addition of
NAD(P)H (100 µM) and dark adapted lucigenin (5 µM). Light emission was recorded
and expressed as mean light unit (MLU) per minute per milligram of protein over
10 min. Using this method, the superoxide anion production also represents
NAD(P)H oxidase activity. The specificity of superoxide measured was confirmed
either by adding superoxide dismutase (200 units/ml) or apocynin (1 mM).
## *In situ* Detection of Superoxide Production in the PVN
Dihydroethidium, an oxidative fluorescent dye, was used to detect in situ
superoxide in the PVN of rats as previously described. At the end of the
experiment, the brains were removed, quickly frozen, embedded into OCT, and
cryostat sectioned (30 µm, coronal) directly onto chilled microscope slides.
Sections were then incubated in a light protected humidified chamber at 37°C for
30 minutes with 1 µmol/L dihydroethidium (Molecular Probes). After washing with
phosphate-buffered saline, red fluorescence was visualized by confocal laser
scanning microscopy using an excitation wavelength of 490 nm and emission
wavelength of 610 nm.
## Statistical Analysis
All results are expressed as mean±SEM. For statistical analysis of the data,
Student’s *t* test, one-way ANOVA or repeated measures ANOVA followed by
Bonferroni’s *post hoc* test was performed using GraphPad Prism version 5.0 for
Windows (Graph Pad Software, San Diego California, USA) to determine differences
among groups. A value of *p*\<0.05 was considered statistically significant.
# Results
## Effect of Central TNF Blockade on Mean Arterial Pressure and Cardiac Hypertrophy
To assess the effect of central TNF blockade on the Ang II-induced hypertensive
response, mean arterial pressure (MAP) was measured using a radio-telemetry
system. After 28 days, chronic Ang II infusion significantly increased the MAP
in rats when compared with control rats (165±5 mmHg vs 108±8 mmHg, respectively;
*p*\<0.05). In contrast, ICV treatment with etanercept attenuated the Ang II-
induced increase in MAP (126±21 mmHg vs 165±5 mmHg, respectively; *p*\<0.05),
while etanercept treatment alone had no effect on MAP (104±4 mmHg).
To evaluate Ang II-induced changes on cardiac hypertrophy in these rats, the
hearts were harvested and weighed at the end of experimental period. The ratio
of heart weight/body weight (HW/BW) was calculated as an indicator of cardiac
hypertrophy. Chronic Ang II infusion lead to increased cardiac hypertrophy
versus controls as indicated by the increased HW/BW ratio. ICV treatment with
etanercept inhibited Ang II-mediated cardiac hypertrophy. Furthermore, mRNA
expression of a molecular marker of cardiac hypertrophy, atrial natriuretic
peptide (ANP), was measured in cardiac tissue using real time RT-PCR. Chronic
Ang II infusion showed increased mRNA expression of ANP in the heart, which was
decreased by ICV treatment with etanercept. These data suggests a role for TNF
in the brain on Ang II-induced blood pressure regulation and cardiac hypertrophy
in the hypertensive state.
## Effect of Central TNF Blockade on the Expression of Pro- and Anti-inflammatory Cytokines
To determine the effect of Ang II on the production of PICs and chemokines, the
mRNA expression of TNF, IL-6, IL-1β, and the chemokine MCP-1 were measured in
the PVN by real time RT-PCR. Ang II infusion induced an increase in the gene
expression of the PICs TNF, IL-6 and IL-1β, and the chemokine MCP-1, in the PVN,
when compared to the control group. These PICs were attenuated in the PVN of Ang
II-infused rats treated ICV with etanercept, demonstrating that chronic Ang II
infusion increases the pro-inflammatory response within the PVN through TNF in
Ang II-induced hypertension.
We also examined the effect of TNF inhibition on the expression of the anti-
inflammatory cytokine IL-10. Chronic subcutaneous Ang II infusion resulted in
significant decrease in PVN mRNA and plasma protein levels of IL-10. However,
Ang II infusion and simultaneous inhibition of TNF in the brain using etanercept
restored the PVN mRNA expression of IL-10 and plasma levels of IL-10.
## Effect of Central TNF Blockade on the Expression of RAS Components in the PVN
Both Ang II and TNF have been shown to modulate RAS component expression. To
determine the manner by which Ang II infusion alters the expression of the pro-
and anti-hypertensive components of the RAS in the PVN, we examined the mRNA and
protein expression levels of ACE, ACE2, AT<sub>1</sub>R, AT<sub>2</sub>R, and
the Mas receptor. The PVN mRNA and protein expression of the RAS pro-
hypertensive components ACE and AT<sub>1</sub>R were significantly increased in
Ang II-infused rats when compared with control rats; this was prevented by ICV
treatment with etanercept. Conversely, the anti-hypertensive components of the
RAS (ACE2, Mas receptor and AT<sub>2</sub>R) showed a decreased gene and protein
expression in Ang II-treated rats. These levels were increased in the Ang II+ETN
group. These data suggest that in Ang II-induced hypertension, the pro- and
anti-hypertensive components of the RAS are differentially regulated within the
PVN in a deleterious manner and TNF inhibition restores the balance between
these RAS components.
## Effect of Central TNF Blockade on the Oxidative Stress Markers in the PVN
Ang II and TNF have both been shown to act through oxidative stress mediated
pathways, especially within the PVN, in inducing elevated sympathetic outflow
and a progressive hypertensive response. Thus, several experiments were
performed to test the effect of central TNF inhibition on oxidative stress
markers in the PVN in response to the Ang II infusion hypertension model. First,
NAD(P)H oxidase dependent superoxide production in the PVN was measured using a
lucigenin enhanced chemiluminiscence method. Consistent with earlier reports,
Ang II caused a significant increase in NAD(P)H oxidase dependent superoxide
production in the PVN homogenates compared with control rats. This response was
abolished by ICV inhibition of TNF using etanercept. Next, in situ
dihydroethidium fluorescence was used to assess Ang II-induced superoxide
production in the PVN of rats receiving systemic Ang II-infusions concomitant
with ICV infusions of either etanercept or aCSF. Consistent with earlier
reports, dihydroethidium fluorescence was increased in the PVN of Ang II-infused
rats compared with control rats. This response was markedly inhibited in rats
receiving ICV etanercept. Furthermore, Ang II infusion significantly increased
the NOX-2 and NOX-4 mRNA and protein expression in the PVN of Ang II infused
rats. These changes were attenuated in Ang II-infused rats treated centrally
with etanercept. To further determine the effect of TNF blockade on Ang II-
induced NO signaling, we analyzed mRNA and protein expression of inducible NOS
(iNOS) and neuronal NOS (nNOS) within the PVN. Four weeks of Ang II infusion
significantly increased iNOS mRNA and protein expression and decreased the nNOS
mRNA and protein expression. These changes were prevented in rats with central
infusion of etanercept. Overall, these data suggest that increased oxidative
stress in Ang II-induced hypertension is potentially through a TNF-driven
mechanism and the effects of central TNF blockade on attenuation of Ang II-
induced hypertension, at least in part, are mediated by a decrease in oxidative
stress.
# Discussion
In the present study, the role of central TNF on Ang II-induced hypertension and
cardiac hypertrophy was investigated. These data suggest for the first time that
inhibition of TNF in the brain using etanercept lowered blood pressure, reduced
inflammation, decreased oxidative stress, and restored the balance between the
pro- and anti-hypertensive axes of the RAS. Our findings demonstrate that the
changes observed in Ang II-induced hypertension are regulated, at least in part,
through the central actions of TNF and potentially via the dysregulation of
components of the RAS within the hypothalamic PVN.
Recent evidence suggests that hypertension is an inflammatory condition where
various PICs such as TNF, IL-6 and IL-1β, both centrally and peripherally, have
been shown to play an important role in the pathogenesis of hypertension. A
recent study from our lab demonstrated that chronic peripheral Ang II infusion
results in increased production of PICs within the PVN. Etanercept is a soluble
recombinant fusion protein that inhibits TNF by posing as a TNF receptor decoy
and acting through competitive inhibition of TNF and an overall reduction in
free TNF to act on endogenous receptors. Blockade of TNF by etanercept has been
shown to prevent renal damage in a genetic hypertensive rat model, as well as
lower blood pressure in rats with Ang II- and salt-induced hypertension,
suggesting a role for TNF in blood pressure regulation and renal injury. Another
study also showed that mice treated with etanercept had an attenuated
hypertension and a blunted increase in superoxide production in response to Ang
II. In our study, using ICV etanercept infusion techniques, we inhibited the TNF
levels specifically in the brain. Our present observations complement those
prior findings and show that blockade of TNF by ICV administration of etanercept
into the brain protects rats against Ang II-dependent cardiac hypertrophy and
hypertension.
Ang II can act as a potent proinflammatory agent and stimulate the production of
chemokines such as MCP-1, and PICs, such as TNF, IL-6 and IL-1β in the brain.
TNF is commonly considered as one of the initiators of the pro-inflammatory
cascade, which can induce production of other cytokines, and inhibition of its
action during inflammatory events abrogates many of the ensuing responses,
including the production of IL-1β and IL-6. A recent study by Shi et al.
demonstrated that Ang II-induced hypertension involves activation of microglia
and increased expression of PICs within the PVN. A previous study from our group
demonstrated that chronic Ang II infusion increases proinflammatory cytokines
expression in the PVN of rats. In our study, TNF blockade with etanercept
decreased the PVN expression not only of TNF, but of other PICs such as IL-6,
IL-1β, and the chemokine MCP-1, supporting the hypothesis that PICs are involved
in the Ang II-induced hypertensive response. It has been shown that central gene
transfer of IL-10 reduces hypothalamic inflammation in heart failure rats after
myocardial infarction. In addition, IL-10 overexpression in the PVN attenuates
Ang II-induced hypertension. In our study, chronic Ang II infusion resulted in
decreased mRNA and protein expression of anti-inflammatory cytokine IL-10, which
was restored by central TNF blockade suggesting that some of beneficial effects
of TNF blockade are mediated by restoring the levels of anti-inflammatory IL-10.
Neurogenic hypertension is characterized by an overactive brain RAS,. In
addition to locally generated Ang II within the brain, blood borne Ang II can
enter the brain via circumventricular organs and modulate the pathogenesis of
hypertension and fluid homeostasis. Elevated activity and expression of RAS
components in central cardiovascular regulatory regions has been shown to be
involved in the pathogenesis of hypertension in several genetic and experimental
models. Treatment with ACE inhibitors and Ang receptor blockers has been shown
to prevent this RAS overactivity and restore normal cardiovascular function. In
the brain, the AT<sub>1</sub>R mediates the central effects of Ang II, including
vasopressin release, water and salt intake and balance, and increased
sympathetic drive, all of which contribute to the development of hypertension.
In many animal models of hypertension, the expression of the AT<sub>1</sub>R is
up-regulated in central cardiovascular regulatory centers, including the
hypothalamic PVN. Both *in vitro* and *in vivo* studies have demonstrated a
cross-talk between Ang II and TNF, a mechanism we have also shown using TNF
knockout mice where Ang II-induced hypertension was attenuated via a decreased
expression of AT<sub>1</sub>R. Within the last decade, the discovery of an
alternate set of components of the RAS which may act as a counterbalance to the
actions of the ACE/Ang II/AT<sub>1</sub>R pathway added complexity to the
understanding of RAS regulation, especially within the brain. These components,
termed anti-hypertensive due to their cardio-protective effects, along with all
the components of the pro-hypertensive RAS axis, are known to be expressed
throughout the various central cardio-regulatory regions, including the PVN. In
various experimental hypertensive models, the components of anti-hypertensive
RAS axis (ACE2, Ang (1–7), the Mas receptor and AT<sub>2</sub> receptors) are
shown to be down-regulated, while the pro-hypertensive components are increased,
. This dysregulation may be the lynch pin trigger towards developing the
hypertensive state, but it remains poorly understood. It has been shown that the
brain ACE2 activity was inhibited in a chronically hypertensive mouse model with
high Ang II levels and this decrease was mediated by AT<sub>1</sub>R. We
previously showed that ACE2 overexpression within the PVN attenuated Ang II-
induced hypertension by abolishing PIC production in the PVN in combination with
restoring the balance between pro- and anti-hypertensive axes of the RAS. In the
present study, ICV treatment with etanercept resulted in reduction of Ang II-
induced pro-hypertensive RAS components expression in the PVN, including ACE and
AT<sub>1</sub>R, as well as the restoration of the anti-hypertensive RAS
components ACE2, Mas receptor, and AT<sub>2</sub> receptors. These results
suggest that this RAS dysregulation and perpetuation of the hypertensive state
may be the result of a pro-inflammatory response through the actions of TNF.
Excessive ROS production in brain cardio-regulatory centers such as the PVN can
contribute to the neurogenic component of the hypertensive response by enhancing
sympathetic activity and outflow. It has been previously shown that NAD(P)H
oxidase is the primary source of Ang II-induced ROS in neurons and that
treatment with Tempol, a cell permeable superoxide dismutase (SOD) mimetic,
inhibits Ang II-mediated superoxide production and hypertension. Enhanced
NAD(P)H oxidase activity associated with increased expression of several NAD(P)H
oxidase subunits including NOX-2 and NOX-4, have been shown to be predominant
homologues expressed in the forebrain, including the PVN. A recent study showed
that in an aldosterone/salt induced hypertensive animal model, both NOX-2 and
NOX-4 are necessary to generate functional NAD(P)H oxidase within the PVN.
Furthermore, TNF can induce activation of NADPH oxidase leading to enhanced
oxidative stress and decreased bioavailability of NO. In the present study, we
found that central TNF inhibition abolished the Ang II-induced oxidative stress
as indicated by decreased super oxide production, NAD(P)H oxidase activity as
well as attenuated NOX-2 and NOX-4 subunit expression within the PVN. These
results indicate that TNF mediates NAD(P)H oxidase-derived superoxide production
during Ang II-induced hypertension. In addition, the decreased nNOS expression
and increased iNOS expression in the PVN indicates NO signaling dysregulation in
Ang II-induced hypertensive rats. Neuronal NOS is an inverse indirect indicator
of sympathoexcitation, in that a decrease in nNOS correlates with an increase in
sympathetic outflow. Therefore, a reduction in beneficial NO not only decreases
with the reduction in nNOS expression and activity, but also due to the rapid
interconversion of NO to peroxynitrite (ONOO<sup>−</sup>) due to the increased
production of NADPH oxidase-derived superoxide. This is further complicated by
the involvement of iNOS, which rapidly uses up available L-arginine for NO
production and commences with superoxide production and thereby increasing free
radical concentrations. This combination of factors can lead to an increased PIC
response, sympathoexcitation and a continued propagation of neurogenic
hypertension. The present study provides further support for these observations
by showing that central TNF blockade with etanercept results in reduced
oxidative stress within the PVN of hypertensive rats.
In summary, chronic Ang II infusion resulted in cardiac hypertrophy and elevated
MAP, and within the PVN, an increased expression of PICs and markers of
oxidative stress. More importantly, Ang II-infused rats had an increased
expression of the injurious pro-hypertensive RAS components ACE and
AT<sub>1</sub>R, and a decreased expression of the protective anti-hypertensive
RAS components ACE2, the Mas receptor and AT<sub>2</sub> receptors. These
findings suggest that elevated TNF levels by Ang II hypertension are associated
with initiation of inflammatory cascade, which in turn, promote downstream
events and further raise blood pressure, leading to severe hypertension. Central
blockade of TNF with etanercept resulted in attenuation of hypertension, cardiac
hypertrophy and PIC expression, decreased oxidative stress, as well as a
restored the balance between the protective and deleterious axes of the RAS,
within the hypothalamic PVN. The beneficial effects of central TNF blockade in
Ang II-induced hypertensive responses appears to be mediated by the returned
balance of the central RAS components, especially within the PVN. It is
important to note, however, that due to the administration of etanercept ICV,
the TNF inhibitory effects may have impacted additional cardio-regulatory
regions in the brain and elicited a similar response as in the PVN, but in light
of its central integrative function versus the other regions, the PVN was of
utmost concern. Future studies should investigate these additional cardio-
regulatory regions, as well as looking more specifically at the pathway between
Ang II, TNF and the differential regulation of the RAS arms in the Ang II
hypertension-induced animal model. Our findings provide further evidence and
insight for the involvement of the RAS within the PVN and its interaction and
mediation through TNF in the neurogenic component of hypertension. Further
exploration of these system interactions within the brain may be beneficial
towards the development of novel hypertensive therapeutics.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: SS JF. Performed the
experiments: SS JC. Analyzed the data: SS. Contributed
reagents/materials/analysis tools: JF. Wrote the paper: SS. Reviewed the
manuscript: JC JF. |
# Introduction
Oxygen supply is essential for physiological functioning and metabolism in human
organs. Retinal neurons need more oxygen than other tissues, and abnormal
retinal oxygenation is thought to play a pivotal role in the pathogenesis of
various retinal diseases such as diabetic retinopathy, retinopathy of
prematurity, and retinal vein or artery occlusion. *In vivo* measurement of
retinal oxygen levels would be of help for the management of such diseases.
Recently, a non-invasive retinal oximeter Oxymap T1 has become commercially
available. Oxymap T1 is based on the principle that deoxyhemoglobin and
oxyhemoglobin have different light absorbance at specific wavelengths of light
(570 nm and 600 nm).
Using Oxymap T1, various investigators have reported the oxygen levels in the
major retinal vessels and studied the repeatability of the measurements in
healthy populations, mainly in Caucasians. In addition, recent studies have
reported the retinal oxygen concentration in eyes with retinal pigmentosa,
glaucoma, high myopia, or diabetic retinopathy. Longitudinal studies on retinal
oximetry would contribute to the early detection and management of such chronic
diseases. In diabetic patients, the level of oxygen saturation is reported to be
increased in the major retinal veins, as early as the stage of background
retinopathy or even no retinopathy. In eyes with retinitis pigmentosa, the
retinal venous oxygen saturation is correlated with the residual visual field.
However, previous studies have indicated that retinal oximetry may be influenced
by the flash intensity, face position, or the degree of fundus pigmentation. So
far, limited information is available on either the normative data for the
Japanese population or the reproducibility of retinal oximetry in the Japanese.
In addition, only one previous report investigated the intervisit
reproducibility of Oxymap T1, which is essential to set up and interpret in
longitudinal studies.
Thus, the purpose of the current study was to establish a normative database for
retinal oximetry using Oxymap T1 in a healthy Japanese population, and study the
reproducibility of the measurements in Japanese.
# Patients and Methods
The Ethics Committee at Kagawa University Faculty of Medicine approved this
prospective study, which was conducted in accordance with the tenets of the
Declaration of Helsinki. Written informed consent was obtained from each subject
before any study procedures or examinations were performed. This study is
registered in ClinicalTrials.gov (NCT02318641).
## Subjects
This prospective study consisted of 252 eyes with no ocular disease of 252
healthy Japanese subjects. Oxygen saturation in the major retinal vessels was
studied using Oxymap T1 (Oxymap ehf, Reykjavik, Iceland) at the Kagawa
University Hospital Department of Ophthalmology between December 2014 and June
2015. Exclusion criteria were as follows: keratoconus, high myopia (more severe
than -6 diopters), high astigmatism (more severe than ± 3 diopters), best-
corrected visual acuity worse than 20/25, prior intraocular surgery, or any co-
existing ocular disease (e.g., glaucoma, diabetic retinopathy, retinal vein or
artery occlusion, hypertension retinopathy, age-related macular degeneration,
retinal degenerative disease, or senile cataract that diminished image quality).
Patients who had suspected adverse effects to pupil dilation, diabetes mellitus,
severe cardiovascular or respiratory diseases, pregnancy, or collagen disease
were also excluded from the current study.
## Image acquisition for retinal oximetry
The principle of a commercially available retinal oximeter Oxymap T1 has been
described previously by other researchers. In brief, Oxymap T1 is composed of
two digital cameras, an image splitter, and two narrow band-pass filters and is
attached to a fundus camera (TRC-50DX, Topcon, Tokyo, Japan). Oxymap T1
simultaneously captures two fundus images at two different wavelengths of light
(570 nm and 600 nm). Light of 570 nm, which is isosorbetic to deoxyhemoglobin
and oxyhemoglobin, is insensitive to oxygen saturation, and light of 600 nm,
which is more sorbetic to oxyhemoglobin than deoxyhemoglobin, is sensitive to
oxygen saturation.
After a comprehensive medical interview, all subjects underwent a thorough
ocular examination, including autorefractometer, best-corrected visual acuity
measurement with a 5-m Landolt chart, slit lamp examination, intraocular
pressure measurement, dilated funduscopy, 50° digital fundus photographs, and
optical coherence tomography (Spectralis HRA+OCT; Heidelberg Engineering,
Heidelberg, Germany). When both eyes met the inclusion criteria, one eye was
selected randomly for the examination.
After mydriasis with 0.5% of tropicamide and 0.5% of phenylephrine hydrochloride
(Mydrin-P; Santen, Osaka, Japan), each included eye was examined with Oxymap T1
in a dark room. Setting of Oxymap T1 for image acquisition was as follows: flash
intensity was 50 W and small aperture and large pupil settings were applied to
the fundus camera. Fifty-degree fundus images were obtained centered at the
optic disc by using Oxymap T1. At each setting, more than 2 images were obtained
per eye.
## Oxygen saturation and vessel diameter measurement from acquired images
Fundus images acquired with Oxymap T1 were processed using a built-in software
Oxymap Analyzer (version 2.4.2, Oxymap Ehf). The software calculates optical
density of retinal vessels at two wavelengths (570 nm and 600 nm). The ratio of
the optical densities at these two wavelengths has an inverse and approximately
linear relationship with oxygen saturation. The resolution of Oxymap T1 is 9 μm.
In each acquired image centered at the optic disc, the oxygen saturation was
calculated in major retinal arteries and veins measuring more than 6 pixels in
vessel width in the measurement zone. The evaluators set analyzed vessel
segments in a standardized manner. For the analysis, vessel selection was done
with the disc centered in a 1.5-to 3-disc diameter area and 1 disc diameter away
from the disc margin to avoid uneven retinal background reflections near the
optic disc margin. All branches and vessel crossings within the measurement area
were also manually excluded from the analysis. After detailed selection of the
vessel section for the analysis, Oxymap Analyzer automatically measured the
levels of oxygen saturation and vessel diameter in each selected vessel.
## Reproducibility of retinal oximetry with Oxymap T1
Reproducibility of retinal oximetry was examined in 20 eyes of 20 healthy
subjects (15 men and 5 women; 5 right and 15 left eyes; mean age, 24.0 ± 1.7
years \[range, 23 to 29 years\]). To investigate interphotograph reproducibility
of retinal oximetry measurements, an evaluator (YN) performed the calculation of
the levels of oxygen saturation in each major retinal vessel with Oxymap
Analyzer, from two different photographs obtained by Oxymap T1 on the same day.
To investigate intervisit reproducibility of retinal oximetry measurements, 2
photographs were obtained by the same photographer at 2 different visits.
Photography was performed 1 week apart. Calculation of the levels of oxygen
saturation in each major retinal vessel was performed by the same evaluator
(YN). To investigate interevaluator reproducibility of retinal oximetry
measurements, two different evaluators (TS, YN) independently performed
calculation of the levels of oxygen saturation in each major retinal vessel from
a single photograph. The intraclass correlation coefficient (ICC) was used to
determine the reproducibility using oxygen saturation in a major retinal vessel
in each quadrant and the mean of all 4 major retinal arteries and veins.
## Statistical analysis
All data were collected in an Excel database (Microsoft Office 2010; Microsoft,
Redmond, WA). All statistical analyses were conducted using Software R (version
2.8.1; R Foundation for Statistical Computing, Vienna, Austria). The oxygen
saturation was calculated in each major retinal artery and vein in 4 quadrants,
the mean of values of 2 vessels in superior or inferior hemispheres, and the
mean of values of 4 vessels in all quadrants. Pearson’s correlation coefficient
was calculated to assess the relationship between age, refractive sphere, or
vessel diameter and the level of oxygen saturation. Stepwise forward
multivariate linear regression analyses were also performed to evaluate the
contribution that each initially identifiable factor made to retinal oximetry. A
p value \<0.05 was considered to be statistically significant.
# Results
In the current study, retinal oximetry was performed in 252 eyes of 252 healthy
Japanese subjects (137 men and 115 women). Mean age was 61.1 ± 18.8 years and
ranged between 20 and 93 years. Oxymap T1 allowed us to capture the
monochromatic fundus images at two wavelengths, and Oxymap Analyzer calculated
the oxygen saturation in each major retinal vessel. Mean oxygen saturation in 4
quadrants was 97.0 ± 6.9% in arteries and 52.8 ± 8.3% in veins. The mean
arteriovenous (A-V) difference in mean oxygen saturation was 44.2 ± 9.2%. shows
the oxygen saturation in each major retinal artery and vein in 4 quadrants, the
mean of values of 2 vessels in each hemisphere, and the mean of values of 4
vessels in all quadrants. Measurements of both arterial and venous oxygen
saturation were significantly lower in the temporal side of the retina,
especially in the temporal-inferior vessels. However, there were limited
differences in A-V difference of oxygen saturation in the 4 quadrants. shows the
mean oxygen saturation and mean vessel diameter in major retinal vessels in all
groups stratified by age.
Reproducibility of retina oximetry was examined in 20 eyes of 20 healthy
subjects. shows the ICCs in arterial and venous oximetry and A-V differences.
Interphotograph, intervisit, and interevaluator ICCs of the retinal oximetry in
one vessel were 0.891–0.970, 0.766–0.949, and 0.379–0.922, respectively.
Although interphotograph ICC was high, interevaluator ICC was relatively low.
However, the mean retinal oximetry in 4 quadrants had high ICCs between two
photographs (0.936–0.979), two visits (0.809–0.837), or two evaluators
(0.732–0.947).
and show the associations between the retinal oxygen saturation and age in
healthy subjects. In the major retinal arteries, oxygen saturation increased
with age (r = 0.18, p\<0.01). Arterial oxygen saturation increased by 0.67% per
10 years. However, the venous oxygen saturation showed no correlation with age
(r = -0.06). A-V difference in retinal oxygen saturation increased with age (r =
0.19, p\<0.01) by 0.92% per 10 years.
shows the correlation between refractive sphere, age, or vessel diameter and
oxygen saturation. There was no association between refractive sphere and oxygen
saturation in the major retinal vessels. Multivariate analysis showed that
arterial retinal saturation was significantly associated with age (β = 0.026, p
= 0.002) and vessel diameter (β = -0.16, p = 0.013). The A-V difference was
significantly associated with age (β = 0.19, p = 0.003).
# Discussion
In the current study, retinal oximetry was performed in 252 eyes of 252 healthy
Japanese subjects, which is the largest normative database of Oxymap T1 to date.
In a study on 120 Caucasians, Geirsdooier et al. reported that retinal
oxygen saturation was 92.2 ± 3.7% in arterioles and 55.6 ± 6.3% in venules, and
the A-V difference was 36.7 ± 5.4%. In a study on 118 Asians, Yip et al.
reported that the oxygen saturation was 93.64 ± 6.9% in retinal arterioles and
54.22 ± 6.9% in venules, and the A-V difference was 39.43 ± 8.9%. In a recent
study on 98 Indians, Mohan et al. reported that the oxygen saturation was
90.3 ± 6.6% in retinal arterioles and 56.9 ± 6.3% in venules, and the A-V
difference was 33.2 ± 5.2%. Thus, it is important to note that the results of
retinal oximetry with Oxymap T1 show variation across races, and this fact
should be kept in mind while obtaining and interpreting measurements.
We used the default formula of Oxymap Analyzer to calculate the retinal oxygen
concentration, which uses the mean oxygen saturation values obtained from
retinal vessels in healthy individuals by Schweitzer et al.. In their
measurement, the mean oxygen saturation was 92.2 ± 4.1% in retinal arteries and
57.9 ± 9.9% in retinal veins. In a recent study of Oxymap T1 by Ueda-Consolve et
al., the mean arterial and venous oxygen saturation values in 14 healthy
Japanese were 99.9 ± 8.9% and 54.6 ± 6.3%, respectively. We can estimate that
the A-V difference was 45.3%, which is relatively higher than that reported in
previous reports, which were mainly on Caucasians. Two fundus images captured at
two different wavelengths of light by Oxymap T1 may be influenced by the degree
of fundus pigmentation. Because the fundus of Japanese subjects is more
pigmented than that of Caucasians, this difference may account for the racial
variations in the measurements of retinal oximetry. We may need to establish a
calibration formula optimized for each race.
In the current study, measurements of both arterial and venous oxygen saturation
were significantly lower in the temporal hemisphere, especially in the temporal-
inferior quadrant. Geirsdooier et al., Mohan et al., and Palsson et
al. reported similar findings. The exact reason for this regional variation
is unclear. Indeed, venous oxygen concentration may be influenced by retinal
metabolism. However, arterial oxygen concentration should not show such a great
variation because all retinal arteries originate from a single central retinal
artery. Rather, this variation may be explained by the measurement error,
perhaps on the photographs by Oxymap T1, because venous oxygen saturation was
also lower in the temporal hemisphere and the A-V difference was almost canceled
out. Recently, Mohan et al. reported a strong negative correlation between
calculated oxygen saturations measured using Oxymap T1 and peripapillar retinal
nerve fiber thickness, which is large in the temporal hemisphere. This finding
would support our current finding that both arterial and venous oxygen
saturation were low in the temporal hemisphere. We measured oxygen saturation in
all 4 quadrants. To minimize the error in photography, it would be most
appropriate to calculate the mean of the values in all quadrants.
In the current study on a healthy Japanese population, interphotograph,
intervisit, and interevaluator ICCs for retinal oximetry in one vessel ranged
from 0.891 to 0.970, from 0.766 to 0.949, and from 0.379 to 0.922, respectively.
Interphotograph ICC in our measurement was high and Palsson et al., Goharian
et al., and Yip et al. also reported high interphotograph (intravisit)
reproducibility. In addition, O'Connell et al. reported relatively high
intervisit reproducibility of retinal oximetry with Oxymap T1. However, it is
suggested that pupil size or face position may influence the measurements in
retinal oximetry. This would account for the slightly lower intervisit ICC in
the current population, compared with interphotograph ICC. In the current study,
interevaluator ICC was lower than interphotograph or interevaluator ICCs. In a
study by Yip et al., intergrader (interevaluator) ICC was 0.77–0.94.
Although Oxymap Analyzer has a built-in, semiautomatic software to calculate the
oxygen concentration, the selection of the vessel segment depends on the
evaluator. In addition to the standardization of retinal image capture by Oxymap
T1, the standardization of analysis with Oxymap Analyzer is also essential to
maximize the reproducibility of retinal oximetry.
In our subjects, while arterial oxygen saturation increased with age, venous
oxygen saturation showed no correlation with age. The A-V difference increased
with age. The age-related changes in retinal oxygen saturation are still
controversial. In the Indian population, it has been reported that both arterial
and venous retinal oxygen saturation increase with age. In a previous study on a
multiethnic population, both arterial and venous retinal oxygen saturation
decreased with age, resulting in an increase in A-V difference. Various factors
including the decrease in pupil dilation, cataract formation, decrease in the
vessel caliber, and atrophy of the retinal pigment epithelium or choroid may be
involved in this discrepancy. Judging from the results of previous reports and
those of the current study, longitudinal changes in individual retinal oximetry
may be small. However, these changes may be crucial in the comparisons of
retinal oximetry between different generations.
One of the major limitations of our study is its cross-sectional nature. Based
on a large healthy population, we investigated the age-dependent changes in
retinal oximetry. However, a long follow-up study would be necessary to
investigate the longitudinal changes in retinal oximetry. In addition, we
excluded patients with severe cardiovascular or respiratory diseases but
subjects with smoking history or systemic hypertension were included. History of
controlled systemic hypertension is reported to be associated with an increased
A-V difference in retinal oxygen saturation. In addition, the mean age of the
subjects recruited in the reproducibility test was substantially lower than that
of the total study population. Subject selection may have had some influence on
the measurement value and its reproducibility.
In spite of these limitations, we constructed a database of retinal oximetry for
healthy Japanese. Although interphotograph repeatability was high,
interevaluator and intervisit repeatability was relatively low. However, mean
retinal oximetry in the 4 quadrants may contribute to higher reproducibility of
measurements. Since the results of retinal oximetry show some variation across
races, we might need to establish a calibration formula optimized for each race.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: YN AT. Performed the
experiments: YN TS NK YM AO MK CS KH AT. Analyzed the data: YN TS.
Contributed reagents/materials/analysis tools: YN AT. Wrote the paper: YN
AT. |
# Introduction
RNA silencing is an ancient network of highly related pathways that repress gene
expression in eukaryotic organisms by means of small regulatory RNAs. The
mechanism is triggered by dsRNA and specifically targets any related RNA (post-
transcriptional pathways) or DNA (transcriptional gene silencing). Key steps in
the process are: 1) the dsRNA trigger is cut into 21–24 nucleotide (nt) short-
interfering (si) RNA duplexes by a ribonuclease III-like enzyme termed Dicer, 2)
one strand of the siRNA duplex associates with an argonaute-like protein to form
the core of a silencing effector complex, and 3) the siRNA directs the complex
to complementary genetic elements. In post-transcriptional gene silencing, the
complex targets mRNA, which is then cleaved by the ribonuclease-H activity of
argonaute (AGO).
Higher plants, *C. elegans*, and fungi additionally amplify silencing via
transitivity, a pathway that produces dsRNA and additional siRNAs from the
targeted mRNA. Production of dsRNA in transitive silencing depends on cellular
RNA-dependent RNA polymerase (RDR) activity, and the siRNAs generated are termed
secondary siRNAs, while those that derive from the initial dsRNA trigger are
termed primary siRNAs. In plants, production of both primary and secondary
siRNAs entails cleavage of dsRNA by the Dicer-like (DCL) family of enzymes. In
plants, therefore, transitivity not only increases the siRNA population, but
also degrades the target mRNA in the process of making secondary siRNAs, thereby
amplifying silencing in two ways. Interestingly, secondary siRNA production
itself is not a mechanism of target degradation in *C. elegans* because
antisense secondary siRNAs are transcribed directly from the target mRNA
template in that organism. The importance of transitive silencing has recently
come to light in studies in *C. elegans* showing that the vast majority of
siRNAs that accumulate during RNA silencing in that organism are secondary
siRNAs. Transitive silencing is also important in plants, where it is essential
for some types of transgene-induced silencing and thought to play a key role in
defense against viruses.
Although RNA silencing was initially considered simply a novel defense mechanism
against viruses and other invading nucleic acids, the subsequent discovery of
endogenous small regulatory RNAs led to the realization that it is a fundamental
genetic regulatory mechanism in eukaryotic organisms. There are three major
classes of endogenous small regulatory RNAs in plants: micro (mi) RNA, trans-
acting (ta) siRNA, and heterochromatin-associated (hc) siRNA. The biogenesis and
mechanism of action of the different silencing-associated small regulatory RNAs
are highly related; however, hc-siRNAs suppress transcription as opposed to
mediating RNA degradation and, in animal systems, miRNAs primarily repress
translation. Many of the enzymes involved in RNA silencing are encoded by
multigene families, allowing for the possibility of diverse, specialized
pathways. In *Arabidopsis*, there are four *DCL*, ten *AGO*, and six *RDR*
genes. The roles of the four *Arabidopsis DCL* genes in small RNA biogenesis and
silencing have been the subject of intense study, and the picture that has
emerged is one of primary roles for many of the enzymes, plus functional
redundancy. Thus, *DCL1* is required for the biogenesis of 21-nt miRNAs, and
*DCL3* is responsible for the biogenesis of 24-nt hc-siRNAs. *DCL4* seems the
most versatile, being required for production of 21-nt ta-siRNAs, which derive
from *RDR6*-generated dsRNA, as well as for production of 21-nt
*RDR6*-independent siRNAs from hairpin transgenes, in which case dsRNA can be
produced directly by transcription of an engineered inverted repeat. *DCL4* also
plays a primary role in production of siRNAs from viruses, with *DCL2* as the
substitute if *DCL4* has been inactivated. Although *DCL2* has been reported to
produce an endogenous siRNA from a convergently transcribed and overlapping gene
pair, it is otherwise considered to play a subordinate and redundant role in
siRNA biogenesis. Another possibility, however, is that the primary role of
*DCL2* has simply not yet been identified.
With this idea in mind, we undertook a systematic study of the impact of
mutations in *DCL* genes and the effect of viral silencing suppressors on siRNA
biogenesis in *Arabidopsis thaliana* carrying different types of silenced
transgene. Our experiments focused on three general cases: 1) sense transgene-
induced silencing, in which case a construct that was designed to express a
reporter gene becomes post-transcriptionally silenced instead, 2) hairpin
transgene-induced silencing of a sense transgene, in which case a transgene that
produces a self-complementary transcript is used to target an expressing,
homologous sense transgene, and 3) processing of the hairpin transcript itself
(hairpin transgene self-silencing). We expected that these cases might differ in
their *DCL* requirements because sense transgene-induced silencing requires a
set of genes that are not required for hairpin-induced silencing of transgenes.
Some of these genes are thought to mediate production of dsRNA from the sense
transgene transcript and include *RDR6*, *AGO1,* and *SGS3*–which encodes a
coiled-coiled domain protein of unknown function, while *HEN1* encodes a
methylase that acts downstream of dsRNA and stabilizes siRNAs.
The transgenes we used differ in several important respects from those in other
studies. First, no other study to date has analyzed the *DCL* requirements for
sense transgene-induced silencing. The exact nature of the silencing trigger is
still unknown in this type of silencing, but it is thought that the locus gives
rise to an aberrant transcript, which might lack normal 5′ or 3′ ends. The
aberrant transcript becomes a template for RDR6, which synthesizes the
complementary strand and produces dsRNA. Second, with respect to hairpin
transgene-induced silencing, the loop portion of the hairpin construct we used
is retained in the mature transcript and has sequence identical to that of the
targeted sense transgene. Thus, this hairpin construct is unusual in that it has
a region that requires *RDR6* to produce dsRNA in addition to one that can
directly produce dsRNA, and siRNAs from both regions can target the sense
transgene. Furthermore, although hairpin transgene-induced silencing has been
the subject of numerous studies, no other study has examined the fate of the
hairpin transcript itself and the *DCL* requirements for self-silencing of the
hairpin. Lastly, we used transgenes driven by the ubiquitously expressed
cauliflower mosaic virus (CaMV) 35S promoter. Consequently, silencing occurs
throughout the plant without the need for a silencing signal.
In all three cases of silencing we examined, accumulation of secondary siRNAs
required *DCL2* but not *DCL4*, providing evidence that DCL2 plays an essential,
as opposed to redundant, role in transitive silencing of transgenes. This result
strongly suggests that there are natural targets of silencing for which DCL2
plays a primary role. Furthermore, we found that viral suppressors of silencing
and a *dcl2* null mutation had similar effects on accumulation of secondary
siRNAs, supporting a primary role for *DCL2* in antiviral defense.
# Results
## *DCL2* but not *DCL4* is Required for Sense Transgene-induced Silencing
To study sense transgene-induced silencing, we used the L1 transgenic line,
which carries a direct repeat of the T-DNA insert and is silenced for the *uidA*
gene encoding β-glucuronidase (GUS). Silencing of the GUS transgene in line L1
initiates at about the time the plant bolts and is characterized by very low
accumulation of GUS mRNA and concomitant accumulation of GUS siRNAs that are
*RdR6*-dependent and 21- and 22-nucleotides (nt) in length (lanes 5–6 and 11–12;
). These siRNA size classes have previously been attributed to the activity of
DCL4 and DCL2, respectively. About equal amounts of the two size classes are
detected using a probe to the 3′-most region of the transcript, whereas only
21-nt siRNAs are detected with a probe to the central region, and no siRNAs of
any size class can be detected using a probe to the 5′-most region (lanes 5–6
and 11–12). This drop-off in accumulation of siRNAs at the 5′ end of the L1 GUS
transcript was not seen in an earlier study , possibly because the 5′ probe used
by that group extended 258-nt further 3′ than ours.
To identify which *DCL* genes are required for sense transgene-induced
silencing, we crossed the L1 line with *dcl* mutant lines and examined the
accumulation of GUS mRNA and siRNAs in F2 progeny that were homozygous for the
*dcl* mutation and also carried at least one copy of the L1 GUS transgene. The
post-transcriptional silencing induced by a single copy of the L1 GUS locus is
comparable to that observed in plants homozygous for the locus (compare lanes 13
and 14). An L1 GUS line that expressed the P1/HC-Pro viral suppressor of
silencing was used as a positive control showing expression of the GUS
transgene. The *dcl2* and *dcl3* mutant lines were null mutants generated by
T-DNA insertion, whereas the *dcl1* mutant lines were partial loss of function
mutants. The *dcl4* mutant lines had point mutations in *DCL4* that eliminated
or greatly reduced accumulation of the 21 nt species of ta-siR255 (lanes 3–4 and
9–10;). Because 21 nt ta-siRNAs are produced by DCL4, this result confirms that
these *dcl4* mutant lines are highly deficient for DCL4 activity. All F2 progeny
homozygous for the *dcl3* mutation were transcriptionally silenced for GUS,
whether they carried one or two copies of the GUS transgene (Mlotshwa and Vance,
manuscript in preparation), preventing examination of the role of *DCL3* in
sense transgene-induced silencing in this system. Transcriptional silencing
could be avoided, however, in the *dcl2* mutant by using progeny that were
hemizygous for the GUS transgene: Run-off transcription assays showed that
whereas *dcl2* mutant progeny homozygous for the L1 locus were transcriptionally
silenced for GUS, ones hemizygous for the L1 locus were not. The transcriptional
silencing induced by T-DNA insertion mutations is likely due to the CaMV 35S
promoter in the engineered T-DNA. This propensity of the L1 locus to become
transcriptionally silenced in the presence of additional copies of the CaMV
promoter highlights the importance of examining siRNA accumulation before
concluding that absence of GUS mRNA or activity indicates that the locus is
post-transcriptionally silenced. Transcriptional silencing was not a problem in
the *dcl1* or *dcl4* mutant lines, which were not T-DNA insertion mutants.
Silencing of the L1 GUS transgene was severely impaired in *dcl2* mutant plants,
as shown by greatly increased accumulation of GUS mRNA and the absence of GUS
siRNAs (compare lanes 14, 16, and 17). Thus, *DCL2* plays a primary role in this
*RDR6*-dependent silencing pathway. In contrast, silencing of the L1 GUS
transgene was enhanced in both *dcl4-2* and *dcl4-10* mutant plants, as shown by
reduced accumulation of GUS mRNA compared to wild type plants (lanes 3–6 and
9–12). Thus, although DCL4 activity produced the major fraction of GUS siRNAs
that accumulated in the wild type background, neither *DCL4* nor
*DCL4*-dependent 21-nt siRNA is required for sense transgene-induced silencing.
The enhancement of silencing seen in the *dcl4* mutants might indicate some
inhibitory role of *DCL4* or an earlier onset of silencing due to the
accelerated juvenile-to-adult transition seen in these mutants. The *dcl1*
mutant plants were slightly impaired for silencing of L1 GUS (lanes 18–20),
suggesting that *DCL1* plays a facilitating role in sense transgene-induced
silencing like that reported for hairpin transgene-induced silencing of an
endogenous gene.
Although accumulation of GUS mRNA in the *dcl2* mutant plants is much greater
than in wild type, it is considerably less than seen in plants expressing P1/HC-
Pro (lanes 14–17). Part of this differential is likely a gene dosage effect due
to the fact that the P1/HC-Pro line is homozygous for L1 GUS, whereas the *dcl2*
mutant is hemizygous for the transgene. The remaining differential might
indicate that absence of DCL2 does not completely eliminate sense transgene-
induced silencing, but only reduces it. Finally, the absence of GUS siRNAs of
all size classes in the *dcl2* mutant plants suggests that DCL2 or the 22-nt
class of siRNAs it generates is necessary for DCL4 and other DCL enzymes to be
active on this type of substrate.
## *DCL2* but not *DCL4* is Required for Transitivity in Self-silencing of a Hairpin Transgene
Our initial studies of hairpin transgene-induced silencing used northern
analysis to examine the fate of the hairpin transcript itself. The self-
complementary ΔGUS-SUG transgene in line 306-1 consists of the GUS coding
sequence with a 231-nt deletion after nucleotide 558 and an inverted duplication
of the 5′ proximal 558-nt at the 3′ end. The 558-nt self-complementary regions
form the stem of the hairpin, while the intervening GUS sequence forms the loop.
Unlike hairpin transgene constructs that have an intron separating the self-
complementary regions, the loop portion of the ΔGUS-SUG transgene is not
eliminated from the mature transcript. As a result, it is possible to examine
siRNA biogenesis not only from the stem of the hairpin, which can pair to form
dsRNA, but also from the loop, which requires the action of an RDR to generate
dsRNA.
Not surprisingly for a transgene that is a strong inducer of silencing, very
little full-length ΔGUS-SUG transcript accumulated in line 306-1 (, lanes 1–4,
band labeled hp). Unexpectedly, however, significant quantities of a smaller RNA
species accumulated. This species could be detected with a probe specific for
the loop (lanes 3–4) but not with one specific for the stem (lanes 1–2), showing
that it corresponded to the loop portion of the hairpin transcript. Its size, as
determined by migration in agarose gels, corresponds to that of the entire loop
(data not shown). The presence of GUS siRNAs confirms that the ΔGUS-SUG
transgene in line 306-1 is silenced (lanes 3–4). Preferential accumulation of
the loop mRNA, however, indicates that degradation of the hairpin transcript
occurs mainly via processing of the stem of the hairpin. Degradation of the loop
portion of the transcript, which requires transitive silencing, apparently does
not occur very effectively in this system. Analysis of ΔGUS-SUG mRNA and siRNA
accumulation in an *rdr6* mutant backgound confirms that elimination of the
full-length ΔGUS-SUG transcript and accumulation of siRNAs from the stem of the
hairpin are *RDR6*-independent, whereas accumulation of siRNAs from the loop is
*RDR6*-dependent: Loop mRNA, but little full-length ΔGUS-SUG transcript,
accumulates in *rdr6* mutant plants, as in wild type; however, siRNAs from the
loop are eliminated by the *rdr6* mutation, while those from the stem are
largely unaffected (lanes 1–2). Thus, as expected, siRNAs from the stem are
primary siRNAs, while those from the loop are secondary siRNAs and part of the
transitive silencing pathway. The increased accumulation of GUS loop mRNA in the
*rdr6* mutant compared to wild type provides additional confirmation that
degradation of the loop occurs via transitive silencing.
The ΔGUS-SUG primary siRNAs are predominantly 21-nt (lanes 1–4), suggesting that
DCL4 is normally responsible for processing the dsRNA stem of the hairpin. In
contrast, secondary siRNAs from the hairpin transcript are approximately equal
parts 21- and 22-nt (lanes 3–4), as seen for the 3′ end of the L1 GUS sense
transgene. To examine the roles of *DCL2* and *DCL4* in self-silencing of the
ΔGUS-SUG transcript, we crossed line 306-1 with *dcl2* and *dcl4* mutant lines
and examined F2 progeny homozygous for the *dcl* mutation. The hairpin transgene
remains silenced in both *dcl* mutants, as indicated by the presence of GUS
siRNAs and little accumulation of full-length ΔGUS-SUG mRNA (lanes 11–13). In
the *dcl4* mutant, however, accumulation of loop mRNA is nearly eliminated
(lanes 12–13), suggesting that impairing DCL4 activity promotes transitive
silencing of the hairpin transcript. Enhanced accumulation of loop siRNAs and
elimination of the majority of primary siRNAs in the *dcl4* mutant compared to
wild type (lanes 12–13) are consistent with a shift to transitive silencing in
the mutant. Increased accumulation of full-length ΔGUS-SUG mRNA in the *dcl4*
mutant compared to wild type (lanes 12–13) indicates that the reduction in loop
mRNA accumulation in the mutant is not due to increased non-specific
degradation. Furthermore, it suggests that transitive silencing of the hairpin
transcript is not as effective as the *DCL4*-dependent pathway that normally
eliminates the stem.
In contrast to the shift to transitive silencing produced by the *dcl4*
mutation, the *dcl2* mutation impairs transitivity: No loop siRNAs accumulate in
the *dcl2* mutant (lanes 5–8). Because loop siRNAs are RDR6-dependent secondary
siRNAs (lanes 1–4), this result implies that *DCL2* is required for transitive
self-silencing of the hairpin transgene. In addition, accumulation of stem
siRNAs, which are predominantly 21-nt, is reduced in the *dcl2* mutant (lanes
5–8), suggesting that DCL2 facilitates DCL4 production of primary siRNAs or
enhances their stability. The *dcl2* mutation blocks transitive silencing even
in a *dcl4* mutant background: Loop siRNAs are absent and high levels of loop
mRNA accumulate in *dcl2 dcl4* double mutant plants (lanes 9–10), indicating
that degradation of the hairpin transcript occurs mainly via processing of the
dsRNA stem of the hairpin in the absence of both *DCL2* and *DCL4*. Thus, the
shift to transitive silencing observed in *dcl4* mutant plants requires *DCL2*.
Processing of the dsRNA stem of the hairpin transcript in the *dcl2 dcl4* double
mutant presumably involves DCL3, DCL1 or residual low-level DCL4 activity, as
24-nt and a small amount of 21-nt primary (stem) siRNAs accumulate in these
plants (lanes 9–10).
Altogether, these results show that *DCL2* is required for production of
*RDR6*-dependent siRNAs from a hairpin transgene as well as from a sense
transgene. In addition, self-silencing of the hairpin transcript provides
another example of the hierarchical action of the DCL proteins and reveals that
different DCL proteins are preferentially associated with different mechanisms
of transcript degradation. In wild type plants, the major pathway for
degradation of the ΔGUS-SUG hairpin transcript appears to be *DCL4*-dependent
processing of the stem into primary siRNAs. In a *dcl4* mutant, transitive
silencing is prominent and involves *DCL2*- and *RDR6*-dependent production of
secondary siRNAs. When both *DCL2* and *DCL4* are defective, degradation again
occurs via processing of the stem into primary siRNAs, but this time involves
*DCL3* and possibly *DCL1*.
## *DCL2* but not *DCL4* is required for transitivity in hairpin transgene-induced silencing of a sense transgene
To analyze hairpin transgene-induced silencing of a sense transgene, we used the
ΔGUS-SUG locus in line 306-1 to target an expressing GUS locus. Line 6b4
carries a GUS sense transgene that is not silenced, and 6b4 plants accumulate
high levels of GUS mRNA but no GUS siRNAs (lane 5). However, the GUS-expressing
locus in line 6b4 is silenced in the presence of the ΔGUS-SUG construct, as
evidenced by the loss of GUS activity. For our experiments, therefore, we used a
6b4/306 transgenic line obtained by crossing the 6b4 and 306-1 lines. The full-
length GUS and ΔGUS-SUG transcripts are similar in size, but the ΔGUS-SUG
sequence is 327-nt longer.
Northern analysis shows that the 6b4/306 line is silenced for both transgenes
because little or no full-length transcript from either one accumulates, but
loop mRNA from the hairpin transcript and GUS siRNAs do (lanes 3–4). To detect
GUS siRNAs in these experiments, we used the same stem and loop probes (1 and 3,
respectively) as in the previous section, but also included probe 2, which
corresponds to the region deleted in the ΔGUS-SUG construct. In line 6b4/306,
probes 1 (stem) and 3 (loop) can detect siRNAs arising from either the ΔGUS-SUG
transgene or the GUS sense transgene, while probe 2 is specific to the GUS sense
transgene. Accumulation of the GUS siRNAs detected by probes 2 and 3 in 6b4/306
plants was eliminated in the *rdr6* mutant background (lanes 1–2), confirming
that these are *RDR6*-dependent secondary siRNAs and part of the transitive
silencing pathway. Accumulation of the GUS siRNAs detected by probe 1, however,
was not reduced by the *rdr6* mutation (lanes 1–4), showing that even in the
presence of the GUS sense transgene, these are mostly primary siRNAs from the
stem of the hairpin transcript. This observation plus the accumulation of loop
mRNA suggests that hairpin transgene-induced silencing of the GUS-sense
transgene in line 6b4/306 does not have a large transitive component. Indeed,
silencing of the GUS-sense transgene in line 6b4/306 is largely unaffected by
the *rdr6* mutation, as little or no full-length GUS mRNA accumulates in the
*rdr6* mutant plants (lanes 1–2), consistent with earlier work showing that
hairpin transgene-induced silencing is *RDR6*-independent.
To examine the roles of *DCL2* and *DCL4* in ΔGUS-SUG-induced silencing of the
GUS sense transgene, we performed crosses to make homozygous *dcl2*, *dcl4*, and
*dcl2 dcl4* mutant progeny of line 6b4/306. The effects of the *dcl* mutations
on silencing of the GUS sense transgene in these lines was very similar to their
effects on self-silencing of the hairpin transgene shown above for lines
carrying the hairpin locus alone. In the 6b4/306 background, the *dcl4* mutation
eliminated accumulation of loop mRNA and greatly increased accumulation of
secondary siRNAs–including those (probe 2) that could be derived only from the
GUS sense transgene transcript–providing additional evidence that impairing DCL4
activity promotes a shift to transitive silencing (, lanes 11–14, probes 2 and
3). The highly abundant secondary siRNAs in the *dcl4* mutant included 24-nt as
well as 22-nt siRNAs, showing that both DCL3 and DCL2 produce secondary siRNAs
from the targeted sense transgene when DCL4 is defective. The much greater
increase in secondary siRNA accumulation caused by the *dcl4* mutation in lines
carrying both transgenes than in those having the hairpin locus alone suggests
that the GUS sense transgene transcript is a much better substrate for
*RDR6*-dependent production of siRNAs than the hairpin transcript.
In contrast to *dcl4* enhancement of secondary siRNA accumulation, the *dcl2*
mutation eliminated secondary siRNAs–including those (probe 2) that could be
derived only from the GUS sense transgene mRNA (lanes 6–9, probes 2 and 3).
Accumulation of siRNAs detected by probe 1, which are mostly primary siRNAs, was
not greatly affected (lanes 6–9). The *dcl2* mutation also eliminated
accumulation of secondary siRNAs in the *dcl4* mutant background (compare lanes
16–19, probes 2 and 3) and restored accumulation of loop mRNA (lanes 16–20).
Thus, *DCL2* is required for transitive silencing of the sense transgene target
of a hairpin transgene, as well as for transitive silencing of the hairpin
itself.
The above results provide a basis for understanding the robust nature of hairpin
transgene-induced post-transcriptional silencing. Whereas a sense transgene
alone activates only an *RDR6*-dependent silencing pathway, a hairpin transgene
activates multiple silencing pathways–including one(s) involving only
*RDRP*-independent primary siRNAs. Consequently, in the presence of a homologous
hairpin transgene, the sense transgene becomes a target for all the silencing
pathways activated by the hairpin construct.
## The P1/HC-Pro and P38 viral suppressors block hairpin transgene-induced silencing of a sense transgene, but do not block processing of the stem of the hairpin transcript into primary siRNAs
Previous studies in our laboratory showed that the tobacco etch virus (TEV)
P1/HC-Pro viral suppressor of silencing altered the accumulation of siRNAs in
tobacco, eliminating those derived from sense transgenes and shifting the size
distribution of ones derived from inverted repeat and amplicon transgenes. To
determine the effect of P1/HC-Pro and other viral suppressors on siRNA
biogenesis in the three cases of silencing examined above, we crossed the L1,
306-1, and 6b4/306 lines with *Arabidopsis* lines transgenic for P1/HC-Pro (from
turnip mosaic virus) or P38 (from turnip crinkle virus). The P38 viral
suppressor carried a C-terminal HA epitope tag, whereas P1/HC-Pro had no tag. In
all cases, progeny carrying a viral suppressor transgene exhibited the
developmental phenotype associated with expression of the suppressor in the
parental line (data not shown).
Both of these suppressors restored accumulation of GUS mRNA and eliminated
accumulation of GUS siRNAs in progeny of crosses with the sense transgene
silenced line L1, similar to the effect of TEV P1/HC-Pro on sense transgene-
induced silencing in tobacco. Neither suppressor, however, enabled accumulation
of high levels of the full-length ΔGUS-SUG transcript in progeny of crosses with
the hairpin-transgenic line 306-1, although accumulation of the full-length
transcript was increased (lanes 1–5). In contrast to the full-length transcript,
high levels of loop mRNA accumulated in both the P1/HC-Pro and P38 lines (lanes
1–4). Thus, P1/HC-Pro and P38 have little or no inhibitory effect on processing
the dsRNA stem of the hairpin, but they suppress the secondary siRNA-dependent
pathway responsible for degradation of the loop. Consistent with this
interpretation, P1/HC-Pro and P38 reduced accumulation of secondary (loop)
siRNAs (lanes 1–5). Interestingly, the suppressors also increased accumulation
of primary (stem) siRNAs (lanes 1–5). The relatively minor accumulation of full-
length transcript in the P1/HC-Pro and P38 lines suggests that although most
ΔGUS-SUG transcripts are degraded via processing of the dsRNA stem into primary
siRNAs, some are degraded as a result of being targeted by primary siRNAs and
that P1/HC-Pro and P38 block this latter pathway. The increase in primary siRNA
accumulation in the viral suppressor lines compared to wild type plants is
consistent with the idea that some full-length hairpin transcripts are degraded
by transitive silencing in wild type plants, thereby reducing production of
primary siRNAs. Alternatively, the suppressors might increase primary siRNA
stability, perhaps by binding siRNA duplexes.
In progeny of crosses with the 6b4/306 line, both P1/HC-Pro and P38 restored
accumulation of full-length mRNA from the GUS sense transgene and eliminated
secondary siRNAs (probes 2 and 3) (lanes 3–8). In addition, both suppressors
enhanced accumulation of loop mRNA and primary siRNAs. The enhancement of loop
mRNA accumulation by P38 was so great that loop mRNA in wild type plants was
barely detectable at the exposure appropriate for the samples from
P38-expressing plants (lanes 7–9), although it was clearly visible at a 3-fold
longer exposure (, image of lanes 7–9 labeled “loop 3x exp”). These results are
consistent with the effects of the suppressors on secondary siRNA-dependent
silencing seen with the hairpin transgene alone (lanes 1, 2, 9). Hybridizing a
duplicate high molecular weight RNA blot with probes specific to the viral
suppressors confirmed that both suppressors were being expressed (panels in
lanes 1–6 directly below rRNA).
The P38 viral suppressor produced several very interesting effects in the above
experiments. P38 transgenic plants accumulated much higher levels of loop mRNA
than P1/HC-Pro plants (lanes 1–4 and 4B, lanes 3–6), suggesting that P38 is much
more effective than P1/HC-Pro at preventing degradation of the loop. P38 also
had a more pronounced effect than P1/HC-Pro on the size distribution of primary
siRNAs, causing the accumulation of a closely spaced smaller species (lanes
5–6), suggesting that altering the processing of small RNAs is part of the P38
mechanism of action.
Some of our viral suppressor results differ from those of another group. P1/HC-
Pro inhibition of ta-siRNA accumulation and P38 inhibition of DCL4 activity is
not evident in our lines (siR255 probe; primary siRNAs). Conversely, the
enhancement of primary siRNA accumulation by P1/HC-Pro and P38 that we observe
in hairpin transgene-induced silencing was not detected by the other group. We
expect that differences in the plant lines as well as in the inducers and
targets of silencing used by the two groups are likely responsible for such
discrepancies. Consistent with this expectation and in agreement with our
result, an independent group using our plant line observed that P1/HC-Pro does
not block ta-siRNA accumulation. It is also interesting to note that P1/HC-Pro
enhancement of siRNA accumulation has previously been observed for a hairpin
promoter sequence construct that induced transcriptional silencing in tobacco.
# Discussion
The present work demonstrates that *DCL2* is required for silencing induced by a
sense transgene locus and for accumulation of secondary siRNAs in three
mechanistically different examples of transgene silencing. Surprisingly, even
*DCL4*-dependent 21-nt and *DCL3*-dependent 24-nt secondary siRNAs are
eliminated by a *dcl2* null mutation, suggesting that DCL2 is required for DCL4
and DCL3 participation in production of *RdR6*-dependent siRNAs from these
transgenes. Our results show that DCL2 plays a primary role in transitive
silencing of transgenes and suggest that there are as-yet-unidentified natural
substrates for which DCL2 is the primary DCL enzyme. One possible origin of
natural substrates of DCL2 is foreign genes that are introduced into the plant
genome during infection with pathogens like *Agrobacterium tumefaciens*. Such
genes constitute a natural analogue of transgenes. In addition, our observation
that a *dcl2* null mutation and two viral suppressors of silencing have the same
effect on secondary siRNA accumulation suggests that natural substrates of DCL2
might be produced in viral infection. The similarity in effect of the *dcl2*
mutation and the viral suppressors applies only to accumulation of secondary
siRNAs, however, and not to suppression of silencing in general: The suppressors
eliminated both hairpin and sense transgene-induced silencing, whereas the
*dcl2* mutation impaired only the latter.
Although DCL2 was initially proposed to play an antiviral role, subsequent
studies suggested that the enzyme functions in only a subordinate and redundant
capacity in antiviral defense. A key piece of evidence for this conclusion is
that 22-nt viral siRNAs accumulated primarily when DCL4 was inactive. Those
studies, however, either involved only primary siRNAs or did not distinguish
between primary and secondary siRNAs. In contrast, our results point to a
requirement for *DCL2* specifically in the production of secondary siRNAs.
*RDR6*-dependent production of secondary siRNAs is thought to be particularly
important in slowing the systemic spread of viruses by allowing systemically
invaded cells to respond before the virus starts replicating. After replication
is established, however, viral siRNA production no longer depends on *RDR6*,
suggesting that any specific requirement for *DCL2* in antiviral defense might
be transient and not detectable in bulk infected tissue.
The inhibition of transitivity by P1/HC-Pro and P38 provides additional evidence
for the importance of this *RDR6*-dependent branch of silencing in antiviral
defense. However, suppression of silencing by these viral proteins must involve
more than inhibition of transitivity because they suppress hairpin transgene-
induced silencing, which does not require *RDR6*. One possibility is that viral
suppressors also inhibit siRNA function by binding to siRNA duplexes. It will be
interesting to determine whether inhibition of transitivity results from the
dsRNA binding activity of the suppressors or reflects some additional activity.
Small RNA pathways have been shown to involve functional modules of specific
gene family members, and modules can act alone or sequentially. Thus, *DCL1*
and *AGO1* are involved in the biogenesis and function of miRNAs, while *RDR2*,
*DCL3*, and *AGO4* are involved in that of hc-siRNAs. *RDR6* and *DCL4*
constitute a module that functions in the biogenesis of ta-siRNAs and works
downstream of miRNA-directed cleavage. Our results suggest that *DCL2* and
*RDR6* also constitute a module in the case of some substrates. These substrates
differ from ones previously identified for the *DCL4/RDR6* module in that
neither DCL4 nor DCL3 appears to process them in the absence of DCL2, perhaps
due to unique structural features of the substrates or their localization in the
cell. One possibility is that DCL2 might be required upstream of dsRNA
production to recognize the substrates and/or recruit RDR6, after which DCL2 and
other DCL enzymes produce siRNAs from the resultant dsRNA. Because the majority
of secondary siRNAs in wild type plants are 21-nt for all three cases of
silencing we examined, DCL4 appears to be overall the most active one of the
enzymes on the *RDR6*-dependent dsRNA substrates produced by transgenes.
Alternatively, 21-nt siRNAs might simply be the most stable. For all of three
transgenes, however, 21-nt and 22-nt secondary siRNAs are equally abundant at
the 3′ end of the GUS sequence, consistent with a specific requirement for
*DCL2* early in the *RDR6*-dependent arm of transgene silencing. The
processivity of DCL4 appears to be greater than that of DCL2 on these substrates
because only 21-nt siRNAs accumulate from the middle region of the GUS sequence.
This difference in processivity of the two enzymes might be at least partially
responsible for the perception that DCL2 plays a purely subordinate role. The
reduced accumulation of loop mRNA and primary siRNA in *dcl2* mutant plants
suggests that DCL2 might also stabilize its substrates against non-silencing
related nucleolytic degradation.
Because transitivity and sense transgene-induced silencing–like ta-siRNA
biogenesis-require *RDR6*, DCL4 has been considered the likely DCL enzyme for
siRNA production in sense transgene silencing. Moreover, siRNA production from a
silenced sense transgene that was engineered to have a miRNA cleavage site was
shown to utilize the *DCL4/RDR6* module after miRNA cleavage and not to require
*DCL2*, providing support for the expected role of DCL4 in transitivity and
sense transgene silencing. The present work, however, shows that
*RDR6*-dependent production of siRNAs from transgenes that do not have a miRNA
cleavage site differs from ta-siRNA biogenesis.
A number of studies have examined the *DCL* requirements of hairpin transgene
induced silencing. The studies variously differ from one another and from our
work with respect to structure of the hairpin transgene, dependence on a
silencing signal, and whether the target of silencing is a transgene or an
endogenous gene. Our study is the only one to date that has focused specifically
on cell-autonomous silencing of transgenes and systematically distinguished
between accumulation of primary and secondary siRNAs. Using this approach, we
find that hairpin transgene-induced silencing occurs in *dcl2*, *dcl4*, and
*dcl2 dcl4* mutants. The mechanism of target degradation varies, however,
depending on which of the DCL enzymes are active. DCL4-dependent processing of
the stem of the hairpin into primary siRNAs is the major pathway utilized in
wild type and *dcl2* mutant plants. Inactivation of DCL4 promotes a shift to
transitive silencing and the production of secondary siRNAs. The very high level
of accumulation of secondary siRNAs in *dcl4* mutant plants transgenic for both
the ΔGUS-SUG and 6b4 GUS loci suggests that under some conditions, secondary
siRNA production itself is a major contributor to degradation of the targeted
transcript, consistent with the observation that *AGO1* is not required for
silencing of a GUS transgene by the ΔGUS-SUG locus. When both DCL2 and DCL4 are
inactive, silencing is again dependent on primary siRNAs, and processing of the
stem of the hairpin by DCL3 becomes evident. Our observation that DCL4
completely processes the 558-bp dsRNA stem of a hairpin construct into primary
siRNAs, leaving only the loop portion, suggests that the enzyme is highly
processive on *RDR6*-independent dsRNA as well as on *RDR6*-dependent dsRNA and
is consistent with the observation that *DCL4* is required when silencing
depends entirely on production of primary siRNAs from a hairpin transgene.
The gene families involved in RNA silencing in plants have evolved to provide a
large degree of functional diversity. It is striking that despite this potential
for diversity, production of secondary siRNAs relies on *DCL2* in both sense and
hairpin transgene silencing, which otherwise differ in their genetic
requirements. The DCL enzymes and functional modules undoubtedly evolved to
efficiently handle a wide variety of natural substrates–many of which have
probably not yet been identified, and it is likely that the enzymes recognize
signature structural features of their preferred substrates. The multiplicity of
functional pairings is just beginning to be elucidated. For example, although
RDR2/DCL3 is a well established module involved in hc-siRNA biogenesis, DCL3
activity without RDR2 and the pairing of RDR2 with DCL4 has recently been
proposed for a particular case of hairpin transgene targeting of an endogenous
gene. The diversity and great versatility of small RNA pathways in plants is
perfectly exemplified by how well prepared plants turned out to be to defend
themselves against the recent evolution of genetic engineers.
# Materials and Methods
## Transgenic and mutant *Arabidopsis* lines
All lines are in the Columbia (Col-0) ecotype. The following transgenic or
mutant lines were described previously: L1, 306-1 and 6b4/306, P38 (CP), P1/HC-
Pro, *sgs2-1 (rdr6)*, *dcl1-7* and *dcl1-8*, *dcl2-1* (SALK_064627) and
*dcl4-2*. The *dcl4-10* mutation is a previously unpublished mutation that arose
in an EMS mutagenesis. It is a single mucleotide mutation that produces a
glycine to arginine change at amino acid 1403, which is located in the RNAse III
domain of DCL4 (data not shown).
## PCR genotyping for mutant and transgene loci
The T-DNA primer LBa1 (tgg ttc acg tag tgg gcc atc g) was used with primers
DCL2p5 (ttg gat tgc atg cac aca tt) and DCL2p6 (ctc aga aat aaa gat aac agt aag
caa at) for *dcl2-1* genotyping. Primer DCL2p5 together with DCL2p6 amplifies a
400-bp product from the wild type locus, while DCL2p5 together with LBa1
amplifies a 600-bp product from the *dcl2-1* locus. Thus, a PCR reaction with
all three primers will amplify only the 600-bp product in the case of homozygous
*dcl2-1*. Genotyping for homozygous *dcl4-2* was performed as described
previously. Homozygous *dcl4-10* plants containing the L1 GUS transgene were
identified by their distinctive leaf phenotype, which is similar to that of the
*dcl4-2* mutant. Primers L1-306-6b4-F (ttg ggg ttt cta cag gac gga c) and
L1-306-6b4-R (cta tcc ttc gca aga ccc ttc c) were used in combination with GUS
staining to screen for the presence of different GUS loci. A 250-bp fragment is
obtained with the 306-1 locus, a 188 bp fragment with the L1 locus and a 127-bp
fragment with the 6b4 locus, due to differences in the 5′ upstream sequences of
the GUS constructs. Homozygous *dcl1-7* and *dcl1-8* plants containing the L1
GUS transgene were identified by their distinctive recessive phenotypic defects.
Primers 2911F7 (gca ggg ata ctt gaa cat ggc c) and 2911R8 (gtt aac aac cta tgc
cac gc) were used for *sgs2-1* genotyping: PCR followed by digestion with BstNI
yields three fragments (sizes 250, 200, and 150 bp) from the wild type locus,
but only two fragments (200 and 400 bp) from the *sgs2-1* locus. P38-derived
primers P38-F (cgc cca atg ggc gat aaa g) and P38-R (cgt ctc ggt cga atg cca gag
c) were used to confirm the presence of the P38 transgene in combination with
phenotypic and phosphoinothricin selection.
RNA isolation, gel blot analysis, and nuclear run-off transcription. RNA was
isolated from a mixture of representative aerial tissues of flowering plants.
For most experiments, tissues from about ten plants of a given genotype were
pooled for RNA isolation. For each genotype, at least two independent RNA
preparations were made from separate plants or pools of plants and
electrophoresed in neighboring lanes on RNA gels. Total RNA isolation and gel
blot analysis of high and low molecular weight RNA were performed as described
previously. Nuclear run-off transcription analysis was performed as described
previously except that nuclei were isolated from aerial tissues of flowering
plants.
The minimal sequence (taa tac gac tca cta tag gg) of the T7 promoter was
incorporated by PCR into the 3′ or 5′ ends of DNA templates to make RNA probes
of antisense polarity to detect mRNAs or of sense polarity to detect antisense
siRNAs, respectively. \[α-<sup>32</sup>P\]UTP-labeled RNA probes were
transcribed in vitro using an Ambion MAXIscript kit with T7 polymerase and
hybridized to mRNA blots at 68°C in Ambion ULTRAhyb buffer, or to siRNA blots at
42°C in Ambion ULTRAhyb-oligo buffer. DNA probes were labeled using an Ambion
DECAprime II kit and hybridized to mRNA blots in Ambion ULTRAhyb buffer at 42°C.
The coding sequence coordinates of the probes for viral suppressors of silencing
were: P38, nucleotides 42 to 472; HC-Pro, entire coding sequence. The siR255
probe was prepared by end-labeling the complementary DNA oligonucleotide with
\[α-<sup>32</sup>P\]ATP using the StarFire™ Oligo Labelling System (Integrated
DNA Technologies) as described previously. The probe was hybridized to small RNA
blots at 42°C in Ambion ULTRAhyb-oligo buffer.
We thank Herve Vaucheret for providing seeds of the L1, 6b4, and 306 lines and
Jim Carrington for seeds of the P38 transgenic line.
[^1]: Conceived and designed the experiments: VV SM LB. Performed the
experiments: SM. Analyzed the data: VV GP SM LB. Contributed
reagents/materials/analysis tools: SP XC SM AP ME JL. Wrote the paper: VV GP
SM LB. Other: Generated the dcl4-10 mutant: SP AP.
[^2]: Current address: Howard Hughes Medical Institute, Skirball Institute
of Biomolecular Medicine, New York University School of Medicine, New York,
New York, United States of America
[^3]: The authors have declared that no competing interests exist. |
# Introduction
Heterosis is defined as the superior performance of crossbred characteristics as
compared with corresponding inbred ones. The utilization of heterosis has become
a major strategy to increase the productivity of plants and animals. Despite the
successful utilization of heterosis in many crops, there still exists a
contradiction between the agricultural practice of heterosis utilization and our
understanding of the genetic basis of heterosis and this hampers the effective
exploitation of this biological phenomenon.
The classical quantitative genetic explanation of heterosis centered on three
hypotheses: dominance, over-dominance and epistasis. Evidence of these genetic
models remained unavailable until very recent advances in molecular marker
technology, high-density linkage maps and genome sequencing. Although much
research into the genetic basis of heterosis in crops and plants has been
conducted, little consensus has emerged. Research has indicated that heterosis
may be attributable to dominance, over-dominance, epistasis or a combination of
all of these, depending on the study materials, traits and analytical approach.
Typically, little is known about the genetic control of heterosis in the complex
polyploid crop rapeseed (*Brasscia napus* L.). Based on the phenotype of the
E×R53-DH population and the corresponding BC population, as well as the mid-
parent heterosis of the BC population, Radoev et al. (2008) mapped 33 QTL (9 of
which showed a significant dominant effect) and a large number of epistatic
interactions for seed yield and the three yield-component traits. They concluded
that epistasis together with all levels of dominance from partial to over-
dominance is responsible for the expression of heterosis in rapeseed. Based on
this E×R53-DH population and another E×V8-DH population with the same parent,
and using the same experimental design, Basunanda et al. (2010) detected a
number of QTL hotspots responsible for seedling biomass and yield-related
traits. Given the key role of epistatic interactions in the expression of
heterosis in oilseed rape, they supposed that these QTL hotspots might harbour
genes involved in regulation of heterosis for different traits throughout the
plant life cycle, including a significant overall influence on heterosis for
seed yield. However, in both studies, all kinds of genetic effects (A, D and AA,
AD/DA, DD) were unable to be estimated in the same population, thus it was
difficult to accurately estimate their mode-of-inheritance and relative
importance in the expression of heterosis.
There were several common patterns described in most of these studies. Firstly,
the QTL for yield and yield-correlated traits tended to be clustered in the
genome in many crop and model plants, such as rice, maize, wheat, rapeseed and
*Arabidopsis*, which suggested the QTL of yield-correlated traits might have
pleiotropic effects. However, this kind of pleiotropy has not been well analyzed
genetically. Secondly, only a few limited traits were investigated and only a
few QTL and epistatic interactions were identified for each trait, so a
relatively comprehensive picture of the genetic architecture of heterosis
remained unavailable. Thirdly, trials were carried out in only one or two
environments and the environmental response of QTL and epistatic interactions
for heterosis was not analyzed and thus remains unclear.
The main objective of this study was to unravel the genetic architecture of
heterosis with QTL mapping in rapeseed, including: (1) determine the level of
heterosis for a range of yield-correlated traits; (2) investigate the
relationship between molecular marker heterozygosity and heterosis/hybrid
performance; (3) identify QTL and epistatic interactions underlying heterosis
and estimate their genetic effect, mode-of-inheritance and environmental
responses; (4) analyze the relative contribution of all kinds of genetic effects
in the expression of heterosis in rapeseed (*Brassica napus* L.).
# Results
## Correlation of trait performance and mid-parent heterosis among the 15 investigated traits
In the same environment, most pair-wise genetic correlations of performance and
mid-parent heterosis were similar. This was understandable since mid-parent
heterosis was calculated from trait performance. In different environments,
pair-wise genetic correlations differed considerably (mostly in degree, a few in
direction), which suggested that genetic correlations depended strongly on the
environments.
Genetic correlations of performance and mid-parent heterosis among the
investigated traits were also calculated across the three environments. In
general, significant correlations were observed for 81.9% and 67.6% of the pair-
wise combinations of the trait performance and mid-parent heterosis,
respectively. Seed yield correlated significantly with the other 14 investigated
traits for both trait performance and mid-parent heterosis; negatively for
flowering time, maturity time and protein content, and positively for the other
11 ones. Interestingly, the mean *r*<sup>2</sup> of trait performance was
somewhat higher than that of mid-parent heterosis for most traits, ranging from
0.04 and 0.03 (for seed development times) to 0.24 and 0.20 (for seed yield),
respectively.
## Traits showing significant heterosis
The analysis of variance (in both populations) revealed that genotype,
environment and the interaction between them had significant effect on the
performance of all the 15 yield-correlated traits, so they were calculated
separately for each environment. The broad-sense heritability of these traits
ranged from 0.58 (for seed yield) to 0.90 (for flowering time), with a mean of
0.73. The two parents showed significant differences in 38 of the 43 trait-
environment combinations. The two populations showed obvious transgressive
variation for all of the trait-environment combinations. It should be noted that
DH and the reconstructed-F<sub>2</sub> population showed over-F<sub>1</sub>
variations for 13 (except seed yield and seed number per plant) and all of the
traits respectively in all environments, which indicated that heterozygosity was
not always favorable for trait performance. There was significant heterosis on
F<sub>1</sub> and F<sub>2</sub> generations compared with the mean of the
parents and the DH population, respectively, for the nine (branch number,
biomass yield, harvest index, plant height, pod number, pod yield, seed number
per pod, seed number per plant and seed yield) and eight (except branch number)
traits. Interestingly, for these traits with significant heterosis, the
performance of F<sub>1</sub> was significantly higher than the mean of the
F<sub>2</sub> population and higher than the mean of the DH population in 19 and
all of the 25 trait-environment combinations respectively, which showed an
obvious trend of inbreeding depression.
According to the significance of heterosis, the 15 yield-correlated traits could
be classified into two groups: the nine traits (seed yield, seed number per
plant, biomass yield, pod number, harvest index, plant height, pod yield, seed
number per pod and branch number) with heterosis and the other six traits (oil
content, protein content, maturity time, flowering time, seed weight and seed
development time) without heterosis. It should be noted that the correlation
coefficients between seed yield and the nine traits with heterosis were all
higher than that between the other six traits without heterosis.
The analysis of variance revealed that genotype, environment and genotype ×
environment interaction had significant effect on mid-parent heterosis of the
nine traits with heterosis, so they were calculated separately for each
environment. For hybrid F<sub>1</sub>, seed yield and seed number per plant
showed strong mid-parent heterosis, biomass yield and pod number per plant
showed moderate mid-parent heterosis, while pod yield, seed number per pod,
harvest index, branch number and plant height showed low mid-parent heterosis.
For the reconstructed F<sub>2</sub> population, the amount of heterosis varied
widely for these traits, from highly negative to highly positive. The average
mid-parent heterosis of the reconstructed F<sub>2</sub> population showed
similar trend with that of F<sub>1</sub> for the nine traits. It should be noted
that in each environment the mid-parent heterosis of some (the proportion is
10.2% for seed yield in S5 environment, data not shown) combinations of
reconstructed F<sub>2</sub> population was higher than that of F<sub>1</sub>,
but the average mid-parent heterosis in the reconstructed F<sub>2</sub>
population was in all cases lower than that in F<sub>1</sub>. This indicated
that heterosis was generally related to the heterozygosity at the population
level but poorly correlated with heterozygosity at the individual level.
It should be noted that, for these yield-correlated traits, the heritabilities
(ranging from 0.40 to 0.60) of mid-parent heterosis were all lower than that
(ranging from 0.58 to 0.90) of trait performance.
## Correlation between heterozygosity and hybrid performance/mid-parent heterosis for the nine traits with significant heterosis
The correlation between heterozygosity and hybrid performance/mid-parent
heterosis was significant for the nine traits with significant heterosis except
branch number and seed number per pod, with mean *r*<sup>2</sup> ranging from
0.001 (branch number) to 0.066 (seed yield) for the different traits, which
accorded well with the heterosis level of these traits. Generally, the mean
*r*<sup>2</sup> between heterozygosity and hybrid performance was similar to
that between heterozygosity and mid-parent heterosis. Whereas, the mean
*r*<sup>2</sup> (0.026/0.022) between special heterozygosity and hybrid
performance/mid-parent heterosis was a little higher than that (0.013/0.014) of
general heterozygosity and hybrid performance/mid-parent heterosis in most
cases. Interestingly, the mean *r*<sup>2</sup> between heterozygosity and hybrid
performance/mid-parent heterosis was stronger in the S5 environment than in the
other two environments, which suggested these correlations were also depended on
the environment. Although 47 of the 100 correlations between heterozygosity and
hybrid performance/mid-parent heterosis were significant, the *r*<sup>2</sup>
were relatively small (from 1.21% to 18.5%), which suggested that molecular
marker heterozygosity could not predict hybrid performance and mid-parent
heterosis.
## Genome-wide detection and meta-analysis of QTL for 15 yield-correlated traits
A total of 967 QTL (579 significant QTL and 388 suggestive QTL) were identified
for the 15 yield-correlated traits in both populations in three environments.
Exclusion of 209 non-overlapping suggestive ones, a total of 758 QTL was
identified finally. Of which 390 identified QTL were from reconstructed
F<sub>2</sub> population (ranging from 11 to 56 for each trait), they were
potentially responsible for heterosis and were the objectives of the following
analysis. The 390 identified QTL explained 1.4-20.8% (mean = 5.6%) of the
phenotypic variance while 92.8% showed only moderate effect, with
*R*<sup>2</sup>\<10% and only one explained \> 20% of phenotypic variance.
Furthermore, for the 13 identified QTL with *R*<sup>2</sup>≥10%, the absolute
values of their dominant degree (∣D/A∣) were all \< 1. This suggested that
heterosis of these yield-correlated traits was typically controlled by numerous
loci with little heterotic effect.
To estimate the environmental response of QTL in natural environments, meta-
analysis was used to integrate the identified QTL trait-by-trait in different
environments. A total of 300 consensus QTL was identified, of which only 77
(25.7%) were repeatedly found in more than two environments and regarded as
repeatable QTL, the other 223 (74.3%) were specifically identified in one of the
three environments and considered as non-repeatable ones. This indicated that
the expression of QTL of yield-correlated traits was strongly dependent on
environmental conditions, which is also confirmed by the result that 55.3%
(166/300) of consensus QTL showed significant QTL × environment interaction in
ANOVA analysis. The proportion of the repeatable QTL was high for flowering
time, development time of seeds, pod yield and seed number per pod, and results
accorded with the high heritability of these traits. Only 77 consensus QTL were
repeatable, whereas 68.8% changed their mode-of-inheritance in different
environments. Only 5.2% of the 77 repeatable consensus QTL changed the direction
of additive-effect, which suggested that the relative superiority of one allele
over the others was stable in different environments. In contrast, 31.2% of the
77 repeatable consensus QTL changed their dominant-effect directions in
different environments. In addition, only 20.8% ( = 16/77) of these repeatable
consensus QTL showed significant interaction with the environment at P≤0.05,
which was lower than that (67.3% = 150/223) of the non-repeatable ones.
Therefore, the expression, direction and effect of QTL were all dependent on
environmental conditions, which suggested the variability of QTL.
The confidence intervals of most consensus QTL determined for each trait
overlapped. The 300 consensus QTL for the 15 yield-correlated traits were
therefore subjected to a second round of meta-analysis, which resulted in the
integration of 220 consensus QTL into 84 pleiotropic unique QTL.
## Genome-wide detection and analysis of epistatic interactions in the reconstructed F<sub>2</sub> population and three environments for 15 yield-correlated traits
A total of 522 statistically significant epistatic interactions were identified
for the 15 yield-correlated traits in two populations and three environments and
most of them were also confirmed by the two-way analysis of variance (data not
shown). Of these significant epistatic interactions, 272 were identified from
the reconstructed F<sub>2</sub> population (ranging from 11 to 29 for the
different traits), potentially responsible for heterosis and were the objectives
of the following analysis. Only two epistatic interactions of seed yield, which
were detected in different environments and located in similar positions, were
considered as repeatable, which suggested epistatic interactions of yield-
correlated traits were extremely sensitive to the environmental variation. A
total of 136, 103 and 33 epistatic interactions belonged to NN (the two loci
involved in epistatic interaction were both with non-significant main-effects),
NS (the two loci involved in epistatic interaction was one with significant
main-effect and the other one with non-significant main-effect,) and SS (the two
loci involved in epistatic interaction were both with significant main-effects)
type of epistatic interactions respectively, which indicated most loci of
epistatic interactions have no significant effect on trait performance alone but
may affect it by epistatic interaction with other loci. The 272 epistatic
interactions explained 1.4–18.3% (mean = 5.1%) of the phenotypic variance, while
95.6% showed only moderate effect, with *R*<sup>2</sup>\<10%. It should be noted
that 91.9% of the 272 epistatic interactions occurred between different
chromosomes.
The proportion of the loci involved in multiple (2–7) epistatic interactions
varied from 52.3% (for plant height) to 88.5% (for harvest index) for different
traits and with a mean of 68.2% on average, which indicated the prevalence of
pleiotropic loci regulating heterosis on an epistatic level. For example, seven
epistatic interactions (*eqOIL.13-16/14-26*, *eqPN.13-16/16-28*,
*eqSN.11-42/13-16*, *eqSP.13-16/19-12*, *eqSP.11-14/13-16***,**
*eqSY.13-16/19-21*, and *eqSY.13-16/19-20*,) shared the common chromosome
interval 13-16 indicating existence of a hotspot.
## Mode-of-inheritance of QTL and epistatic interactions
Four kinds of QTL mode-of-inheritance (A; PD; D; OD) and three kinds of
epistatic interactions mode-of-inheritance (AA; AD/DA; DD) were found for the 15
yield-correlated traits, which accounted for 24.6%, 49.0%, 13.8%,12.6%, and
63.0%, 26.0%, 11.0% respectively. For the same trait, the QTL and epistatic
interactions showed an unequal distribution among different mode-of-inheritance
categories. For the same mode-of-inheritance category of QTL or epistatic
interactions, unequal distribution was also observed among different traits,
which suggested that the genetic mechanism underlying the heterosis of different
traits might be different. Seed yield and seed number per plant clearly showed
the highest proportion of +D/+OD mode-of-inheritance, which accorded well with
the highest mid-parent heterosis of both traits. The dominant-effect direction
of 41.8% QTL, 54.0% (48 out of 89, 48 from negative and 41 from positive) AD/DA
and 48.7% (19 out of 39, 19 from negative and 20 from positive) DD epistatic-
effect was negative, which was consistent with the low correlation between
marker heterozygosity and mid-parent heterosis/hybrid performance.
To test whether the mode-of-inheritance of identified QTL and/or epistatic
interactions was associated with the significance of heterosis, a *t* test was
used for each mode-of-inheritance category between the nine traits with
heterosis and the other six traits without heterosis and no significant
differences were found. However, between the 15 yield-correlated traits and 9
seed-quality/metabolic traits (glucosinolates, erucic acid, linolenic acid,
linoleic acid, palmitic acid, oleic acid, stearic acid, α-tocopherol and
γ-tocopherol contents in seeds, which were not significantly correlated with
seed yield and unpublished in the current research), significant and extremely
significant differences were found for +D and +OD mode-of-inheritance,
respectively. In addition, for the nine traits with significant heterosis, the
direction of OD effect was more frequently found to be positive than to be
negative.
## Phenotypic effect of QTL and epistatic interactions
To test the effect of identified QTL and epistatic interactions on the trait
performance of the reconstructed F<sub>2</sub> population for 15 yield-
correlated traits, the performance of all kinds of genotypes was calculated
(using the marker that was closest to the peak position of the identified QTL
and epistatic interactions), compared and sorted. For the single-locus analysis,
a homozygote was frequently the best and also the worst genotype, while a
heterozygote was the most unlikely best and also worst genotype. For the two-
locus analysis, a complementary homozygote (two loci were homologous for Tapidor
and Ningyou7 respectively) was frequently the best genotype, followed by a
parental homozygote (two loci were homologous for Tapidor or Ningyou7
respectively), a single heterozygote and a double heterozygote for almost all
traits. For example in the case of seed yield, it was deduced that, in order to
get the best genotype only 39.1% and 8.8% loci of identified QTL and epistatic
interactions (21.1% for all loci involved) respectively, should be heterozygous.
This accorded well with the previous finding that the seed yield of many lines
in the reconstructed F<sub>2</sub> population was higher than that of the
F<sub>1</sub> hybrid.
# Discussion
## Reconstructed F<sub>2</sub> population is very suitable for heterosis study
The reconstructed F<sub>2</sub> population used here holds several unique
characteristics for dissecting the genetic architecture of heterosis. Firstly,
it is well known that the F<sub>2</sub> population was theoretically the most
complete and informative source for most genetic analysis. The genotype of the
reconstructed F<sub>2</sub> population was basically the same to that of the
F<sub>2</sub> population because the genotype of double haploid lines used in
making the reconstructed F<sub>2</sub> population was essentially the same as
that of the gamete produced by the F<sub>1</sub> hybrid (except for the
possibility that genotypic selections existed in the process of microspore
culture). In this sense, the reconstructed F<sub>2</sub> population is more
similar to the F<sub>2</sub> population than the immortalized F<sub>2</sub>
population produced by the random intercross of recombinant inbred lines.
Secondly, each genotype of the reconstructed F<sub>2</sub> population was
represented by many individuals and thus permitted replicated experiments in
multiple environments, so the reconstructed F<sub>2</sub> population was better
than the F<sub>2</sub> and F<sub>2:3</sub> populations. This also increased the
power (or decreased experimental error) and reproducibility of QTL detection,
and especially facilitated the analysis of environmental response of QTL in
natural environments. Thirdly, additive, dominant and all kinds of epistatic
effects (including AA, AD/DA and DD) can be well estimated in one population,
thus increasing the accuracy of the estimation of dominant degree, mode-of-
inheritance and especially the relative importance of all kinds of genetic
effects in the expression of heterosis. Therefore, for heterosis study
reconstructed F<sub>2</sub> population is also better than BC, TC, NCIII and TTC
populations in this sense. However, it should be noted that among all of the
available experimental designs, TTC population has the unique potential to
identify QTL that is directly linked to heterosis.
## Level of heterosis across traits and species
In all environments, seed yield showed the strongest heterosis among the 15
yield-correlated traits, consistent with the findings in other rapeseed
research, as well as in other crops and plants, such as rice, , maize,
*Arabidopsis*, and tomato. This confirmed the hypothesis that complex traits
usually express higher heterosis than component traits. Interestingly, the
theoretical mid-parent heterosis of seed yield was calculated as: (1 + 18.4%) ×
(1 + 10.1%) × (1 + 2%) - 1 = 30.6%, a value which was very clear to the true
value (31.4%) of mid-parent heterosis of seed yield (18.4%, 10.1% and 2% was the
mid-parent heterosis mean in the reconstructed F<sub>2</sub> population,
respectively, for the three yield component traits). In addition, the yield
heterosis of the tomato +/*sft* heterozygote could be traced back to component
traits, number of flowers per plant and fruit weight. This suggested that the
heterosis of complex trait (such as yield) can be well explained by that of the
component traits, because the middle and/or weak heterosis of the component
traits may result in high heterosis of the complex traits in a multiplicative
manner,
Generally, the level of mid-parent heterosis for similar traits in the current
research as well as other research in rapeseed, rice, wheat, *Arabidopsis*, and
tomato were all much lower than that of the corresponding traits in maize. This
may be attributable to differences in reproductive biology. Maize is an
allogamous species and was supposed to have more deleterious alleles than
autogamous species (because in autogamous species, deleterious alleles are
possibly eliminated by natural and artificial selection since the individuals
are homozygous), so the extent of inbreeding depression in maize was greater
than that in rice, wheat, tomato and *Arabidopsis*, the autogamous species, and
rapeseed, a partially allogamous crop.
## Mode-of-inheritance of QTL and epistatic interactions
No significant difference was found for the proportion of the eight model-of-
inheritance categories of QTL between the nine traits with heterosis and the
other six traits without heterosis. This suggested that the presence or absence
of heterosis was not associated with QTL mode-of-inheritance in the current
research, which may be because the dominant effect only accounted for a small
proportion of variance when compared with the epistatic effect of these traits.
However, between the 15 yield-correlated traits and the 9 seed-quality/metabolic
traits, significant and extremely significant differences were found for +D and
+OD mode-of-inheritance. This indicated +OD/+D mode-of-inheritance was
associated with the traits of yield category, which may be because the
occurrence of +OD/+D QTL for yield-correlated traits will increase crop
productivity during the processes of domestication. Thus, OD may be an
essentially pseudo-OD that involves linked loci with dominant alleles in
repulsion. We detected A, PD and D QTL for both yield-correlated and seed-
quality/metabolic traits, but OD was basically absent in seed-quality/metabolic
traits. This indicated that pseudo-OD due to random linkage is unlikely to be
the major genetic basis underlying OD QTL, and thus we favored the true OD
model. In fact, +OD/+D QTL was prevalent in almost all research regarding the
genetic basis of yield, life-history and reproductive traits in crops. In a
tomato introgression line population, +OD QTL was more prevalent for the
reproductive traits than nonproductive traits. In a summary research, the
dominance effect was found to be larger in life-history traits than in
morphological traits. Although only a few studies reported the QTL mapping of
metabolic traits, the results all showed that only a few metabolic-QTL showed OD
mode-of-inheritance. This suggested that different phenotypic classes may have
different dominance relationships among variable alleles, possibly due to
differences in the complexity underlying the molecular networks. More
importantly, the sign of dominant-effect of OD QTL for the nine traits with
heterosis was more frequently found to be positive than to be negative, which
suggested that selection also has changed the frequency of the direction of OD
effect for these traits of heterosis. This is understandable, since a positive
OD effect may undoubtedly increase the heterosis and yield of hybrids.
However, no mode-of-inheritance categories and their direction of epistatic
interactions showed significant difference in proportion among different
phenotypic categories. In fact, this phenomenon seemed to be typical in other
crops. In a two-year experiment conducted in an “immortalized F<sub>2</sub>”
population of an elite rice hybrid known as Shanyou63, the proportions of three
kinds of epistatic interactions (AA, AD/DA and DD) were almost the same between
reproductive (grain yield, tillers per plant, grains per panicle *etc.*) and
non-reproductive (heading date, plant height and panicle length *etc.*) traits.
In a two-location experiment conducted in an F<sub>2:3</sub> population in
maize, no significant difference was also found in the proportion of three kinds
of epistatic interactions between yield traits (such as grain yield, rows
number, kernels per row *etc.*) and morphological (ear length, ear diameter and
axis diameter *etc.*) traits. This suggested that selection was not effectual at
epistatic level during the domestication of rapeseed, as well as other crops.
This was understandable: since epistatic interactions were more dependent on the
genetic background and environmental variations than QTL, their role was
variable, and thus capturing the best gene combination(s) was difficult for
breeders.
It should be noted that the relative proportion of the four kinds of mode-of-
inheritance of QTL showed great differences in different traits and studies. For
example, in the same QTL mapping experiment of nine yield traits, the
predominant mode-of-inheritance of QTL was over-dominant and additive, in an
intraspecific and intrasubspecific rice hybrid. However, in all research in
which the three kinds of epistatic effects could be resolved, AA interaction
occurred at the highest frequency for all traits, followed by AD/DA and the DD
interaction at the middle and lowest frequency, respectively. This confirmed
that selection has great but little or no impact on mode-of-inheritance of QTL
and epistatic interactions, respectively. In addition, in all cases the
practical proportions (usually \>50%, \<40% and \<8%) of AA, AD/DA and DD
interactions were all quite different with their theoretical proportions of 25%,
50% and 25%, respectively. This provided the evidence that the identified
epistatic interactions were absolutely not the results of chance events.
## Environmental response of QTL and epistatic interactions
The meta-analysis of QTL identified in different environments facilitated the
exact estimation of the environmental response of QTL. Totally, 74.3% (223) of
the consensus QTL and 99.3% (270) of the epistatic interactions for the 15
yield-correlated traits was specifically identified in one of the three
environments, which indicated the great impact of natural environments on the
genes underlying the heterosis of these yield-correlated traits. These
proportions were much higher than the corresponding ones (48.4% and 91.6%) of
the other 9 seed-quality/metabolic traits (unpublished data), which accorded
well with the broad-sense heritability of these traits. In fact, the high
dependency on environment seemed to be a common character of the QTL and
epistatic interactions for heterosis in other research. In a two year experiment
conducted in an F<sub>2:3</sub> population derived from an elite rice hybrid
(Shanyou63), 62.5% QTL and 90.6% digenic interactions for grain yield and the
three yield component traits were observed in only one year. In another two year
experiment conducted in an “immortalized F<sub>2</sub>” population derived from
the same rice hybrid, 67.5% QTL and 91.5% digenic interactions for the same four
yield traits were detected in only one year. In a two-location experiment
conducted in an F<sub>2:3</sub> population derive from an elite maize hybrid,
62.1% QTL and 91.8% of digenic interactions for grain yield and the three yield
component traits were detected in only one location. It should be noted that the
proportion of environment-specific epistatic interactions was much higher than
that of QTL in all cases, which was understandable since the epistatic
interactions involved two genetic loci which were also dependent on
environmental conditions. It should also be noted that the proportions of
environment-specific QTL and epistatic interactions in the current research as
well as other rapeseed research were all higher than that in rice, and maize,
possibly due to the genome plasticity of polyploids,. This indicated the high
variability and plasticity of the genetic architecture of heterosis in rapeseed.
Furthermore, of the 77 repeatable consensus QTL for 15 yield-correlated traits,
68.8% changed their mode-of-inheritance in different environments. This
proportion was also much higher than that (46.9%) of the 9 seed-
quality/metabolic traits (data not shown). This indicated that the relative
importance of dominant vs additive effect of QTL of different phenotypic
categories may have different sensitivity to environmental variations, possibly
due to differences in the intrinsic mechanism of regulation. Interestingly, the
additive-effect direction of the repeatable consensus QTL was usually the same
in different environments, which was consistent with previous research. This has
great significance for genetics and crop breeding: since the relatively
favorable alleles identified in one environment were usually relatively
favorable in another environment, the actual effect of selection might be well
ensured. From an evolutionary point of view, these retained alleles all
experienced the processes of far-flung natural or artificial selection, and
alleles that were adaptable to changed environments could be successfully
retained. Whereas, 24 of the 77 repeatable consensus QTL changed their dominant-
effect direction in different environments, this proportion (31.2%) was much
higher than that (5.2%) of the additive-effect direction. Furthermore, for the
other 53 repeatable consensus QTL with a consistent dominant-effect direction,
54.7% changed their mode-of-inheritance in different environments. For example,
the mode-of-inheritance of *qSY.A1-5* was changed from +PD in N6 environment to
+OD in an S5 environment. This indicated that the favorable heterozygote
identified in one environment was not always favorable in another environment.
## Genetic architecture of heterosis in rapeseed and other species
Using a reconstructed F<sub>2</sub> population (that has the maximum similarity
to an F<sub>2</sub> population), a multiple-environment experiment and a high-
density linkage map, we identified hundreds of QTL and epistatic interactions
responsible for the heterosis of 15 yield-correlated traits. Surprisingly, 92.8%
identified QTL and 95.6% epistatic interactions explained \<10% of variance.
This indicated that heterosis of yield-correlated traits in this cross was
mainly controlled by numerous loci with very little effect. In addition, the
maximum variances explained by individual QTL and epistatic interactions were
20.8% and 18.3% respectively. Therefore, the development of QTL and epistatic
interactions near-isogenic lines, toward fine-mapping and finally cloning the
genes responsible for heterosis in this cross would be very challenging.
In contrast with the high variability of QTL and epistatic interactions, their
most important feature was the high proportion (73.3% and 68.2% respectively)
that co-localized at the genomic level. This accorded well with the
comprehensive correlation of the mid-parent heterosis/hybrid performance among
these yield-correlated traits. These co-localizations indicated the existence of
pleiotropic loci regulating heterosis. In fact, most published fine-mapped QTL
or genes identified for yield heterosis exhibit pleiotropic effects on at least
one or multiple yield-correlated traits. Fifteen of the 21 consensus QTL of seed
yield co-localized with other consensus QTL and 7 of them co-localized with more
than two consensus QTL. This indicated that, in addition to pleiotropy, the
effect of the QTL for seed yield could be a synthetic effect of several tightly-
linked QTL of different yield-correlated traits. The multiple co-localized QTL
might come from the different environments, which indicated that the
environmental conditions contribute to the variability and plasticity of the QTL
for seed yield. It should be noted that more than half of the loci of the QTL
and epistatic interactions were clustered in several chromosomes.
Research from autogamous species, such as *Arabidopsis*, rice, and barley,
usually showed that epistasis played a more important role than main-effect as
the genetic basis of heterosis. In contrast, data from allogamous crops, such as
maize, exhibited the reverse result, demonstrating that main-effect is more
important than epistasis. This is not surprising, since co-adapted gene
complexes exhibiting favorable epistatic effects can be more easily maintained
in autogamous species than in allogamous species. Therefore, it is reasonable
for our result to show that epistasis was somewhat more important than main-
effect as the genetic basis of heterosis in rapeseed (a partially allogamous
crop with an out-crossing rate of 10-30%), and is consistent with other research
in rapeseed. According to the theory of classical genetics, only D, AA and DD
effect are the genetic components of mid-parent heterosis. Furthermore, the
average \|D\| was smaller than the average \|A\|, and their ratios ranged from
0.40 (for branch number) to 0.73 (for seed yield) and with a mean of 0.51. This
suggested that dominant effect only accounted for a minor proportion of
*R*<sup>2</sup> of QTL, whereas, AA and DD effects explained a major proportion
(67.1%) of *R*<sup>2</sup> of epistatic interactions. In conclusion, our
research showed that epistasis (especially AA epistasis) was the major genetic
basis of heterosis in rapeseed (*Brassica napus* L.).
## Implications for evolution and crop breeding
The two parents used in this study, Tapidor and Ningyou7, are the representative
of two highly diverse gene pools, the European winter-type rapeseed gene pool
and the Chinese semi-winter type rapeseed gene pool, both adaptable to their
corresponding agro-ecological areas. The proportion of positive (54.9%) and
negative (45.1%) additive-effect was basically equal, which indicates that both
gene pools harboured alleles adaptable to other agro-ecological areas. One
hundred and three epistatic interactions showed significant positive AA
interactions, which indicated co-adapted gene complexes retained during the
evolution of rapeseed, a phenomenon also found in other species. Oilseed rape
(AACC, 2n = 38) originated from the natural hybridization of *Brassica rapa*
(AA, 2n = 20) and *Brassica oleracea* (CC, 2n = 18) and the following chromosome
doubling, both of which also experienced an evolutionary process of
triploidization. Therefore, each gene has an average of 6 copies in rapeseed. If
these duplicated genes favorably interacted with each other, this would result
in ectopic heterozygosis and the fixed heterosis in inbred lines **.** In fact,
many epistatic interactions identified in reconstructed F<sub>2</sub> and DH
populations occurred between homologous intervals/blocks (data not shown), which
indicated the existence of fixed heterosis loci in rapeseed. Since a high-
density linkage map together with detailed chromosome block information was
available, it was possible to study the hypothesis of fixed heterosis and
demonstrate its advantage in the evolution of polyploids using two-segment near-
isogenic lines chosen from the backcross progenies in our laboratory. One
hundred and twenty epistatic interactions of the 15 traits showed significant
and negative AA interactions, which indicated the complementary homozygote of
these epistatic interactions tended to enhance fitness. This also suggested that
complementary loci played an important role in the maintenance of genetic
variation in the rapeseed population. Therefore, reserving the adapted genes and
co-adapted gene complexes (including fixed heterosis loci) in per se gene pool
while further pyramiding the favourable genes and gene combinations (including
fixed heterosis loci) in another gene pool may be an effective strategy to
further improve rapeseed conventional cultivars in both agro-ecological areas.
Consistent with the findings in other research in rapeseed as well as other
species, a considerable proportion of dominant effect (41.8%) and DD interactive
effect (48.7%) was negative, which indicated the general existence of hybrid
weakness genes across species. This suggested that heterozygote was not always
advantageous for the hybrid performance and mid-parent heterosis in rapeseed.
This conclusion was also confirmed by the comparison of phenotypic effects of
all kinds of genotypes both at the single and two locus level. Therefore, the
knockout or substitution of hybrid weakness genes represents a new avenue to
further improve hybrid cultivars. It should also be noted that 58.2% of dominant
effect and 51.3% of DD interactive effect was positive, which indicated
heterozygosis played an important role in the fitness of natural populations by
providing a heterozygous advantage to buffer against recessive alleles and
providing genetic plasticity to variable environmental conditions.
Although homozygotes of the detected QTL and epistatic interactions were usually
the best genotypes in rapeseed as well as in rice and maize, the proportion
still needs to be well demonstrated. The most striking finding in this research
is that to be the best hybrid, most heterozygous loci (83.2% in this experiment)
of all QTL and epistatic interactions in hybrid F<sub>1</sub> should be
homozygous, which accorded well with the results that only 19.2% of QTL and
17.4% of epistatic interactions showed positive OD/D and DD/AD(DA) mode-of-
inheritance respectively. This suggested that, in most cases, homozygotes were
more advantageous for trait performance than heterozygotes. At first view, this
conclusion seemed unbelievable, a truth usually neglected, is that, heterosis
(usually defined as mid-parent heterosis) and hybrid performance are related but
essentially two different concepts, because the latter is more complex and equal
to the former plus the parental mean. The cryptic meaning is that a hybrid
showing the strongest mid-parent heterosis for a given trait did not always
exhibit the best per se manifestation of the same trait. Similarly, a
heterozygote may enhance mid-parent heterosis value but decrease per se hybrid
performance. Therefore, our conclusion is not intricate, and this has great
significance for genetics and crop breeding. Because heterosis usually coincides
with the genetic distance between parents, to maximize heterosis, breeders
usually adopted parents with greater genetic distance, and as a result, the
unadapted germplasm was also adopted in the hybrid breeding scheme. Therefore,
the final result is that the breeders get the combinations of max heterosis but
not the best hybrids. To avoid the occurrence of this embarrassing situation, we
suggest an adapted germplasm with relatively large genetic distance would be a
better choice in a hybrid breeding scheme. In addition, our result also
suggested the utilization of the residual heterosis of inbred and backcross
progenies (such as F<sub>2</sub>, F<sub>3</sub> and BC<sub>x</sub> *etc*) in
rapeseed as well as other partially-allogamous and autogamous crops would be
feasible, because the over-F<sub>1</sub> phenomenon for yield and/or biomass was
usually found in the subsequent inbred and backcross progenies even for elite
hybrids.
This research revealed that epistasis played an important role in the genetic
architecture of trait performance and heterosis in autogamous and partially-
allogamous crops. The research also showed that epistasis is very sensitive to
environment, and the epistatic effect varied from one environment to another,
thus artificial selection seemed to have little or no effect on it, though it
has proved to be effectual at the single-locus level (illustrated by the
association between +OD/+D QTL and the traits of yield category, and between
positive signs of OD effects and traits with heterosis). This suggested that
while challenging, marker-assisted selection to significantly improve the
heterosis/hybrid performance of yield traits in the aforementioned crops has
great potential.
# Materials and Methods
## Design and development of a reconstructed F<sub>2</sub> population
A double haploid (DH) population of 202 lines was developed by microspore
culture from the F<sub>1</sub> cross between Tapidor (an European winter-type
rapeseed cultivar) and Ningyou7 (a Chinese semi-winter type rapeseed cultivar)
and named as TNDH. A reconstructed F<sub>2</sub> population was made by making
101 crosses per round between pairs of DH lines randomly chosen from the 202
lines of the TNDH population. In the spring of 2004 and 2005, three and four
rounds of crossing were made by hand emasculation and hand pollination,
resulting in 303 and 404 crosses respectively.
## Field experiments and trait measurements
The two populations (TNDH and reconstructed F<sub>2</sub>), two parents (Tapidor
and Ningyou7) and F<sub>1</sub> (Tapidor × Ningyou7) were grown in 3 different
environments (year-location combinations) in China. The field planting followed
a randomized complete block design with three replications. Each plot was 3.0
m<sup>2</sup> with 30 plants in N6 and S6 environments and 4.0 m<sup>2</sup>
with 40 plants in S5 environments, with a distance of 40 cm between rows and 25
cm between individuals. The seeds were hand sown and the field management
followed standard agricultural practice. Twelve representative individuals from
the middle of each row in each plot were hand harvested from ground level at
maturity.
A total of 15 traits were investigated: (1) seed yield (SY, kg/ha), (2) biomass
yield (BY, kg/ha), (3) pod number per plant (PN); (4) seed number per pod (SN);
(5) seed weight/1000 seeds (SW, g); (6) flowering time (FT, days); (7) maturity
time (MT, days); (8) plant height (PH, cm); (9) branch number (BN); (10)
development time of seeds (DT, days), calculated from maturity time and
flowering time by the formula, DT = MT - FT; (11) seed number per plant (SP),
calculated from SY and SW by the formula, SP = 10 × SY (kg/ha)/SW (g/1000);
(12) pod yield/100 pods (PY), calculated from SN and SW by the formula, PW =
SN × SW/10; (13) harvest index (HI), calculated from BY and SY by the formula,
HI = SY/(SY + BY)); (14) protein content in seeds (PRO), (15) oil content in
seeds (OIL).
Seed yield per plant was measured as the average dry weight of seeds of the
harvested individuals in a plot. Biomass yield per plant was measured as the
average total above-ground (except the seeds) dry weight of the harvested
individuals in a plot. Pod number was the number of well-filled, normally
developed pods on each harvested individual in a plot. Seed number per pod was
the average number of well-filled seeds from 100 well-developed pods, sampled
from the primary branch in the middle of the harvested individuals in a plot.
Seed weight was the average dry weight of 1000 well-filled seeds from three
replicate samples, taken from the mixed seeds of the harvested individuals in a
plot. Flowering time was measured as the interval between the date of sowing and
the date when the first flowers emerged on 50% of the plants in a plot. Maturity
time was measured as the interval between the date of sowing and the date when
pods on most of the plants in a plot were yellow. Plant height was the height of
each harvested individual in a plot, measured from the base of the stem to the
tip of the main shoot. Branch number was the number of branches arising from the
main shoot of each harvested individual in a plot. The oil and protein content
of seeds was measured by Near Infrared Spectroscopy (NIR) using standard
methods.
## Statistical analysis
Year-location combinations were treated as independent environments. Environment
was treated as a fixed effect while genotype (DH or reconstructed F<sub>2</sub>
lines) was treated as a random effect. The broad-sense heritability was
calculated as: *h*<sup>2</sup> =
*σ*<sup>2</sup><sub>g</sub>/(*σ*<sup>2</sup><sub>g</sub> +
*σ*<sup>2</sup><sub>ge</sub>/n + *σ*<sup>2</sup><sub>e</sub>/nr). Where,
*σ*<sup>2</sup><sub>g</sub> is the genetic variance,
*σ*<sup>2</sup><sub>ge</sub> is the interaction variance of genotype with
environment, *σ*<sup>2</sup><sub>e</sub> is the error variance, n is the number
of environments and r is the number of replications. The genetic correlation was
calculated as: *r*<sub>G</sub>
= *cov*<sub>Gxy</sub>/(*σ*<sup>2</sup><sub>Gx</sub> ×
*σ*<sup>2</sup><sub>Gy</sub>)<sup>1/2</sup>, where, *cov*<sub>Gxy</sub>,
*σ*<sup>2</sup><sub>Gx</sub> and *σ*<sup>2</sup><sub>Gy</sub> were the genetic
covariance and variance of the pair-wise traits respectively. The significance
of each genetic correlation was determined using a *t* test of the correlation
coefficients. The estimation of variance and covariance components were obtained
using an SAS GLM procedure. The mean value for three replications in each
environment for both populations was used in subsequent QTL analysis for all
traits. General heterozygosity was calculated as
N<sub>H</sub>/(N<sub>T</sub>+N<sub>N</sub>+N<sub>H</sub>). N<sub>T</sub>,
N<sub>N</sub> and N<sub>H</sub> were the number of markers with genotypes of
Tapidor, Ningyou7 and both parents, respectively. Special heterozygosity was
calculated using the same formula but the statistics were restricted to the
marker that was significantly associated with phenotype (data not shown).
## Genetic linkage map
A total of 786 markers were mapped to the new linkage map generated with the
TNDH population using JoinMap 3.0 (<http://www.kyazma.nl/index.php/mc.JoinMap>).
This covered 19 chromosomes identified as A1–A10 and C1–C9, with an average
distance of 2.7 cM between markers. The threshold for goodness of fit was set to
≤5.0 with logarithm of the odds ratio (LOD) scores 1.0 and a recombination
frequency\<0.4. The order of the markers on the linkage map agreed well with our
published maps. The genotype of each RC-F2 line was deduced from the
corresponding genotype of their parents.
## Genome-wide detection of QTL, meta-analysis and test the result of QTL meta-analysis
QTL were detected by composite interval mapping using WinQTL cartographer 2.5
software (<http://statgen.ncsu.edu/qtlcart/WQTLCart.htm>). The number of control
markers, window size and walking speed were set to 5, 10 cM and 1 cM
respectively. The default genetic distance (5 cM) was used to define a QTL in a
specific experiment. The threshold of experiment wise error rate was determined
by permutation analysis with 1000 repetitions. LOD values corresponding to
P = 0.05 were used for identifying “significant” QTL. To avoid missing QTL with
very small effects, a lower LOD value corresponding to P≤0.50 was adopted for
the presence of “suggestive” QTL. The overlapping “suggestive” QTL and all the
“significant” QTL were admitted and named as “identified-QTL”.
The dominant degree of an identified-QTL was defined as d/\|a\|. For mode-of-
inheritance of identified-QTL the QTL was defined as additive (\|d/a\|\<0.2),
partially-dominant (0.2≤\|d/a\|\<0.8), dominant (0.8≤\|d/a\|\<1.2) and over-
dominant (\|d/a\|≥1.2).
Since QTL of the same traits or related ones detected in different experiments
and mapped to the same region of a chromosome, might in fact be several
estimations of the position of one single QTL, algorithms for QTL meta-analysis
were used to estimate the number and positions of the meta-QTL underlying the
analyzed QTL. This approach, using the *Akaike* information criterion (AIC),
provided the basis on which to determine the number of meta-QTL that best fitted
the results on a given linkage group. It also grouped the QTL detected in the
different experiments into classes that correspond to the same QTL and provided
a consensus estimation of QTL positions. Computations were conducted using the
*BioMercator2.1* software. At present, the method used in this software cannot
distinguish between models with more than four meta-QTL on the same linkage
group. If the estimated number of meta-QTL is more than four, *Biomercator2.1*
declares the most probable model as one with a number of meta-QTL equal to the
number of the analyzed QTL. Then the *Delete* function of the software was used
to select specific segments of a linkage group separated by regions with no QTL
and separately apply QTL meta-analysis to these segments. The software also
provides a method to calculate 95% confidence intervals for the meta-QTL:Where,
*S<sub>i</sub><sup>2</sup>* is the variance of position of the QTL<sub>i</sub>
and k is the total number of QTL integrated into the meta-QTL.
A two-round strategy of QTL meta-analysis was adopted. The QTL identified in
different experiments were first integrated into consensus QTL, trait by trait.
In the second round of QTL meta-analysis, the consensus QTL for the different
traits was integrated into unique QTL.
To test the result of QTL meta-analysis, ANOVA implemented in SAS/Stat version
8e was utilized to identify QTL × environment interaction by GLM (generalized
linear model) model: P = G + E + G × E. Where, P, G, E and G × E represent the
phenotype and the effects of genotype, environment and genotype by environment
interaction, respectively. The genotype of each consensus QTL was estimated by
that of the molecular marker closest to it's peak position. The significant
threshold was set as p≤0.05.
## Genome-wide detection of epistatic interactions
The maximum-likelihood estimation method in QTLmapper V2.0 software
(<http://www.cab.zju.edu.cn/ics/faculty/zhujun.htm>) was employed to detect the
epistatic interactions. It was based on mixed linear model and performs
composite interval mapping. The walking speed was set to 1 cM. The LR value
corresponding to P = 0.005 was used as the threshold for claiming the presence
of putative epistatic interactions. The significance of the epistatic effect was
further tested by running the submenu of the Bayesian test (using P≤0.005).
# Supporting Information
The authors thank Mr. Dianrong Li and Mr. Hao Wang (Hybrid Rapeseed Centre of
Shaanxi, Dali 715105, China) for the field work and collecting of the phenotypic
data.
[^1]: Conceived and designed the experiments: JS JM. Performed the
experiments: JS. Analyzed the data: JS RL JM. Contributed
reagents/materials/analysis tools: JS JM RL YL. Wrote the paper: JS RL JM
JZ.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
Brucellosis is considered one of the most important zoonosis. It is responsible
for important economic losses due to abortion and culling of infected animals.
In man, it causes a systemic febrile illness with a wide spectrum of symptoms,
although arthritis is among the most frequent manifestations. In cattle the main
causative agent of brucellosis is *Brucella abortus* and during pregnancy it can
infect and multiply intensely within trophoblastic cells of the placenta at late
gestation causing necrotizing placentitis associated with abundant neutrophilic
infiltrate.
The capacity of *B*. *abortus* to cause disease is related to its ability to
invade host cells, survive intracellularly, and evade antimicrobial defenses of
the host. However, the mechanisms related to this ability are not yet fully
understood. It is known, however, that once *Brucella* spp. reaches the
intracellular environment, the pathogen actively interferes with the host cell
metabolism and defense favoring its survival and intracellular multiplication.
*Brucella* spp. modulate intracellular trafficking by preventing maturation of
phagosomes and blocking endosome-lysosome fusion, which prevents the degradation
of bacteria.
Although *Brucella* spp. do not have classical virulence factors, these
organisms have several known mechanisms associated with pathogenicity. One of
these mechanisms is associated with LPS, which differs from other Gram-negative
bacteria. *Brucella* spp. LPS is a poor inducer of oxidative burst, reactive
nitrogen intermediates, and secretion of lysozyme. Some studies have also shown
that *Brucella* spp. LPS reduces TLR4 (Toll-like receptor 4) agonistic activity
and that despite being recognized by the receptor, this interaction does not
induce cytokine production. The *virB* operon-encoded type IV secretion system
(T4SS) is a key virulence factor for *Brucella* spp. This T4SS secretes effector
proteins through the envelope of the bacterial cell, and it is required for
intracellular survival and *in vivo* persistence of *Brucella* and this
expression is induced by acidification of the phagosome after phagocytosis.
Effector proteins secreted through the T4SS modulates maturation of the
*Brucella*-containing vacuole (BCV), preventing fusion of the BCV with lysosomes
at early stages of infection, and driving the interaction between BCV and
endoplasmic reticulum (ER) and subsequent maturation of BCV at later stages of
infection. Two *Brucella* proteins containing a TIR (Toll intracellular
domain/interleukin-1) domain, namely BtpA and BtpB, have been identified in
*Brucella* spp. BtpA and BtpB modulate the host inflammatory responses during
*Brucella* sp. infection by interfering with TLR signaling.
Proteomic analyzes in the context of the interaction between *B*. *abortus* and
its target cells are also scarce. These studies are very challenging due to the
high complexity of samples and very low concentrations of certain proteins,
requiring the use of highly sensitive analytical techniques. All proteomic
studies reported to date have used the model of infection of phagocytic cells,
whereas the profile of protein expression by trophoblastic cells infected with
*B*. *abortus* have not been previously studied. In this study, *ex vivo*
culture of trophoblastic cells in CAM explants was associated with proteomic
analysis to study the interaction between *B*. *abortus* and trophoblastic
cells.
# Materials and Methods
## Bacterial strain and growth conditions
The inoculum was prepared from cultures of *B*. *abortus* 2308 grown in 20 mL of
tryptic soy broth (Difco, USA) for 12–15 h at 37°C under agitation (200 rpm).
After incubation, optical density of bacterial suspensions was determined by
spectrophotometry (OD<sub>600</sub>) and adjusted to 1.0 x 10<sup>8</sup>
bacteria/mL. Number of bacterial cells was confirmed by serially diluting in PBS
(pH 7.4), and plating 100 μL of each dilution on tryptic soy agar (Difco) in
duplicate. After 48 h of incubation at 37°C with 5% CO<sub>2</sub>, colonies
were counted and the number of colony forming units (CFU) was obtained by
averaging the duplicates. CFU numbers were determined by the drop count method.
Manipulation of *B*. *abortus* was performed under biosafety level 3 conditions.
## Infection of chorioallantoic membrane (CAM) explants with *Brucella abortus* 2308
CAM explants were obtained from three intact bovine uteruses at the final third
of gestation collected at a local slaughterhouse (Frigorífico Uberaba Ltda,
Sabará, Minas Gerais), as previously described. Gestational age was estimated by
measuring the cephalococcygeal length (Crown-rump length). Only placentas from
*Brucella*-free fetuses, based on rose Bengal plate agglutination test using
amniotic fluid, were included in these experiments. This study was approved by
the Committee for Ethical use of Experimental Animals of the Universidade
Federal de Minas Gerais, Brazil (CETEA), under protocol 183/2010.
Chorioallantoic membranes were aseptically removed from the uterus and
immediately placed into RPMI 1640 (Roswell Park Memorial Institute) medium
(Invitrogen, USA) with 50 U/mL of penicillin and 50 g/mL of streptomycin
(Invitrogen, USA) for 20 min and were then washed two times with RPMI 1640 to
remove antibiotic residues. Explants were prepared using snapwell inserts
(Snapwell<sup>™</sup> Inserts—Corning, USA) and placed into six-well cell
culture plates (Corning) with supplemented medium (RPMI 1640 with 4 mM
glutamine, 1 mM pyruvate, 1 mM non-essential amino acids, 2.9 mM sodium
bicarbonate, 15% fetal bovine serum) in contact with the trophoblastic and
allantoic or amniotic surfaces. The central area of each explant was inoculated
with 200 μL of culture medium (supplemented RPMI 1640) containing 2.0 x
10<sup>7</sup> CFU, which correspond to a multiplicity of infection (MOI) of
approximately 1000. The explants were centrifuged for 15 min at 1000 xg and
maintained at 37°C in 5% CO<sub>2</sub> for 30 min to allow internalization of
bacteria. Extracellular bacteria were eliminated by adding 200 μL of medium with
gentamicin (50 mg/mL) (Invitrogen), followed by incubation for 1 h at 37°C under
5% CO<sub>2</sub>. After incubation, medium containing gentamicin was removed,
the explants were gently washed twice with PBS (phosphate buffered saline—pH
7.4) and then, 200 μL of supplemented RPMI 1640 medium was added in each
explant. In this assay, each group was evaluated in triplicate at 0.5, 2, 4, and
8 h after the removal of the gentamicin supplemented RPMI 1640. Explants in the
control group were inoculated with sterile supplemented RPMI 1640 medium and
submitted to the same conditions.
## Internalization of *Brucella abortus* by trophoblastic cells of CAM explants
Three explants from each group (uninfected and infected) obtained from the
chorioallantoic membrane of three fetuses were used to determine the number of
internalized bacteria. After removal of the RPMI culture medium with gentamycin,
the explants were washed twice in PBS pH 7.4 and then lysed with 200 μL of
sterile 0.1% Triton X-100 (Roche, Germany). Lysates were serially diluted in PBS
pH 7.4 and 100 μL of each dilution were plated on Tryptic Soy Agar (Difco, USA)
in duplicate. After 48 h of incubation at 37°C in 5% CO<sub>2</sub>, colony
counts were performed and the number of CFU was obtained from the average of
triplicates. CFU numbers underwent logarithmic transformation followed by
analysis of variance (ANOVA). Comparison of means was performed using the
Tukey's multiple comparison test with significance level of P≤0.05.
## Extraction of proteins from CAM explants
At 0.5, 2, 4, and 8 h after the removal of the medium containing gentamicin, CAM
explants were washed three times with RPMI 1640 medium without bicarbonate and
without fetal bovine serum. Protein extraction was performed using 200 μL of
lysis buffer (8 M urea, 2 M thiourea, 4% w/v CHAPS, 40 mM Tris 1M) containing
protease inhibitors (GE Healthcare, UK), which was added to the trophoblastic
surface of CAM explants. The explants were maintained under agitation (50 rpm)
for 30 min on an orbital shaker (IKA Labortechnik, Germany). Lysates were
centrifuged for 30 min at 20.000 xg at room temperature. The supernatant was
collected and kept at -80°C. Protein concentration was measured using the 2D
Quant Kit (GE Healthcare, UK).
## Two-dimensional gel electrophoresis (2DE)
In order to evaluate reproducibility and to determine the time points to be
evaluated by DIGE, the proteomic profile was assessed initially using 7 cm gels,
pH 4–7. Extracted proteins (50 μg) from CAM explants were added to the
rehydration solution (1.25 μL IPG buffer, pH 4–7, 10 μL/L—GE Healthcare, UK) and
IEF buffer (8 M urea, 2 M thiourea, 4% CHAPS, 0.0025% bromophenol blue, 10 mg/mL
dithiothreitol—GE Healthcare) in a total volume of 125 μL per IPG strip (7 cm,
pH 4–7) (GE Healthcare). Samples were incubated with IPG strips on rehydration
apparatus (Immobiline DryStrip Reswelling Tray, GE Healthcare, UK) for 12 h and
subjected to isoelectric focusing using the Ettan<sup>™</sup>
IPGphor<sup>™</sup> 3 Isoelectric Focusing System (GE Healthcare, UK) and the
program IPGphor (GE Healthcare, UK). Focused IPG strips were incubated for 15
min in equilibration solution (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol,
2% SDS, 0.002% bromophenol blue, and 125 mM DTT) and then alkylated for further
15 min in an equilibration solution containing 13.5 mM iodocetamide (GE
Healthcare, UK) instead of DTT. Electrophoresis was performed in 12%
polyacrylamide gel containing SDS in a vertical electrophoresis apparatus (Ettan
DALTsix electrophoresis Unit -GE Healthcare, UK) at 30 mA per gel. Bench Mark
Protein<sup>®</sup> Lader (Invitrogen, USA) was used as molecular mass marker.
Gels were stained with Colloidal Coomassie Brilliant blue G-250 (GE Healthcare,
UK) and digitized using the Image Scanner (Amersham Biosciences, England).
## Differential gel electrophoresis (DIGE)
Protein samples extracted from CAM explants were labeled using the CyDye DIGE
Fluors (minimal dyes) for Ettan DIGE kit (GE Healthcare, UK) according to the
manufacturer. To a pool containing 50 μg of an equal mixture of proteins
extracted from infected and uninfected CAM explants produced from three fetuses,
400 pmol of dyes were added. Dye swap was performed and Cy2 dye was used as
internal standard. Labeled samples and 800 μg of mixture of unlabeled proteins
were incubated with the IPG strip (18 cm, pH 4–7—GE Healthcare) in a rehydration
apparatus (Immobiline DryStrip Reswelling Tray, GE Healthcare, UK) for
approximately 12 h and subjected to isoelectric focus using Ettan<sup>™</sup>
IPGphor<sup>™</sup> 3 Isoelectric Focusing System (GE Healthcare, UK) and the
program IPGphor (GE Healthcare, UK). Electrophoresis was performed in 12%
polyacrylamide gel containing SDS in vertical electrophoresis apparatus (Ettan
DALTsix Electrophoresis Unit—GE Healthcare, UK) and scanned using Typhoon Trio
(GE Healthcare, UK) with excitation/emission wave lengths specific for Cy2
(488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). After scanning, gels were
stained with Coomassie Brilliant blue G-250 (Thermo Scientific, USA) for marking
the spots of interest.
## Image analysis of the gels
The 2D Image Master Platinum<sup>™</sup> software (version 6.0, GE Healthcare,
UK) was used for image analysis of the two-dimensional gels (2DE) stained with
Coomassie Brilliant blue G-250 by a combination of automatic detection and
manual detection of spots. To determine the relative amount of each spot was
used the method of normalization volume. Mean volumes of each spot were
calculated by the software, and spots with at least two-fold increase or
decrease were considered for further analysis. Means were compared by the
Student t test and considered significant when P≤0.05. For the fluorescent gels,
DeCyder<sup>™</sup> 2-D Differential Analysis v7.0 software (GE Healthcare, UK)
was used according to the manufacturer's instructions. The spots containing
proteins labeled with different fluorescent dyes were co-detected and quantified
in the three images obtained from each gel. Based on the ratio of the average
volume normalized spots that had at least a two-fold increase or decrease was
evaluated for statistical significance. Data was subjected to analysis of
variance (ANOVA) and means compared using the Student t test with significance
level of P≤0.05. Spots with significant changes were selected for identification
by mass spectrometry.
## In gel proteolysis and mass spectrometry (MS)
Selected spots were cut in pieces of approximately 1.0 mm<sup>3</sup> using
sterile scalpel. Discoloration of the gel with 400 μL of a solution containing
25 mM ammonium bicarbonate (NH<sub>4</sub>CO<sub>3</sub>) pH 8.0 (Synth, Brasil)
and 50% acetonitrile (Sigma-Aldrich, USA) was repeated three times for 15 min at
room temperature under vigorous agitation. Gel fragments were dehydrated with
200 μL of acetonitrile for 5 min and dried under vacuum centrifugation. Proteins
were digested using 20 μL of a solution containing 2.0 mM of
NH<sub>4</sub>CO<sub>3</sub> (Synth, Brasil) and 20 ng/μL trypsin (Promega,
USA), followed by incubation for 16–24 h at 37°C. Peptides were extracted with
5% formic acid (Sigma-Aldrich, USA) and 50% acetonitrile. Protein extracts were
concentrated (final volume of 10 μL) in ZipTip<sup>®</sup> C<sup>18</sup>columns
(Millipore, USA) and the final volume was reduced to 5.0 μL in a vacuum
centrifuge. For sample analysis in the mass spectrometer, 0.3 μL of sample
solution was mixed with the same volume of saturated matrix solution \[10 mg/mL
R-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid\]
(Sigma-Aldrich, USA). Raw data for identifying proteins were obtained from
ABSCIEX Proteomics Analyzer MALDITOF/TOF<sup>™</sup> System 5800 (Applied
Biosystems, USA). The external calibration mode MS was performed using a mixture
of four peptides: des-arg1-bradykinin (m/z = 904,468), angiotensin I (m/z =
1296.685); Glu1-fibrinopeptide B (m/z = 1570.677) and adrenocorticotropic
hormone (18–39) (m/z = 2465.199) (Applied Biosystems, USA). The MS/MS spectra
were calibrated externally using known masses of fragmentations observed in the
MS/MS angiotensin I. After data acquisition, a list of peaks was obtained from
the raw data of the MS/MS using the Mascot to function Peaks 4000 Series Explore
software (Applied Biosystems, USA).
## Identification of proteins in the database
All information acquired for each spot, i.e. the mass/charge ratio and intensity
of peaks, obtained in the spectrum and the reasons mass/charge and intensities
of the peaks relating to each of the five spectra MS/MS has been compiled into
one text file. This file was used by MASCOT (Matrix Science, USA)
(<http://www.matrixscience.com>) to perform the search in databases. Once
complete the search, MASCOT reports the results in a bar graph in which there is
a value taken as the limit. Scores below this value indicates random events,
i.e. no statistical value. On the other hand, if the score assigned to a given
protein exceeds the threshold value identifying the chance of an event being
generated randomly is 5% and the larger this value the greater the probability
of being correct.
The parameters used in the search were: no restriction for protein molecular
weight, a trypsin cleavage site lost, variable modifications of methionine
(oxidation) and cysteine (carbamidomethylation), formation of pyroglutamate at
the N-terminal glutamine without other post-translational modifications. The
mass tolerance for peptides in searches was 0.8 Da for MS spectra and 0.6 Da for
MS/MS spectra. The data bases of *Bos taurus* and *Brucella abortus* available
at NCBI (National Center for Biotechnology Information, USA) were used.
# Results
## Internalization of *Brucella abortus* 2308 in bovine trophoblastic cells of CAM explants
Considering that placentitis and abortion are the most important manifestation
of *B*. *abortus* infection in cattle, and that those are key steps for
transmission of the disease, in this study we used a previously developed bovine
CAM explant model of infection to evaluate differentially expressed host
proteins during the early stages of infection of trophoblastic cells with *B*.
*abortus*. In order to ensure that the three independent experiments were in
fact comparable, the number of bacteria located within trophoblastic cells, as
previously demonstrated, was determined after inoculation of CAM explants with
*Brucella abortus* 2308, centrifugation for 15 min at 1000 xg and incubation at
37°C in 5% CO<sub>2</sub> for 30 min, followed by 1 h of incubation with
gentamicin. The shows the average number of internalized bacteria (Log CFU/mL)
in three explants (triplicates) prepared from three independent experiments
(different fetuses). There were no significant differences in the number of
*Brucella abortus* internalized in CAM explants from different fetuses under
these conditions.
## Protein expression profile of CAM explants infected with *Brucella abortus*
The 2D gels (7 cm, pH 4–7) were compared to verify the reproducibility and the
occurrence of experimental variation related to the quantity of protein extract
added to the gel or/and intensity of the dye. According previous study, 2D gels
are considered reproducible when presenting percentage of matching spots higher
than 80%, and correlation ratio higher than 0.75. We observed that at each time
point after infection, 2D gels had high reproducibility when the triplicates of
uninfected CAM explants (% matching spots\> 84%; correlation ration \> 0.77) and
triplicate of infected CAM explants (% matching spots\> 88%; correlation ratio
\> 0.80) were compared. The proteomic profile of uninfected CAM explants
remained stable throughout the course of the experiments (% matching spots =
77%). The same was observed in the profile of infected CAM explants (% matching
spots = 80%). No significant changes were observed when the proteomic profile of
uninfected CAM explants at various time points were compared. In contrast, four
spots with significant changes in volume over time were detected in gels of
infected explants: one of the spots was significantly increased at 2 h when
compared to 4h, and the other three spots were significantly increased at 4 h
when compared to 8 h.
To determine the times points to be used for DIGE, an inter-class analysis in
which the gels of uninfected and infected samples were compared in the same time
post infection, was performed. There were no significant differences in volume
of spots when infected and uninfected explants were compared, in each times
evaluated. However, spots present in a single experimental group were detected
(qualitative differences). Thus, 0.5 and 4 h post infection were chosen to be
evaluated by the DIGE since these time points had the highest qualitative
differences.
## Differential gel electrophoresis (DIGE)
In the analysis by DIGE, there were no statistically significant differences in
the volume of the spots when gels from infected or uninfected explants were
compared, considering significant in this analysis, values of fold change \> 2
and P≥0.05. Interestingly, although no significant differences were observed in
the volume of spots detected in both experimental groups at both time points,
several spots were detected in only one experimental group (qualitative
differences), as well was observed in 2D gels analysis. These spots were
selected and numbered for identification by mass spectrometry (MALDI TOF/TOF).
From a total of 103 spots analyzed by mass spectrometry, 74 (72%) were
identified, from which 73 (98.64%) corresponded to proteins produced by the host
(*Bos taurus*). Only peptides from the spot number 129, identified as
adenosylhomocysteinase, matched with significant scores *B*. *taurus* and *B*.
*abortus*. Importantly, in this case, search in *Bos taurus* database resulted
in identification of seven peptides with high value score (378–17% coverage),
while search of the *B*. *abortus* data base identified one peptide with a low
score (61–4% coverage), which indicates a greater probability that this protein
was in fact expressed by bovine cells. The 74 spots identified corresponded to
51 proteins, i.e. the same protein was identified in different spots. All of
them were classified into 11 functional categories by Funcat (Functional
Classification of Proteins) (MIPS—Munich Information Center for Protein). In
order to better understand the distribution of proteins according to their
function and experimental group from which they were identified, a Venn diagram
showing the identified proteins in uninfected and infected CAM explants at 0.5
and 4 h was constructed.
In uninfected CAM explants group (0.5 and 4 h) were mainly identified proteins
related to the biogenesis of cellular components (cytokeratin 8, dynein light
chain roadblock-type 1, F-actin-capping protein subunit beta, transgelin),
proteins with binding or cofactor function (3-hydroxyisobutyrate dehydrogenase,
mitochondrial precursor, albumin protein, alpha-actinin-4, alpha-fetoprotein
precursor, beta actin, gelsolin isoform a, gelsolin isoform b, hemoglobin
subunit beta, prostaglandin reductase 2, transitional endoplasmic reticulum
ATPase) but also related to the cell structure (gelsolin isoform a, gelsolin
isoform b, alpha-actinin-4), proteins related to the metabolism
(3'(2'),5'-nucleotidase bisphosphate 1 3-hydroxyisobutirate dehydrogenase,
creatine kinase B-type, inositol-3-phosphate synthase 1, precursor
transthyretin) and related to protein fate (activator of 90 kDa heat shock
protein ATPase homolog 1, cathepsin D, endoplasmin precursor, heat shock cognate
71 kDa protein). It is also important to emphasize that most of the spots in
which these proteins were identified, were selected based on the comparison
between the two groups of uninfected CAM explants (77,5%, 38/49). Together these
findings suggest that the differentially expressed proteins observed in
uninfected CAM explants are related to the maintenance and stabilization of
cellular organization and adaptation to cell culture after a stress condition
related to the process of CAM explants preparation.
In addition, in *B*. *abortus*-infected CAM explants group the highest number of
proteins identified was classified as related to metabolism or cell rescue
defense and inflammation. Proteins with cell rescue, defense and inflammation
functions (n = 15) were identified only in explants infected with *B*. *abortus*
when compared to uninfected explants: 8 (53,3%) proteins identified 0,5 h post
infection (aldose 1-epimerase, aldose reductase, biliverdin reductase A, heat
shock protein beta-1, high-mobility group box 1, protein disulfide-isomerase A3
precursor, thioredoxin-dependent peroxide reductase and toll-interacting
protein) and 7 (46,7%) proteins identified 4 h post infection (aldose reductase,
complement component 1 Q subcomponent-binding protein, heat shock protein
beta-1, high-mobility group box 1, NADH dehydrogenase \[ubiquinone\] iron-sulfur
protein 8, protein disulfide-isomerase A3 precursor and thioredoxin-dependent
peroxide reductase). Moreover, the proteins aldose 1-epimerase, heat shock
protein beta-1, high-mobility group box 1-like protein disulfide-isomerase A3
precursor thioredoxin-dependent reductase peroxide, mitochondrial precursor, has
been identified in both groups of infected explants. The proteins albumin,
alpha-fetoprotein precursor and gelsolin isoform b were identified in more than
one experimental group but with different values of pI or molecular mass
indicating possible post-translational modifications.
# Discussion
Despite being bovine brucellosis a zoonotic disease that causes significant
economic losses worldwide due to abortion and culling of infected animals, the
pathogenic mechanisms are not yet fully understood.
Considering our results with what has been reported in the literature, we
suggest the hypothesis that at earlier stages of infection with virulent *B*.
*abortus* 2308, bovine CAM explants exhibited increase abundance of proteins
directed to recognition of bacteria, mostly activation of the innate immune
response, involving proteins related to TLR signaling and ROS production, as
well as expression of proteins associated with intracellular trafficking and
inflammation. Accordingly, we will direct our discussion to the simultaneous
involvement of the proteins identified here and that have been previously linked
to *Brucella* and their interactions.
One of the most interesting proteins identified in CAM explants during the
initial stages of infection with *B*. *abortus* was the Toll-interacting protein
(TOLLIP), a protein that modulates TLR signaling and also control membrane
trafficking processes by its interaction with proteins and phosphoinositides. As
TOLLIP is a protein that negatively modulates the inflammatory response,
expression of this protein in CAM explants infected by *B*. *abortus* at 0.5 h
post inoculation may be related to the state of immunosuppression observed at
early stages of infection, condition that can contribute to bacteria evasion of
initial immune response, important step in the establishment of infection. The
suppression of proinflammatory response by trophoblastic cells at early stages
of *B*. *abortus* infection has been previously shown since transcriptomic
analysis demonstrated a reduction of transcription of genes associated with TNF
superfamily, e.g. lymphotoxin beta, tumor necrosis factor, and ligand
chemokine—CXC motif at 4 h post infection. Recent published data from our group
indicated that this active suppression of proinflammatory responses induced by
*B*. *abortus* in trophoblastic cells requires a functional T4SS and the BtpB
TIR-containing protein. Increased expression of TOLLIP maybe a host mechanism
for controlling inflammation or it may be induced by the pathogen in order to
evade an effective immune response. It is known that *Brucella* spp. is able to
subvert immune response by the producing BtpA and BtpB that bind directly to
MyD88 thereby preventing TLR signaling.
Another protein that participates in TLR signaling, and that was identified in
infected trophoblastic cells in this study was the high-mobility group box 1
(HMGB1), a DAMP (damage associated molecular pattern), which participates in
signaling danger to other cells, activates innate and adaptive immune responses
and promotes tissue regeneration. HMGB1 is an endogenous ligand of TLR, which
may explain its ability to induce cellular activation and inflammatory responses
similar to those initiated by LPS. Considering that HMGB1 is passively released
by necrotic cells or actively secreted by activated cells of the immune system,
its ligation to several receptors that induce inflammation including TLR2, TLR4,
TLR9, and RAGE, activating NF-kB and inducing secretion of proinflammatory
cytokines, expression of HMGB1 by cell of CAM explants infected with *B*.
*abortus* can have a significant immunomodulatory effect and can potentially
impact the outcome of pregnancy, and along with other mechanisms may play a
relevant role in the development of necrotizing placentitis that is associated
with *B*. *abortus*-induced abortion in cattle.
The Ras-related protein Rab-11A (RAB11A) was upregulated in explants infected
with *B*. *abortus*. This protein is related to transport of TLR to phagocytic
vesicles containing Gram-negative bacteria and consequently with mechanisms of
pathogen recognition and immune response. Another protein related to
intracellular trafficking that was identified in infected CAM explants was
SCAMP2 (Secretory carrier membrane proteins 2), which is part of a group of
transmembrane proteins expressed in most eukaryotic cells that participate in
traffic vesicles between Golgi apparatus and plasma membrane and also plays a
role in exocytosis and endocytosis. The involvement of SCAMPs in formation of
*Salmonella* sp. containing vacuole (SCV) has been described. Several
intracellular pathogens are able to manipulate the secretory pathways of the
host cell, including *Brucella* spp. which interacts with the endoplasmic
reticulum to establish a niche for multiplication and to evade the host immune
response.
The proteomic screening in this study also identified proteins related to
generation of reactive oxygen species (ROS), and inflammatory responses induced
by oxidative stress (Aldose reductase—AKR1B1, NADH dehydrogenase \[ubiquinone\]
iron-sulfur protein 8—NDUFS8). Induction of ROS causes loss of intracellular
redox homeostasis, with altered cell signaling and development of pathological
processes. Moreover, *Brucella* spp. is capable to produce superoxide dismutase
(SOD), which protects against endogenous superoxide produced by aerobic
metabolism and respiratory burst from the host cell. Production of SOD prevents
bacterial death and enables the establishment and maintenance of intracellular
bacteria, and thus it is important for the survival of *Brucella* spp. in its
intracellular niche. Conversely, biliverdin reductase (BVR), thioredoxin-
dependent peroxide reductase (PRDX3—peroxiredoxin 3) and heat shock protein
beta-1 (HSPB1) or Hsp27 are proteins related to cytoprotection against oxidative
stress and were also identified in *B*. *abortus*-infected CAM explants.
Detection of these proteins in infected explants suggests an attempt of host
cells to reduce the damaging effects of ROS, although these proteins may also
favor intracellular survival of *Brucella* spp.
*B*. *abortus* is capable of inducing a strong neutrophilic and necrotizing
placentitis that is associated with transcription of CXCL8 and CXCL6 in CAM
explants and *in vivo* in the placenta of experimentally infected cows. In
present proteomic analysis of infected CAM explants resulted in the
identification of several proteins potentially related to recognition of
pathogens and tissue inflammation that had not been reported in previous
studies. Among these proteins, there was upregulation of the complement
subcomponent1 Q-binding protein (C1QBP), an intracellular protein found
predominantly in association with mitochondria and the nucleus whose expression
can be activated by the action of proinflammatory cytokines such as IFN-γ, TN
F-α, or LPS; protein disulfide isomerase A3 (PDIA3), a protein that belongs to
the superfamily of enzymes thioloxiredutases that is specifically associated
with peptide presentation via class I MHC and whose overexpression has been
recently related to immunological processes; aldose 1-epimerase or
galactosemutarotase (GALM), an enzyme that catalyzes interconversion of β-D-
galactose to α-D-galactose, whose increase in expression has been described in
inflammatory processes.
Interestingly, in *B*. *abortus* CAM explants there was low abundance of
adenosylhomocysteinase (AHCY), a highly conserved enzyme that catalyzes the
reversible hydrolysis of S-adenosyl-L-homocysteine to adenosine and homocysteine
so it is considered a key enzyme in immune response. The use of AHCY inhibitors
for therapeutic purposes in inflammatory and immune diseases is being studied,
since inhibition of AHCY is associated with immunosuppression.
# Conclusions
This study clearly demonstrated changes in the protein expression profile of
bovine trophoblastic cells in CAM explants in early stages of *B*. *abortus*
infection and that infection induces increase or production of proteins that may
be associated with the necrotic placentitis seen in infection of cattle
placenta. Several of the proteins that were upregulated during infection are
associated with modulation of the innate host immune response to infection with
*Brucella abortus*. Therefore, this study contributes to improving our
understanding of the mechanisms related to abortion caused by infection with
*B*. *abortus* in cattle.
# Supporting Information
This study was funded by the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), and the Fundação de Amparo a Pesquisa do Estado de Minas
Gerais (FAPEMIG—PRONEX). JPSM, APL and RLS have fellowships from CNPq.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JPSM HMA APL. Performed the
experiments: JPSM SFP. Analyzed the data: JPSM SFP ADC JP. Contributed
reagents/materials/analysis tools: JP RLS HMA APL. Wrote the paper: JPSM RLS
HMA APL. |
# Introduction
One of the striking patterns in geographic distribution of terrestrial
biodiversity is the increase in species richness towards lower latitudes in
several groups of organisms, including birds. The possible causes for this
pattern is one of the highly debated topics in ecology and evolution, even
though no definitive conclusion was yet been achieved,. The Neotropical area
alone holds a third of the recognized extant bird species (about 3,300 out of
10,000), with a biodiversity hotspot in the tropical forests. Moreover, recent
phylogenies suggest the number of species in the area is underestimated because
reproductively isolated lineages are frequently described in these studies. In
stark contrast to bird taxonomy in temperate zones, genetic evidence for species
limits in the Neotropics is often discordant with traditional taxonomy due to
the high incidence of species complexes. These complexes commonly feature
gradual variation in morphological and behavioural characters, masking the
occurrence of similar species that can be uncovered with genetic analyses.
DNA barcodes based on the 5′ portion of the cytochrome oxidase I gene (*COI*)
linked with specimens vouchers and locality information provides a rapid and
inexpensive method to identify species and detect ‘provisional new species’.
Pilot DNA barcode surveys in birds of North America, sister-species pairs, and
birds of Korea were successful in either identifying recognized species of
birds, and detecting some potential new species, except for a minor proportion
of cases where species are very recently diverged or hybridize. Critics
questioned if the success observed in North American birds could be extrapolated
to the tropics, where species clearly exhibit a higher level of phylogeographic
subdivision. However, DNA barcoding has subsequently proved to be highly
successful in identifying Neotropical species of birds; all 16 species (100%) of
antbirds (Thamnophilidae) that were barcoded and 494 of 500 (95.8%) species of
birds of Argentina had distinguishable *COI* signatures. The screening of
Argentinean birds also detected 21 species with deep intraspecific structure,
and revealed more complex patterns of regional divergence in the Neotropical
than in the North American avifauna. Even though more species will doubtlessly
be shown to share barcodes when complete coverage of species and genera is
available, it is clear that large-scale sequencing of *COI* associated with
vouchered specimens and locality information is a valuable tool in understanding
genetic differentiation within and among species of birds.
In this study we increased the coverage of Neotropical bird species that have
been barcoded by adding 637 samples from 431 species, with higher representation
in tropical forest areas of Brazil and Guyana, but also including samples from
localities ranging from Mexico to Argentina and Chile. We compared these
sequences with previously published sequences of congeneric species of
Neotropical birds, totaling 1,431 samples from 561 different species of birds,
296 of which were represented by multiple individuals. We showed that a high
success rate in species identification (93%) with DNA barcodes can be achieved
in this large sample of avian biodiversity from the mega-diverse Neotropical
region similar to that obtained in broad geographic surveys in the Nearctic and
Palearctic regions of the world. Additionally, a higher percentage (12%) of
species had multiple deep phylogeographic splits than in previous surveys, some
of which are likely reproductively isolated lineages.
# Results
## Species identification in Neotropical birds
About 93% of the species in our sample (520 out of 561) did not share sequences
with any other species included in the analysis, and when multiple individuals
were sampled (296 species), mean genetic distances among individuals were lower
than to the closest species from the same genus. Kimura 2-Parameter genetic
distances (K2P) within-species had a wide range (0 up to 13.7%), with more than
75% of the observations below 1% K2P. Conversely, 10% of the pairwise
comparisons were higher than 3% K2P (range 3.1–13.7%), overlapping considerably
with among-species variation. Pair-wise comparisons among-species of the same
genus were distributed from 0.08 to 20.3% K2P with most of the comparisons
observed between 5–15% K2P. Extremely high genetic distances suggestive of
higher rates of evolution or ancient divergences were observed among species
within *Trogon* (Trogoniformes) and *Crypturellus* (Tinamiformes), with maximum
distances of 19, and 20.3%, respectively. One specimen identified as
*Nothoprocta ornata* differed from other species in the genus by 24.7%, but was
only 0.34% divergent from specimens of **Tinamotis pentlandii.** Hence it was
either incorrectly identified in the field or possibly is a hybrid between the
two genera, as both species occur near to the collecting locality in Chile.
A total of 41 species did not have unique barcodes, of which 21 share sequences
with other species. Eight of those species despite being reciprocally
monophyletic, or represented by one sample, only differed from their sister
species by 1 to 6 diagnostic characters, or 0.14 to 0.86% K2P distance.
Aggregation of closely related haplotypes in phylogenetic trees can either
represent distinct taxonomic units, or random branches of lineages within the
same taxonomic group. To distinguish between the two scenarios, we applied a
statistical test of taxonomic distinctiveness proposed by Rosenberg for sister
species differing by less than 1% K2P. With the current limited sampling of
individuals, chance occurrence of reciprocal monophyly between species could not
be rejected (p\>0.05), so they were considered not distinguishable by *COI*
barcodes. Species pairs differed by few nucleotide substitutions, with marginal
values for the test of chance occurrence of reciprocal monophyly
(0.01\<p\<0.05). Thus the following species groups were considered to be
distinguishable by *COI* barcodes: the ducks *Anas puna*/*versicolor*
(p = 0.01), the greenfinches *Carduelis atrata/barbata/versicolor* (p = 0.01)
and the orioles *Icterus cayanensis*/*chrysocephalus* (p = 0.03).
Sixteen species had multiple divergent clusters (K2P genetic distances between
1.54 up to 13.7%), not recovered monophyletic with COI, that often corresponded
to samples from different areas of endemism or ecoregions (- cat. IV). A few
exceptions were observed, where paraphyletic divergent specimens were found in
the same geographic locality. For instance, specimens from the long-tailed
hermit (*Phaethornis superciliosus*) from Aripuanã and Juruena, both within the
Rondonian area of endemism, were 8% divergent, and the specimen from Juruena
differed from a scale-throated hermit (*P. eurynome*) from Southern Atlantic
forest by 7.4%. Even more strikingly, two samples of the yellow-margined
flycatcher (*Tolmomyias assimilis*) from Napo were 8.3% divergent. The species
pair of thrushes *Turdus albicollis/leucomelas* were paraphyletic in their *COI*
sequences, sharing barcodes in their Amazonian distribution.
## Deep genetic structure within Neotropical bird species
Deep intraspecific divergences in 48 species overlapped widely with among-
species distances (K2P 1.6 to 7.8%). These genetically structured species belong
to 21 bird families from nine different bird orders, most frequently represented
by antwrens (Thamnophilidae, Passeriformes). Most of the species with deep
genetic structure were broadly distributed in the Neotropics, and several are
subdivided into multiple subspecies. Often samples from different areas of
endemism or different ecoregions were the most divergent within species. Some
species showed genetic discontinuities in some pairs of geographic areas, but
not in others, such as the ochre-bellied flycatcher (*Mionectes oleagineus*).
Samples from the Napo, Imeri and Guyanian areas of endemism were not very
distinct genetically, but specimens from Belém were 2.76% divergent from the
others. All samples of the white-shouldered antshrike (*Thamnophilus aethiops*)
from different areas of endemism (Belém, Rondonian, Imeri, and Napo) had deep
instraspecific genetic variation. The deepest split was between Belém and the
other areas, and then the next split was between Rondonian, Imeri, and Napo.
## Phylogeographic patterns
Deep intraspecific divergences in different species were often located between
the same pairs of areas of endemism or ecoregions. The most common pattern
observed was between the Napo and Rondonian areas of endemism, followed by Belém
and Rondonian. However, phylogeographic splits between areas varied in depth in
different species. For example, distances between Napo and Rondonian were 8% in
*Tolmomyias assimilis*, *Myrmotherula longipennis*, and *Conopophaga aurita*, 5%
in *Thamnomanes caesius*, *Cyphorhinus arada*, *Hylophylax naevius*,
*Schiffornis turdina*, and *Jacamerops aureus*, and 2% in *Myrmoborus
myotherinus*, *Hylophylax poecilinotus*, *Deconychura longicauda*, *Deconychura
stictolaema*, *Hemithraupis flavicollis*, *Thamnophilus aethiops*, and *Monasa
morphoeus*. Between Guyanian and Napo areas of endemism instraspecific
divergences of 5 and 2% were observed in *Dendrocincla fuliginosa* and
*Jacamerops aureus*, respectively. Conversely, K2P genetic distances were close
to zero within the species *Dixiphia pipra* and *Nyctidromus albicollis*.
Genetic divergences between Belém and Napo were from 2 to 6%, with a wide range
of intermediate levels: 2.4 (*Phaethornis ruber*), 2.5 (*Formicarius colma*),
2.8 (*Mionectes oleaginous*), 3.3 (*Xiphorhynchus guttatus*), 3.6 (*Thamnophilus
aethiops*), 4.2 (*Jacamerops aureus*), 5.6 (*Dendrocincla fuliginosa*) and 5.7%
(*Turdus leucomelas*).
# Discussion
## Identification of Neotropical species with DNA barcodes
Despite the high success we obtained in Neotropical bird species identification
with DNA barcodes (93%), comparable to previous barcode surveys in birds, most
of the genera and species were not sampled across their entire distribution,
which overestimates its potential to differentiate species. This was observed
for at least two species previously distinct with DNA barcodes, *Anas
sibilatrix*, and *Celeus lugubris*, who were shown to be sharing sequences with
*Anas flavirostris* and *Celeus elegans*, respectively, when samples from other
areas of their geographic range were included in this study. When comprehensive
genus and species coverage becomes available in Neotropical birds, more species
are likely to not have unique DNA barcodes. Nonetheless, more certainty will be
achieved overall in the identification of species with *COI* barcodes because we
will be able to better address monophyly of lineages and to verify the frequency
with which individuals from different populations within species complexes are
exchanging genes. In most of the genera for which we had better species coverage
for *COI*, such as *Paroaria*, *Coryphospingus*, *Hemithraupis*, *Cyanerpes*,
*Cyanocompsa*, *Mimus*, *Phacellodomus*, and *Dendrocincla*, species did not
share barcodes. Even though we obtained only single sequences for many species,
they will contribute to future systematic efforts as part of the public
standardized DNA barcode library. They also will aid in faster identification of
specimens that are difficult to identify morphologically, such as embryos and
eggs, which will positively impact the conservation of avian wildlife in the
Neotropical region.
## Species not identified by DNA barcodes
Among the species we considered not identifiable with COI barcodes, some were
very closely related with very similar barcode sequences (category III). Our
sampling was not comprehensive enough to reject their monophyly by chance, but
once more individuals from different areas of their range are included stronger
support might be adduced for their reciprocal monophyly. On the other hand some
species might be recovered as not monophyletic with increased sample sizes, due
to unsorted ancestral polymorphism or hybridization. In that case they would not
be identified by DNA barcodes at the species level, suggesting that future
studies should employ multilocus phylogenetic inference with faster evolving
nuclear sequences in a coalescent framework to try to resolve species lineages.
Once larger sample sizes are available for closely related species, character-
based approaches implemented automatically, such as in CAOS, are preferable to
genetic distance levels to determine their distinctiveness, as distance levels
within and among species can overlap considerably even when substitutions among
species are fully sorted.
The species recovered as non-monophyletic (category IV) are strong candidates
for taxonomic revision, and some of their divergent lineages might correspond to
different species. For instance, the divergent lineages within the bearded
flycatcher (*Myiobius barbatus*) belong to different recognized subspecies:
*amazonicus*, *insignis*, and *mastacalis*. They currently are allopatric, have
morphological differences and differ in their K2P genetic distances by
12.6–13.7%. The three subspecies clades were not recovered as monophyletic with
COI barcodes because the ruddy-tailed flycatcher (*Terenotriccus_erythrurus*)
and the black-tailed myiobius (*Myiobius_atricaudus*) were included in the
species clade. Similarly, specimens from North and South Atlantic forest of the
rufous gnateater (*Conopophaga lineata*) differ by 9.6%. However, the lineages
from the two localities are not monophyletic because the chestnut-belted
gnateater (*Conopophaga aurita*) and the hooded gnateater (*Conopophaga
roberti*) are embedded in this group, as shown previously with more
comprehensive sample sizes and mitochondrial markers. The morphological
characters used to define these lineages as members of a single species could be
under strong stabilizing selection, and thus not mirroring the accumulation of
mutations through time in neutral genes like *COI*. Most cases of paraphyly in
birds are caused by incorrect taxonomy. Alternatively, paraphyletic species can
arise when geographically isolated lineages merge in part of their distribution
before complete reproductive isolation has evolved. Phylogeographic studies
including samples from their entire geographic range and from the closely
related species are needed to properly understand their diversification
patterns, and establish their taxonomic status.
The 17 species that shared barcodes with closely related species in sympatry
likely experienced hybridization, or recent speciation and incomplete lineage
sorting, or could simply be examples of incorrect taxonomy or sample
misidentification. For instance, the flightless steamer duck (*Tachyeres
pteneres*) shares barcodes with the flying steamer duck (*Tachyeres
patachonicus*) in Argentina, even though these species are very distinct
morphologically. In this example, misidentification of the sample is less
likely. A multigene phylogeny of four duck genera also reported difficulty in
resolving the relationships among species of *Tachyeres*, and attributed this to
a rapid diversification of the group, with possible incomplete lineage sorting,
founder effects, and introgression. The tawny-crowned greenlet (*Hylophilus
ochraceiceps*) had intraspecific clusters differing by almost 7% sequence
divergence between Napo/Imerí and Rondonian endemic areas, and shared barcodes
with the grey-chested greenlet (*H. semecinereus*) in their Rondonian
distribution. Both species are comprised of multiple subspecies, and some of
their variants are morphologically alike. The current taxonomy of the genus
might not be an accurate reflection of lineage relationships, but
misidentification of samples cannot be ruled out.
Two species pairs occurring in allopatry were not reciprocally monophyletic: the
bicolored (*Gymnopithys leucaspis*) and rufous-throated (*Gymnopithys rufigula*)
antbirds, and the ochre-collared piculet (*Picumnus temminckii*) and spotted
piculet (*Picumnus pygmaeus*). In both cases they are morphologically distinct
and do not share identical barcodes with the other species; genetic distances
among samples were around 0.5% and 1.0%, respectively. In these examples the
lack of reciprocal monophyly could be result of recent speciation and shared
ancestral polymorphism, and hybridization. A faster evolving marker such as the
control region or larger mitochondrial sequences might recover their reciprocal
monophyly.
## Complex patterns of population structure detected with DNA barcodes
Our results agree with previous hypotheses that complex patterns of speciation
were responsible for the high diversity in Neotropical bird species, and
strongly supports the view that most avian species in the region are narrowly
endemic rather than widely distributed. Several hypotheses were proposed to
explain the patterns of taxon distribution in the Amazonian lowland region. The
forest *refugia* hypothesis, suggested that cycles of expansion and retraction
of dry patches within forest areas were associated with interglacial and glacial
periods, and this could create multiple events of isolation among widely
distributed groups, promoting speciation. The *riverine* hypothesis suggested
that the formation of the rivers in the Amazon region could have acted as
important geographic barriers to promote speciation, as they delimit most areas
of endemism. This would have started at least by the end of the Miocene with the
uplift of the Northern portion of the Andes. Another proposal is the *marine
incursions* hypothesis, in which sea-level rises of about 100 m in the
Quaternary and late Tertiary are suggested to have fragmented the Amazonian
lowland into a large number of true islands and archipelagos, favoring active
allopatric speciation. The wide range of divergence levels we observed within
the 61 non-monophyletic and monophyletic species with deep intraspecific
variation (1–13% K2P distances), together with the high incidence of recently
evolved species, is consistent with speciation events starting well before the
Pliocene and Pleistocene, and extending to more recent geologic periods.
Although several groups of species have similar patterns of genetic and
geographic breaks among the same areas of endemism, different levels of genetic
distances between the same areas were also recovered in other species. The wide
range of intraspecific genetic distances observed between a pair of geographical
localities might reflect multiple vicariant events that have occurred at
different geological times, or they could reflect multiple dispersal events that
followed a major isolation process, or variation in rates of evolution across
different species, whose populations were isolated by a single vicariant event.
Additionally, a significant relationship was observed in previous studies
between interspecific levels of cross-barrier genetic differentiation within the
forest stratum at which a species forages in Neotropical rain forest. More
comprehensive taxon sampling and estimates of times of diversification that take
into account variation in rates of evolution across lineages are needed to
properly associate the diversification of a particular taxon with geographical
events.
We have chosen not to flag divergent lineages as provisional new species,
because our sampling was not comprehensive enough to properly quantify genetic
variation in each locality in different species, such as the red-eyed vireo
(*Vireo olivaceus*) and the ultramarine grosbeak (*Cyanocompsa brissonii*).
Specimens of red-eyed vireo from Puna+Napo and Atlantic Forest were genetically
divergent (2–3%), but haplotypes from the Atlantic Forest and Puna were observed
in the Chaco. Similarly, specimens of ultramarine grosbeak from Caatinga and
Puna were also divergent (2.7%), and both haplotypes are also found in Chaco.
Both species may have reinvaded the Chaco after being isolated on the borders of
this area. To check if these lineages deserve species recognition it is
important to investigate if the highly divergent specimens in sympatric zones
are reproductively isolated. Some of the deep intraspecific lineages we
described in this study were reported previously, such as the difference among
thrush-like Schiffornis (*Schiffornis turdina*) from Rondonian and Napo areas of
endemism. Others, such as the whiskered myiobius (*Myiobius barbatus*) from
Belém, Para2 and Atlantic forest will likely prove to be different species.
DNA barcodes of several new species of Neotropical birds will contribute to a
deeper understanding of the systematics and diversification of these taxa in the
area. Assuming the current species taxonomy, studies of historical patterns of
diversification of species in the area can be obscured since many species were
revealed not to be monophyletic. Moreover, a high number of species in the
Neotropical realm are comprised of multiple divergent lineages, thus the sample
sizes of barcoded individuals and other markers within and among species in the
area need to be higher than in other biogeographic areas that are not as taxon-
diverse. This can be achieved by complementary efforts of independent research
groups. Common and divergent patterns of genetic distances observed within and
among closely related species suggest that multiple geographic processes have
shaped the distribution of avian taxa in the Neotropics, and DNA barcodes
surveys will continue to reveal many more interesting geographic patterns in the
region.
# Materials and Methods
## Taxon sampling
We analyzed 637 individuals from 431 species of Neotropical bird species from
two tissue collections: Laboratório de Genética e Evolução Molecular de Aves
(LGEMA) in the Universidade de São Paulo, São Paulo, and The Royal Ontario
Museum in Toronto (ROM), with high representation in the Amazon lowlands and
Atlantic Forest. Whenever available, individuals from different localities of
their distribution range were sampled (GenBank numbers JN801479 - JN802115,
project “Neotropical-BRAS” in the completed projects section of the Barcode of
Life Data System- BOLD). To increase intraspecific sampling and to compare more
closely related congeneres, we added sequences of individuals from the same
species and same genera of Neotropical birds from the study of birds from
Argentina (project “Birds of Argentina-Phase I-BARG” in the completed projects
section of BOLD), thus extending our survey to 1,431 samples from 561 different
species.
## DNA extraction and amplification
DNA was extracted by a membrane purification procedure in glass fiber-filtration
plates (Acroprep 96 Filter Plate- 1.0 µm Glass, PALL Corporation), and collected
in PCR plates. Sequences of about 700 base pairs (bp) were obtained from the
5′end of the mitochondrial gene Cytochrome oxidase I (*COI*). Polymerase Chain
Reaction (PCR) amplifications were performed in 12.5 µL reactions in a buffer
solution containing 10 mM Tris-HCl (pH8.3), 50 mM KCl, 2.5 mM MgCl<sub>2</sub>,
0.01% gelatin, 0.4 mM dNTPs, 0.2 µM of each primer, 1 U *Taq* Polymerase
(Invitrogen) and 20–25 ng of DNA. Cycle conditions were: an initial denaturation
at 94°C for 5 min, 36 cycles of 94°C for 40 sec, 50°C for 40 sec and 72°C for 1
min, and a final extension at 72°C for 7 min. Bird universal primers used in
*COI* amplifications were LTyr (forward – TGTAAAAAGGWCTACAGCCTAACGC) and
COI907aH2 (GTRGCNGAYGTRAARTATGCTCG) resulting in a long but very stable
amplified product of about 910 bp. This primer set successfully amplified the 5′
end of *COI* across a wide range of bird species. The amplified segments were
purified by excising bands from agarose gels and centrifuging each through a
filter tip. Sequences were obtained on an ABI3730 (*Applied Biosystems*)
according to the manufacturers' suggested protocols using the same primer LTyr
to sequence the 5′end, and the internal primer COI748Ht (reverse-
TGGGARATAATTCCRAAGCCTGG) to sequence the reverse 3′end, resulting in a
sequenced product of about 750 bp. Sequences were checked for ambiguities in
CodonCode Aligner (*CodonCode Corporation*), and Geneious 5.3.
## Data analyses
Sequences were aligned in Geneious 5.3 using the Geneious alignment algorithm,
with gap penalty set as 12.8, and gap extension penalty set as 3. Species and
genera counts were performed in the software environment R 2.12. Genetic
distances were calculated under the Kimura 2 –Parameter model (K2P) for all
pair-wise comparisons in the matrix using PAUP4b10. Two datasets of genetic
distances were built in R: the first, including all within-species comparisons;
and the second, including among-congener comparisons (excluding within-species
ones). We wrote R scripts to summarize the mean, variance, maximum, and minimum
genetic distances per species and among congeners, respectively, using the first
two datasets. Frequency plots of pairwise genetic distances for congeners of
different species, and with only within species comparisons were built in R. The
maximum likelihood tree topology for the complete dataset was calculated in
Geneious 5.3 using PHYML. The best fit-model (General Time Reversible with
proportion of invariable sites and gamma, GTR+I+Γ, I = 0.5, Γ = 0.42) was
selected with jModelTest with a sample of the original dataset including one or
two representative samples of each bird family. Species were considered not
distinguishable by DNA barcode if: a) they were not monophyletic; b) they shared
barcodes with other species; or c) their intraspecific variation overlapped with
the lowest 5% of among-species variation, and reciprocal monophyly of sampled
individuals could not be distinguished from random branching at p = 0.05 with
the test for chance occurrence of reciprocal monophyly.
Within-species clusters with minimum pairwise distances higher than 1.5% K2P
were considered for analyses, because this level of genetic distances overlapped
with more than 5% of among congeners comparisons , but information on clades
differing by less than 1.5% K2P distance is also available. Species without
unique barcodes were sorted into the following non-exclusive categories: I) they
share barcodes with species occurring in sympatry or II) they share barcodes
with species occurring in allopatry, or III) were monophyletic differing from
their sister species by few mutations, or IV) paraphyletic species with lineages
more than 1.5% divergent.
For all the paraphyletic and monophyletic species with deep intraspecific
divergences, we compared the genetic discontinuities with the geographic
locality of the samples. Because areas of endemism are known to harbor unique
biota, and many subspecies of birds are delimited also by these zones, we
classified the sample localities of individuals according to the areas of
endemism in the Amazon and in the Atlantic forest where they occur. We adopted
the revised areas of endemism in Amazon and Atlantic forest from Bates *et al.*
and Borges. Samples collected in other localities were classified according to
their respective ecoregion according to the simplified map from Haffer.
# Supporting Information
We thank A. C. Mendez, Alexandre Aleixo, Alexande Martensen, Alexandre Uezu,
Camila Ribas, E. Machado, Fernando d'Horta, Fernando Nodari, Guilherme Brito,
Gustavo Gabanne, Luis Fabio Silveira, Pedro Develey, Renato Gaban Lima, and
Rodrigo Pessoa for collecting the biological samples deposited at LGEMA and used
in this study. For permission to barcode loaned samples we thank the American
Museum of Natural History and Field Museum of Natural History; Nicole Leung and
Nicolle Domnick for their help with sample processing, Bernd Schierwater and
three anonymous reviewers for valuable suggestions on the manuscript, and Cedrik
Juillet for advice in developing the R scripts.
[^1]: Conceived and designed the experiments: EST PG. Performed the
experiments: EST PG CYM. Analyzed the data: EST PG. Contributed
reagents/materials/analysis tools: CYM AJB. Wrote the paper: EST PG CYM AJB.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
Quantum dots (QDs) are semiconductor nanocrystals that are gaining in popularity
over organic fluorophores in applications ranging from bioimaging and
analytical assays to electronic displays, solid-state lighting and
photovoltaics. QDs commonly consist of a CdSe, CdTe, ZnSe, or PbSe core
coated with a ZnS shell to enhance stability and optical properties. In some
cases, the shell is further functionalized with thiols or amphiphilic polymers
to make the nanocrystals soluble in aqueous solvents and to allow for
biomolecule conjugation.\[–\] As QDs become more prevalent in consumer products
that will be used, reused, recycled, and landfilled, concerns have been rising
about their impact on humans and the environment.
Studies conducted with cultured eukaryotic cells have revealed that QDs can
exert cytotoxic effects through a variety of mechanisms. These include leaching
of toxic heavy metals from the inorganic core, (photo)generation of reactive
oxygen species that induce oxidative stress, and direct or indirect damage to
genomic DNA and biological membranes.\[,–\] Parameters such as size, shape,
composition, and surface coating(s) can all impact cytotoxicity outcomes and do
so in a mechanism-specific (and cell-specific) manner. For instance, while ZnS
shells or polymer coatings can reduce the cellular toxicity of CdSe
nanocrystals, they do little to prevent photo-induced DNA damage.
Similar observations have been made in animals, where the situation is further
complicated by the route of exposure and where long-term retention in the liver,
spleen, kidney, and lymph nodes is of primary concern.
There is considerably less information on how QDs interact with prokaryotes
although these abundant microorganisms will be first to come into contact with
engineered nanomaterials that find their way into the environment. Furthermore,
most studies conducted to date have focused on cadmium-based QDs (CdS, CdSe and
CdTe cores) produced with different synthesis schemes and coatings, and used at
different doses with a variety of strains and culture conditions.\[–\]
To answer a growing demand for the production of functional nanomaterials
through environmentally friendly processes, we previously described a set of
“designer” proteins that support the low-temperature and aqueous fabrication of
undoped and transition metal-doped ZnS QDs to which antibodies can be conjugated
by simple mixing.\[–\] Because zinc is not as toxic as cadmium, these
particles should exhibit low cellular toxicity upon core dissolution, and
because they are capped by proteins as part of the manufacturing process, their
shell is already biologically-relevant.
Here, we used *E*. *coli* as a model organism to investigate the penetration
requirements, subcellular localization, induction of stress responses, and long-
term fate of luminescent ZnS:Mn nanocrystals fabricated with a minimized
designer protein. Our results suggest that such protein-coated fluorophores
are environmentally benign because their uptake requires membrane
destabilization, they only induce oxidative stress at high doses, and they are
rapidly diluted by cell division.
# Results and Discussion
## Uptake by *Escherichia coli* requires membrane destabilization
We recently reported that BB-CT43, a minimized designer protein consisting of a
linear ZnS binding peptide (CT43) fused to an antibody-binding domain derived
from *S*. *aureus* Protein A (BB) is suitable for the one-pot synthesis of
ZnS:Mn QDs. These luminescent nanocrystals are produced when the designer
protein caps the growth of the inorganic core at about 4 nm, a process that
is schematically illustrated in. With their protein shell, the particles have an
overall hydrodynamic diameter of 9.5 ± 2 nm and a zeta potential of -16.5 ± 6
mV. They exhibit a strong emission peak at 590 nm under UV illumination, can be
decorated with antibodies by simple mixing and are stable for months without
aggregation or degradation of optical properties. Unlike traditional QDs,
these fluorophores are manufactured using mild aqueous conditions, do not
contain highly toxic heavy metals such as cadmium, and sport a built-in protein
shell coat. Thus, they should have a minimal impact on microbial life and the
environment.
As a first test of this hypothesis, we studied the uptake of BB-CT43-stabilized
QDs by *Escherichia coli*, a well-studied gram-negative organism whose envelope
consists of a 5 nm-thick phospholipid bilayer inner membrane, a 12 nm-thick
interstitial space known as the periplasm, a 1 to 2 nm-thick peptidoglycan
layer, and a 13 nm-thick, negatively charged outer membrane composed of a
lipopolysaccharide outer leaflet and a phospholipid inner leaflet.
Like all prokaryotes, *E*. *coli* lacks the endocytosis pathways responsible for
nanoparticle uptake by eukaryotes. In addition, bacterial porins, which allow
free diffusion of small molecules across prokaryotic membranes through 1–2 nm
pores, should be too small to allow even the smallest QDs to enter the cell.
Nevertheless, Hirschey and coworkers reported that CdSe/CdS QDs stabilized by
citrate, isocitrate, succinate, or malate readily penetrate *E*. *coli* when
their inorganic core is smaller than 6 nm. By contrast, Wenhua *et al*. found
that the uptake of mercaptoacetic acid-stabilized QDs with 3 to 4 nm CdSe/CdS
cores require chemical destabilization of the outer membrane, while Nadeau
and coworkers reported that internalization of adenine-coated CdSe QDs strictly
depends on photo-induced membrane damage and purine metabolism.
To determine whether BB-CT43-stabilized nanocrystals would be uptaken by
unmodified *E*. *coli*, we incubated 0.5 μg/mL of nanoparticles with mid-
exponential phase *E*. *coli* cells for 2h at room temperature. Cells were
washed to remove nonspecifically bound particles, pelleted by centrifugation and
exposed to UV light. Under these conditions, there was no detectable nanocrystal
uptake. However, in agreement with the results of Wenhua and coworkers,
fluorescent material colocalized with sedimented cells if they were first made
chemically competent by incubation with 100 mM CaCl<sub>2</sub> at low
temperature. This treatment transiently affects the integrity of the outer
membrane and is routinely used for introducing naked DNA into cells, although
the precise mechanisms at play remain unknown.
Not unexpectedly, uptake was dose-dependent and we observed a linear increase in
cell fluorescence at 590 nm (the QD emission maximum) when the particle
concentration was increased from 0.5 to 2.5 μg/mL. We conclude that chemical
disruption of the outer membrane is required for the uptake of QDs coated with
protein shells and whose inorganic cores are less photo reactive than CdSe/CdS.
## Internalized QDs localize to the cytoplasm
To confirm that the nanocrystals were not simply adsorbed to the surface of the
outer membrane, competent cells incubated with 0.5 μg/mL of BB-CT43-stabilized
QDs as above were subjected to spheroplasting. This procedure strips *E*. *coli*
of its outer membrane and peptidoglycan layer, cause release of periplasmic
contents in the surrounding medium, and leads to loss of rod shape and the
formation of spherical vesicles bounded by the inner membrane. shows that
spheroplasts remained fluorescent, indicating that the QDs were either
associated with the inner membrane or had translocated to the cytoplasm.
Because confocal microscopy does not allow one to unambiguously distinguish
between these two possibilities, we took advantage of the fact that the emission
spectrum of UV-excited ZnS:Mn nanocrystals overlaps the absorption spectrum of
the fluorescent protein mCherry. Thus, Forster Resonance Energy Transfer
(FRET) should occur between the two fluorophores if they co-localize to the same
cellular compartment and are separated by distances smaller than 10 nm.
To test this idea, we first recorded the fluorescence emission spectra of
competent cells that had taken up QDs or had been exposed to buffer alone
following excitation at 280 nm. Subtraction of the two spectra eliminated the
contribution of background fluorescence and revealed a weak but clear peak
centered at 590 nm and corresponding to ZnS:Mn emission (orange). Next, we
introduced a plasmid expressing mCherry at high level in the cytoplasm of *E*.
*coli* and confirmed that the emission spectrum of these cells exhibited the
characteristic shape and 610 nm emission maximum of mCherry upon excitation at
590nm (, inset). Finally, we made competent mCherry-producing cells, exposed
them to QDs or buffer, and collected fluorescence emission data following
excitation at 280 nm. The subtracted spectrum (red) shows that the QD-associated
peak at 590 nm peak completely disappeared to the profit of a 610 nm peak
corresponding to mCherry emission. We conclude that nonradiative energy transfer
occurs between QDs (donor) and mCherry (acceptor) and therefore that both
species are located within a few nanometers of each another in the cytoplasm.
How QDs (or plasmid DNA for the matter) translocate across the peptidoglycan
layer, periplasm and inner membrane to reach the cytoplasm remains unclear. One
possible explanation is that they rely on the transient and
CaCl<sub>2</sub>-induced opening of sites where the outer and inner membranes
come into intimate contact. Such adhesion zones, known as Bayer’s patches, were
identified microscopically over 40 years ago, but their existence has
remained controversial in spite of supporting biochemical evidence.
## High doses of ZnS:Mn nanocrystals are required to induce an oxidative stress responses
We next investigated how the presence of BB-CT43-stabilized nanocrystals in the
cytoplasm would impact cell physiology. Prokaryotes have evolved complex and
redundant mechanisms to survive exposure to environmental stresses. Many of
these processes rely on increasing the synthesis of protective proteins (e.g.,
molecular chaperones, proteases, DNA repair enzymes and reductases) through
upregulation events that are often controlled at the transcriptional level.
Previously, we described *E*. *coli* cells harboring single-copy gene fusions
between the stress-inducible *ibp* or *sulA* promoters and the *lacZ* gene
(which encodes β-galactosidase). These strains report on the amount of
stress experienced by the cell as a result of cytoplasmic protein misfolding
(*ibp*::*lacZ* fusion) or DNA damage (*sulA*::*lacZ* fusion) by producing the
easily assayed enzyme, β-galactosidase. Because QD cytotoxicity has repeatedly
been correlated with oxidative damage,\[,–\] we constructed an additional
isogenic strain bearing a single-copy gene fusion between the oxidative stress
responsive promoter of the major *E*. *coli* catalase (the OxyR-regulated *katG*
gene product) and *lacZ*.
The functionality of the reporter panel was first confirmed using hydrogen
peroxide, nalidixic acid and ethanol at concentrations known to cause extensive
oxidative stress, DNA damage, or protein misfolding, respectively. These
chemicals caused an about 3-fold induction of the corresponding promoters
(positive controls). Next, the three strains were made chemically competent,
exposed to QDs, and cultures were assayed for β-galactosidase activity after 3h.
While there was no detectable induction of any of the stress promoters when QDs
were supplied at the concentration used in all above experiments (0.5 μg/mL),
addition of 2.5 μg/mL nanocrystals was as effective as the use of 10 μM
H<sub>2</sub>O<sub>2</sub> in inducing the *katG* promoter. Of note, however,
there was no statistically significant activation of either the *ibp* or *sulA*
promoter under the same conditions. While the dependency of toxicity on QD dose
is not particularly surprising, our results indicate that it takes highly
concentrated solutions of nanocrystals to fully induce the bacterial oxidative
stress response and that BB-CT43-stabilized QDs do not cause appreciable protein
misfolding or DNA damage under the same conditions.
## QD fluorescence is rapidly lost in growing cells
The persistence of toxicants in the environment can lead to their long-range
transport and bioaccumulation at toxic doses in animal and human tissues. To
gain information on the *in vivo* stability of BB-CT43-stabilized QDs, we first
incubated cells that had internalized nanocrystals in phosphate buffered saline
(PBS) for 24h at temperatures ranging from 4 to 42°C. There was no significant
change in the fluorescence of cell pellets indicating that protein-capped
nanocrystals are stable for extended periods of time in quiescent cells exposed
to a physiologically relevant range of temperatures.
To determine if growth or metabolic activity would influence this outcome, QD-
loaded cells were taken in LB medium or PBS and incubated at 37°C, the optimum
growth temperature for *E*. *coli*. While non-growing cells held in PBS did not
lose their initial fluorescence, we observed a linear decrease in fluorescence
over time and nearly complete disappearance of the signal after 3h of
cultivation in LB medium (, closed symbols). Because cells experienced a 1h lag
phase and exponential growth only started about 2h after transfer to LB (open
symbols), the nearly 50% loss of fluorescence that occurs over the first 1.5h of
cultivation cannot be attributed to QD dilution by cell division. Indeed, when
the experiment was repeated in the presence of the translational inhibitor
kanamycin, we observed a similar fluorescence loss over the first 1.5h but,
remarkably, no further decrease thereafter. Thus, although *de novo* protein
synthesis and/or cell growth are not implicated in initial signal loss, they are
necessary for complete elimination of QD fluorescence.
There are several possible explanations for the initial fluorescence loss:
dissolution or extrusion of about 50% of the internalized nanocrystals or
substitution of the BB-CT43 shell by host species that change the QD optical
properties. We do not believe that chemical dissolution of the nanocrystals is a
likely mechanism since it would be unlikely to abruptly stop in kanamycin-
treated or quiescent cultures ( and Figs).
To directly test the possibility that active extrusion was involved, we repeated
the experiment of in isogenic cells containing or lacking TolC, a trans-
periplasmic protein that functions as an exit duct for the expulsion of a wide
variety of small drugs and proteins from the cytoplasm to the growth medium.
The lack of significant difference in the kinetics and extent of fluorescence
loss in *tolC*<sup>+</sup> and *tolC* ruled out the involvement of TolC-
dependent QD export.
While we cannot rule out extrusion through other systems, we favor a mechanism
in which endogenous species replace at least some of the bound BB-CT43 at the
ZnS:Mn surface and cause a decrease in emission intensity through fluorescence
quenching. Such quenching phenomena have previously been described and exploited
for ZnS:Mn QDs. This explanation is consistent with our previous finding
that ZnS:Mn nanocrystals fabricated with BB-TrxA::CT43 have about 30% lower
emission intensity than those synthesized with BB-CT43 owing to fluorescence
quenching by the TrxA domain. It is also in agreement with the fact that the
fluorescence of kanamycin-treated cultures reaches a plateau after 1.5h, a time
that is presumably needed to modify the surface of all internalized
nanocrystals. Why samples taken in PBS do not experience a similar initial
decrease in fluorescence remains unclear but the process appears to require
metabolic activity. Irrespective of the precise mechanism of initial
fluorescence loss, the data of shows that the QD signal is rapidly lost in
actively growing cultures due to dilution by cell division.
# Conclusions
We have shown here that protein-coated ZnS:Mn nanocrystals can translocate in a
dose-dependent manner to the cytoplasm of *E*. *coli*. The process requires
transient destabilization of the cell outer membrane and is reminiscent of
bacterial transformation. Once in the cytoplasm, biofabricated QDs do not cause
a significant induction of the unfolded protein or SOS responses. However, they
lead to oxidative stress when supplied at very high concentrations (2.5 μg/mL).
Although internalized QDs are stable over a broad range of temperature in
quiescent cells, they are rapidly diluted in dividing cells. Taken together, our
results suggest that biomimetic fluorophores designed with low toxicity cores
and biologically-relevant shells are unlikely to cause significant damage to the
microbial ecosystem. These design principles may prove useful for the production
of other environmentally benign nanomaterials.
# Materials and Methods
## QD uptake by competent cells
AB734, an *E*. *coli* K-12 strain containing a mutation in the *lacZ* gene but
otherwise wild type was obtained from the *E*. *coli* Genetic Stock Center. To
prepare competent cells, 500 mL cultures were grown in LB medium at 37°C to
*A*<sub>600</sub> ≈ 0.4, and cells were sedimented by centrifugation at 8,000*g*
for 8 min and resuspended in 100 mL of 100 mM CaCl<sub>2</sub> or phosphate
buffered saline (PBS;150 mM NaCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, 2 mM
KH<sub>2</sub>PO<sub>4</sub>). After 30 min incubation on ice and centrifugation
at 8,000*g* for 8 min, cells were taken into 12.5 mL of 100 mM CaCl<sub>2</sub>
(or PBS for a non-competent control) and held on ice overnight. Glycerol was
added to a 10% (v/v) final concentration and aliquots (200 μL) were stored at
-80°C for future use. For uptake experiments, competent or control cells were
thawed at room temperature, washed twice with PBS with intervening cycle of
centrifugation at 4,000 rpm for 5 min in a microfuge, and resuspended in 900 μL
of the same buffer. QDs (approximately 100 μL for a dose of 0.5 μg/mL) were
added and the mixture was incubated at room temperature for 2h without shaking.
Cells were washed twice with PBS to remove unincorporated QDs.
## QD subcellular localization
Cells that had uptaken QDs were stripped of their outer membrane and
peptidoglycan layer by spheroplasting. Briefly, samples prepared as above
were resuspended in 200 μL of buffer A (100 mM Tris-HCl, pH 8.0, 0.5 M sucrose,
0.5 mM EDTA), and 10 μL of a 2 mg/mL solution of lysozyme was added, followed by
400 μL buffer A, and 400 μL of ddH<sub>2</sub>O. After 20 min at room
temperature, spheroplasts were recovered by centrifugation at 12,800*g* for 30 s
and resuspended in 100 mM Tris-HCl, pH 8.0, 0.3 M sucrose, 10 mM
MgCl<sub>2</sub>. Samples were visualized on an optical microscope at 50x
magnification.
## FRET experiments
AB734 (pmCherry-mut2) cultures were grown to *A*<sub>600</sub> = 0.4 at 37°C in
LB medium supplemented with 50 μg/mL kanamycin. Production of mCherry was
induced by addition of 0.2% L-arabinose and cultures were collected after 3 h of
growth at 37°C. Cells were made competent by CaCl<sub>2</sub> and ice treatment
as above and stored in 200 μL aliquots. After two wash cycles and resuspension
in 900 μL PBS, one sample was incubated for 2 h with 100 μL of BB-
CT43-stabilized QDs or the same volume of PBS to serve a control. After 2 wash
cycles with PBS, samples were diluted 20-fold in PBS and fluorescence emission
spectra were recorded with excitation at 280 nm or 590 nm. Control samples of
AB734 cells lacking the pmCherry-mut2 plasmid and incubated or not with QDs were
prepared as above and fluorescence spectra were recorded following excitation at
280 nm. The spectra of show QD-free AB734 emission subtracted from QD-treated
AB734 emission with excitation at 280 nm (orange), and QD-free mCherry-producing
AB734 subtracted from QD-treated mCherry-producing AB734 with excitation at 280
nm (red).
## QD fate
Aliquots (200 μL) of AB734 cells that had uptaken QDs as above were used to
inoculate 2 mL of LB media in multiple 15 mL culture tubes supplemented or not
with 50 μg/mL of the translational inhibitor kanamycin. Cultures were
transferred to 37°C water bath. At the indicated time points, culture absorbance
was recorded at 600 nm and samples (2 mL) were subjected to centrifugation at
5,000 rpm for 5 min in a microfuge. Cells were resuspended in 50 μL of PBS,
deposited on quartz microscope slide and photographed on a UV table with
excitation at 303 nm. Median fluorescence in square areas encompassing about 60%
of the droplets and excluding their edges was quantified in the red channel
using the histogram function of Adobe Photoshop. Fluorescence loss was
quantified by subtracting the fluorescence of control samples from that of QD-
loaded cells at the indicated time points.
## Stress responses
Strains ADA110 (AB734 λϕ*ibp*::*lacZ*)) and ADA510 (AB734 λϕ*sulA*::*lacZ*))
have been described previously. ADA710 (AB734 λϕ*katG*::*lacZ*)) was
constructed by lysogenizing AB734 with a bacteriophage λ derivative bearing the
oxidative stress-responsive *katG*::*lacZ* translational fusion and isolated
from BGF931 (a kind gift from Dr. Gonzalez-Flecha) through standard
protocols. The three strains were made chemically competent by
CaCl<sub>2</sub> treatment and incubated or not with QDs as above. After
resuspension in buffer, 200 μL of culture was used to inoculate 5 mL of LB
medium. Samples were either exposed to buffer (negative control), known stress
response inducers (4% ethanol, 15 μg/mL nalidixic acid, or 10 μM
H<sub>2</sub>O<sub>2</sub>) or 0.5 μg/mL or 2.5 μg/mL of BB-CT43-stabilized QDs.
After 3h incubation at 37°C, cells were lysed and β-galactosidase activities
determined as described.
## Analytical techniques
UV-visible absorption spectra were recorded on a Beckman DU640
spectrophotometer. Fluorescence and phosphorescence emission spectra were
recorded using 1 mL of sample on a Hitachi F4500 fluorescence spectrophotometer
with excitation at 280 nm and excitation and emission slit widths set at 2.5 nm
(fluorescence) or excitation at 316 nm and excitation and emission slit width at
2.5 nm and 10 nm, respectively (phosphorescence). The wavelength region
corresponding to the second order diffraction peak of the excitation light was
omitted.
# Supporting Information
We are grateful to Elyse Shapiro for constructing ADA710.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: FB BJFS. Performed the
experiments: BJFS. Analyzed the data: FB BJFS. Wrote the paper: FB BJFS. |
# Introduction
Recent investigations into the pathogenesis of colorectal cancer (CRC) in the
general population have indicated that 15% to 35% of CRCs arise through the
serrated neoplasia pathway, which differs from the adenoma–carcinoma sequence.
Among serrated polyps, sessile serrated lesions (SSLs) and traditional serrated
adenomas (TSAs) are considered premalignant lesions. In the general population,
the prevalences of SSLs and TSAs are 5%–15% and \<1%, respectively. *BRAF*
mutations, CpG island hypermethylation phenotype (CIMP)-positive status, and
microsatellite instability are associated with SSL development, whereas *KRAS*
mutations are involved in TSA development.
Ulcerative colitis (UC) is associated with an increased risk of developing CRC.
CRC in patients with UC develops through a carcinogenesis pathway distinct from
the dominant pathway in sporadic CRC. Risk factors for CRC include a long
disease duration, extensive colitis, and more severe or persistent inflammation.
Serrated polyps in patients with UC have been reported in several recent
articles. In these studies, serrated polyps were found in 1.2%–1.7% of patients
with UC and accounted for 11%–23% of the neoplasias in patients with UC.
Serrated epithelial change has been described as a potential risk factor and
precursor of colorectal dysplasia and cancer in patients with UC. Serrated
polyps in patients with UC have been recognized as histologically and
biologically distinct from serrated epithelial change, which shows a high
frequency of *TP53* mutations and low frequency of *KRAS/BRAF* mutations. Ko et
al. examined serrated polyps in 78 patients with inflammatory bowel disease
(IBD), including 56 with UC. The authors reported that SSLs occurred mainly in
the proximal colon and contained the *BRAF* mutation, whereas TSAs occurred
mainly in the distal colon of men and contained *KRAS* mutations. Miller et al.
examined TSA-like lesions in 30 patients with IBD, including 22 with UC, and
reported that *KRAS* and *BRAF* mutations were detected in 59% and 16% of the
patients, respectively. With the increasing number of reports of serrated polyps
in patients with UC, these polyps have become recognized as dysplastic variants
that are histologically distinct from conventional dysplasia. The most recent
histological studies classified serrated polyps in patients with UC into three
subtypes: SSL-like dysplasia, TSA-like dysplasia, and serrated dysplasia not
otherwise specified (SD NOS). However, the histological terminology
differed among the previous studies, and few studies have examined the clinical
characteristics (including risk factors for development) and biological
characteristics (including the *KRAS*, *BRAF*, and CIMP status) of serrated
polyps in patients with UC based on such accurate histological classification.
Therefore, the present study was performed to clarify the yet unknown
prevalence, risk factors, and clinical and biological characteristics of
serrated polyps in patients with UC.
# Materials and methods
## Data collection, patients, and neoplasia
Consecutive patients with UC who underwent total colonoscopy at Yokohama City
University Medical Center, a tertiary IBD center in Japan, from 2000 to 2020
were identified in this retrospective single-center study. Colonoscopies were
performed by experienced endoscopists with knowledge of IBD. In particular, for
patients with a \>7-year disease duration, surveillance colonoscopies with
panchromoendoscopy were performed by one of three experienced endoscopists (MN,
TO, or RK). The inclusion criteria for endoscopic resection in UC patients were
as follows: neoplasias that were well-circumscribed endoscopically, neoplasias
that had no evidence of invisible dysplasia in the surrounding mucosa on the
basis of confirmational biopsies, and neoplasias that had no evidence of
submucosal invasion. When the lesions were diagnosed as neoplasia on the basis
of endoscopic observation or biopsy, endoscopic resection was performed
regardless of size. When the neoplasia could not be resected endoscopically,
surgical resection was selected.
Established UC databases and endoscopy and pathology reports were reviewed to
ensure complete case capture. Patients in whom the diagnosis of UC was
uncertain, or who had an IBD unclassified status or underwent only flexible
sigmoidoscopy, were excluded. Among the patients with UC who underwent total
colonoscopy, data for those with colonic neoplasia were extracted. Indefinite
for dysplasia and hyperplastic polyps were not considered neoplastic. Data on
the demographic and clinical parameters (sex, age at diagnosis of neoplasia and
UC, disease duration, disease extent, course of disease, location and endoscopic
features of the neoplasia, and treatment of the neoplasia) were obtained from
the medical charts.
To compare the clinical and endoscopic findings between serrated polyps in
patients with UC and without IBD, we reviewed 249 consecutive serrated polyps in
patients without IBD (216 SSLs and 30 TSAs) that were resected endoscopically or
surgically at Yokohama City Medical Center during the study period.
## Classification and histological evaluation of neoplasia
All pathological slides of neoplasia were re-reviewed and classified in
accordance with the most recent World Health Organization classification by two
expert gastrointestinal pathologists (YA and SC); one of whom (YA) was highly
specialized in UC-associated dysplasia or cancer.
We classified neoplasia into five categories on the basis of the histological
findings and whether the neoplasia was located in colitis-affected segments, as
follows: serrated polyps in colitis-affected segments, conventional dysplasia
(intestinal-type dysplasia in colitis-affected segments), serrated polyps in
colitis-unaffected segments (usually proximal to the extent of the colitis),
sporadic adenomas in colitis-unaffected segments, and invasive carcinoma.
Additionally, serrated polyps in colitis-affected segments were histologically
classified into three subtypes in accordance with previous studies, as follows:
SSL-like dysplasia, TSA-like dysplasia, and serrated SD NOS. Briefly, SSL-like
dysplasia is characterized by distorted serrated crypts with prominent basal
crypt dilatation (i.e., dilated L- or inverted T-shaped crypts) at the interface
with the muscularis mucosa. TSA-like dysplasia is characterized by a villiform
growth pattern with columnar cells with intensely eosinophilic cytoplasm and
ectopic crypts, creating a prominent serrated profile. SD NOS was defined as
serrated dysplasia without definite features of SSL-like or TSA-like dysplasia,
with a complex serrated architecture and evidence of dysplasia. The
representative endoscopic and histological features of SSL-like dysplasia, TSA-
like dysplasia, and SD NOS are shown in. Serrated polyps in colitis-unaffected
segments were classified as hyperplastic polyps, SSLs, TSAs, or unclassified
serrated adenomas.
## Definitions
Persistent active colitis was defined as endoscopically active colitis lasting
\>6 months. Whether neoplasia was located in colitis-affected segments was
determined on the basis of clinical and endoscopic data for the most active
inflammation during the course of UC treatment. The proximal colon comprised the
cecum, ascending colon, and transverse colon, whereas the distal colon comprised
the descending colon, sigmoid colon, and rectum.
## DNA extraction and genetic and epigenetic analyses
DNA samples were purified from archived formalin-fixed, paraffin-embedded blocks
of serrated polyps and invasive cancer that had been endoscopically or
surgically resected from patients with UC. The pathologists selected the
appropriate tissue blocks for DNA extraction. We used a laser microdissection
system for selective isolation of the neoplastic sections, avoiding foci of
inflammation, and then extracted the DNA. Neoplasia specimens containing
adequate DNA quantity and quality were provided for genetic analysis. *KRAS* and
*BRAF* mutations were detected using a droplet digital polymerase chain reaction
system. The CIMP status was evaluated in accordance with previous reports \[,
\]. The details of the laser microdissection system, DNA extraction, and genetic
and epigenetic analyses are shown in.
## Statistical analysis
Data were analyzed using JMP Pro 12 (SAS Institute Inc., Cary, NC). The
prevalence and clinical characteristics were compared among neoplasia groups
using Fisher’s exact test or the Wilcoxon rank sum test. Statistical
significance was set at *P* \< 0.05.
## Ethical considerations
This study was approved by the Ethics Committee of Yokohama City University
Medical Center (Protocol number: A130926011). All patients with UC whose
neoplastic DNA was extracted and genetically analyzed provided written informed
consent in accordance with the tenets of the Declaration of Helsinki. An opt-out
for the present study was published on the Web.
# Results
## Prevalence of serrated polyps in patients with UC
shows an overview of the neoplasias among the consecutive patients with UC
included in the present study. During the study period, 2035 patients with UC
underwent total colonoscopy, and 252 neoplasms from 187 patients were
identified. Of 219 neoplasms (after excluding 33 invasive cancers), 26 serrated
polyps and 132 cases of conventional dysplasia were observed in colitis-affected
segments, whereas 10 serrated polyps and 51 sporadic adenomas were found in
colitis-unaffected segments.
## Clinical characteristics of patients with serrated polyps and other neoplasms
shows the endoscopic characteristic of serrated polyps in patients with UC and
without IBD. Serrated polyps in patients with UC were more frequent in the
distal colon and significantly smaller in size compared with those in patients
without IBD (42% vs. 24%, respectively, *P* = 0.04, and 10 mm vs. 16 mm,
respectively, *P* \< 0.01). However, there was no significant deference in
neoplasia morphology and border description. Regarding the subtype of serrated
polyps (SSL/SSL-like dysplasia and TSA/TSA-like dysplasia), there were no
significant differences in neoplasia location, neoplasia morphology, and border
description ( and Tables).
shows the patient and neoplasia characteristics of serrated polyps in colitis-
affected and -unaffected segments. shows representative results of the
comparisons among the categories of neoplasia (serrated polyps in colitis-
affected and colitis-unaffected segments, conventional dysplasia in colitis-
affected segments, and sporadic adenomas). The detailed characteristics of each
category are shown in. The age at diagnosis of neoplasia, age at diagnosis of
UC, and duration of UC did not differ between patients with serrated polyps and
those with conventional dysplasia found in colitis-affected segments (50 vs. 57
years, *P* = 0.11; 37 vs. 40 years, *P* = 0.055; and 12.1 vs. 10.0 years, *P* =
0.21, respectively). However, the age at diagnosis of neoplasia and age at
diagnosis of UC in patients with sporadic adenomas (67 and 52 years,
respectively) were significantly older than those in patients with each
neoplasia in colitis-affected segments (*P* values are shown). Serrated polyps
in colitis-affected segments were more frequent in men than in women, while
those in colitis-unaffected segments were more frequent in women than in men
(percentages in men: 88% vs. 20%, respectively; *P* \< 0.001).
Patients with either type of neoplasia in colitis-affected segments were more
likely to have extensive colitis (serrated polyps: 88%, conventional dysplasia:
74%) and a history of persistent active colitis (serrated polyps: 58%,
conventional dysplasia: 52%) compared with patients with either type of
neoplasia in colitis-unaffected segments (serrated polyps: 40% and 0%,
respectively; sporadic adenomas: 10% and 22%, respectively) (*P* values are
shown). Neoplasia size was similar (serrated polyps in colitis-affected
segments: 9 \[interquartile range: –\] mm, conventional dysplasia: 9 mm,
serrated polyps in colitis-unaffected segments: 10 mm), with the exception of
sporadic adenomas (4 mm) (*P* values are shown). Regarding previous treatment,
immunomodulators were used significantly more frequently in patients with
serrated polyps in colitis-affected segments than in those with polyps in
colitis-unaffected segments.
## Clinical characteristics of serrated polyps in colitis-affected segments
Of 26 serrated polyps in colitis-affected segments, 15 (58%), 6 (23%), and 5
(19%) were categorized as SSL-like dysplasia, TSA-like dysplasia, and SD NOS,
respectively. The characteristics of these polyps are shown in. All subtypes of
serrated polyps in colitis-affected segments were common in men. SSL-like
dysplasia was common in the proximal colon, whereas TSA-like dysplasia and SD
NOS were common in the distal colon. Although statistical analysis was not
performed owing to the small number of samples, SD NOS was more likely to be
associated with large neoplasia size (23 mm) than with SSL-like dysplasia (12
mm) or TSA-like dysplasia (8 mm). TSA-like dysplasia was more likely to have a
polypoid morphology (67%) compared with SSL-like dysplasia (7%) and SD NOS
(20%).
## Locational and biological characteristics of serrated polyps in patients with UC
shows the locational distribution of serrated polyps in patients with UC,
including 26 polyps in colitis-affected segments and 10 polyps (9 SSLs and 1
TSA) in colitis-unaffected segments. SSL-like dysplasia was distributed in all
segments of the colorectum, and 10 lesions (67%) were located in the proximal
colon. All cases of TSA-like dysplasia were located in the distal colon (2
\[33%\] in the rectum and 4 \[67%\] in the sigmoid colon). Four (80%) SD NOS
lesions were located in the distal colon (one in the descending colon and three
in the rectum), and one was located in the proximal colon. In contrast, all
serrated polyps in colitis-unaffected segments were located in the proximal
colon.
Of 36 serrated polyps in both colitis-affected and -unaffected segments in
patients with UC, genetic and epigenetic evaluations were performed in 18
specimens (13 affected and 5 unaffected) from which DNA of adequate quality and
quantity was obtained. *BRAF* mutations were observed in 75% (3/4) of cases of
SSL-like dysplasia in colitis-affected segments and in all SSLs in colitis-
unaffected segments, whereas *KRAS* mutations were observed in all cases of TSA-
like dysplasia in colitis-affected segments and in all TSAs in colitis-
unaffected segments. In SD NOS, 75% (3/4) of the specimens showed *KRAS*
mutations and 25% (1/4) showed *BRAF* mutations. CIMP-positive status for
serrated polyps in colitis-affected segments was observed in 60% (3/5) of cases
of TSA-like dysplasia but in only 25% of cases of SSL-like dysplasia and SD NOS.
The genetic and epigenetic status of 16 invasive cancers in colitis-affected
segments were also evaluated. *KRAS* and *BRAF* mutations and CIMP-positive
status were observed in 13% (2/16), 0% (0/16), and 19% (3/16) of invasive
cancers in colitis-affected segments.
# Discussion
This study showed the prevalence of serrated polyps in patients with UC and is
the first, to our knowledge, to suggest the increasing prevalence of these
polyps, which have been observed more frequently recently. The clinical and
biological characteristics of SSL-like dysplasia and TSA-like dysplasia in
patients with UC are basically similar to those of each counterpart in
individuals without IBD. However, SSL-like dysplasia in patients with UC was
more frequently observed in men than in women, in the present study, a finding
that contrasts the known female dominance of SSLs in individuals without IBD.
Although many other issues remain unknown, our findings suggest a new pathway in
the development of UC-associated neoplasia.
The present study showed that the prevalence of serrated polyps was 1.8%
(36/2035) in patients with UC, and these polyps accounted for 14% (36/252) of
all neoplasias in patients with UC. This proportion is similar to that in a
previous study that reported a prevalence of serrated polyps of 1.2%–1.7% in
patients with IBD. Additionally, serrated polyps accounted for 11%–23% of all
neoplasias in the study.
Regarding ethnicity, the present study is the first, to our knowledge, to
investigate the prevalence of serrated polyps in the Asian population. Meta-
analyses of the non-IBD population have reported that the prevalence of serrated
polyps in Western countries was higher than those in Eastern countries. The
possible reason for the similar proportions of serrated polyps in patients with
UC between the present study and previous studies is differences in the study
periods. Two previous studies included serrated polyps detected between 2005 and
2007 and 2000 and 2013. Because serrated polyps (SSLs and TSAs) were considered
hyperplastic polyps before 2010, SSLs and TSAs may have been overlooked without
biopsy or endoscopic resection. To clarify the differences in the prevalence
between Eastern and Western countries, an accumulation of recent cases in both
ethnic groups is desirable.
Previous studies have shown that serrated polyps in patients with UC have
clinical and biological characteristics similar to those in individuals without
IBD. For instance, SSLs are usually found in women and in the proximal colon,
whereas TSAs are usually found in men and in the distal colon. Our results also
showed similar neoplasia location, neoplasia morphology, and border description
for both SSLs/SSL-like dysplasias and TSAs/TSA-like-dysplasias in patients with
UC and without IBD. The difference in the neoplasia size between the groups may
be associated with different indications for endoscopic resection between
patients with UC and without IBD. Notably, our results demonstrated a unique
clinical characteristic in SSL-like dysplasia in colitis-affected segments. SSLs
in colitis-unaffected segments showed clinical and biological similarity to
those in patients without IBD (all SSLs in colitis-unaffected segments were
found in women and in the proximal colon). In contrast, most cases of SSL-like
dysplasia in colitis-affected segments were found in men, although their
locational and biological characteristics were similar to those of SSLs in
colitis-unaffected segments (predominantly in the proximal colon and with *BRAF*
mutation). These sex-related differences may arise from differences in the
biological mechanisms of development between serrated pathways with and without
background inflammation. In this context, although previous studies indicated
that SSLs in patients with UC were dominant in women and in the proximal colon,
the distribution of SSLs was not always based on the presence or absence of
background inflammation. Conversely, TSA-like dysplasia in colitis-affected
segments showed clinical and biological characteristics similar to those of TSAs
in colitis-unaffected segments (predominantly in men, in the distal colon, and
with *KRAS* mutations). These results suggest that the specific development of
SSL-like dysplasia in men with UC is derived from a unique tumorigenic pathway.
Serrated polyps in colitis-affected segments were more common in patients with a
long disease duration and a history of persistent active colitis than in
patients without these characteristics, and this was also true in the
development of UC-related dysplasia. Therefore, chronic inflammation must be
involved in the development of both dysplasia and serrated polyps in patients
with UC. Notably, both patients who have IBD with dysplasia and patients without
IBD who have serrated polyps are likely to develop synchronous and metachronous
CRCs. Considering this similarity, serrated polyps in patients without IBD may
also develop on a background of chronic inflammation. In fact, previous studies
have shown that the concentrations of some key inflammatory factors, such as
tumor necrosis factor α, cyclooxygenase-2, interleukin-4, and interleukin-1β,
are increased in patients with serrated polyps without IBD. These studies
suggest that chronic inflammation is correlated with the development of serrated
polyps in both patients with UC and individuals without IBD. Long-term follow-up
of our patients and other observational studies are needed to further examine
the multiple developmental pathways of serrated polyps in patients with UC.
Recently, SD NOS was newly described as serrated polyps without definite
features of SSL-like and TSA-like dysplasia. However, only one study reported a
small number of cases of SD NOS, and the clinical and biological characteristics
were not assessed. The present study is the first, to our knowledge, to show the
clinical and biological characteristics of SD NOS. Our study showed a similar
distribution (predominantly located in the distal colon) and similar biological
characteristics between TSA-like dysplasia and SD NOS, suggesting that SD NOS is
a subtype of TSA-like dysplasia. Additionally, considering the longer duration
of UC and larger size of SD NOS compared with the duration and size of TSA-like
dysplasia, SD NOS may be an enlarged lesion of TSA-like dysplasia induced by
long-term inflammation. Similarly, Ko et al. reported that 32% of the serrated
polyps in patients with IBD were TSA-like, and this rate is consistent with our
rate of the combination of TSA-like dysplasia and SD NOS (42%). Although the
reason for the higher proportion of TSA-like dysplasia (with SD NOS) than the
proportion of TSAs in patients without IBD is unclear, chronic inflammation may
also be involved in the development of TSA-like dysplasia.
No previous studies have assessed the CIMP status of serrated polyps in patients
with UC. Although CIMP-positive status is important in the serrated neoplasia
pathway, recent studies showed that CIMP-positive status was exclusively
associated with SSLs in the proximal colon and advanced age. The low rate of a
CIMP-positive status of SSL-like dysplasia in our study was considered to be
associated with the neoplasia location (33% were in the distal colon) and
relatively young age of our study population. In patients without IBD, TSAs in
the proximal colon frequently show *BRAF* mutation and CIMP positivity, whereas
TSAs in the distal colon show *KRAS* mutations and CIMP negativity. In our
study, although all cases of TSA-like dysplasia were located in the distal colon
and showed *KRAS* mutations, 60% (3/5) were also CIMP-positive. The reason for
this discrepancy is unclear, but TSA-like dysplasia arising from chronic
inflammatory mucosa may develop through a pathway distinct from TSAs in non-
inflamed mucosa. The proportion of *KRAS* and *BRAF* mutations and CIMP-positive
status in serrated polyps in colitis-affected segments tended to be higher in
invasive cancer in colitis-affected segments in this study. These results
suggest that most serrated polyps may not be precursors of colitis-associated
invasive cancers. Although the optimal management of serrated polyps in patients
with UC is still unclear, our results suggest that colectomy is excessive for
serrated polyps in colitis-affected segments. However, considering that serrated
polyps are known precursors of CRCs in patients without IBD, serrated polyps in
patients with UC should be treated endoscopically as for those in patients
without IBD. Although there was no UC-associated dysplasia within SSLs or TSAs
in the present study, high-grade dysplasia with a hyperplastic polyp was
detected in a 19-year-old man in our hospital (this case was not included in the
present study because the lesion did not meet the diagnostic criteria for SSL,
TSA, or SD NOS). This case suggests that some serrated polyps in patients with
UC, although rare, have malignant potential. To determine the appropriate
management of serrated polyps in patients with UC, further accumulation of cases
is desirable.
Our study has some limitations. The setting was a single center, and the study
had a small sample size, which included only Japanese patients. Undoubtedly, the
number of serrated polyps in patients with UC was smaller than those of
conventional dysplasia and sporadic adenomas. Another limitation is the lack of
data regarding a family histology of CRC, obesity, smoking, and alcohol intake,
which are risk factors for SSLs.
# Conclusions
The present study showed the detailed clinicopathological and biological
characteristics of serrated polyps in patients with UC. Serrated polyps in
colitis-affected segments were common in men with extensive colitis and a long
duration of UC, suggesting that chronic inflammation might be involved in the
development of serrated polyps in patients with UC.
# Supporting information
10.1371/journal.pone.0282204.r001
Decision Letter 0
Suzuki
Hiromu
Academic Editor
2023
Hiromu Suzuki
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
3 Jan 2023
PONE-D-22-31131Serrated Polyps in Patients with Ulcerative Colitis: Prevalence
and Unique Clinicopathological and Biological CharacteristicsPLOS ONE
Dear Dr. Kunisaki,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
a revised version of the manuscript that addresses the points raised during the
review process.
This manuscript was carefully reviewed by 2 experts. Although both reviewers
highly evaluated this study, they suggested several points which need to be
addressed before acceptance. For instance, it is important to see whether
endoscopic findings of serrated lesions in UC-patients differ from those in non-
UC patients. Reviewer 1 also suggested additional mutation and methylation
analyses, while the editor considers it is not mandatory because KRAS, BRAF
mutations and CIMP status are already determined in the samples. Reviewer 2
indicated possible difficulties in comparing samples collected in different time
periods because of the inconsistent clinical definition of serrated lesions.
Reviewer 2 also suggested additional molecular analysis in in invasive cancer.
Analysis of KRAS/BRAF mutations and CIMP status in the invasive cancers may help
us to understand whether those cancers develop through the serrated pathway.
Please respond to each of the reviewer comments.
Please submit your revised manuscript by Feb 17 2023 11:59PM. If you will need
more time than this to complete your revisions, please reply to this message or
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\[Note: HTML markup is below. Please do not edit.\]
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Reviewer \#1: Yes
Reviewer \#2: Partly
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: Yes
Reviewer \#2: Yes
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Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: The paper is potentially interesting and seems to be convincing,
however, I have several concerns with this paper as shown below.
1\. Comparison should be made between the endoscopic findings of serrated
lesions in UC patients compared to non-UC patients.
2\. The authors should determine mutations of driver genes of colorectal
serrated lesions such as APC, GNAS, and RNF43.
3\. The authors should analyze all 36 serrated lesions for genetic alterations
and methylation statuses.
4\. The difference between Asian patients and non-Asian patients is of interest
of readers. Are there ethnic differences? The authors are encouraged to add more
discussion in details on this point.
5\. Grammatical and typographical errors in the text need to be corrected by a
native English speaker.
Reviewer \#2: In this manuscript, the authors study clinical and molecular
features of serrated polyps in patients with ulcerative colitis (UC),
retrospectively. Clinicopathological and biological characteristics of serrated
polyps in colitis-affected segments is potentially interesting, but several
concerns need to be addressed.
1, Neoplasias enrolled in this study was classified into five categories based
on histological findings. Were all neoplasias endoscopically or surgically
resected? In fig3G, median size of sporadic adenoma was less than 5mm. Criteria
of endoscopic resection should be shown.
2, It is difficult to compare the prevalence of serrated polyps in patients with
UC from 2000 to 2009 and from 2010 to 2020. Endoscopic findings of serrated
polyp were not established, and almost serreted polyps were classified into
hyperplastic polyp before 2010, as the authors are discussing. If the authors
want to show the increasing incidence of serrated polyps, the data after 2010
should be used.
3, Molecular features of serrated polyp in colitis affected and unaffected
segments were interesting. Molecular features of invasive cancer should be
examined.
\*\*\*\*\*\*\*\*\*\*
6\. PLOS authors have the option to publish the peer review history of their
article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-
and-peer-review-process#loc-peer-review-history)). If published, this will
include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may
still be made public.
**Do you want your identity to be public for this peer review?** For information
about this choice, including consent withdrawal, please see our [Privacy
Policy](https://www.plos.org/privacy-policy).
Reviewer \#1: No
Reviewer \#2: No
\*\*\*\*\*\*\*\*\*\*
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Please note that Supporting Information files do not need this step.
10.1371/journal.pone.0282204.r002
Author response to Decision Letter 0
26 Jan 2023
Responses to the Reviewers’ Comments
Reviewer \#1:
The paper is potentially interesting and seems to be convincing, however, I have
several concerns with this paper as shown below.
1\. Comparison should be made between the endoscopic findings of serrated
lesions in UC patients compared to non-UC patients.
Response:
We thank Reviewer \#1 for the insightful comment, which has helped us improve
the Results and Discussion sections of our manuscript.
As Reviewer \#1 pointed out, a comparison of the clinical and endoscopic
characteristics between serrated polyps in patients with UC and without IBD is
important. Accordingly, we reviewed 249 consecutive serrated polyps in patients
without IBD (216 SSLs and 30 TSAs) that were resected endoscopically or
surgically at our hospital during the study period. We compared the endoscopic
characteristics of serrated polyps in patients with UC and without IBD. Serrated
polyps in patients with UC were more frequent in the distal colon and
significantly smaller in size than those in patients without IBD (42% vs. 24%,
respectively, P = 0.04, and 10 mm vs. 16 mm, respectively, P \< 0.01). However,
there was no significant deference in neoplasia morphology and border
description. Next, we compared the endoscopic characteristics on the basis of
the subtype of serrated polyps (SSL/SSL-like dysplasia and TSA/TSA-like
dysplasia). There was no significant deference in neoplasia location, neoplasia
morphology, and border description between patients with UC and without IBD for
both SSL/SSL-like dysplasia and TSA/TSA-like dysplasia. These results support
the finding in previous studies that serrated polyps in patients with UC were
similar to those in patients without IBD. However, our results showed a unique
characteristic of SSL-like dysplasia in colitis-affected segments: these were
common in men.
We added this information in the methods and discussion of the revised
manuscript. We also created new Table 1, S1 Table, and S2 Table. Previous Tables
1, 2, and 3, and S1 Table have been renumbered accordingly.
2\. The authors should determine mutations of driver genes of colorectal
serrated lesions such as APC, GNAS, and RNF43.
Response:
We thank Reviewer \#1 for the insightful comment.
As Reviewer \#1 pointed out, APC, RNF43, and GNAS mutations have been reported
in addition to KRAS or BRAF mutations in TSAs in patients without IBD.
Accordingly, we examined somatic mutations in serrated polyps in colitis-
affected segments using next-generation sequencing (NGS). However, sufficient
quality and quantity of DNA were obtained in only six samples. After excluding
samples that did not meet the inclusion criterion of successful sequencing, only
three samples remained (two TSA-like dysplasias and one SD NOS). Among the three
samples that could be evaluated, KRAS mutations were detected in all cases,
whereas APC and GNAS mutations were not detected.
In our study, RNF43 mutations were not evaluated because the Cancer Hotspot
Panel v2 (Thermo Fisher Scientific) that we used did not include RNF43.
Because only three samples could be examined, the above data were insufficient
to discuss somatic mutations other than KRAS and BRAF mutations of serrated
polyps in patients with UC. Therefore, we presented only the data for NGS in
this response.
3\. The authors should analyze all 36 serrated lesions for genetic alterations
and methylation statuses.
Response:
We thank Reviewer \#1 for the insightful comment.
As Reviewer \#1 pointed out, analysis of all 36 lesions is desirable for more
accurate genetic and epigenetic characterization. However, in our study, only 18
samples had sufficient quality or quantity of DNA for analysis. Therefore, it is
difficult to perform additional examinations. The possible reasons for not
obtaining sufficient quality or quantity of DNA are as follows: First, a long
time has passed since the samples were collected. Second, before 2017, the
specimens were fixed in 20% formalin buffer, which caused tissue damage.
We would like to examine the DNA of more cases under improved preservation
conditions, in the future.
4\. The difference between Asian patients and non-Asian patients is of interest
of readers. Are there ethnic differences? The authors are encouraged to add more
discussion in details on this point.
Response:
We thank Reviewer \#1 for the insightful comments, which have helped us improve
the Discussion section of our manuscript.
As Reviewer \#1 have pointed out, the prevalence of serrated lesions in the non-
IBD population differs between ethnic groups, and it is very important to
consider ethnic differences in serrated polyps in patients with UC.
To the best of our knowledge, no study has compared the prevalence of serrated
polyps in patients with UC between Eastern and Western countries. All previous
studies that reported the prevalence of serrated lesions in patients with UC
were from Western countries. Therefore, to our knowledge, the present study is
the first to report the prevalence in the Asian population.
Two meta-analyses of the non-IBD population reported that the prevalence of
serrated polyps in Western countries was higher than those in Eastern countries.
In comparison, the prevalence of serrated polyps in patients with UC in our
study (1.8%) was similar to those in previous studies from Western countries
(1.2%–1.7%). The possible reason for this similarity is differences in the study
periods. The two previous studies included serrated polyps detected between 2005
and 2007 and 2000 and 2013, respectively. Because serrated polyps (SSLs and
TSAs) were considered hyperplastic polyps before 2010, SSLs and TSAs may have
been overlooked without biopsy or endoscopic resection. To clarify the
differences in the prevalence between Eastern and Western countries, the
accumulation of more recent cases in both ethnic groups is desirable.
We added the above information in the revised discussion.
5\. Grammatical and typographical errors in the text need to be corrected by a
native English speaker.
Response:
We thank Reviewer \#1 for the insightful comment, which has helped us improve
our manuscript.
The revised manuscript has been edited by a native English-speaking Medical
Editor.
Reviewer \#2:
In this manuscript, the authors study clinical and molecular features of
serrated polyps in patients with ulcerative colitis (UC), retrospectively.
Clinicopathological and biological characteristics of serrated polyps in
colitis-affected segments is potentially interesting, but several concerns need
to be addressed.
1, Neoplasias enrolled in this study was classified into five categories based
on histological findings. Were all neoplasias endoscopically or surgically
resected? In fig3G, median size of sporadic adenoma was less than 5mm. Criteria
of endoscopic resection should be shown.
Response:
We thank Reviewer \#2 for the insightful comments.
All neoplasias evaluated in the present study were resected endoscopically or
surgically.
As Reviewer \#2 pointed out, the Japanese guidelines recommend endoscopic
resection for adenomas ≥ 6 mm in size or for superficial depressed-type lesions
even when the lesion measures ≤ 5 mm in non-IBD patients. However, no indication
for endoscopic resection on the basis of size has been established for colitis-
associated dysplasia. In patients with UC, the distinction between sporadic
adenoma and colitis-associated dysplasia is sometimes difficult even after
observation with magnifying endoscopy and biopsy. Therefore, in our hospital,
when the lesions are diagnosed as neoplasia on the basis of endoscopic
observation or biopsy, the lesions are resected regardless of size. In this
study, the inclusion criteria for endoscopic resection in UC patients were as
follows: neoplasias that were well-circumscribed endoscopically, neoplasias that
had no evidence of invisible dysplasia in the surrounding mucosa with
confirmational biopsies, and neoplasias that had no evidence of submucosal
invasion. When the neoplasia could not be resected endoscopically, surgical
resection was performed.
We added the criteria for endoscopic resection in patients with UC in the
Methods section.
2, It is difficult to compare the prevalence of serrated polyps in patients with
UC from 2000 to 2009 and from 2010 to 2020. Endoscopic findings of serrated
polyp were not established, and almost serreted polyps were classified into
hyperplastic polyp before 2010, as the authors are discussing. If the authors
want to show the increasing incidence of serrated polyps, the data after 2010
should be used.
Response:
We thank Reviewer \#2 for the insightful comments.
As Reviewer \#2 pointed out, data after 2010 should be used to investigate the
increasing incidence of serrated polyps. However, we think that it is
inappropriate to discuss the increasing incidence of serrated polyps in
post-2010 data on the basis of the present data because of the short study
period. Additionally, considering that the definition of serrated polyps was
revised in 2019, further accumulation of serrated polys detected after 2019 is
needed to investigate the prevalence of serrated polyps.
Therefore, we deleted text that mentioned the increasing incidence of serrated
polyps in the abstract, results, and discussion. We also deleted original Fig 2,
and current Figs 3 and 4 have been renumbered accordingly.
3, Molecular features of serrated polyp in colitis affected and unaffected
segments were interesting. Molecular features of invasive cancer should be
examined.
Response:
Reviewer \#2 for the insightful comments, which have helped us improve the
results and discussion in our manuscript.
In accordance with your recommendation, we examined the KRAS and BRAF mutations
and CIMP-positive status of invasive caner in colitis-affected segments.
Sixteen invasive carcinoma samples with adequate DNA quality and quantity were
examined. KRAS and BRAF mutations and CIMP-positive status were observed in 13%
(2/16), 0% (0/16), and 19% (3/16) of invasive carcinomas, respectively, in
colitis-affected segments. Considering that the proportions of KRAS and BRAF
mutations and CIMP-positive status of invasive cancers tended to be lower than
those of serrated polyps in colitis-affected segments, most serrated polyps may
not be precursors of colitis-associated invasive carcinoma. These results
suggest that colectomy is excessive for serrated polyps in colitis-affected
segments. However, considering that serrated polyps are known precursors of CRCs
in patients without IBD, serrated polyps in patients with UC should be treated
endoscopically, as for those in patients without IBD.
In our hospital, high-grade dysplasia with a hyperplastic polyp was detected in
a 19-year-old man (this case was not included in the present study because the
lesion did not meet the diagnostic criteria for SSL, TSA, and SD NOS). This case
suggests that some serrated polyps in patients with UC, although rare, have
malignant potential. To determine the appropriate management of serrated polyps
in patients with UC, further accumulation of cases is desirable.
We added the details of these additional examinations in the methods, results,
and discussion. We also created a new supplementary table (S4 Table) explaining
the results the of genetic and epigenetic examinations of invasive carcinoma.
Additionally, we revised the total number and percentage of CIMP-positive
serrated polyps in colitis-affected segments in Table 4 (original Table 3)
because of a text error in the original manuscript (wrong: 6 (50%) → correct: 5
(38%)).
10.1371/journal.pone.0282204.r003
Decision Letter 1
Suzuki
Hiromu
Academic Editor
2023
Hiromu Suzuki
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
10 Feb 2023
Serrated polyps in patients with ulcerative colitis: Unique clinicopathological
and biologial characteristics
PONE-D-22-31131R1
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suitable for publication and will be formally accepted for publication once it
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Hiromu Suzuki, M.D., Ph.D.
Academic Editor
PLOS ONE
Additional Editor Comments (optional):
Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. If the authors have adequately addressed your comments raised in a previous
round of review and you feel that this manuscript is now acceptable for
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Reviewer \#1: (No Response)
Reviewer \#2: All comments have been addressed
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
3\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
4\. Have the authors made all data underlying the findings in their manuscript
fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
manuscript fully available without restriction, with rare exception (please
refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
participant privacy or use of data from a third party—those must be specified.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
5\. Is the manuscript presented in an intelligible fashion and written in
standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
articles must be clear, correct, and unambiguous. Any typographical or
grammatical errors should be corrected at revision, so please note any specific
errors here.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
6\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: The authors have responded appropriately to my concerns, providing
additional data.
This reviewer thinks this paper is improved.
Reviewer \#2: Clinicopathological and biologial characteristics of serrated
polyps in colitis-affected segments is interesting. The revised manuscript is
improved and acceptable for publication.
\*\*\*\*\*\*\*\*\*\*
7\. PLOS authors have the option to publish the peer review history of their
article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-
and-peer-review-process#loc-peer-review-history)). If published, this will
include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may
still be made public.
**Do you want your identity to be public for this peer review?** For information
about this choice, including consent withdrawal, please see our [Privacy
Policy](https://www.plos.org/privacy-policy).
Reviewer \#1: **Yes: **Yasushi Sasaki
Reviewer \#2: No
\*\*\*\*\*\*\*\*\*\*
10.1371/journal.pone.0282204.r004
Acceptance letter
Suzuki
Hiromu
Academic Editor
2023
Hiromu Suzuki
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
15 Feb 2023
PONE-D-22-31131R1
Serrated Polyps in Patients with Ulcerative Colitis: Unique Clinicopathological
and Biological Characteristics
Dear Dr. Kunisaki:
I'm pleased to inform you that your manuscript has been deemed suitable for
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Thank you for submitting your work to PLOS ONE and supporting open access.
Kind regards,
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on behalf of
Dr. Hiromu Suzuki
Academic Editor
PLOS ONE
[^1]: The authors have declared that no competing interests exist.
[^2]: ‡ KH, AT, YI, WU, KO, HO, TO, HK, YS and HK also contributed equally
to this work. |
# Introduction
*Plasmodium falciparum* is the species responsible for the vast majority of
malaria-related morbidity and mortality. Serious clinical complications
frequently arise due to dramatic modification of the structural and functional
properties of *P. falciparum*-infected erythrocytes (IEs). During the
intraerythrocytic stage, malaria parasites interact with and affect both the
plasma membrane and the membrane skeleton of the IEs. Alterations in
biochemical, structural and adhesive properties of the host membrane occur as
the parasite develops within the erythrocyte. Recently, a large study showed
that many parasite proteins contribute at different degrees to remodel the host
erythrocyte. Among parasite-secreted proteins, several types of enzymes (like
kinases, several still uncharacterized and phosphatases) are trafficked to the
erythrocyte membrane indicating that post-translational modifications may
contribute to establish successful intracellular parasite proliferation.
Phosphorylation of membrane skeleton proteins of both parasite and host origin
have been described during *P. falciparum* infections. Selective phosphorylation
of host membrane skeleton proteins include protein 4.1, β-spectrin, ankyrin and
band 3,. It has also been established that phosphorylation of some of these
proteins modulate their interactions with other membrane proteins , and,
consequently, the membrane mechanical functions and membrane stability. In
addition, erythrocyte membrane skeleton phosphorylation was suggested to be
involved in the regulation of malaria parasite invasion and development.
However, the molecular events involved in the phosphorylation of membrane
skeleton proteins have not yet been identified.
Recently we showed that some members of the *apicomplexa*-specific FIKK kinase
family are transported to the erythrocyte membrane via Maurer's clefts. *Fikk*
is a single copy gene in most *Plasmodium* species but has expanded in *P.
falciparum* to 20 related members dispersed mostly on subtelomeric regions of 11
of the 14 parasites chromosomes. Nineteen *Pf fikk* genes possess the
*Plasmodium* exported element/host targeting motif downstream of a signal or
anchor sequence required for transport across the parasitophorous vacuole.
Despite the fact that these proteins share a common structure, the N-terminal
regions are highly variable, suggesting that individual members of this family
may have access to distinct substrate pools since their variable N- terminal can
probably target them to different locations. Due to the restricted homology with
well-characterized kinase domains, the FIKK proteins did not cluster within any
of the kinase groups described in higher eukaryotes.
In this work, we have analyzed the biological role of two members of the *P.
falciparum* FIKK kinase family (FIKK7.1 and FIKK12) in IEs. We show that both
FIKK kinases are non-essential for parasite growth *in vitro*. However, the
absence of functional copies of either *Pf fikk7.1*(*MAL7P1.144*) or *Pf fikk12*
(*PFL0040c*) resulted in altered rigidity of the IEs using single cell
micromanipulation and both mutant parasites showed changes in the
phosphorylation pattern of two distinct proteins of the erythrocyte membrane
skeleton.
# Results
## Single targeted disruption of two *fikk* genes in *P. falciparum* leads to viable blood stage development
We have previously reported that FIKK proteins are exported to different
locations in the IE. Using both GFP-tagging and specific antibodies against
FIKK12 we noticed that this protein was transported to the erythrocyte membrane.
In this initial analysis we also noticed that *Pf fikk7.1* was more than 3-fold
up-regulated in ring stage FCR3-CSA-selected (chondroitin sulfate A) parasites
when compared to CD36-selected parasites. To investigate the biological role of
these two members of the *Pf fikk* gene family, we established two parasite
lines with single gene disruption by double crossover recombination. The pHTK-
FIKK7.1 and the pHTK-FIKK12 vectors contain the human dihydrofolate reductase
(*hdhfr*) gene flanked by 5′ and 3′ segments of the *Pf fikk7.1* and *Pf fikk12*
genes, respectively. FCR3 parasites were transfected with the pHTK-FIKK
constructs and selected on WR99210 and ganciclovir to generate two insertional
disruptant mutants, the FIKK7.1-KO and the FIKK12-KO. After drug selection, the
mutants were cloned by limiting dilution and genetically characterized. Clones
were screened by polymerase chain reaction (PCR) analysis for the disruption of
the *Pf fikk7.1* or *Pf fikk12* gene as well as for the absence of contaminating
wild type gene (data not shown). To confirm that the pHTK-FIKK vector had
integrated into the respective *Pf fikk* gene, Southern blots were performed
using genomic DNA derived from parental FCR3 or recombinant parasites previously
digested with AluI to test FIKK12-KO or HindIII to test FIKK7.1-KO.
Radiolabelled probes from *Pf fikk7.1* 5′ and 3′ segment or *Pf fikk12* 5′ and
3′ segment were used for hybridization. These hybridizations showed bands of the
expected size, indicating that the integration occurred at the predicted site
within the *Pf fikk* genes. Transcription of the specific genes was analysed by
real-time PCR. No transcript was detected in the respective KO-line; i.e. no *Pf
fikk7.1* transcript was detected in the FIKK7.1-KO line and no *Pf fikk12*
transcript was detected in the FIKK12-KO line.
The KO parasites were viable and there was no obvious difference in the parasite
growth and multiplication rates between the KO parasites and wild type FCR3
strain (data not shown), suggesting the FIKK7.1 and FIKK12 proteins are non-
essential for the parasite growth of laboratory isolates.
## Analysis of the adhesive properties of IEs and trafficking process in FIKK-KO parasites
Given the transit of several FIKK kinases via Maurer's clefts and the presence
of a variable N-terminal domain in each protein we hypothesized that the kinase
activity of these proteins might be involved in the trafficking of parasite-
encoded variant surface molecules, such as the adhesion molecule *Plasmodium
falciparum* erythrocyte membrane protein 1 (PfEMP1). To test this hypothesis we
analyzed the adhesive properties of the transgenic lines to endothelial cell-
expressed receptors by static *in vitro* adhesion assays. The FIKK7.1-KO and
FIKK12-KO parasite lines were selected to adhere to human placenta
choriocarcinoma Bewo cells expressing CSA receptor or Chinese hamster ovary 745
cell expressing CD36 receptor. The levels of parasite cytoadherence were
quantified after the third round of selection. Both KO parasite lines
cytoadhered to CSA and CD36 in similar numbers to the wild type parasites,
suggesting that the disruption of these *Pf fikk* genes neither prevented the
trafficking of PfEMP1 to the surface of the infected erythrocyte membrane nor
their ability to bind to different receptors.
To examine if the PfEMP1 switching rates in the KO parasites were altered, we
performed binding assays with receptors immobilized on plastic over a 2-month
period after which the numbers of bound IEs per mm<sup>2</sup> were quantified.
The binding phenotype of the CSA-selected KO parasites was analyzed with two
different receptors (purified bovine CSA and human recombinant CD36) at three
different intervals: 1 week, 5 weeks and 7 weeks post-selection (Supplementary
). Both KOs and wild type parasites maintained similar CSA-binding phenotypes
during the long *in vitro* 7 weeks cultivation and they did not bind to CD36,
though as expected the binding efficiency to CSA decreased gradually in the
three CSA-selected parasite lines. CD36-selected parasites conserved a strong
affinity for CD36 during the all experiment (data not shown). These data suggest
that FIKK12 and FIKK7.1 do not interfere with parasite adhesion to CSA and CD36
receptors. Furthermore, the assay we used did not reveal any apparent alteration
in the switching rate to other adhesion phenotypes.
We also tested the possibility that the FIKK7.1 and/or FIKK12 might be involved
in the trafficking of other parasite-encoded variant multi-gene families. To
address this question, the localization of Rifin (repetitive interspersed
family), Stevor (subtelomeric variable open reading frame) and Surfin (surface-
associated interspersed family) proteins was analyzed in the transgenic
parasites and compared to wild type parasites by immunofluorescence assay using
specific antibodies. The genes of these three families are located close or
within the subtelomeric chromosomic region. And different protein members have
been localized in the IEs Maurer's clefts. Similar immunofluorescence patterns
were detected in IEs with mature stages of FIKK7.1-KO, FIKK12-KO and FCR3
parental parasites. We were unable to detect differences in the Maurer's cleft
localizations of A-type Rifin, Surfin 4.2 and Stevor between the KO and wild
type lines, suggesting that FIKK7.1 and FIKK12 do not control trafficking of
these multigene families into the erythrocyte membrane.
## Erythrocytes infected with mutant FIKK7.1-KO and FIKK12-KO parasites have altered membrane rigidity
To evaluate whether FIKK proteins play a role in alteration of the mechanical
properties of the IE membrane skeleton and, therefore overall erythrocyte
deformability, we measured and compared the shear elastic modulus of uninfected
erythrocytes and highly synchronized erythrocytes infected with pigmented
trophozoites of either parental FCR3 parasites or mutant parasite lines. Using
single cell micromanipulation, we observed that infection of erythrocytes with
*P. falciparum* FCR3 caused an approximately 4-fold increase in the rigidity of
the erythrocyte membrane. In contrast, the overall level of rigidification of
erythrocytes infected with either of the FIKK-KO parasites was reduced when
compared to erythrocytes infected with parental FCR3 parasites. When compared to
the level of rigidification of the erythrocyte membrane induced by FCR3 parental
parasites, the rigidity of erythrocytes infected with FIKK7.1-KO parasites was
significantly lower (P\<0.0005 by Mann Whitney U test). Similarly, the median
level of rigidity of erythrocytes infected with FIKK12-KO parasites was also
lower than those infected with FCR3 but the difference did not quite reach
statistical significance at the 95% confidence level (P = 0.06) These suggest
that both FIKK7.1 and FIKK12 play a role in modulating the overall level of
membrane rigidification induced by *P. falciparum* infection.
## Phosphorylation levels of two different proteins in the erythrocyte membrane skeleton are altered in FIKK12-KO and FIKK7.1-KO parasites
We took a phospho-proteomics approach in an attempt to identify proteins which
are regulated by the FIKK kinases. Since most of the FIKK members are
transported into the erythrocyte membrane via Maurer's clefts, we investigated
the level of phosphorylation in ghost fractions prepared from IEs. We first
evaluated the purity of the ghost samples, confirming that only erythrocyte
membranes and Maurer's cleft were present without contamination by other
parasite components. The punctate pattern of Maurer's cleft proteins revealed by
antibodies against the Pf332 antigen was observed in the ghost preparations as
previously described and the intraparasitic maker Pfhsp70 was detected in the
total IEs extracts but not in the ghost fraction, indicating no contamination of
parasite cytosolic proteins in the ghosts (Supplementary).
The purified ghost fractions from ring (10–14 hours after infection),
trophozoite (18–22 hours after infection) and schizont (32–36 hours after
infection) stages of the FIKK12-KO and the parental FCR3 parasites were analyzed
by gel-based 1 dimensional electrophoresis (1-D). Given that phosphorylation in
*P. falciparum* blood stages occurs in a stage-specific manner, tightly
synchronized parasites, within a window of +/− 2 hours, were used in this
analysis. FIKK12-KO and FCR3 ghosts from the three intraerythrocytic stages were
lysed in the presence of protease and phosphatase inhibitors, after which the
same protein amount was subjected to 1-D SDS-PAGE. The gel was sequentially
stained with the phosphoprotein-specific Pro-Q Diamond fluorescence dye,
followed by SYPRO Ruby staining to detect total protein. The experiment was
performed independently three or four times in each stage. Comparison of the
phosphorylation pattern between the KO and wild type parasites preparations
showed a clear distinct profile. A protein with an apparent molecular weight of
80 kDa was significantly less stained in FIKK12-KO trophozoites than in wild
type parasites. This differential phosphorylation level was reproducibly
observed at the trophozoite stage but neither in ring nor schizont stages.
To investigate if the detected change in phosphorylation could result from non-
specific protein staining by Pro-Q Diamond, the FCR3 ghost sample was treated
with λ–protein phosphatase (λ-PPase), a Mn<sup>2+</sup>-dependent enzyme with
activity towards phosphorylated serine, threonine, tyrosine, and histidine.
Treated and untreated ghost samples were resolved by 1-D and compared by Pro-Q
Diamond and Coomassie blue staining. With the exception of one protein with high
molecular weight (over 250 kDa), all proteins were sensitive to λ-PPase
treatment and showed a strong reduction, or no reactivity to the phospho-
specific Pro-Q Diamond staining, validating the specificity of the Pro-Q Diamond
staining.
FIKK7.1-KO ghost extracts from highly synchronized parasites were also analyzed
for changes in the phosphorylation pattern. We detected a band with an estimated
molecular weight of 300 kDa in schizont extracts from wild type ghosts, which
was absent in FIKK7.1-KO extracts. No difference was seen in the 80 kDa band,
indicating that FIKK7.1 and FIKK12 target distinct proteins in the erythrocyte
membrane skeleton. This differential phosphorylation level was reproducibly
observed at the schizont stage but neither in ring nor trophozoite stages.
# Discussion
In this work, we demonstrate that two members of the FIKK kinase family are
involved in the remodeling of erythrocyte membrane skeleton proteins.
Importantly, each analyzed FIKK member apparently targets a distinct protein at
a different time point of the asexual blood cycle. Our experimental evidence
suggests that FIKK12 targets a protein of 80 kDa at trophozoite stage and
FIKK7.1 targets another protein of approx. 300 kDa at schizont stage. A recent
study reported changes in the phosphoproteome of IEs and identified numerous *P.
falciparum* phosphorylated proteins and 77 human proteins as phosphorylated in
IEs whereas only 48 were detected in uninfected erythrocytes. This indicates an
elevated level of post-translational modifications of the host cell by parasite
kinases. Given that most of the *Pf fikk* genes were shown to be transcribed in
blood stages and N-terminal regions are unique to each paralog, this raises the
possibility that each FIKK protein might have different functions in the IEs and
that other FIKK proteins could have other biological roles including
trafficking, adhesion and antigenic variation.
A reverse genetic screen identified different parasite proteins that are related
with changes in the deformability of the erythrocyte membrane. Inactivation of
several genes (none of the *fikk* genes was analyzed) showed moderate decreases
in the deformability of the IEs, validating that several proteins contribute to
the overall IEs rigidity. We may expect that these proteins interact with the
erythrocyte membrane skeleton or may facilitate the trafficking of skeleton-
related proteins. We previously showed that FIKK12 is exported into the
cytoplasm of the erythrocyte via Maurer's cleft at ring stage and disassociates
from the Maurer's cleft at trophozoite stage. Taken together, this raises the
possibility that the exported FIKK12 protein might interact with the membrane
skeleton of IEs at the trophozoite stage and be involved in the phosphorylation
of proteins of the erythrocyte membrane skeleton. Potential candidates for
FIKK12 substrates include the erythrocyte protein protein 4.1 with a molecular
weight of 80 kDa. Protein 4.1 plays different roles in protein attachment to the
membrane and is involved in regulating the membrane mechanical stability, which
is decreased in the protein 4.1-deficient erythrocytes. Interestingly
phosphorylation of protein 4.1 has been suggested to affect its role in protein-
protein interaction at the erythrocyte membrane skeleton and erythrocyte
membrane deformability. Our results show that disruption of *Pf fikk12* resulted
in less phosphorylation of the band at 80 kDa, however this was not completely
blocked. One possibility is that this substrate might be a substrate for a
variety of kinases in the erythrocyte and that FIKK12 might phosphorylate a
particular site.
For FIKK7.1 we identified a potential substrate with a high molecular weight,
although the parasite protein PfEMP1 has a molecular weight in this range and is
only detectable after radioactive labelling, at this stage we cannot conclude
that a parasite protein in the erythrocyte membrane is the target of FIKK7.1.
Furthermore, we do not know if these FIKK kinases target directly erythrocyte
membrane proteins or are involved in a phosphorylation cascade. At this stage,
we can not rule out the possibility that differences in the phosphoproteomes of
the KO lines are derived from the lack of activity of non FIKK kinase regulated
by FIKK12 or 7.1.
Deletion of both *Pf fikk7.1* and *Pf fikk12* in the FCR3 strain demonstrates
that they are not crucial for parasite replication *in vitro*, and since the
transgenic parasite lines show normal multiplication rates they are probably not
involved in the invasion process. However, our erythrocyte deformability data
strongly suggest that these proteins are involved in changes of the erythrocyte
membrane skeleton to meet the needs of the growing intracellular parasite. The
resulting modification of the rheological properties of IEs and may have a role
in splenic clearance by IEs *in vivo*.
When we commenced this work, we hypothesized that the FIKK proteins could be
involved in the process of IE adhesion and antigenic variation of *var* genes,
since this process is unique to *P. falciparum*. It was recently reported that
the phosphorylation of PfEMP1 cytoplasmic domain by casein kinase II alters the
association of this domain with knob associated histidine-rich protein and
interestingly that inhibition of phosphorylation reduced the cytoadherence of
IEs to two endothelial receptors. We, however, could not see any differences in
the adhesion properties, switching rate and trafficking of multigene families
between the two KO parasite lines and the wild type FCR3 strain.
In conclusion, we present for the first time that FIKK kinases are likely
involved in remodelling the membrane of IEs and our data point to changes in the
cellular mechanical properties in a stage-specific and target specific manner.
Work is ongoing to identify the specific proteins that are phosphorylated by
these FIKK kinases. In the future it will be interesting to investigate if the
*Pf fikk* genes perform similar functional roles in gametocytes and infected
hepatocytes.
# Materials and Methods
## *Plasmodium falciparum* cultures and adhesion assays
*P. falciparum* blood stage parasites from the FCR3 strains were cultured using
modifications to the method described by Trager and Jensen. IEs were selected on
the human placental derived BeWo cell line (European Collection of Cell
Cultures) to obtain the FCR3-CSA parasite line or on Chinese Hamster Ovary
cells-745 (CHO-745; American Type Culture Collection) expressing CD36 to obtain
the FCR3-CD36 parasite line. Pannings were repeated three times, and parasites
were tested for their ability to bind purified CSA (Sigma) or recombinant human
CD36 (R&D Systems). Cytoadhesion assays on receptors immobilized on plastic
petri dishes were carried out as described. Briefly, plastic Petri dishes were
coated overnight at 4°C with phosphate-buffered saline (PBS) containing 1 mg/ml
CSA sodium salt from bovine trachea (Sigma), 1 mg/ml chondroitin sulfate C
sodium salt from shark cartilage (Sigma), 10 µg/ml recombinant human CD36 (R&D
Systems). All spots were blocked with 1% bovine serum albumin (BSA) for 1 h at
room temperature (RT) before trophozoite-IE (5×10<sup>7</sup> IEs/ml) were
allowed to adhere. The average number of adherent IE (±SEM) for four different
fields in duplicate spots was determined in two to three independent experiments
after fixing with 2% glutaraldehyde in PBS for 2 h at RT and staining the plates
with Giemsa. Pictures were taken with a Nikon camera. Lucia software was used to
count the number of bound IEs per mm<sup>2</sup>.
## Plasmids and transfection
*Pf fikk7.1* and *Pf fikk12* were targeted using fragments amplified by PCR from
FCR3 strain genomic DNA with the following oligonucleotides: *fikk7.1* 5'
segment 5′-GAG*ccgcgg*TAGTACATTGTATAATAAAATATAACGC-3′ and
5′-CGC*agatct*CAAGAGATTATCATTTTTATTTTGC-3′ and 3′ segment
5′-ACGC*ccatgg*CTGTGGATATGTTGTAATGGTATC-3′and
5′-CGA*cctagg*CTATAAATATAATATTATGTATGCAC-3′; *fikk12* 5′ segment
5′-GAG*ccgcgg*ATGTATATTTTGAGAAATATGTTCTG-3′ and
5′-CGC*actagt*TCGTCCTCTTTTAAATTAGACATAC-3′ and 3′ segment
5′-ACGC*ccatgg*CAGATAAATTAAGACATATAGATAAAAAG-3′ and
5′-CGA*cctagg*TTATGTTTCGTTAAACCATGGGTGTG-3′ (enzyme restriction sites in
italics). These PCR fragments were sequentially cloned into pHTK using the
SacII/BglII and NocI/AvrII sites for *fikk7.1* and SacII/SepI and NocI/AvrII
sites for *fikk12*, to derive pHTK-*FIKK7.1* and pHTK-*FIKK12*, respectively.
Ring-stage FCR3-CSA *P. falciparum* parasites were transfected with 100 µg
plasmid DNA and cultured with WR99210 (10 nM) (Jacobus Pharmaceutical Co. Inc.)
after cultures were established parasites where double crossover homologous
recombination events had occurred were selected with 4 µM ganciclovir (Roche).
The resistant parasites were cloned by limiting dilution.
## Southern blot analysis of the two KO lines
Genomic DNA was digested using the following enzymes: HindIII (FIKK7.1
transfectants) and AluI (FIKK12 transfectants) and size fractionated on 0.8%
agarose gels. Southern blot were performed, as described previously. Specific
probes were amplified with the same set primers used for the initial cloning.
Membranes were hybridized at high-stringency conditions at 60°C overnight and
washed twice with 0.2x saline-sodium citrate (SSC) and 0.1% SDS at 60°C for 30
min.
## Immunofluorescence microscopy
Synchronized IEs were washed in PBS, cell pellets were resuspended in 10 vols of
PBS and a monolayer was set onto microscope slides. Parasites were air-dried and
fixed for 30 min at RT in 4% paraformaldehyde and 0.0075% glutalaldehyde. Slides
were washed with PBS and incubated with the primary antibodies diluted in 0.1%
BSA: rabbit anti-Rifin-A 565 antibody 1∶100 (serum raised against a peptide in
the highly conserved C-terminus of RIFIN-A, recognizes this type of RIFIN
specifically), rabbit anti-Stevor antibody 1∶400 (obtained from rabbits
immunized with a peptide designed on the basis of conserved STEVOR regions and
recognizes the subset of *stevor* genes transcribed in parasite population) and
rabbit anti-Surfin 4.2 antibody 1∶800 (antibody against PFD1160w that detects
specifically Surfin 4.2) for 45 min at RT. After washing cells were incubated
for 30 min with mouse anti-rabbit secondary antibody conjugated with
fluorochrome (Invitrogen) in a 1∶500 dilution. Slides were washed thoroughly in
PBS and mounted in Vectashield anti-fading with DAPI (Vector Laboratories).
Images were captures using a Nikon Elipse 80i optical microscope.
## Ghost preparation
Erythrocyte ghosts were essentially prepared as previously described with some
modifications. Synchronized ring (10–14 hours after infection), trophozoite
(18–22 hours after infection) or schizont (32–36 hours after infection)-infected
erythrocytes were extensively washed in RPMI 1640 medium and lysed for 15 min at
4°C in 40 vols of hypotonic buffer \[10 mM sodium-phosphate buffer, pH7.4
containing protease inhibitor cocktail tablet (Roche) and phosphatase
inhibitors, 400 nM Okadaic acid (Sigma) and phosphatase inhibitor cocktail 2
(Sigma)\]. The lysates were then separated by centrifugation at 15,000×g for 30
min at 4°C into a cytosolic fraction and a pellet fraction containing ghosts and
free parasites. The erythrocyte ghosts were collected at the top of the free
parasite pellet, washed extensively five times in the hypotonic buffer. Ghosts
were also prepared from uninfected erythrocyte for a mocked control. Ghost
samples from each parasite stage were prepared in three or four independent
experiments and analyzed by phospho-proteomics. Twenty µg of the purified ghosts
were analyzed by SDS-PAGE using 4–12% polyacrylamide gel followed by immunoblot
assay using anti-Pf332 and anti-Pfhsp70 antibodies to confirm the purity of the
ghost preparation. In some experiments, 20 µg of ghost proteins were subjected
to dephosphorylation prior SDS-PAGE analysis. For this samples were incubated
for 30 min at 25°C with 4,000 U of λ-PPase (New England BioLabs) in reaction
buffer (50 mM Tris-HCl pH7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35,
and 2 mM MnCl<sub>2</sub>).
## Staining procedures
Polyacrylamide gels were fixed in 50% methanol, 10% acetic acid, and stained
using Pro-Q Diamond phosphoprotein gel stain (Molecular Probes) in accordance
with the manufacturer's instructions. The stained gel was visualized on a
Typhoon 9400 scanner (Amersham Biosciences) using excitation at 532 nm and 560
nm longpass emission filter, normal sensitivity (600 V), 3 mm focal plane and 50
mm resolution. Total protein was restained with SYPRO Ruby total protein gel
stain (Molecular Probes), and visualized on the scanner using excitation at 457
nm and 610 nm bandpass emission filter and the same settings as above. Images
were acquired as gel file format, imported into ImageJ software and stored as
tiff file format for subsequent analysis.
## Measurement of membrane shear elastic modulus by micropipette aspiration
Single cell micropipette aspiration was used to determine the shear elastic
modulus of erythrocyte membranes as previously described. Briefly, the membrane
of individual erythrocytes was aspirated progressively into glass micropipettes
(internal diameter 1.3 µm) over a range of increasing negative hydrostatic
pressures. The shear elastic modulus of the membrane skeleton was determined by
measuring the length of a membrane tongue (L) aspirated from the erythrocyte
into the pipette for a range of aspiration pressures (P) and calculated from the
linear regression of dL/dP. The range of aspiration pressures was 1.0 - 4.5
mmH<sub>2</sub>O for uninfected erythrocytes or 1.0–12.0 mmH<sub>2</sub>O for
infected erythrocytes. All measurements were performed at RT (approximately
20–25°C).
# Supporting Information
We would like to thank Denise Mattei and Ross Coppel for helpful discussions. We
are grateful to the generous gift of the Rifin and Surfin antibodies by Mats
Wahlgren and Stevor antibodies by Peter Preiser. We also thank Fiona Glenister
for assistance with the micromechanical measurements.
[^1]: Conceived and designed the experiments: MCN MO BMC AS. Performed the
experiments: MCN MO CSB. Analyzed the data: MCN MO BMC. Wrote the paper: MCN
MO BMC AS.
[^2]: Current address: Respiratory and Meningeal Pathogens Research Unit,
University of the Witwatersrand, Johannesburg, South Africa
[^3]: Current address: National Center for Global Health and Medicine,
International Medical Center of Japan, Tokyo, Japan
[^4]: The authors have declared that no competing interests exist. |
# Introduction and background
Proton cancer therapy (PCT) is an approved type of radiotherapy that utilizes
high-energy H<sup>+</sup> projectiles to fight cancer. The ultimate effect of
this radiation is to damage the DNA of cancerous cells. If left unrepaired, this
damage produces various anomalies in cancerous cells that eventually lead to
their death (apoptosis). While PCT damages both healthy and cancerous cells, the
latter have a high rate of division and a reduced ability to repair damaged DNA.
Thus, cancer cells are particular vulnerable to radiation attacks.
PCT radiation is applied as collimated beams of H<sup>+</sup> projectiles at an
initial kinetic energy of 70–250 MeV. As they penetrate the patient’s body,
these projectiles lose their energy through molecular interactions until they
reach thermal energy in deep tissues. The radiation energy deposited in the
tissues is a measure of its potential for DNA damage. Typically, a plot of the
radiation energy loss *vs*. the radiation travelled distance exhibits a maximum
of energy deposition. Conventional X-ray therapy exhibits a broad deposition
maximum not far after the photons’ penetration into the body, followed by a
gradual energy loss at deeper penetrations. In contrast, PCT (and other ion
therapies) exhibits a sharp maximum peak known as the Bragg peak; that peak
occurs just before the H<sup>+</sup> projectiles are stopped in deep tissues.
Thus, by focusing Bragg peaks on a deep tumor, PCT inflicts a maximum DNA damage
on that region and a minimum DNA damage on the surrounding healthy tissues.
In all radiotherapies, the radiation predominantly interacts with cellular
H<sub>2</sub>O because the latter constitutes about 70% of the human cell mass.
This interaction triggers water radiolysis reactions—i.e. a series of cascade
hydrolytic reactions producing DNA-damaging species. In PCT, water radiolysis
generates various secondary species: (a) free radicals (e.g. H<sup>+</sup> +
H<sub>2</sub>O → H<sup>+</sup> + H·+ OH·)\[–, \], (b) secondary ions (e.g.
H<sup>+</sup> + H<sub>2</sub>O → 2H<sup>+</sup> *+* OH<sup>-</sup>)\[–, \], (c)
reactive molecules (e.g. H<sup>+</sup> + 2H<sub>2</sub>O → H<sup>+</sup> +
H<sub>2</sub> + H<sub>2</sub>O<sub>2</sub>), and (d) localized heat in the
medium. These species can react with H<sub>2</sub>O and generate similar
tertiary species and so forth. All these reactive products eventually reach
cellular DNA and cause various types of damage: DNA bases’ fragmentations and
deletions, and single-, double- and clustered-strand DNA breaks.
PCT comprises various processes spanning different space (*l* =
10<sup>−10</sup>–10<sup>0</sup> m) and time (*t* = 10−21–10<sup>5</sup> s)
scales. For instance, water radiolysis reactions, DNA damage at the genome level
and tumor remissions lie at the microscopic (10<sup>−10</sup>
≤*l*≤10<sup>−8</sup> m), mesoscopic (10<sup>−8</sup> ≤*l*≤10<sup>−3</sup> m) and
macroscopic (10<sup>−3</sup> ≤*l*≤10<sup>0</sup> m) scales, respectively. The
scale of a process determines the appropriate methods for its study. Thus, in
theoretical/computational studies, microscopic water radiolysis reactions can be
feasibly simulated with *ab initio* quantum-mechanics methods. In contrast,
mesoscopic energy-loss and Bragg peak processes are only tractable with Monte
Carlo (MC) models. Quantum-mechanics and MC methods act in synergy: the former
predict properties (e.g. reaction cross sections) required as input data for MC
simulations \[, –\], and the latter calculate proper radiation doses for
treatments.
Although PCT is clinically approved, various PCT details at the microscopic
scale remain uncertain. Knowledge of those details is essential for a rational
design of PCT seeking to maximize its therapeutic power and minimize its side
effects. While the predominant paradigm for cancer research is
experimental/clinical, theoretical/computational methods can reveal microscopic
details of PCT more exhaustively than experimental/clinical techniques and
without putting human subjects at risk. Therefore, time-independent scattering
and time-dependent dynamics methods have been applied to computationally
feasible prototypes of PCT reactions (e.g. H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1–4</sub> to model water radiolysis reactions and
H<sup>+</sup> + DNA/RNA bases to model DNA proton damage).
Among quantum-mechanics methods for PCT, the electron nuclear dynamics (END)
theory offers distinctive capabilities to study PCT reactions. END is a (1)
time-dependent, (2) direct and (3) non-adiabatic method to simulate chemical
reactions. These attributes are valuable for PCT simulations because they
afford: (1) time-dependent detail, (2) independence from predetermined potential
energy surfaces, and (3) capability of describing high-energy non-adiabatic
processes \[e.g. electron transfers (ETs)\]. Among different END versions \[,
\], the simplest-level (SL) END (SLEND) describes the nuclei and electrons in
terms of classical mechanics and a Thouless single-determinantal wavefunction,
respectively. Thus, SLEND possesses a suitable balance between accuracy and
computational feasibility to simulate large PCT prototypes (cf. previous SLEND
studies of H<sup>+</sup> + (H<sub>2</sub>O)<sub>*n*</sub>, *n* = 1, 2, and 3–4
and of H<sup>+</sup> + DNA/RNA bases and DNA base pairs).
Following the aforesaid precedents, we present herein an exploratory SLEND study
of PCT water radiolysis reactions with the H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1-6</sub> prototypes at *E*<sub>*Lab*</sub> = 100 keV. This
energy is selected because it corresponds to the average Bragg peak energy in
bulk water, the medium where water radiolysis occurs. However, no *ab initio*
quantum-mechanics methods can simulate bulk water due to prohibitive
computational cost and, therefore, those methods treat the above-mentioned
water-clusters prototypes. Surprisingly, most quantum-mechanics studies of water
radiolysis have utilized the smallest prototype: H<sup>+</sup> + H<sub>2</sub>O,
whose “cluster” is the farthest from being bulk water. While these H<sup>+</sup>
+ H<sub>2</sub>O studies were indeed useful for investigating radiolysis
processes, they could not completely capture the processes occurring in bulk
water. For that reason, previous SLEND studies explored H<sup>+</sup> +
(H<sub>2</sub>O)<sub>*n*</sub> prototypes with n = 2 and 3–4. However, these
studies still concentrated on the smallest possible clusters and involved a
limited number of proton-cluster orientations and simulations. Therefore, to
overcome all the discussed limitations, we study herein the H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1-6</sub> prototypes that include ten isomers in a larger
series of clusters (H<sub>2</sub>O)<sub>1-6</sub> (cf.) and involve a larger
number of proton-cluster orientations (60) and simulations (25,020).
The selected (H<sub>2</sub>O)<sub>1-6</sub> series contains the initial terms of
the long progression from molecular H<sub>2</sub>O to bulk water. Specifically,
the first terms in this progression, (H<sub>2</sub>O)<sub>1-5,</sub> have mono-
and di-cyclic quasi-planar/multiplanar structures exhibiting no “bulky” shapes
(cf.), but two isomers in the last term—the prism and cage isomers of
(H<sub>2</sub>O)<sub>6</sub>—have multi-cyclic, three-dimensional structures
exhibiting drop-like shapes (cf.). In fact, these two
(H<sub>2</sub>O)<sub>6</sub> isomers, particularly the prism, are considered the
smallest possible drops of water—i.e. the minimum water structures that manifest
the three-dimensional hydrogen-bond structure and solubility properties of bulk
water. Based on those facts, we expected that this series would reveal the
earliest manifestations of bulk water effects on PCT properties; however, the
present results do not display such manifestations and suggest that even larger
water clusters should be considered (cf. Results and Discussion Section).
Despite that outcome, all the predicted properties and reactions of
H<sup>+</sup> + (H<sub>2</sub>O)<sub>2-6</sub> have never been measured or
calculated before; therefore, the present results are truly predictive and fill
a gap in the medical physics literature. Furthermore, these results are
important to understand PCT more thoroughly and to model water radiolysis
processes and radiation dosages with MC methods. Finally, it should be noticed
that our simulated phenomena are the first processes occurring upon a short-time
direct collision of a proton with moderate size clusters. Other post-collision
phenomena contributing to PCT such as local temperature increases and
hypothesized shock waves in water require for their modelling longer simulation
times, larger clusters and even different theoretical methods; therefore, those
phenomena are not reproduced by the current simulations.
# Methodology
The END theory and its SLEND version have been reviewed in detail in Refs.;
therefore, we provide a brief outline of them. END is a variational, time-
dependent, direct, and non-adiabatic dynamical method. END prescribes a total
trial wavefunction $\left| \Psi_{Total}^{END} \right\rangle = \left|
\Psi_{N}^{END} \right\rangle\ \left| \Psi_{e}^{END} \right\rangle$, which
consists of nuclear $\left| \Psi_{N}^{END} \right\rangle$ and electronic $\left|
\Psi_{e}^{END} \right\rangle$ wavefunctions, and treats $\left|
\Psi_{Total}^{END} \right\rangle$ under the time-dependent variational principle
(TDVP). The various versions of END differ in the kind of descriptions for the
nuclei and electrons (e.g., density functional theory for electrons). In
SLEND, the nuclear wavefunction $\left| \Psi_{N}^{SLEND} \right\rangle$ for a
system having *N*<sub>*N*</sub> nuclei is the product of 3*N*<sub>*N*</sub>
frozen, narrow Gaussian wave packets: $$\left\langle \mathbf{X} \right.\left|
{\Psi_{N}^{SLEND}(t)} \right\rangle = \left\langle \mathbf{X} \right.\left|
{\mathbf{R}(t),\ \mathbf{P}(t)} \right\rangle = {\prod\limits_{A =
1}^{3N_{N}}{\exp\left\{ {- \left\lbrack \frac{X_{A} - R_{A}(t)}{2\Delta R_{A}}
\right\rbrack^{2} + iP_{A}(t)\left\lbrack {X_{A} - R_{A}(t)} \right\rbrack}
\right\}}}$$ with average positions **R**<sub>*A*</sub>(*t*), average momenta
**P**<sub>*A*</sub>(*t*) and widths Δ*R*<sub>*A*</sub>. To lower computational
cost, the zero-width limit, Δ*R*<sub>*A*</sub> → 0, is applied to the nuclear
wave packets after constructing the SLEND quantum Lagrangian (cf. next
paragraph). That procedure generates a classical nuclear dynamics but with full
retention of the nucleus-electron non-adiabatic coupling terms (cf.). As proven
previously, classical nuclear dynamics does not impair the accuracy of PCT
simulations because they happen at high collision energies. The SLEND electronic
wavefunction $\left| \Psi_{e}^{SLEND} \right\rangle$ is a spin-unrestricted,
single-determinantal wavefunction in the Thouless representation. Specifically,
taking *N*<sub>*e*</sub> occupied {*ψ*<sub>*h*</sub>}, and *K* –
*N*<sub>*e*</sub> virtual {*ψ*<sub>*p*</sub>} molecular spin-orbitals (MSOs),
$\left| \Psi_{e}^{SLEND} \right\rangle$ is: $$\left| \Psi_{e}^{SLEND}
\right\rangle = \left| {\mathbf{z}(t);\mathbf{R}(t),\mathbf{P}(t)} \right\rangle
= \det\left\{ {\chi_{h}\left\lbrack {\mathbf{x}_{h};\
\mathbf{z}(t),\mathbf{R}(t),\mathbf{P}(t)} \right\rbrack} \right\} =
\text{exp}\left\lbrack {\sum\limits_{h = 1,\ p = N_{e} + 1}^{N_{e},\
K}{z_{ph}(t)b_{p}^{\dagger}b_{h}}} \right\rbrack\left| 0 \right\rangle;$$ where
$\left| 0 \right\rangle = \left| \psi_{N_{e}} \right.\ldots\left. \psi_{1}
\right\rangle$ is an unrestricted Hartree-Fock (UHF) reference state and
{*χ*<sub>*h*</sub>} are dynamical spin-orbitals (DSOs) $$\chi_{h}\left\lbrack
{\mathbf{x};\ \mathbf{z}(t),\mathbf{R}(t),\mathbf{P}(t)} \right\rbrack =
\psi_{h}\left\lbrack {\mathbf{x};\ \mathbf{R}(t),\mathbf{P}(t)} \right\rbrack +
{\sum\limits_{p = N_{e} + 1}^{K}{z_{ph}(t)\psi_{p}}}\left\lbrack {\mathbf{x};\
\mathbf{R}(t),\mathbf{P}(t)} \right\rbrack;\ \left( {1 \leq h \leq N_{e}}
\right)$$ with complex-valued molecular coefficients {*z*<sub>*ph*</sub>(*t*)}.
The MSOs are obtained at initial time at the UHF level. The MSOs are constructed
with travelling atomic basis set functions $\phi_{A_{i}}\left(
{\mathbf{r}_{i};\mathbf{R}_{A},\mathbf{P}_{\mathbf{A}}} \right)$—i.e., Slater-
type orbitals in terms of contracted Gaussian-type orbitals on the moving
nuclear centers **R**<sub>*A*</sub>(*t*) and augmented with electron translation
factors (ETFs) to include explicit nuclear momenta **P**<sub>*A*</sub>(*t*)
effects. MSOs and DSOs are spin-unrestricted; therefore, the unrestricted
determinant \|**z**(*t*);**R**(*t*),**P**(*t*)⟩ can reasonably describe bond-
breaking/-forming processes. The Thouless representation provides a non-
redundant and singularity-free parameterization of an evolving single-
determinantal state.
The SLEND dynamical equations are obtained according to the TDVP. First, the
quantum Lagrangian $L_{SLEND} = \left\langle \Psi_{Total}^{SLEND}\ \right|\
\left( {i/2} \right)\ \left( {\overset{\rightharpoonup}{\partial}/\partial t}
\right. - \left. {\overset{\leftarrow}{\partial}/\partial t} \right) -
{\hat{H}}_{Total}\ \left| \ \Psi_{Total}^{SLEND} \right\rangle/\left\langle
\Psi_{Total}^{SLEND}\ \right|\ \left. \Psi_{Total}^{SLEND} \right\rangle^{- 1}$
is constructed and then the zero-width limit is applied to the nuclear wave
packets. Subsequently, the stationary condition is imposed to the quantum action
*A*<sub>*SLEND*</sub>, $\delta A_{SLEND} =
\delta{\int_{t_{1}}^{t_{2}}{L_{SLEND}(t)dt}} = 0$. The resulting SLEND dynamical
equations are: $$\begin{bmatrix} {i\mathbf{C}} & 0 & {i\mathbf{C}_{\mathbf{R}}}
& {i\mathbf{C}_{\mathbf{P}}} \\ & & & \\ 0 & {- i\mathbf{C}^{*}} & {-
i\mathbf{C}_{\mathbf{R}}^{*}} & {- i\mathbf{C}_{\mathbf{P}}^{*}} \\ & & & \\
{i\mathbf{C}_{\mathbf{R}}^{\dagger}} & {- i\mathbf{C}_{\mathbf{R}}^{T}} &
\mathbf{C}_{\mathbf{R}\mathbf{R}} & {- \mathbf{I} +
\mathbf{C}_{\mathbf{R}\mathbf{P}}} \\ & & & \\
{i\mathbf{C}_{\mathbf{P}}^{\dagger}} & {- i\mathbf{C}_{\mathbf{P}}^{T}} &
{\mathbf{I} + \mathbf{C}_{\mathbf{P}\mathbf{R}}} &
\mathbf{C}_{\mathbf{R}\mathbf{P}} \\ \end{bmatrix}\begin{bmatrix}
\frac{d\mathbf{z}}{dt} \\ \frac{d\mathbf{z}^{*}}{dt} \\ \frac{d\mathbf{R}}{dt}
\\ \frac{d\mathbf{P}}{dt} \\ \end{bmatrix} = \begin{bmatrix} \frac{\partial
E_{Total}}{\partial\mathbf{z}^{*}} \\ \frac{\partial
E_{Total}}{\partial\mathbf{z}^{}} \\ \frac{\partial
E_{Total}}{\partial\mathbf{R}} \\ \frac{\partial E_{Total}}{\partial\mathbf{P}}
\\ \end{bmatrix}$$ where *E*<sub>*total*</sub> is the total (nuclear and
electronic) energy and the dynamic metric matrices are $$\left( \mathbf{C}_{XY}
\right)_{ik,\ jl} = \left. {- 2{Im}\frac{\partial^{2}\ln S}{\partial
X_{ik}^{'}\partial Y_{jl}}} \right|_{\begin{array}{l} {\mathbf{R}^{\prime} =
\mathbf{R}} \\ {\mathbf{P}' = \mathbf{P}} \\ \end{array}};\mspace{9mu}\left(
\mathbf{C}_{X_{ik}} \right)_{ph} = \left. \frac{\partial^{2}\ln S}{\partial
z_{ph}^{*}\partial X_{ik}} \right|_{\begin{array}{l} {\mathbf{R}^{\prime} =
\mathbf{R}} \\ {\mathbf{P}' = \mathbf{P}} \\
\end{array}};\mspace{9mu}\mathbf{C}_{ph,\ qg} = \left. \frac{\partial^{2}\ln
S}{\partial z_{ph}^{*}\partial z_{qg}} \right|_{\begin{array}{l}
{\mathbf{R}^{\prime} = \mathbf{R}} \\ {\mathbf{P}' = \mathbf{P}} \\
\end{array}}$$ where *X*<sub>*ik*</sub> and *Y*<sub>*jl*</sub> denote either
**R**<sub>*A* = *i*,*k*</sub> or **P**<sub>*A* = *j*,*l*</sub> and *S* =
⟨**z**(*t*), **R**′(*t*), **P**′(*t*)\|**z**(*t*), **R**(*t*), **P**(*t*)⟩.
**C**<sub>**R**</sub> and **C**<sub>**RR**</sub> are equivalent to the ordinary
non-adiabatic coupling terms. Neglect of these terms seriously impairs the
accuracy of the SLEND non-adiabatic dynamics; therefore, those terms are kept in
the present calculations. However, to lower computational cost, ETFs are not
included in the present basis set so that **C**<sub>**P**</sub> =
**C**<sub>**PR**</sub> = 0. The accelerated SLEND equations thus obtained have
been successfully applied to various reactive and non-adiabatic processes, from
a few eV to the keV regime\[–\] and up to 900 keV (cf. also the seminal END
Ref., especially page 948, where this approximation is applied exhaustively to
high-energy non-adiabatic processes). At initial time, the reactants are
prepared with positions $\left\{ \mathbf{R}_{A}^{i} \right\}$, momenta $\left\{
\mathbf{P}_{A}^{i} \right\}$ and Thouless electronic state
\|**z**<sup>(*i*)</sup> = **0**, **R**<sup>(*i*)</sup>⟩ = \|0⟩, i.e. the UHF
ground state of the reactants’ super-molecule. When the reaction accesses the
non-adiabatic regime, **z**(*t*) ≠ **0** and \|**z**(*t*),**R**(*t*)⟩ becomes a
superposition of ground \|0⟩ and excited \|… *h* → *p* …⟩ UHF states (cf.)
$$\left| {\Psi_{e}^{SLEND}(t)} \right\rangle = \left|
{\mathbf{z}(t);\mathbf{R}(t),\mathbf{P}(t)} \right\rangle = {\sum_{h = 1,\ p =
N_{e} + 1}^{K,\ N_{e} + 1}{z_{ph}(t)\left| \ldots\ h\rightarrow p\ \ldots
\right\rangle}} + \ \ldots$$
# Computational details
## Software
All the present SLEND simulations were computed with our END program <span
class="smallcaps">PACE</span> (Python-Accelerated Coherent states Electron-
nuclear dynamics, T. V. Grimes, E. S. Teixeira and J. A. Morales, Texas Tech
University, 2010–2016; cf. Ref., Sect. 4). PACE combines several advanced
computer science techniques such as a mixed programming language (Python for
logic flow and Fortran and C++ for calculations), intra- and internode
parallelization, and the fast OED/ERD atomic integral package from the ACES
III/IV program. In addition, the water clusters’ geometries at initial time were
computed with the NWChem <span class="smallcaps">\[</span><span
class="smallcaps">\]</span> and <span class="smallcaps">GAMESS \[</span><span
class="smallcaps">\]</span> programs.
## Water cluster structures at the initial states
Present SLEND simulations start with the super-molecular systems H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1-6</sub> optimized at the UHF level and having projectile-
to-target \[H<sup>+</sup>-to-(H<sub>2</sub>O)<sub>1-6</sub>\] separations ≥
30.00 a.u. (cf.). Integration of the SLEND requires the evaluation of their
various terms at numerous adaptive time steps on a total of 25,020 trajectories.
Thus, for feasibility’s sake, the medium size basis sets 6-31G\* \[for
H<sup>+</sup> + (H<sub>2</sub>O)<sub>1-6</sub>\] and 6-31G\*\* \[for
H<sup>+</sup> + (H<sub>2</sub>O)<sub>1-5</sub>\] are adopted. These basis sets
provide good water clusters’ geometries and energies (cf. Refs.; for these basis
sets’ dynamical performance, cf. Electron Transfer Properties and Further
Analysis Sub-Sections).
Numerous theoretical and experimental studies have been devoted to determine the
geometries and energies of water clusters because these systems are prototypes
to study the structural and solubility properties of bulk water. In fact, the
scientific literature about water clusters is vast and growing. Therefore, for
brevity’s sake, we limit ourselves to cite herein only the water clusters
studies closely related to this investigation. It is well-known that the
(H<sub>2</sub>O)<sub>2-3</sub> clusters present one isomer each; however, the
(H<sub>2</sub>O)<sub>*n*</sub>, *n* ≥ 4, clusters present a variable number of
isomers that rapidly increase with the cluster size *n*.
(H<sub>2</sub>O)<sub>1-5</sub> present mono- and di-cyclic quasi-
planar/multiplanar structures, whereas (H<sub>2</sub>O)<sub>*n*</sub>, *n* ≥ 6,
present multi-cyclic, three-dimensional structures in addition to quasi-
planar/multiplanar ones (cf.). The three-dimensional
(H<sub>2</sub>O)<sub>6</sub> isomers (e.g. the prism and cage isomers named
hexamer a and b) are considered the smallest drops of water. In general, theory
and experiments agree in regard to the structures and relative energies of the
(H<sub>2</sub>O)<sub>1-5</sub> isomers, but discrepancies arise in the energy
orders of the (H<sub>2</sub>O)<sub>*n*</sub>, *n* ≥ 6, isomers. Recent
spectroscopy experiments have identified the cage isomer as the lowest-energy
(H<sub>2</sub>O)<sub>6</sub> structure. However, *ab initio* calculations
at various levels of accuracy have disagreed on whether the prism, the cage,
both of them, or the chair isomer(s) is(are) the lowest-energy
(H<sub>2</sub>O)<sub>6</sub> structure(s). Ultimately, the most accurate
calculations with the coupled-cluster with singles, doubles and perturbative
triples \[CCSD(T)\] method have identified the prism isomer as the lowest-energy
(H<sub>2</sub>O)<sub>6</sub> structure.
For this investigation, we selected ten representative isomers in the
(H<sub>2</sub>O)<sub>1-6</sub> series: each single isomer of
(H<sub>2</sub>O)<sub>1-3,</sub> the two isomers of (H<sub>2</sub>O)<sub>4,</sub>
two isomers of (H<sub>2</sub>O)<sub>5</sub> out of a total of at least four
<sub>,</sub> and three isomers of (H<sub>2</sub>O)<sub>6</sub> out of a total of
at least twelve (cf.). The isomers were calculated at the UHF/6-31G\*
\[(H<sub>2</sub>O)<sub>1-6</sub>\] and /6-31G\*\*
\[(H<sub>2</sub>O)<sub>1-5</sub>\] levels. When two or more isomers of a
(H<sub>2</sub>O)<sub>n</sub> cluster are considered, they are depicted (cf.)
and listed (cf.) in their increasing order of energies \[e.g. hexamer a
(prism), hexamer b (cage), and hexamer c since $E_{\text{Hexamer\
b}}^{\text{Cage}} - E_{\text{Hexamer\ a}}^{\text{Prism}}$ = 4.2 kJ/mol and
$E_{\text{Hexamer\ c}}^{} - E_{\text{Hexamer\ a}}^{\text{Prism}}$ = 15.4 kJ/mol
with UHF/6-31G\*). Notice that the UHF prism isomer is the lowest-energy
(H<sub>2</sub>O)<sub>6</sub> structure as predicted by CCSD(T). The first
depicted/listed isomer is also the lowest-energy structure in its whole set of
isomers. The present UHF calculations of (H<sub>2</sub>O)<sub>1-6</sub> are not
intended to contribute to the resolution of the discrepancies about the
(H<sub>2</sub>O)<sub>6</sub> energies because these calculations do not attain
the accuracy of CCSD(T). Instead, these optimizations provide a good description
of the water clusters for subsequent SLEND/6-31G\* and /6-31G\*\* simulations.
## Initial states preparation and simulation times
Once the water clusters are optimized at the UHF/6-31G\* and /6-31G\*\* levels,
the super-molecular systems H<sup>+</sup> + (H<sub>2</sub>O)<sub>1-6</sub> are
assembled for the initial conditions (cf.). The (H<sub>2</sub>O)<sub>1-6</sub>
targets are prepared at rest in their equilibrium geometries with their centers
of mass placed at the origin of the laboratory-frame coordinate axes; the
(H<sub>2</sub>O)<sub>1-6</sub> major (pseudo-)planes of symmetry are placed with
maximum coincidence with the x-y plane. The H<sup>+</sup> projectile is first
prepared with position $\mathbf{R}_{\text{H}^{+}}^{0} = \left( {b \geq 0}
\right.,\ 0,\mspace{9mu}\left. {+ 30\ \text{a}.\text{u}.} \right)$ and momentum
$\mathbf{P}_{\text{H}^{+}}^{0} = \left( 0,\ 0, - \left. p_{\text{H}^{+}}^{z}
\right) \right.$, where *b* ≥ 0 is the projectile impact parameter measured from
the (H<sub>2</sub>O)<sub>1-6</sub> centers of mass, and $p_{\text{H}^{+}}^{z}$
corresponds to *E*<sub>*Lab*</sub> = 100 keV (cf. , panel I). Having set
$\mathbf{R}_{\text{H}^{+}}^{0}$ and $\mathbf{P}_{\text{H}^{+}}^{0}$, various
projectile-target relative orientations can be generated by rotating a
(H<sub>2</sub>O)<sub>1-6</sub> target according to ordinary Euler angles
prescriptions. However, such a procedure involves the electronic re-optimization
of the (H<sub>2</sub>O)<sub>1-6</sub> targets at each new orientation.
Therefore, since the H<sup>+</sup> bare ion requires no electronic optimization,
we adopted the easier and equivalent procedure of keeping a
(H<sub>2</sub>O)<sub>1-6</sub> target fixed while rotating the H<sup>+</sup>
projectile around. The definite initial conditions of the H<sup>+</sup>
projectile $\mathbf{R}_{\text{H}^{+}}^{i}$ and $\mathbf{P}_{\text{H}^{+}}^{i}$
are therefore obtained by rotating $\mathbf{R}_{\text{H}^{+}}^{0}$ and
$\mathbf{P}_{\text{H}^{+}}^{0}$ through the *extrinsic* Euler angles in the
order: 1<sup>st</sup>, 0<sup>0</sup> ≤ *γ* \< 360<sup>0</sup>, 2<sup>nd</sup>,
0<sup>0</sup> ≤ *β* ≤ 180<sup>0</sup>, and 3<sup>rd</sup>, 0<sup>0</sup> ≤ *α*
\< 360<sup>0</sup>, around the *space-fixed* z, y, and z axes, respectively
(cf., panels I, II and III for each angle rotation). *α* and *γ* are measured
from the +x axis employing the $\mathbf{R}_{\text{H}^{+}}^{0}$ and
$\mathbf{P}_{\text{H}^{+}}^{0}$ projections on the x-y plane and *β* is measured
directly from the +z axis (cf.). These rotations define projectile-target
relative orientations Ω<sub>*i*</sub> = (*α*<sub>*i*</sub>, *β*<sub>*i*</sub>,
*γ*<sub>*i*</sub>). The definite initial conditions of the H<sup>+</sup>
projectile for the simulations, $\mathbf{R}_{\text{H}^{+}}^{i}$ and
$\mathbf{P}_{\text{H}^{+}}^{i}$, are shown in, panel IV. *α* and *β* determine
the direction of an axis of incidence whereby an incoming H<sup>+</sup>
trajectory runs parallel to that axis with a lateral separation *b* ≥ 0; *γ* is
the polar angle of that trajectory around the axis of incidence. In all
simulations, the selected values of the Euler angles correspond to a 60-point
grid: Ω<sub>*i*</sub>, 1≤*i*≤60, developed in Ref.. This grid displays a uniform
sampling of the orientation space and provides a numerical quadrature that
ensures the invariance of Euler-angle integrals under several rotation
operations (e.g. Wigner D-matrices satisfy ${\int\limits_{0}^{2\pi}{\int\limits_
{0}^{\pi}{\int\limits_{0}^{2\pi}D_{M^{\prime}M}^{J}}}}\ \left( {\alpha,\ \beta,\
\gamma} \right)\ \sin\beta d\alpha\ d\beta d\gamma = 0$ for 2 ≤ *J* ≤ 5). For a
given orientation Ω<sub>*i*</sub>, *b* is varied according to the grids defined
in. Simulations for the calculation of 1-ET ICSs utilize the grids denoted as
\[*b*\]<sub>1</sub> in and run for a total time of 30.00 a.u. (0.7257 fs); this
simulation time ensures that the final projectile-target separation is at least
equal to the initial one (30.00 a.u.). Simulations for the prediction of
fragmentations utilize the grids denoted as \[*b*\]<sub>2</sub> in and run for a
total time of 1,000 a.u. (24.19 fs); this much longer simulation time permits
the manifestation of post-collision fragmentations that have longer time scales
than those of the 1-ET processes. The described initial conditions generate a
total of 25,020 trajectories to complete the H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1-6</sub> study.
## Final states analysis and properties calculations
By the end of a simulation, various auxiliary codes in the PACE package identify
and analyze the final products and calculate dynamical properties. The most
important properties calculated herein are the cluster-to-proton 1-ET total
ICSs, *σ*<sub>1−*ET*</sub>, corresponding to H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1–6</sub> → H + different cluster products $$\sigma_{1
- ET} = \frac{1}{4\pi}{\int\limits_{0}^{\infty}{\int\limits_{0}^{2\pi}{\int\limi
ts_{0}^{\pi}{\int\limits_{0}^{2\pi}{b\mspace{9mu} P_{1\text{-ET}}\ \left(
{\alpha,\ \beta,\ \gamma,\ b} \right)db\mspace{9mu}\sin\beta}}}}}\ d\alpha\
d\beta\ d\gamma$$ where *P*<sub>1−*ET*</sub>(*α*, *β*, *γ*, *b*) is the
probability of a 1-ET process from a bound electronic state of the cluster to a
bound electronic state of the projectile, henceforth named bound-to-bound 1-ET
for brevity’s sake. Notice that for atom-atom collisions involving spherically
symmetric potentials, *P*<sub>1−ET</sub> (*α*, *β*, *γ*, *b*) →
*P*<sub>1−ET</sub> (*b*), and Eq simplifies to the familiar expression:
$\sigma_{1 - \text{ET}} = 2\pi{\int\limits_{0}^{\infty}b}\mspace{9mu} P_{1 -
\text{ET}}(b)\ db$. In the present systems, an outgoing projectile can in
practice capture up to two electrons: H<sup>+</sup> + A → H<sup>1−*n*</sup> +
A<sup>−*n*</sup>, 0 ≤ *n* ≤ 2, because the probability of forming unstable
H<sup>1−*n*</sup> with *n* ≥ 3 is negligible. Under these conditions,
*P*<sub>*n*−ET</sub> (*α*, *β*, *γ*, *b*), 0 ≤ *n* ≤ 2, are $$\begin{array}{l}
{P_{0 - \text{ET}}\left( {\alpha,\ \beta,\ \gamma,\ b} \right) = \left( {1 -
N_{\alpha}} \right)\left( {1 - N_{\beta}} \right);} \\ {P_{1 - \text{ET}}\left(
{\alpha,\ \beta,\ \gamma,\ b} \right) = N_{\alpha}\left( {1 - N_{\beta}} \right)
+ N_{\beta}\left( {1 - N_{\alpha}} \right);} \\ {P_{2 - \text{ET}}\left(
{\alpha,\ \beta,\ \gamma,\ b} \right) = N_{\alpha}N_{\beta}} \\ \end{array}$$
where *N*<sub>*α*</sub> and *N*<sub>*β*</sub> are the number of *α*- and
*β*-spin electrons in the outgoing projectile calculated from their respective
electron densities $\rho_{\alpha}^{out.\ proj.}\left( \mathbf{r} \right)$ and
$\rho_{\beta}^{out.\ proj.}\left( \mathbf{r} \right)$: $$N_{\alpha} =
{\int{\rho_{\alpha}^{out.\ proj.}\left( \mathbf{r} \right)}}\
d\mathbf{r};\mspace{9mu} N_{\beta} = {\int{\rho_{\beta}^{out.\ proj.}\left(
\mathbf{r} \right)}}\ d\mathbf{r}$$ Eqs and are evaluated at the final
simulation time, when the outgoing projectile and the clusters are well
separated by at least 30.00 a.u. of length; therefore, *N*<sub>*α*</sub> and
*N*<sub>*β*</sub> are the number of electrons unequivocally assigned to the
distant outgoing projectile. Moreover, at those separations, *N*<sub>*α*</sub>
and *N*<sub>*β*</sub> from and those from any ordinary electron population
analyses (Mulliken, Löwdin, etc.) become identical; this assures that
*N*<sub>*α*</sub> and *N*<sub>*β*</sub> are free of any arbitrary criterion for
electrons’ distributions over the projectile and clusters. Other calculated
properties are the orientation-averaged 1-ET probabilities ${\overline{P}}_{1 -
\text{ET}}(b)$, ${\overline{P}}_{1 - \text{ET}}(b) = \left( {8\pi^{2}}
\right)^{- 1}{\int_{0}^{2\pi}{\int_{0}^{\pi}{\int_{0}^{2\pi}P_{1 -
\text{ET}}}}}\ \left( {\alpha,\ \beta,\ \gamma,\ b} \right)\ \sin\beta$, and
their *b* – weighted counterparts, $b{\overline{P}}_{1 - \text{ET}}(b)$, where
$\sigma_{1\text{-ET}} = 2\pi{\int_{0}^{\infty}b}\mspace{9mu}{\overline{P}}_{1 -
\text{ET}}\ (b)\ db$. ${\overline{P}}_{1 - \text{ET}}(b)$ and
$b{\overline{P}}_{1 - \text{ET}}(b)$ reveal more mechanistic details than
*σ*<sub>1-*ET*</sub>.
# Results and discussion
## Electron-transfer properties
The first property calculated in this investigation is the cluster-to-proton
total 1-ET ICS, *σ*<sub>1−*ET*</sub>, for the H<sup>+</sup> +
(H<sub>2</sub>O)<sub>n</sub> systems, *n* = 1–6, at *E*<sub>*Lab*</sub> = 100
keV at the SLEND/6-31G\* (*n* = 1–6) and SLEND/6-31G\*\* (*n* = 1–5) levels.
Both SLEND *σ*<sub>1−*ET*</sub> for the monomeric system (*n* = 1) are listed in
along with their available counterparts from four experiments denoted as Exp. A
to D, respectively. In addition, includes results from two alternative theories,
namely, Theory A: the basis generator method (BGM), and Theory B: the continuum
distorted wave-eikonal initial state (CDW-EIS) approximation. From, one finds
that the average experimental ICS, ${\overline{\sigma}}_{1 - ET}^{Exp.}$, and
its average relative error, *e*<sub>*Exp*.</sub>, are 1.27 Å<sup>2</sup> and ±
10.62%, respectively. The theoretical $\sigma_{1 - ET}^{Theo.}$ ‘s and their
average relative deviations ${\overline{\Delta}}_{Theo.}$ from the experimental
values are: 1.54 Å<sup>2</sup> and +21.8% for SLEND/6-31G\*, 1.00 Å<sup>2</sup>
and +21.0% for BGM, and 0.589 Å<sup>2</sup> and -53.4% for CDW-EIS. Only the BGM
result is within the error bars of one experiment, Exp. D, with
Δ<sub>*Theo*.</sub> = 11.5%, but it lies on the lowest fringe part of the error
bar range. The SLEND/6-31G\* result is very close to the result from Exp. C with
Δ<sub>*Theo*.</sub> = 11.6% and not far from entering the upper part of the
error bar range. In absolute quantitative terms, the BGM and SLEND/6-31G\*
results are at the same level of accuracy and their agreement with the
experimental data should be deemed satisfactory given the difficulty to both
measure and predict the present 1-ET processes. Deviations of the obtained
magnitude are not uncommon in measurements and predictions of similar complex
processes (cf. Ref. for the case of one experiment and four different
theories including SLEND, where even higher deviations are observed). The CDW-
EIS result compares less favorably with the experimental ones, being roughly a
half of its experimental counterparts (${\overline{\Delta}}_{Theo.}$ = -53.4%).
The SLEND/6-31\*\* result also compares less favorably with the experimental
ones, but, opposite to CDW-EIS, its value is roughly twice as much as the
experimental one (${\overline{\Delta}}_{Theo.}$ = 63.0%). The reason and
remediation of the SLEND/6-31\*\* *σ*<sub>1−*ET*</sub> overestimation will be
discussed in detail in the Further Analysis Sub-section. It suffices to say here
that this overestimation results from the *σ*<sub>1−*ET*</sub> contamination
with electron transfers to the continuum of unbound states.
The SLEND *σ*<sub>1−*ET*</sub> for the polymeric systems (*n* = 2–6) are listed
in. Unfortunately, to the best of our knowledge, there are no alternative
experimental or theoretical ICSs for H<sup>+</sup> +
(H<sub>2</sub>O)<sub>2-6</sub>; therefore, current SLEND *σ*<sub>1−*ET*</sub>
for these systems are predictive. To facilitate the comparison among all the
considered *σ*<sub>1−*ET*</sub>, we plot them as a function of the cluster size
*n* in. There, each set of SLEND/6-31G\* and /6-31G\*\* *σ*<sub>1−*ET*</sub> is
fit to the scaling formulae *σ*<sub>1−*ET*</sub> (*n*) = *cn*<sup>2/3</sup>,
where *c* are fitting coefficients reported in. These formulae are by no means
arbitrary because they can be justified on physical grounds as follows. The
volume *V*(*n*) of the (H<sub>2</sub>O)<sub>*n*</sub> clusters should be
approximately proportional to their size *n*, *V*(*n*) ∝ *n*. If the clusters
are represented by the minimal spheres enclosing all their atoms, then their
volume *V*(*n*) and external area *A*(*n*) are $V\ (n) = \left( {4/3} \right)\
\pi\mspace{9mu} R_{n}^{3}$ and $A(n) = 4\pi\mspace{9mu} R_{n}^{2}$,
respectively, where *R*<sub>*n*</sub> is the radius of the
(H<sub>2</sub>O)<sub>*n*</sub> sphere; therefore, *A*(*n*) ∝ *V*
(*n*)<sup>2/3</sup> ∝ *n*<sup>2/3</sup>. In turn, the *σ*<sub>1−*ET*</sub> are
proportional to the effective external area *A*(*n*) of the
(H<sub>2</sub>O)<sub>*n*</sub> exposed to the incident H<sup>+</sup> for ET
processes; therefore, *σ*<sub>1−*ET*</sub> ∝ *A*(*n*) ∝ *n*<sup>2/3</sup> ⇒
*σ*<sub>1−*ET*</sub> (*n*) = *cn*<sup>2/3</sup>. In relative quantitative terms,
the SLEND/6-31G\* and /6-31G\*\* *σ*<sub>1−*ET*</sub> fit remarkably well into
the physically justified formulae *σ*<sub>1−*ET*</sub> (*n*) =
*cn*<sup>2/3</sup> with correlation factors *R*<sup>2</sup> = 0.983 in both
cases (cf.). This indicates that regardless of their absolute quantitative
performance, the SLEND *σ*<sub>1−*ET*</sub> scale correctly with the number of
water molecules in the clusters. Therefore, with these fitting formulae, one can
estimate the *σ*<sub>1−*ET*</sub> of the immediately larger clusters: e.g.,
$\sigma_{1 - ET}^{6 - 31G^{*}}$ = 5.96 and 6.51 Å for *n* = 7 and 8,
respectively; these estimated values should be interpreted as average
*σ*<sub>1−*ET*</sub> over the various (H<sub>2</sub>O)<sub>7</sub> and
(H<sub>2</sub>O)<sub>8</sub> isomers, respectively. Inspection of Tables and and
reveals that the SLEND/6-31G\*\* *σ*<sub>1−*ET*</sub> are always higher in value
than the SLEND/6-31G\* ones for each cluster as was the case with the
H<sub>2</sub>O monomer. Inspection of and reveals that the SLEND
*σ*<sub>1−*ET*</sub> for the various isomers appearing at a given *n* ≥ 4 do not
significantly differ in their values; this implies that these
*σ*<sub>1−*ET*</sub> are rather insensitive to the varying isomers’ structures
as targets. A similar finding was observed in the *σ*<sub>1−*ET*</sub> of
H<sup>+</sup> + DNA/RNA bases at *E*<sub>*Lab*</sub> = 80 keV, where similar
bases differing in their structure even more than isomers exhibited close values
of *σ*<sub>1−*ET*</sub>. We expected that various *σ*<sub>1−*ET*</sub> (*n*) =
*cn*<sup>2/3</sup> formulae differing in their coefficient *c* would exclusively
connect values from different sets of clusters—e.g. a single
*σ*<sub>1−*ET*</sub> (*n*) = *cn*<sup>2/3</sup> formula might have only fit well
with results from the quasi-planar clusters (monomer, dimer, trimer, tetramer a,
pentamer a and hexamer c), another single formula with other type of clusters,
etc., but that is not case. In fact, the uniformity among the SLEND
*σ*<sub>1−*ET*</sub> (*n*) values for isomers at a given *n* led us to fit all
of them with a single formula per basis set. Furthermore, we expected that the
*σ*<sub>1−*ET*</sub> of the drop-like prism and cage
(H<sub>2</sub>O)<sub>6</sub> isomers would differ sharply from the
*σ*<sub>1−*ET*</sub> of the quasi-planar/multiplanar isomers so that it would be
impossible to fit drop-like and non-drop-like *σ*<sub>1−*ET*</sub> with a single
formulae. Such a hypothetical fitting failure would manifest as a “phase
transition” discontinuity from non-drop-like to drop-like *σ*<sub>1−*ET*</sub>.
However, no such “phase transition” is observed in the selected series of
(H<sub>2</sub>O)<sub>1-6</sub> isomers. Thus, unlike the case of structural and
solubility properties, the “magic number” of six waters in the prism and cage
(H<sub>2</sub>O)<sub>6</sub> isomers do not bring about any hint of water
radiolysis processes in bulk water. Likely, an extension of the current
(H<sub>2</sub>O)<sub>1-6</sub> series with the (H<sub>2</sub>O)<sub>7-20</sub>
isomers may bring about some type of bulk-water manifestations.
Total 1-ET ICSs *σ*<sub>1−*ET*</sub> are not very detailed properties and cannot
reveal some dynamical details of 1-ET processes. Therefore, Figs and show the
orientation-averaged 1-ET probabilities ${\overline{P}}_{1 - ET}^{}(b)$ and
their *b* – weighted counterparts $b{\overline{P}}_{1 - ET}^{}(b)$ vs. *b* for
the considered (H<sub>2</sub>O)<sub>1-6</sub> isomers. With both basis sets, the
${\overline{P}}_{1 - ET}^{}(b)$ are high in value at small impact parameters
(roughly, *b*≤6 a.u.) corresponding to close projectile-cluster encounters but
they decrease rapidly at larger impact parameters. The ${\overline{P}}_{1 -
ET}^{}(b)$ vs. *b* plots show a variable number of maximum peaks (from one to
three) depending on the considered water cluster. The $b{\overline{P}}_{1 -
ET}^{}(b)$ vs. *b* plots exhibit similar patterns to those of the
${\overline{P}}_{1 - ET}^{}(b)$ but modulated by the *b* value. As the
integrands of the *σ*<sub>1−*ET*</sub>, the $b{\overline{P}}_{1 - ET}^{}(b)$
plots indicate that the most important contributions to the *σ*<sub>1−*ET*</sub>
come from 1-ET processes arising from intermediate impact parameters (roughly, 2
a.u. ≤ *b* ≤ 9 a.u.).
## Fragmentation reactions
Unlike time-independent scattering methods applicable to PCT, SLEND simulations
can reveal the reactants-to-products time evolution of the chemical reactions
underlying the 1-ET processes. To study those reactions, the SLEND/6-31G\*
simulations to calculate *σ*<sub>1−*ET*</sub> of H<sup>+</sup> +
(H<sub>2</sub>O)<sub>1-4</sub> (only tetramer a for *n* = 4) were prolonged from
their simulation times of 30.00 a.u. (0.7257 fs) to 1,000 a.u. (24.19 fs) using
the impact parameter grids \[*b*\]<sub>2</sub> in. This much longer simulation
time allows for the manifestation of fragmentation reactions that occur at
longer time scales. In fact, no fragmentation was observed within the original
time of 30.00 a.u. As shows, the final SLEND electronic wavefunction
\|**z**(*t*),**R**(*t*)⟩ is a superposition of various UHF states corresponding
to various products’ channels, e.g. $\left. \text{H}_{proj.}^{+} +
\text{H}_{2}\text{O}\rightarrow\text{H}_{proj.}^{q_{1} = + 1} +
\text{H}_{2}^{q_{2} = 0} + \text{O}_{}^{q_{3} = 0} \right.$ or $\left.
\rightarrow\text{H}_{proj.}^{q_{1} = 0} + \text{H}_{2}^{q_{2} = + 1} +
\text{O}_{}^{q_{3} = 0} \right.$ or $\left. \rightarrow\text{H}_{proj.}^{q_{1} =
- 1} + \text{H}_{2}^{q_{2} = + 1} + \text{O}_{}^{q_{3} = + 1} \right.$, etc.,
where $\text{H}_{proj.}^{q_{1} = 0, + 1, - 1}$ is the incoming/outgoing
projectile; these channel states occur with different probabilities. Therefore,
when is applied to all the well-separated fragments at final time,
*N*<sub>*α*</sub> and *N*<sub>*β*</sub> and their corresponding charges
*q*<sub>*i*</sub> (e.g. *q*<sub>1</sub> = 1−*N*<sub>*α*</sub>−*N*<sub>*β*</sub>
for the final $\text{H}_{proj.}^{q_{1}}$) are not necessarily integer numbers
corresponding to canonical chemical species (e.g. $\text{H}_{proj}^{q_{1} = +
1}$, $\text{H}_{proj}^{q_{1} = 0}$ and $\text{H}_{proj}^{q_{1} = - 1}$) but
fractional numbers as the averages of the number of electrons and charges over
the channels’ probabilities. This was always the case in all the present
simulations not leading to clusters fragmentations (i.e. $\left.
\text{H}_{proj.}^{+ 1} + \left( {\text{H}_{2}\text{O}}
\right)_{n}\rightarrow\text{H}_{proj.}^{q_{1}} + \left( {\text{H}_{2}\text{O}}
\right)_{n}^{1 - q_{1}} \right.$, with *q*<sub>*i*</sub> continuously varying in
the range −1≤*q*<sub>*i*</sub>≤+1). However, as in previous SLEND studies of
H<sup>+</sup> + (H<sub>2</sub>O)<sub>1–4</sub> at *E*<sub>*Lab*</sub> = 1 keV,
the present simulations leading to clusters fragmentations always bring about
outgoing projectiles $\text{H}_{proj.}^{q_{1}}$ and clusters fragments
$\text{A}^{q_{2}}$ with *q*<sub>1</sub> = +1 and *q*<sub>2</sub> = 0,
respectively (cf. caption). Thus, the present SLEND simulations predict that the
fragmentation channel leading to outgoing H<sup>+</sup> and neutral fragments
predominates over the others. However, it is known experimentally that proton-
water collisions lead to fragmentations into ions. SLEND can properly describe
fragmentations into ions as shown in previous studies \[e.g. cf. Refs. \].
Therefore, to allow the manifestation of those types of fragmentations here, it
will be necessary to prolong even further the simulation time of the present
calculations or, more likely, increase the number of total simulations by using
a finer grid. Such improvements entail further computational cost and will be
attempted later.
The predicted fragmentations are:
**H**<sup>**+**</sup> **+ H**<sub>**2**</sub>**O simulations:** 9 out of 252
simulations exhibited the H<sub>2</sub>O target fragmenting into: H + OH (2
simulations), H + H + O (6 simulations) and H<sub>2</sub> + O (1 simulation, cf.
).
**H**<sup>**+**</sup> **+ (H**<sub>**2**</sub>**O)**<sub>**2**</sub>
**simulations:** 9 out of 540 simulations exhibited the
(H<sub>2</sub>O)<sub>2</sub> target fragmenting into: H<sub>2</sub>O + HO + H (2
simulations), H<sub>3</sub>O + O + H (1 simulation), H<sub>2</sub>O + 2H + O (5
simulations) and H<sub>3</sub>O + OH (1 simulation, cf.).
**H**<sup>**+**</sup> **+ (H**<sub>**2**</sub>**O)**<sub>**3**</sub>
**simulations:** 3 out of 540 simulations exhibited the
(H<sub>2</sub>O)<sub>3</sub> target fragmenting into: H<sub>3</sub>O + OH +
H<sub>2</sub>O (2 simulations) and H + OH + 2H<sub>2</sub>O (1 simulation).
**H**<sup>**+**</sup> **+ (H**<sub>**2**</sub>**O)**<sub>**4**</sub> **(tetramer
a) simulations:** 10 out of 748 simulations exhibited the
(H<sub>2</sub>O)<sub>4</sub> target fragmenting into:
(H<sub>2</sub>O)<sub>2</sub> + 2H<sub>2</sub>O (4 simulations), H<sub>3</sub>O +
OH + 2H<sub>2</sub>O (4 simulations), an H + OH + 3H<sub>2</sub>O (2
simulations)
In conclusion, present SLEND/6-31G\* simulations predict the DNA-damaging
radicals H, OH, O and H<sub>3</sub>O, and the innocuous species H<sub>2</sub>O
and (H<sub>2</sub>O)<sub>2</sub> as water radiolysis products.
To illustrate the predicted fragmentations, we present a few animation stills
from some representative simulations. shows five sequential stills of the
animation of $\left. \text{H}_{proj.}^{+} +
\text{H}_{2}\text{O}\rightarrow\text{H}_{proj.}^{+} + \text{H}_{2} + \text{O}
\right.$ and a sixth panel plotting the Mulliken populations of the
H<sub>*proj*.</sub>, H<sub>2</sub> and O moieties vs. time. This last panel
permits the observation of the time evolution of the electrons. Mulliken
populations are basis-set dependent and, more importantly, somewhat arbitrary in
the way they distribute electrons over neighboring fragments. However, in
previous SLEND studies, Mulliken populations were good predictors for the time-
evolution of the electrons over atoms and fragments. Furthermore, when all the
fragments are well-separated at final time, the Mulliken populations converge to
the unequivocal *N*<sub>*α*</sub> and *N*<sub>*β*</sub> in (cf. caption). In ,
the colored spheres represent the classical nuclei (white = H and red = O), and
the colored clouds represent selected electron density iso-surfaces (from red =
lowest density to blue = highest density). The incoming projectile
$\text{H}_{proj.}^{+}\ $ passes in between the H atoms of H<sub>2</sub>O, hits
the O atom and bounces back. As a result of this collision, the H<sub>2</sub>
and O moieties of H<sub>2</sub>O break apart; the ejected H<sub>2</sub> moiety
undergoes a series of strong oscillations ranging from the near dissociation of
H<sub>2</sub> into H atoms to these atoms’ recombination back to H<sub>2</sub>.
Finally, shows the nine predicted fragments in H<sup>+</sup> +
(H<sub>2</sub>O)<sub>2</sub>.
## Further analysis and improvements
SLEND/6-31G\*\* *σ*<sub>1−*ET*</sub> compares unsatisfactorily with experiments
in contrast to SLEND/6-31G\* *σ*<sub>1−*ET*</sub>. Indeed, it is surprising that
SLEND/6-31G\*\* performs worse than SLEND/6-31G\* in these calculations since
common knowledge dictates that the 6-31G\*\* basis set is better than the
6-31G\* one. 6-31G\*\* is constructed from 6-31G\* by augmenting the latter with
p-type basis functions on the hydrogen atoms; thanks to this augmentation,
6-31G\*\* provides better time-independent molecular properties than 6-31G\*.
For instance, the UHF/6-31G\*\* energies and geometries of
(H<sub>2</sub>O)<sub>1–6</sub> are more accurate than the UHF/6-31G\* ones.
However, this comparative time-independent performance does *not* necessarily
extend to time-dependent calculations since these basis sets were designed to
calculate static properties. To explain the SLEND/6-31G\*\* *σ*<sub>1−*ET*</sub>
overestimation, one should remember that the main component in the bound-to-
bound SLEND *σ*<sub>1−*ET*</sub> is the 1-ET probability *P*<sub>1−ET</sub> (cf.
Eqs). However, as derived in Ref. and supposed in previous SLEND studies, the
*P*<sub>*n*−ET</sub>, 0 ≤ *n* ≤ 2, in assume that the probabilities of ETs from
the target A to the projectile H<sup>+</sup> are dominated by transitions with
electrons transferring into the localized, discrete, bound states of H:
H<sup>+</sup> + A: → H⋅ + A⋅<sup>+</sup> \[pure charge-transfer (CT)
processes\]; instead, transitions with electrons scattering into the
delocalized, continuous, unbound states of H are considered negligible:
H<sup>+</sup> + A: → (H<sup>+</sup> + *e*<sup>−</sup>) + A⋅<sup>+</sup> \[direct
ionization (DI) processes\]. Typical quantum chemistry basis sets, such as
6-31G\* and 6-31G\*\*, are ultimately based on localized primitive Gaussian
functions so that occupied spin-orbitals {*ψ*<sub>*h*</sub>} below the Fermi
level represent localized, bound states in the discrete part of the spectrum.
However, as a by-product of the UHF procedure, diffuse virtual spin-orbitals
{*ψ*<sub>*p*</sub>} above the Fermi level approximately represent some of the
delocalized, unbound states in the continuous part of the spectrum. Therefore,
the virtual space constitutes the so-called quasi-continuum that may accommodate
DI processes. However, if the basis set is not large, the DI contributions of a
small quasi-continuum to the ET processes become negligible in comparison to the
CT contributions of the occupied space. Under those conditions, the ET
probabilities *P*<sub>*n*−ET</sub> in basically correspond to bound-to-bound
(occupied-space-to-occupied-space) CT processes. For that reason, with
relatively small basis sets, those *P*<sub>*n*−ET</sub> consistently rendered
correct bound-to-bound CT *σ*<sub>1−*ET*</sub> in various systems. However, if
the basis set is large, the DI contributions of an enlarged quasi-continuum may
become substantial. If so, the *P*<sub>*n*−ET</sub> in and resulting
*σ*<sub>*n*−*ET*</sub> no longer correspond to pure bound-to-bound CT processes
since they get contaminated with bound-to-quasi-continuum DI contributions.
Therefore, one can hypothesize that SLEND with the smaller 6-31G\* basis set can
predict genuine CT *σ*<sub>*n*−*ET*</sub> via but not with the larger 6-31G\*\*
one.
To verify the above hypothesis, we performed a series of SLEND simulations on
the simple model system: H<sup>+</sup> + H at 40 keV ≤ *E*<sub>*Lab*</sub> ≤ 100
keV with the 6–31<sup>++</sup>G\*\* basis set. The latter produces the best DI
results for H<sup>+</sup> + H as shown shortly. However, instead of using
*P*<sub>1−*ET*</sub> in for *σ*<sub>1−*ET*</sub>, the final-time electronic
wavefunction $\left| {\Psi_{e}^{SLEND}\left( t_{f} \right)} \right\rangle$, was
projected onto the ground \|0⟩ and excited states \|… *h* → *p* …⟩ of the target
and the projectile. In this way, the evaluation of ET probabilities could
distinguish the cases with the electron transferring into bound or unbound
states of H. In the 40 ≤ *E*<sub>*Lab*</sub> ≤ 80 keV range, where experimental
results are available, the DI ICSs, *σ*<sub>*DI*</sub>, deviate less than 10%
from experimental data, with the best agreement at *E*<sub>*Lab*</sub> = 60 keV:
$\sigma_{DI}^{SLEND}$ = 13.9Å and $\sigma_{DI}^{EXPT.}$ = 13.8 Å ⇒ deviation
Δ<sub>*Theo*.</sub> = 0.7%. Notably, these calculations produced accurate
results even though ETFs were neglected as in ; this gives extra support to the
ETFs’ neglect in the H<sup>+</sup> + (H<sub>2</sub>O)<sub>*n*</sub> simulations
and ruled it out as a source of the SLEND/6-31G\*\**σ*<sub>1−*ET*</sub>
overestimation. For H<sup>+</sup> + H at *E*<sub>*Lab*</sub> = 100 keV, SLEND
*σ*<sub>*DI*</sub> is 0.92 Å<sup>2</sup>. If *P*<sub>1−*ET*</sub> in is used to
calculate CT ICSs, *σ*<sub>*CT*</sub>, a *σ*<sub>*DI*</sub> contribution of 0.92
Å will be spuriously added to the *σ*<sub>*CT*</sub> making it overestimated. If
this DI contribution is assumed to be similar to that in H<sup>+</sup> +
H<sub>2</sub>O, the overestimated SLEND/6-31G\*\* $\sigma_{CT}^{SLEND}$ via can
be corrected by subtracting the $\sigma_{DI}^{SLEND}$ part from it:
$\sigma_{CT}^{SLEND}$ = 2.06 Å2–0.92 Å<sup>2</sup> = 1.14 Å<sup>2</sup>; this
places SLEND/6-31G\*\* $\sigma_{CT}^{SLEND}$ within the range of the four
experimental values and closest to that from Exp. D: $\sigma_{CT}^{Expt.D}$ =
1.13 Å ⇒ Δ<sub>*Theo*.</sub> = 0.8%. A similar correction might occur with the
SLEND/6-31G\* $\sigma_{CT}^{SLEND}$ but it will be far smaller due to a smaller
virtual space as suggested by previous calculations with comparable basis sets.
The calculation of the CT *σ*<sub>*CT*</sub> in H<sup>+</sup> + H<sub>2</sub>O
is more complicated than that of H<sup>+</sup> + H because the former has more
than one electron. For H<sup>+</sup> + H<sub>2</sub>O, numerous excited states
\|… *h* → *p* …⟩ from \|0⟩ forming a full CI expansion should be generated so
that $\left| {\Psi_{e}^{SLEND}\left( t_{f} \right)} \right\rangle$ in can be
projected on all those states. This more demanding capability is not currently
available in PACE but is under development.
# Conclusions
To model microscopic processes in PCT, the SLEND method was applied to the
H<sup>+</sup> + (H<sub>2</sub>O)<sub>*n*</sub> systems at *E*<sub>*Lab*</sub> =
100 keV with the 6-31G\* (*n* = 1–6) and 6-31G\*\* (*n* = 1–5) basis sets. Ten
(H<sub>2</sub>O)<sub>1–6</sub> clusters were selected for this study: eight
exhibit mono- and di-cyclic quasi-planar/multiplanar structures and two others,
the prism and cage (H<sub>2</sub>O)<sub>6</sub> isomers, exhibit multi-cyclic,
three-dimensional, drop-like structures. These “smallest-drop” clusters were
purposely included in this study in an attempt to reproduce early manifestations
of bulk-water properties in PCT. Short-time SLEND/6-31G\* (*n* = 1–6) and
/6-31G\*\* (*n* = 1–5) simulations render cluster-to-projectile total 1-ET ICS,
*σ*<sub>1−*ET*</sub>, and 1-ET probabilities. In absolute quantitative terms,
SLEND/6-31G\**σ*<sub>1−*ET*</sub> compares satisfactorily with alternative
experimental and theoretical results only available for *n* = 1, and
exhibits almost the same accuracy of the best alternative theoretical result
from BGM calculations. SLEND/6-31G\*\* overestimates *σ*<sub>1−*ET*</sub> and a
detail account about the cause and remediation of this effect was presented. In
relative quantitative terms, both SLEND/6-31\* and /6-31G\*\*
*σ*<sub>1−*ET*</sub> precisely fit into physically justified scaling formulae
*σ*<sub>1−*ET*</sub> (*n*) = *cn*<sup>2/3</sup> as a function of the cluster
size *n*. Long-time SLEND/6-31G\* (*n* = 1–4) simulations predict the formation
of the DNA-damaging radicals H, OH, O and H<sub>3</sub>O. While “smallest-drop”
isomers were included, no incipient manifestations of bulk-water PCT properties
are observed. Therefore, to capture bulk-water effects, simulations with larger
water clusters are currently underway.
The authors thank Prof. Takao Oi (Sophia University, Japan) for providing the
optimized geometry of the prism isomer of (H<sub>2</sub>O)<sub>6</sub>. Present
calculations were performed at the Texas Tech University High Performance
Computer Center and the Texas Advanced Computing Center at the University of
Texas at Austin.
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** JAM. **Data curation:** AJP EST JAM. **Formal
analysis:** AJP CS EST JAM. **Funding acquisition:** JAM. **Investigation:**
AJP CS EST JAM. **Methodology:** AJP JAM. **Project administration:** JAM.
**Resources:** JAM. **Software:** AJP EST JAM. **Supervision:** JAM.
**Validation:** AJP CS EST. **Visualization:** AJP EST. **Writing – original
draft:** AJP JAM. **Writing – review & editing:** JAM.
[^3]: Current address: Department of Chemistry and Biochemistry, Lipscomb
University, Nashville, Tennessee
[^4]: Current address: Department of Chemistry and Industrial Hygiene,
University of North Alabama, Florence, Alabama, United States of America |
# Introduction
Intuitively, we all grasp the concept of cognitive effort. We all know what it
is like to feel that a cognitive task is demanding and effortful. However,
defining cognitive effort is not straightforward. Cognitive effort refers to the
degree of engagement in demanding cognitive tasks, as opposed to tasks that can
be completed using routine or habitual behavior, which require little effort. An
influential neuroeconomics approach to effort postulates that deciding whether
to invest cognitive effort comes down to investigating the relevant costs and
benefits (see for example). More specifically, several theories have proposed
that effort may be primarily implicated in the decision to engage cognitive
control resources (\[–\]; for a review see).
The broad concept of cognitive control comprises cognitive operations such as
planning a new strategy, evaluating it, controlling its execution and correcting
possible errors. It kicks in when routine activation of behavior is no longer
sufficient for optimal performance. Cognitive control thus allows us to perform
intelligent, purposive behavior. When performing basically any task or when we
aim to achieve a goal, we need to stay focused and inhibit dominant, yet
irrelevant information to prevent being distracted from our task. Whenever an
irrelevant source of information interferes with our task performance, we will
exert additional cognitive control in order to preserve task performance and
resolve conflict. Conflict can be defined as the simultaneous activation of
incompatible representations. In an experimental context, conflict tasks such as
the Eriksen flanker task are often used to measure cognitive control. This task
consists of a central target arrow flanked by distractor arrows. Congruent
(i.e., flankers and target point in the same direction) and incongruent (i.e.,
flankers and target point in opposite directions) stimuli are presented and
participants must indicate the direction of the central target arrow as fast and
as accurately as possible. Responses to incongruent trials are slower and are
more error prone than to congruent trials. This difference in performance
between incongruent and congruent trials constitutes the flanker effect. Here,
dominant yet task irrelevant stimuli (i.e., flanker arrows) need to be
suppressed in order to perform this task well.
When people encounter interference, such as conflict as defined above, cognitive
control allows them to adjust their behavior to overcome it. However, exerting
cognitive control comes at a cost and this is where cognitive effort comes in.
The neuroeconomic theories treat cognitive effort as the opportunity cost of
exerting cognitive control (i.e., the decision to expend cognitive control is
accompanied by the missed benefit of avoiding effort; see for a review).
Cognitive control usually drives cognitive effort and exerting more cognitive
control would lead to a higher effort cost.
While we are performing a demanding cognitive task, not only do we have a sense
of cognitive effort, we are also subjectively aware that we are initiating,
executing and controlling our thoughts and actions. The feeling of being in
charge when we perform voluntary actions is called the sense of agency. The
brain appears to actively construct the sense of agency using motor actions,
sensory feedback, previous experiences, cause-and-effect inferences, and so on.
The core of sense of agency is the association between a voluntary action (e.g.,
pressing a light switch) and an outcome (e.g., the light in the room goes on).
Experimentally, sense of agency can be explicitly measured by simply asking
participants to judge whether their action caused an outcome. Alternatively,
implicit measures can be used, such as the compression of perceived time between
action and outcome. In the intentional binding paradigm, participants judge the
perceived time of a voluntary action or a subsequent outcome. It has been shown
that voluntary actions (as compared to involuntary movements) are perceived as
shifted in time towards their subsequent outcomes and that the outcomes are
perceived as shifted towards the voluntary actions that caused them. Based on
this implicit measure, sense of agency is defined as the compression (or
underestimation) of the perceived time interval between action and outcome.
Crucially, effort and sense of agency have been linked to each other, dating
back even to the 19<sup>th</sup> century (for an overview see). It has been
argued that without the subjective experience of effort there could not be any
feeling of agency or causality, but only mere facts of behavior. According to
this view, it is the conscious experience of effort that makes self-knowledge
possible. If effort is a crucial determinant of sense of agency, then
experimental manipulations of cognitive effort should also influence the
accompanying sense of agency. A few studies have shown that sense of agency can
indeed be influenced by increased cognitive effort.
Howard et al. instructed participants to memorize two (low effort) or eight
(high effort) letters. After encoding, a maintenance period started by a self-
initiated action which ended after a variable duration with an outcome (i.e., a
tone). Participants’ estimation of the duration of this maintenance period was
used to measure intentional binding. The results showed that a high cognitive
effort context decreased intentional binding compared to a low cognitive effort
context. Similar results were obtained by Hon et al. who used an explicit
instead of an implicit measure of sense of agency. Contrarily, Demanet et al.
observed increased intentional binding in a high physical effort context. In the
study of Sidarus and Haggard, participants responded to flanker trials (i.e.,
the action) which triggered the appearance of a colored circle after a variable
delay (i.e., the outcome). They judged how much control they felt over the
colored circles that were triggered by their actions. Results indicated that
incongruent flanker trials led to lower sense of agency compared to congruent
and neutral flanker trials. Vastano et al. reached the same conclusion using an
implicit intentional binding measure of sense of agency. However, although Wang
et al. also found that sense of agency ratings were lower for incongruent
trials, this appeared to be the case only when the previous trial was a
congruent trial and thus did not contain conflict. Similarly, Di Costa et al.
observed that sense of agency measured with intentional binding is increased
*after* making an error.
Thus, previous studies have shown that cognitive effort can be both detrimental
and facilitative for the experienced sense of agency. We hypothesize that the
reason for the nuances in these findings might lie in the use of differential
time windows in which cognitive effort operates. Indeed, the findings seem to
depend on whether the effort that was taken into account was exerted on the
current trial, the previous trial or across a block of trials. When the
*current* trial contained a high level of effort (e.g., incongruent or error
trial), sense of agency on that trial decreased. However, sense of agency seemed
to be increased on trials *following* high effort. In *contexts* with high
effort, the results are mixed: some studies found increased sense of agency
while others observed decreased sense of agency. Sidarus and Haggard argued that
when an increase of effort can be anticipated, the required cognitive control
and expected self-engagement in the task can become part of action planning and
prediction. In contrast, effort investment that is sudden and unexpected, for
example triggered by unexpected or unpredictable conflict, cannot be predicted
and included in action planning. Based on this, we speculate that when high
effort is required unpredictably or unexpectedly (e.g., on the current trial),
sense of agency will decrease; contrarily, when high effort is anticipated
(e.g., after a conflict or in a high conflict context), it should be accompanied
by increased sense of agency. We formulated three specific hypotheses. First, in
a block of trials where effort is required unpredictably, encountering a high-
effort trial will lead to a decreased sense of agency *on that same trial*
compared to a low-effort trial. Second, in a block of trials where effort is
required unpredictably, encountering a high-effort trial will lead to an
increased sense of agency *on the next trial* compared to a low-effort trial.
Third, in a block of trials where effort can be anticipated (i.e., frequent
high-effort trials), sense of agency will be increased compared to a block of
trials where effort cannot be anticipated (i.e., scarce high-effort trials).
In order to assess these hypotheses we will use a flanker task to manipulate
conflict and hence the required cognitive control and, in turn, the required
effort (i.e., on congruent trials, no conflict is present and thus the required
cognitive control and the effort cost is minimal; contrarily, on incongruent
trials, conflict is present and thus the required cognitive control and effort
cost is larger). In Experiment 1 sense of agency will be measured implicitly
using the intentional binding paradigm.
# Experiment 1
## Method
### Participants
Sixty participants were recruited through the Experiment Management System of
the Katholieke Universiteit Leuven. They received course credit for
participation. All participants provided written informed consent. All of them
had normal or corrected-to-normal eyesight, were not colorblind and were able to
operate a keyboard and mouse. We used the following exclusion criteria for
participants: response times and/or intentional binding exceeding 2.5 *SD*,
and/or error rates above 20%. However, none of the participants met these
exclusion criteria. Thus, all 60 participants were included for the analyses (6
males, mean age = 18.52, *SD* = 1.13, range 18–26). This study was approved by
the Social and Societal Ethics Committee (SMEC) from KU Leuven (G-2019 01 1493).
The study was also preregistered on the Open Science Framework (OSF, osf.io;
doi:[10.17605/OSF.IO/EM3GQ](https://doi.org/10.17605/OSF.IO/EM3GQ)) and the raw
data can be retrieved from <https://osf.io/em3gq/>
(doi:[10.17605/OSF.IO/EM3GQ](https://doi.org/10.17605/OSF.IO/EM3GQ)).
### Apparatus
The experiment was administered in a computer room in small groups. Stimuli were
presented on a 22” monitor with a refresh rate of 60 Hz located approximately 60
cm from the participant. Stimulus presentation and response registration were
controlled by PsychoPy v.3.1.0 (Psychology software for Python;).
### Design and procedure
The experiment was composed of active and passive trials. During *active trials*
an Eriksen flanker task was used in which a string of numbers was presented in
white against a grey background in the center of the screen on each trial (font
= Consolas, height = 1.2 deg). A central target number was flanked by two
distractor numbers on both sides. Participants were instructed to respond as
fast and as accurately as possible to the central target arrow and to ignore the
flankers. The flanker stimuli could either trigger the same response as the
target (i.e., congruent trials; e.g., “22222”), or trigger a different response
as the target (i.e., incongruent trials; e.g., “33233”). The stimuli used were
1, 2, 3 and 4. Participants had to respond by pressing the corresponding key (1,
2, 3 or 4) on an Azerty keyboard. Specifically, the keys of interest were
indicated by stickers: sticker of the number “1” was placed on the “d” key, a
“2” on the “f” key, a “3” on the “j” key and a “4” on the “k” key. To speed up
response time, participants were instructed to keep their left middle finger on
the “1”, their left index finger on the “2”, their right index finger on the “3”
and right middle index finger on the “4”. Active trials were announced by a
white fixation cross (1000ms) that was followed by the flanker stimulus until a
response was provided. After a variable delay of between 500 and 1250ms (in
steps of 250ms;), a colored circle was presented on the screen for 200ms (size =
(2, 2) deg). The color of the circle depended on the response made on the
current trial. Specifically, different colors were linked to passive trials (see
below) and to button presses on “1”, “2”, “3” and “4”, leading to five colors
(i.e., RGB color space values of pink \[1.000, -1.000, 1.000\], yellow \[1.000,
1.000, -1.000\], cyan \[-1.000, 1.000, 1.000\], orange \[1.000, 0.000, -1.000\]
and blue \[-1.000, -1.000, 1.000\]). Thus, on active trials an action (i.e., a
button press) triggered the appearance of a colored circle, whereas on passive
trials no action triggered the appearance of a colored circle. Note that we
avoided the use of green and red to prevent any associations with
correct/incorrect connotations. Which color was related to which response was
randomized across participants. After the disappearance of the circle,
participants performed an Interval Reproduction Task in which they reported the
estimated length of the delay between the disappearance of the flanker stimulus
and the appearance of the circle. They responded by continuously pressing the
space bar for the estimated duration. For a schematic overview of an active
trial, see.
During *passive trials* a string of neutral stimuli (i.e., “00000”) was
presented in black against a grey background in the center of the screen.
Participants were instructed that these trials did not require a response. As
participants did not respond to these stimuli, they were therefore non-agentic.
If participants did respond to a passive trial, this was registered as an error.
Passive trials were announced by a black fixation cross (1000ms) followed by a
neutral stimulus (i.e., “00000”) that was presented for a duration jittered
between 500 and 1500ms. After a variable delay of between 500 and 1250ms a
colored circle was again presented. Next, participants again had to perform the
Interval Reproduction Task in which they estimated and reproduced the length of
the delay between the neutral stimulus disappearance and the circle appearance.
For a schematic overview of a passive trial, see. Note that on passive trials
both the fixation cross and the neutral stimulus were presented in a different
color (i.e., black) compared to the active trials (i.e., white) to make the non-
agentic nature of these trials very clear to the participants.
The experiment started with several practice phases. First, participants
received the instructions about the active trials and practiced only the flanker
task for 10 trials (five congruent and five incongruent trials). Next,
instructions were provided about the presence of the passive trials and
participants practiced this on 10 trials where both active and passive trials
were presented (six passive, two congruent and two incongruent trials). During
these two practice phases, participants received feedback about their responses
(i.e., “correct” or “wrong” and their response time in seconds). After this,
participants were informed that a circle would appear after the disappearance of
the flanker or neutral stimulus and that the color of this circle would depend
on their response. They received instructions about the Interval Reproduction
Task. Finally, they were also told that after each block they would receive a
few additional questions. The first question assessed the level of experienced
effort after each block (i.e., “how much effort did you have to exert during the
previous block?”). Three additional questions assessed the subjective feeling of
agency over congruent, incongruent and passive trials, respectively, during the
previous block. For each type of trial (e.g., congruent trials), the trials were
listed on the screen (e.g., 11111, 22222, 33333 and 44444) and participants were
asked: “To what extent did you have the feeling that you caused the colored
circle to appear on the following trials?”. For all of these post-block
questions, participants indicated their response using a continuous rating scale
ranging from “very little” to “a lot” by a mouse button click on the selected
location on the rating scale. In a final practice block, they practiced the full
procedure for 10 trials (4 passive, 3 congruent and 3 incongruent trials).
After these practice phases, the experimental phase began where feedback was no
longer provided. Active and passive trials were presented intermixed and the
inter-trial interval was 1000ms. Experimental trials were divided into three
blocks, each containing 216 trials. The blocks varied depending on the ratio of
incongruent, congruent, and passive trials: one block with ratio 4:1:1 (i.e., MI
block), one with ratio 1:4:1 (i.e., MC block), and one 5:5:2 (i.e., EQ block)
ratio of incongruent to congruent to passive trials. The block that had mostly
incongruent trials (MI block) served as the high effort context and the bock
that had mostly congruent trials (MC block) served as the low effort context.
The block with an equal ratio of congruent and incongruent trials (EQ block)
served as the block where effort was required unpredictably, to assess the
impact of current and previous trial effort on sense of agency. The order of
trials in each block was randomized. The order of the blocks was counterbalanced
across participants. Finally, after each block, participants received the
additional questions to assess exerted effort and experienced agency over each
trial type, as described above. In between blocks, participants could take a
self-paced break. The whole experiment did not take more than one hour. The
experiment was followed by a short debriefing explaining the goal of the study.
### Statistical analysis
Active trials exceeding 2.5 *SD* of the overall mean RT were excluded from all
analyses (1.7% of trials). For RT and intentional binding analyses, erroneous
flanker trials were removed (5.9% of trials). For trial-by-trial analyses
assessing hypotheses 1 and 2, the first trial (0.46% of trials) and trials
following an erroneous flanker trial (5.8% of trials) were also excluded in the
EQ block. No trials met our exclusion criterion with regards to intentional
binding (i.e., trials exceeding 2.5 *SD* of the overall mean intentional
binding).
First, we calculated the measure of intentional binding for active and passive
trials based on the Interval Reproduction Task. Specifically, for active trials,
the participant’s estimated delay (in milliseconds) was subtracted from the
actual delay between their response on the flanker stimulus (i.e., response) and
the appearance of the colored circle (i.e., outcome) for each trial (i.e.,
actual delay—estimated delay). For the passive trials, we subtracted the
participant’s estimated delay from the actual delay between the disappearance of
the neutral stimulus and the appearance of the colored circle for each trial.
Note that a value of zero indicates a perfect estimate, and hence no intentional
binding. Positive values indicate an underestimation of the time period, and
thus intentional binding. As a control check, we assessed whether our
manipulation of intentional binding was successful by looking at the difference
in average attentional binding between active (i.e., congruent and incongruent)
and passive trials using a paired-samples *t*-test. If our task was indeed
successful in eliciting intentional binding, we should observe that intentional
binding in active trials was larger than in passive trials (where no action is
present). Note that comparing active (i.e., action) and passive (i.e., no-
action) trials is an established paradigm in this field (e.g.,).
Second, to test hypotheses 1 and 2, we focused on the EQ block where effort is
required unpredictably (i.e., equal proportion of incongruent and congruent
trials). For hypothesis 1, we examined whether encountering a high-effort trial
leads to a decreased sense of agency *on that same trial* compared to a low-
effort trial. For hypothesis 2, we examined whether encountering a high-effort
trial leads to an increased sense of agency *on the next trial* compared to a
low-effort trial. We conducted a 2x2 repeated measures ANOVA analysis with
Current and Previous trial congruency as within-subjects factors (both with 2
levels: congruent and incongruent) on intentional binding (in ms), RTs (in ms)
and error rates (in %) as (separate) dependent variables.
Third, to test hypothesis 3, we examined whether sense of agency was increased
in a block of trials where effort can be anticipated (i.e., MI block), compared
to a block of trials where effort cannot be anticipated (i.e., MC block). For
this purpose, a block analyses was conducted. We performed a 2x2 repeated
measures analysis with Block (2 levels: MC and MI) and Current trial congruency
(2 levels: congruent and incongruent) as within-subjects factors on intentional
binding, RTs and error rates as (separate) dependent variables.
Note that in the analyses described above, we did not pool all trials across
blocks to assess the general effect of current trial congruency on intentional
binding. As the different contexts that were created by manipulating the
proportion of congruent trials (i.e., EQ, MC and MI) trigger different cognitive
control mechanisms, and hence differential congruency effects (see for example),
we a priori decided not to pool across blocks. However, an additional
exploratory repeated measures analysis across all blocks with current trial
congruency as within-subjects factor revealed that the main effect of congruency
on intentional binding was not significant (*F*(1,59) = 0.58, *p* =.46,
*η*<sup>2</sup><sub>p</sub> = 0.010).
Finally, we looked at the subjective ratings of experienced sense of agency and
cognitive effort reported after each block for exploratory purposes. We
conducted a repeated measures analysis with block (3 levels: EQ, MC or MI) as
within-subjects factor on the reported experienced effort after each block and a
repeated measures analysis with trial type (3 levels: congruent, incongruent or
passive) as within-subjects factor on the reported explicit sense of agency
after each block.
## Results
### Intentional binding check
A paired-samples *t*-test confirmed that intentional binding was larger on
active compared to passive trials (407 versus 293ms, *t*(59) = -7.41, *p*
\<.001). This ensures that our implicit measurement of sense of agency was
successful. presents the participants’ average estimated delays as a function of
block and actual delay (i.e., 500, 750, 1000 or 1250ms) for the correct active
trials (excluding trials exceeding 2.5 *SD* of the overall mean RT) and the
passive trials.
### Trial-by-trial analyses
These analyses were conducted on the EQ block. We conducted a 2x2 repeated
measures analysis with Current and Previous trial congruency as within-subjects
factors (both with 2 levels: congruent and incongruent) on intentional binding
(in ms), RTs (in ms) and error rates (in %) as (separate) dependent variables.
Means and *SD*s for each of these dependent variables in relation to current and
previous congruency are reported in. The results are also depicted on.
With regard to *intentional binding*, we found no significant main effects of
current or previous congruency (resp. *F*(1,59) = 0.15, *p* =.70,
*η*<sup>2</sup><sub>p</sub> = 0.003 and *F*(1,59) = 0.011, *p* =.92,
*η*<sup>2</sup><sub>p</sub> \< 0.001), nor a significant interaction (*F*(1,59)
= 0.16, *p* =.69, *η*<sup>2</sup><sub>p</sub> = 0.003). This indicates that we
observed no difference in intentional binding depending on current or previous
congruency.
With regard to *RT*, we found a significant main effect of current congruency
(*F*(1,59) = 90.65, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.61) indicating
that participants were slower on incongruent compared to congruent trials (on
average 719.5ms versus 664.5ms). Additionally, we observed a significant
interaction between current and previous congruency (*F*(1,59) = 8.85, *p*
=.004, *η*<sup>2</sup><sub>p</sub> = 0.13). This interaction reflects a typical
Gratton effect: a decreased congruency effect after an incongruent compared to a
congruent trial (40ms versus 70ms). The observation of this Gratton effect
ensures that our manipulation of current and previous congruency was successful
in order to trigger trial-by-trial adaptations. The main effect of previous
congruency did not reach significance (*F*(1,59) = 0.95, *p* =.33,
*η*<sup>2</sup><sub>p</sub> = 0.016).
With regard to *error rates*, we only observed a significant main effect of
current congruency (*F*(1,59) = 21.56, *p* \<.001, *η*<sup>2</sup><sub>p</sub> =
0.27) indicating that participants made more errors on incongruent compared to
congruent trials (on average 7.38% versus 4.39%). The main effect of previous
congruency and the interaction between current and previous congruency did not
reach significance (resp. *F*(1,59) = 1.32, *p* =.26,
*η*<sup>2</sup><sub>p</sub> = 0.022 and *F*(1,59) = 0.77, *p* =.38,
*η*<sup>2</sup><sub>p</sub> = 0.26).
As an exploratory analysis that was not included in our preregistration, we
additionally conducted a 4x2x2 repeated measures analysis with Actual delay (4
levels: 500, 750, 1000 or 1250ms), Current and Previous trial congruency (both
with 2 levels: congruent and incongruent) as within-subjects factors on
intentional binding (in ms). We only observed a main effect of Actual delay
(*F*(3,56) = 285.67, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.94), with more
intentional binding for longer delays (specifically, 132.5, 313, 501 and 670ms
for the increasing delays). None of the other main effects or interactions
reached significance.
### Block analyses
These analyses were conducted on the MC and MI blocks. We conducted a 2x2
repeated measures analysis with Block (2 levels: MC and MI) and Current trial
congruency (2 levels: congruent and incongruent) as within-subjects factors on
intentional binding (in ms), RTs (in ms) and error rates (in %) as (separate)
dependent variables. Means and *SD*s for each of these dependent variables in
relation to current and previous congruency are reported in. The results are
also depicted on.
With regard to *intentional binding*, we found no significant main effect of
congruency (*F*(1,59) = 1.56, *p* =.22, *η*<sup>2</sup><sub>p</sub> = 0.026),
nor a significant interaction between block and congruency (*F*(1,59) = 0.048,
*p* =.83, *η*<sup>2</sup><sub>p</sub> = 0.001). The main effect of block also
did not reach significance, but showed a slight trend (*F*(1,59) = 3.00, *p*
=.089, *η*<sup>2</sup><sub>p</sub> = 0.048). Indeed, we observed slightly more
binding in the MI compared to the MC block, as hypothesized (419.5 versus
398.5ms).
With regard to *RT*, we found a significant main effect of congruency (*F*(1,59)
= 286.29, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.83) indicating that
participants were slower on incongruent compared to congruent trials (on average
738ms versus 671.5ms). Additionally, we observed a significant interaction
between block and congruency (*F*(1,59) = 45.80, *p* \<.001,
*η*<sup>2</sup><sub>p</sub> = 0.44). This interaction reflects a typical
proportion congruency effect: a decreased congruency effect in an MI block
compared to an MC block (44ms versus 89ms). The observation of this proportion
congruency effect ensures that our manipulation of block was successful in order
to trigger blockwise adaptations. The main effect of block did not reach
significance (*F*(1,59) = 0.056, *p* =.81, *η*<sup>2</sup><sub>p</sub> = 0.001).
With regard to *error rates*, we observed a significant main effect of
congruency (*F*(1,59) = 31.93, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.35)
indicating that participants made more errors on incongruent compared to
congruent trials (on average 7.80% versus 4.77%). A main effect of block was
also observed (*F*(1,59) = 10.91, *p* =.002, *η*<sup>2</sup><sub>p</sub> =
0.16), indicating that participants made more errors on the MC block compared to
the MI block (7.17% versus 5.40%). Finally, the interaction between block and
congruency also reached significance (resp. *F*(1,59) = 1.32, *p* =.26,
*η*<sup>2</sup><sub>p</sub> = 0.022), again indicating a proportion congruency
effect: a decreased congruency effect in an MI block compared to an MC block
(1.38% versus 4.68%).
As an exploratory analysis that was not included in our preregistration, we
additionally conducted a 4x2x2 repeated measures analysis with Actual delay (4
levels: 500, 750, 1000 or 1250ms), Block (2 levels: MC and MI) and Congruency (2
levels: congruent and incongruent) as within-subjects factors on intentional
binding (in ms). We only observed a main effect of Actual delay (*F*(3,56) =
372.89, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.95), with more intentional
binding for longer delays (specifically, 128, 328, 518.5 and 682ms for the
increasing delays). None of the other main effects or interactions reached
significance.
### Post-block subjective effort and agency analyses
With regard to *subjective effort* experienced during each block (EQ, MC or MI),
we conducted a repeated measures analysis with block (3 levels: EQ, MC or MI) as
within-subjects factor on the reported experienced effort after each block. We
observed no significant differences between the three blocks (*F*(2,58) = 0.14,
*p* =.87, *η*<sup>2</sup><sub>p</sub> = 0.005). Indeed, the reported experienced
effort was very similar in the EQ, MC and MI blocks (resp. 6.44, 6.53 and 6.36).
With regard to the *subjective sense of agency* experienced for congruent,
incongruent and passive trials, we conducted a repeated measures analysis with
trial type (3 levels: congruent, incongruent or passive) as within-subjects
factor on the reported explicit sense of agency after each block. We observed
significant differences between the experienced agency over congruent,
incongruent and passive trials (*F*(2,58) = 16.83, *p* \<.001,
*η*<sup>2</sup><sub>p</sub> = 0.37). Specifically, participants reported a
higher sense of agency over incongruent trials (4.81) compared to congruent
(4.2, *F*(1,59) = 24.61, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.29) or
passive trials (4.1, *F*(1,59) = 7.98, *p* =.006, *η*<sup>2</sup><sub>p</sub> =
0.12). The explicit sense of agency did not differ between congruent and passive
trials (4.1, *F*(1,59) = 0.12, *p* =.73, *η*<sup>2</sup><sub>p</sub> = 0.02).
Note that this effect did not interact with block: a repeated measures analysis
with trial type (3 levels: congruent, incongruent or passive) and block (3
levels: EQ, MC or MI) as within-subjects factors again only revealed a main
effect of trial type (*F*(2,58) = 16.83, *p* \<.001, *η*<sup>2</sup><sub>p</sub>
= 0.37).
## Discussion
Experiment 1 confirmed that our manipulation of cognitive control was
successful: incongruent trials led to a slower and more erroneous response than
congruent trials (i.e., flanker effect). After an incongruent trial,
participants also increased their exertion of cognitive control compared to
after a congruent trial (i.e., Gratton effect). Finally, when conflict could be
anticipated (i.e., MI block), the flanker effect was reduced (i.e., proportion
congruency effect). Our implicit intentional binding measure also seemed to be
successful: participants reported shorter time intervals on active trials
compared to passive trials. However, we were unable to confirm the results of
previous studies: we observed no decrease in binding on incongruent trials, nor
an increase in binding after incongruent trials. We did observe a slight trend
towards increased binding in a context where conflict could be anticipated
(i.e., MI block).
Interestingly, our explicit measure of sense of agency, administered after each
block, did indicate that participants experienced more agency over incongruent
trials than over congruent trials. According to the two-step account of agency,
the implicit and explicit approaches to measure sense of agency, capture
separate aspects of the sense of agency, namely the implicit feeling of agency
and the explicit judgement of agency. The implicit feeling of agency stems from
a low-level comparator involving motor action planning and prediction, and
sensory feedback processes. The explicit judgement of agency relies on higher-
order causality judgements based on contextual factors and beliefs. Furthermore,
although correlations between intentional binding and subjective sense of agency
have been reported, whether binding truly reflects sense of agency is debated.
Based on this, we decided to conduct the experiment again, but now using an
explicit measure of sense of agency on each trial (see also).
# Experiment 2
## Method
### Participants
Sixty participants were recruited from the Experiment Management System of the
Katholieke Universiteit Leuven. They received course credit or a monetary reward
for participation. All participants provided written informed consent. None of
the them had participated in Experiment 1. All of them had normal or corrected-
to-normal eyesight, were not colorblind and were able to operate a keyboard and
mouse. We used the following exclusion criteria for participants: response times
and/or error rates above 20%. Two participants did not meet these criteria based
on their response time. Thus, 58 participants were included for the analyses (9
males, mean age = 20.67, *SD* = 4.57, range 17–40). This study was approved by
the Social and Societal Ethics Committee (SMEC) from KU Leuven (G-2019 05 1652).
The study was also preregistered on OSF (osf.io;
doi:[10.17605/OSF.IO/EM3GQ](https://doi.org/10.17605/OSF.IO/EM3GQ)) and the raw
data can be retrieved from <https://osf.io/em3gq/> (doi:
[10.17605/OSF.IO/EM3GQ](https://doi.org/10.17605/OSF.IO/EM3GQ)).
### Apparatus, design, procedure and statistical analyses
The apparatus, design and procedure were identical to Experiment 1, except for
the following changes. Instead of using an implicit measure of sense of agency,
as we did in Experiment 1 (i.e., intentional binding based on the Interval
Reproduction Task), we now used an explicit measure of sense of agency.
Specifically, after the disappearance of the colored circle, participants now
had to indicate their explicit sense of agency (i.e., “I felt like I caused the
circle to appear”) using a 9-point scale ranging from “strongly disagree” to
“strongly agree”. This was the case for both active and passive trials. For a
schematic overview of both trial types, see. Statistical analyses were also the
same as in Experiment 1, except that this explicit sense of agency was now used
as dependent variable instead of intentional binding.
Note that, as explained in Experiment 1, we did not pool all trials across
blocks to assess the general effect of current trial congruency on sense of
agency ratings. However, an additional exploratory repeated measures analysis
across all blocks with current trial congruency as within-subjects factor
revealed that the main effect of congruency on sense of agency ratings was not
significant (*F*(1,57) = 1.81, *p* =.18, *η*<sup>2</sup><sub>p</sub> = 0.031).
Active trials exceeding 2.5 *SD* of the overall mean RT were excluded from all
analyses (i.e., 1.9%). For RT and intentional binding analyses, erroneous active
flanker trials were removed (i.e., 4.5%). For trial-by-trial analyses assessing
hypotheses 1 and 2, the first trial of the block (0.46%) and trials following an
erroneous flanker trial (i.e., 3.8%) were also excluded in the EQ block. For one
participant, the sense of agency and effort ratings after each block were not
recorded. Therefore, this participant was excluded from the post-block analyses.
## Results
### Sense of agency rating check
A paired-samples *t*-test confirmed that the reported sense of agency was larger
on active compared to passive trials (5.51 versus 2.56ms, *t*(57) = -9.79, *p*
\<.001). This ensures that our explicit measurement of sense of agency was
successful. presents the participants’ sense of agency ratings as a function of
block and delay (i.e., 500, 750, 1000 or 1250ms) for the correct active trials
(excluding trials exceeding 2.5 *SD* of the overall mean RT) and the passive
trials.
### Trial-by-trial analyses
These analyses were conducted on the EQ block. We conducted a 2x2 repeated
measures analysis with Current and Previous trial congruency as within-subjects
factors (both with 2 levels: congruent and incongruent) on sense of agency
(score on a scale from 1 to 9), RTs (in ms) and error rates (in %) as (separate)
dependent variables. Means and *SD*s for each of these dependent variables in
relation to current and previous congruency are reported in. The results are
also depicted on.
With regard to *sense of agency*, we found no significant main effect of
previous congruency (*F*(1,57) = 0.045, *p* =.83, *η*<sup>2</sup><sub>p</sub> =
0.001), nor a significant interaction (*F*(1,57) = 0.16, *p* =.69,
*η*<sup>2</sup><sub>p</sub> = 0.003). The main effect of current congruency was
not significant, but showed a trend (*F*(1,57) = 3.29, *p* =.075,
*η*<sup>2</sup><sub>p</sub> = 0.055): incongruent trials received a slightly
higher agency rating than congruent trials (5.51 versus 5.39).
With regard to *RT*, we found a significant main effect of current congruency
(*F*(1,57) = 41.57, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.42) indicating
that participants were slower on incongruent compared to congruent trials (on
average 800ms versus 728.5ms). The main effect of previous congruency did not
reach significance (*F*(1,57) = 0.75, *p* =.39, *η*<sup>2</sup><sub>p</sub> =
0.013). We also did not observe a significant interaction between current and
previous congruency (*F*(1,57) = 1.82, *p* =.18, *η*<sup>2</sup><sub>p</sub> =
0.031). This implies that we did not observe a typical Gratton effect: the
congruency effect was not significantly decreased after an incongruent compared
to a congruent trial (60ms versus 83ms).
With regard to *error rates*, we observed no significant effects (all *p*
\>.55).
As an exploratory analysis that was not included in our preregistration, we
additionally conducted a 4x2x2 repeated measures analysis with Delay (4 levels:
500, 750, 1000 or 1250ms), Current and Previous trial congruency (both with 2
levels: congruent and incongruent) as within-subjects factors on sense of agency
ratings (score between 1–9). We observed a main effect of Actual delay
(*F*(3,55) = 3.41, *p* =.024, *η*<sup>2</sup><sub>p</sub> = 0.16), with slightly
decreasing sense of agency for longer delays (specifically, 5.63, 5.51, 5.38 and
5.28 for the increasing delays). We also observed a main effect of Current trial
congruency (*F*(1,57) = 4.28, *p* =.043, *η*<sup>2</sup><sub>p</sub> = 0.070)
with a slightly higher sense of agency for incongruent trials compared to
congruent trials (5.51 and 5.38, respectively). None of the other main effects
or interactions reached significance.
### Block analyses
These analyses were conducted on the MC and MI blocks. We conducted a 2x2
repeated measures analysis with Block (2 levels: MC and MI) and Current trial
congruency (2 levels: congruent and incongruent) as within-subjects factors
sense of agency (score on a scale from 1 to 9), RTs (in ms) and error rates (in
%) as (separate) dependent variables. Means and *SD*s for each of these
dependent variables in relation to current and previous congruency are reported
in. The results are also depicted on.
With regard to *sense of agency*, we found a significant main effect of
congruency (*F*(1,57) = 6.94, *p* =.011, *η*<sup>2</sup><sub>p</sub> = 0.11),
indicating that participants reported slightly more agency on incongruent
compared to congruent trials (5.64 versus 5.46). The main effect of block did
not reach significance (*F*(1,57) = 0.42, *p* =.52, *η*<sup>2</sup><sub>p</sub>
= 0.007), nor did the interaction between block and congruency (*F*(1,57) =
0.024, *p* =.88, *η*<sup>2</sup><sub>p</sub> \< 0.001).
With regard to *RT*, we found a significant main effect of congruency (*F*(1,57)
= 113.85, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.67) indicating that
participants were slower on incongruent compared to congruent trials (on average
811ms versus 734ms). Additionally, we observed a significant interaction between
block and congruency (*F*(1,57) = 5.52, *p* =.022, *η*<sup>2</sup><sub>p</sub> =
0.088). This interaction reflects a typical proportion congruency effect: a
decreased congruency effect in an MI block compared to an MC block (60ms versus
94ms). The observation of this proportion congruency effect ensures that our
manipulation of block was successful in order to trigger blockwise adaptations.
The main effect of block did not reach significance (*F*(1,57) = 0.56, *p* =.46,
*η*<sup>2</sup><sub>p</sub> = 0.010).
With regard to *error rates*, we observed a significant main effect of
congruency (*F*(1,57) = 6.65, *p* =.013, *η*<sup>2</sup><sub>p</sub> = 0.10)
indicating that participants made more errors on incongruent compared to
congruent trials (on average 4.86% versus 3.78%). The main effect of block
(*F*(1,57) = 2.19, *p* =.14, *η*<sup>2</sup><sub>p</sub> = 0.037) and the
interaction between block and congruency (*F*(1,57) = 0.50, *p* =.48,
*η*<sup>2</sup><sub>p</sub> = 0.009) did not reach significance.
As an exploratory analysis that was not included In our preregistration, we
additionally conducted a 4x2x2 repeated measures analysis with Actual delay (4
levels: 500, 750, 1000 or 1250ms), Block (2 levels: MC and MI) and Congruency (2
levels: congruent and incongruent) as within-subjects factors on sense of agency
ratings (score between 1–9). We observed a main effect of Actual delay
(*F*(3,55) = 6.32, *p* =.001, *η*<sup>2</sup><sub>p</sub> = 0.269), with
slightly decreasing agency ratings for longer delays (specifically, 5.77, 5.61,
5.48 and 5.35 for the increasing delays). We also observed a main effect of
Congruency (*F*(1,57) = 6.61, *p* =.013, *η*<sup>2</sup><sub>p</sub> = 0.10),
with a slightly higher sense of agency for incongruent trials compared to
congruent trials (5.65 and 5.46, respectively). None of the other main effects
or interactions reached significance.
### Post-block subjective effort and agency analyses
With regard to *subjective effort* experienced during each block (EQ, MC or MI),
we conducted a repeated measures analysis with block (3 levels: EQ, MC or MI) as
within-subjects factor on the reported experienced effort after each block. We
observed no significant differences between the three blocks (*F*(2,55) = 0.82,
*p* =.44, *η*<sup>2</sup><sub>p</sub> = 0.029). Indeed, the reported experienced
effort was very similar in the EQ, MC and MI blocks (resp. 4.86, 4.87 and 5.20).
With regard to the *subjective sense of agency* experienced for congruent,
incongruent and passive trials, we conducted a repeated measures analysis with
trial type (3 levels: congruent, incongruent or passive) as within-subjects
factor on the reported explicit sense of agency after each block. We observed
significant differences between the experienced agency over congruent,
incongruent and passive trials (*F*(2,55) = 48.90, *p* \<.001,
*η*<sup>2</sup><sub>p</sub> = 0.64). Specifically, participants reported a
higher sense of agency over incongruent trials (5.71) compared to congruent
(5.03, *F*(1,56) = 18.78, *p* \<.001, *η*<sup>2</sup><sub>p</sub> = 0.25) or
passive trials (2.63, *F*(1,56) = 96.65, *p* \<.001, *η*<sup>2</sup><sub>p</sub>
= 0.63). The explicit sense of agency also differed between congruent and
passive trials (*F*(1,56) = 60.03, *p* \<.001, *η*<sup>2</sup><sub>p</sub> =
0.52). Note that this effect did not interact with block: a repeated measures
analysis with trial type (3 levels: congruent, incongruent or passive) and block
(3 levels: EQ, MC or MI) as within-subjects factors again only revealed a main
effect of trial type (*F*(2,55) = 45.32, *p* \<.001, *η*<sup>2</sup><sub>p</sub>
= 0.62).
## Discussion
Next to confirming again that our manipulation of cognitive control and our
measurement of sense of agency were successful, we only obtained a small effect
of congruency on the sense of agency, both in the trial-by-trials and the block
analyses: incongruent trials received a slightly higher agency rating than
congruent trials. This is in line with the post-block reported explicit sense of
agency in Experiment 1, but contrary to our hypothesis. In the general
discussion we will elaborate this further.
# General discussion
The aim of this study was to examine the effect of cognitive effort on sense of
agency. We were interested in shedding light on the contradictory literature
about the facilitative or/and detrimental nature of cognitive effort on sense of
agency. Based on previous studies we suggested that temporal aspects might
determine when effort facilitates or impedes sense of agency. We formulated
concrete hypotheses. First, during a block of trials where effort is required
unpredictably, encountering a high-effort trial would lead to a decreased sense
of agency *on that same trial*, but an increased sense of agency *on the next
trial* compared to a low-effort trial. Second, in a block of trials where effort
can be anticipated (i.e., frequent high-effort trials), sense of agency would be
increased compared to a block of trials where effort cannot be anticipated
(i.e., scarce high-effort trials). Furthermore, we aimed to explore the effect
of cognitive effort on both implicit and explicit measures of sense of agency.
We used a flanker task while also varying the proportion of conflict present in
a block of trials, creating three conditions (MC, MI and EQ block) in which the
required cognitive control (and hence effort) varied.
Our results showed that our manipulations of cognitive control, intentional
binding and sense of agency were successful. With regards to the trial-by-trial
analyses, and contrarily to our expectations, we observed no decreased
intentional binding or sense of agency *on* incongruent trials, nor an increased
intentional binding or sense of agency *after* incongruent trials. Remarkably,
in the trial-by-trial analyses of Experiment 2, we observed a trend towards an
increase of sense of agency on current incongruent trials compared to congruent
trials (which became significant when actual delay was taken into account). This
increased sense of agency for incongruent trials was also observed in the block
analyses of Experiment 2 and in the post-block analyses of both experiments.
With regards to the block analyses of Experiment 1, we observed a slight trend
towards increased intentional binding in a context where conflict could be
anticipated (i.e., MI block) compared to a context where conflict was scarce
(i.e., MC block). This trend was not present for sense of agency in Experiment
2.
Our results are not in line with previous studies observing lower sense of
agency for incongruent compared to congruent trials. A striking difference
between our study and these previous studies, is the interval between action and
outcome and hence when participants are asked to introspect about their sense of
agency. Previous studies used short intervals (ranging from 100 to 500ms),
whereas our intervals ranged from 500 to 1250ms. We chose these longer intervals
(see also) to be able to use the spacebar Interval Reproduction Task, as a
measure of intentional binding, in Experiment 1. But the moment when sense of
agency is tapped into, might be crucial. After shorter action-outcome intervals,
perhaps the experience of conflict is still predominant, leading to smaller
agency ratings on incongruent trials. After longer intervals, perhaps more
metacognitive processes might come into play, leading participants to reappraise
their action as an accomplishment of successfully solving an incongruent trials,
leading to higher agency ratings on incongruent trials. It has already been
shown that conflict experience precedes metacognitive experience, which might
only occur at a later time. In order to further elucidate this, we should
include a broader range of action-outcome intervals in the experimental design.
Interestingly, our results are in line with Damen et al., who, using quite a
different free choice priming task, found that clearly visible incongruent
primes increased agency compared to congruent primes. Based on this observation,
another possible explanation for the discrepancy in results might be the exact
way in which sense of agency is probed. Both in our study and the study of Damen
et al. we particularly asked participants whether they felt they had *caused*
the outcome. In contrast, Sidarus and Haggard and Wang et al. asked participants
how much *control* they felt over the outcome that was triggered by their
actions. This subtle difference in instructions, might have shifted
participants’ focus from how much control they felt (which arguably might be
larger for congruent trials) to how much causality they experienced (which might
be higher for incongruent trials which require the overruling of habitual
behavior). A similar argument can be made for the post-block assessment of sense
of agency. In previous studies, participants had to rank the colored circles and
rate their sense of control for each of them after each block of trials. In our
study, in contrast, participants reflected on the extent to which they felt they
had caused the colored circles to appear on congruent, incongruent and passive
trial specifically. Next to the difference between the focus on control versus
causality, additionally, we specifically highlighted the aspect of congruency
here. This in turn might have led to a reappraisal of their actions in response
to the clearly more difficult, incongruent trials. A follow-up study
manipulating instructions might be very informative with regards to the role of
even subtle nuances in sense of agency instructions.
In this study, we departed from the view that sense of agency and effort are
tightly related and that a subjective experience of effort might be a
prerequisite for any feeling of agency or causality. From this, we speculated
that experimental manipulations of cognitive effort should also influence the
accompanying sense of agency. However, whether our manipulation of effort was
successful, is doubtful. Although only assessed after each block and only per
block type and not per trial-type, participants reported equal experiences of
effort for MC, MI and EQ blocks in both experiments. Thus, although our
manipulation of cognitive control was successful (e.g., reduced congruency
effects in MI compared to MC blocks, indicative of increased cognitive control
in MI blocks), this might not have been accompanied by differences in
experienced effort. Indeed, the constructs of cognitive effort and cognitive
control are not identical, and the possibility of effortless exertion of
cognitive control (i.e., “flow”) has been suggested. Alternatively, even though
it is expected that in an MI block the overall cognitive control, and hence
effort experience, is larger, perhaps the intense effort experienced on the rare
incongruent trials in an MC block cancelled out the effort differences at the
block level. Finally, it could also be the case that the experienced differences
in effort in our experiments were too subtle to be crudely captured only after a
block of trials. Indeed, despite the absence of crude differences in effort
between blocks, we still observed a trend of a difference in intentional binding
between blocks. On the other hand, the lack of clear effort differences between
the blocks, might also explain why our block-based results are minimal. Contrary
to what we expected, we did not find an increased explicit sense of agency in a
context where conflict could be anticipated (i.e., MI block) compared to a
context where conflict was rare (i.e., MC block), but we did observe a slight
trend towards increased intentional binding in the MI block compared to the MC
block. These results could be linked to the fact that the MI block probably was
not effortful enough. In any case, replicating the experiments using a task that
induces stronger differences in experienced effort between the different
conditions (e.g., Stroop task), and regularly assessing the experienced effort
for different trial types and blocks seems advisable.
Our findings seem to imply a dissociation between our intentional binding and
sense of agency measures. First, despite no apparent crude difference in
experienced effort, the explicit sense of agency measure was still able to pick
up some (trends towards) differences between experienced sense of agency for
congruent and incongruent trials, whereas the intentional binding measure was
not. It could be that intentional binding is a less sensitive measure
(especially when there are no strong differences in experienced effort).
Alternatively, the lack of clear effort differences between congruent and
incongruent trials, might also inhibit differences at the assumed implicit, pre-
reflective level of sense of agency, but not the at the explicit reflective,
inferential, belief-like level. Second, across both experiments, the delay
between the action and the outcome had an effect on our measures of sense of
agency: participants showed more intentional binding, but lower sense of agency
ratings when the delay increased (i.e., 500, 750, 1000 or 1250ms). This is in
line with previous studies. Temporal binding measured using interval estimation
is typically larger for longer intervals ( but see for the reversed
result), whereas higher agency ratings are often found for shorter intervals. We
are not the first to highlight differences between different measures of sense
of agency. Recently, Imaizumi and Tanno have suggested that intentional binding
measured using time interval might originate from a different mechanism. Suzuki
et al. even showed that intentional binding might not necessarily reflect sense
of agency, but might be accounted for by multisensory causal binding, without
necessarily being related to intention or agency.
We sought to combine methods typically used in intentional binding tasks, with
methods employed to study the effect of effort on explicit sense of agency. More
specifically, in line with most binding studies , we compared an active
condition to a passive condition to assess the baseline level of intentional
binding. This differs from previous studies that have assessed the effect of
action selection fluency (effort) on explicit sense of agency, which typically
do not include passive trials. While comparing active and passive trials remains
the most common way of assessing intentional binding, it should be noted that
this has been criticised for not adequately controlling for other processes that
differ between these two types of trials. In addition, the inclusion of passive
trials in our studies, leading to a clearly felt difference in agency between
active and passive trials, might have obscured more subtle differences between
the different active trial types (e.g., congruent and incongruent trials).
Therefore, an experiment presenting only active trials might be useful to expose
perhaps elusive differences in agency between active trial types.
The current study examined the effect of cognitive effort on sense of agency
using explicit and implicit measures of sense of agency. We showed that trials
requiring the exertion of more cognitive control lead to a higher sense of
agency. This result was contrasted to previous studies to establish potential
reasons for this contradictory finding. Future studies should ensure that
conditions are sufficiently effortful, use a broad range of action-outcome
intervals and contrast (even subtle) different ways of probing sense of agency.
The authors wish to thank Katleen Vandist and Elke Van Lierde for their help
with the collection of the data.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
The geometric dilution of precision (GDOP) describes the accuracy of positioning
from the geometry of the satellites visible to the receiver. Errors in a GPS
receiver’s determination of its position are typically the GDOP times the
measurement error; in other words, the GDOP is an error amplification factor,
which means that a smaller GDOP generally results in a more accurate position.
Therefore, a smaller GDOP is better. It has been shown that the number of
satellites cause the GDOP to move in the opposite direction, i.e., a higher
number of satellites leads to a lower GDOP value, and a lower number of
satellites generally produces a bad GDOP and results in a corresponding
reduction in the positioning error. The simplest method is to choose a
combination of all of the satellites, all of which must be in view to achieve
optimal position accuracy with a minimal GDOP; however, some GPS receivers can
receive and parse navigation signals from only a limited number of satellites in
real time because of restrictions on hardware resources. Therefore, at least
four satellites should be selected for three-dimensional positioning to balance
hardware resources and positioning performance, however, a minimum of 5
satellites is required to evaluate the consistency and reliability for the GPS
receiver autonomous integrity monitoring(RAIM), meanwhile, more than five
pseudo-ranges are need to be used to identify the contaminating measurement in
the procedure of fault detection and exclusion (FDE).
Calculating the GDOP typically involves multiple mathematical matrix operations,
such as inversions and transformations, particularly for the optimal satellite
selection method, which involves selecting the subset with the lowest GDOP using
all of the satellites. However, the approach used for GDOP calculation creates a
heavy burden for the receiver because the calculation’s complexity increases
exponentially with the number of visible satellites.
Satellite selection is a classical combinatorial optimization problem, the
solution methods of which have been the subject of intense study in recent
years. One solution method includes geometric methods, such as the fast
satellite selection approach, quasi-optimal satellite selection, and optimal
selection. Miaoyan Zhang proposed a novel algorithm based on selecting the
subset of in-view satellites that has a closer distance to optimal solution from
the aspect of geometry when there are more than four satellites for multiple
constellations.
The other solution methods include statistical and machine learning approaches
based on neural networks, support vector machines and fuzzy logic. Simon
initially proposed the neural network-based approach as a way to predict the
GDOP and complete the classification, and back-propagation neural networks and
optimal interpolative nets have been employed to achieve the two objectives,
respectively. Jwo proposed several types of neural network mapping relationships
for different classes of relationships between inputs and outputs and compared
the performance of different network architectures. Mosavi studied the
relationships of eigenvalues between the visibility matrix and its inverse
matrix for GDOP. Azami developed several improved neural network training
algorithms, including the Levenberg Marquardt (LM), modified LM and resilient BP
(RBP) methods. ChihHung addressed the approximation of the GPS GDOP using
support vector machines (SVMs).
In this paper, we develop a novel modified genetic algorithm to select the
optimal subsets of satellites for GPS receivers. Because of the global
optimization capability and fast convergence of the modified genetic algorithm,
this approach effectively searches the entire solution spaces and determines the
optimal satellite subset after evolving through a number of generations without
the number limitation of required visible satellites, which improve the
adaptability and flexibility of the algorithm under a unified framework.
# Materials
## The Geometric Dilution of Precision
The mathematical model behind GPS is based on pseudo-range measurements between
the receiver and visible satellites. Without loss of generality, supposing that
(*x*<sub>*u*</sub>, *y*<sub>*u*</sub>, *z*<sub>*u*</sub>) represents the three-
dimensional coordinates of the receiver’s position in the ECEF system,
(*x*<sub>*j*</sub>, *y*<sub>*j*</sub>, *z*<sub>*j*</sub>) denotes the
coordinates of the *j*th satellite’s position in the ECEF system, and
*t*<sub>*b*</sub> indicates the equivalent distance of the time difference
between the receiver arrival time and the satellite signal sent time. The
receiver acquires information from *n*(*n* \> 4) satellites and determines the
pseudo-range value. $$\rho_{j} = \left( {\left( x_{j} - x_{u} \right)^{2} +
\left( y_{j} - y_{u} \right)^{2} + \left( z_{j} - z_{u} \right)^{2}}
\right)^{1/2} + ct_{b}(j = 1,2,\cdots,n)$$
It is shown that is a nonlinear function because of the square root mathematical
operation. In general, this class of problem can be linearized using the
mathematical principle of Taylor series and be written in a concise matrix form
as $$\begin{array}{r} {{\Delta\rho} \approx {G\Delta x} = {G\Delta x} +
\epsilon} \\ \end{array}$$ where the order of measurement matrix *G* is *n* × 4
(*n* ≥ 4), which can be represented as $$\begin{array}{r} {G = \begin{pmatrix}
a_{x_{1}} & a_{y_{1}} & a_{z_{1}} & 1 \\ a_{x_{2}} & a_{y_{2}} & a_{z_{2}} & 1
\\ \cdots & \cdots & \cdots & \cdots \\ a_{x_{n}} & a_{y_{n}} & a_{z_{n}} & 1 \\
\end{pmatrix}} \\ \end{array}$$
To simplify this problem, we assume that the measurement error has a Gaussian
distribution independently. The least squares solution for the super definite
matrix is typically given by $$\begin{array}{r} {{\Delta x} = \left( G^{T}G
\right)^{- 1}G^{T}{\Delta\rho}} \\ \end{array}$$
To illustrate the position accuracy, the covariance of Δ*x* is
$$\begin{array}{ll} {cov\left( {\Delta x} \right)} & {= E\left\lbrack {\left(
{\Delta x} \right)\left( {\Delta x} \right)^{T}} \right\rbrack} \\ & {=
E\left\lbrack {\left( {G^{T}G} \right)^{- 1}G^{T}\left(
{\Delta\rho\Delta\rho^{T}} \right)G\left( {G^{T}G} \right)^{- 1}} \right\rbrack}
\\ & {= \left( {G^{T}G} \right)^{- 1}cov\left( {\Delta\rho} \right)} \\
\end{array}$$
It is reasonable to assume that all of the errors in the pseudo-range
measurements are stationary random processes for short time intervals, which are
independent and identically distributed with a variance of $\sigma_{uere}^{2}$.
Let $$\begin{array}{r} {cov\left( {\Delta x} \right) = \begin{pmatrix}
\sigma_{x_{u}}^{2} & \cdot & \cdot & \cdot \\ \cdot & \sigma_{y_{u}}^{2} &
\cdot & \cdot \\ \cdot & \cdot & \sigma_{z_{u}}^{2} & \cdot \\ \cdot & \cdot &
\cdot & \sigma_{ct_{b}}^{2} \\ \end{pmatrix}} \\ \end{array}$$
The GDOP, which represents the amplification of the equivalent ranging errors in
the measurement into the receiver’s position solution, is $$\begin{array}{r}
{GDOP = \sqrt{\sigma_{x_{u}}^{2} + \sigma_{y_{u}}^{2} + \sigma_{z_{u}}^{2} +
\sigma_{ct_{b}}^{2}}/\sigma_{uere}^{2}} \\ \end{array}$$
According to linear algebra and matrix theory, when *λ*<sub>*i*</sub> are the
eigenvalues of an invertible matrix *A*, $\lambda_{i}^{- 1}$ are assumed to be
the eigenvalues of the inverse of the matrix *A*<sup>−1</sup>. The measurement
matrix $H = \left( G^{T}G \right)^{- 1}$ is always revertible, and the
eigenvalues $\lambda_{i}^{- 1}$ of $H = \left( G^{T}G \right)^{- 1}$ can be
found using the eigenvalues *λ*<sub>*i*</sub> of (*G* *<sup>T</sup>* *G*). This
leads to a significant reduction in the number of calculations required to find
the matrix inverse; the eigenvalues *λ*<sub>*i*</sub> of (*G* *<sup>T</sup>*
*G*) can be quickly calculated using QR decomposition. Let $$\begin{aligned}
{GDOP =} & \sqrt{tr\left( \left( G^{T}G \right)^{- 1} \right)} \\ = &
\sqrt{tr\left( (H) \right.} \\ = & \sqrt{\lambda_{1}^{- 1} + \lambda_{2}^{- 1}
+ \lambda_{3}^{- 1} + \lambda_{4}^{- 1}} \\ \end{aligned}$$ where *tr*(⋅)
represents the trace function of the matrix and *c* is the constant for the
speed of light.
The satellite selection process is to select a subset of satellites visible in
the current view for the purpose of the best positioning accuracy, which
corresponds to selecting the rows of the visibility matrix *G* that produce the
minimum GDOP as follows: $$\begin{array}{r} {S = {\left\{ s_{i} \right\},1 \leq
i \leq k,k \in (4,5,6,7)}} \\ \end{array}$$ where *s*<sub>*i*</sub> is the
identification number of the satellite currently in view such that
$$\min\limits_{\forall{({G_{i},G_{j},G_{l},G_{m}})} \in G}\,\left( {GDOP}
\right) = \sqrt{tr\left( \left( {G_{k}^{T}G_{k}} \right)^{- 1} \right)} =
\sqrt{\lambda_{1}^{- 1} + \lambda_{2}^{- 1} + \cdots + \lambda_{k}^{- 1}}$$
## Genetic Algorithm Preliminaries
A genetic algorithm (GA) is a robust global optimization approach that operates
according to the principles of evolution, such as inheritance, selection,
crossover and mutation.
In a GA, chromosomes are used to encode a candidate solution, and the fitness
function is the indication factor that represents the quality of the
individuals. As the population evolves, its average fitness gradually increases
based on the principle of the ‘survival of the fittest’; excellent genes that
are more fit are usually selected stochastically. The selected individuals are
operated through the process of the genetic operator, such as selection,
crossover and mutation, until the algorithm reaches termination.
In previous studies, GA was primarily used for micro-strip antenna optimization,
resolving ambiguity and multi-path mitigation in satellite navigation. This
paper proposes a novel modified genetic algorithm (MGA) for GPS satellite
selection.
# Methods
Because individuals evolve from generation to generation, the optimal solution
is found in a short period of time. Several strategies were used to improve the
performance of the algorithm. First, the elitist strategy reserves the most fit
gene of the previous generation to improve the average fitness of the next
generation. Second, adaptive mechanisms are used to adjust the mutation and
crossover rates to maintain each generation’s diversity. Finally, a hybrid
method accelerates the convergence. Without loss of generality, we establish the
following mathematical definitions:
1. *MaxGenNumber* represents the maximum number of evolution cycles.
2. *PopSize* represents the size of the population.
3. *Pop* represents the population.
4. *Ind*<sub>*i*</sub> represents the *i*th chromosome.
5. *Fitness*(*Ind*<sub>*i*</sub>) represents the fitness of the *i*th
chromosome.
## Representation
The integer code includes a clear description of the satellite selection problem
with population diversity followed by binary formation.
It is assumed that *N* GPS satellites are visible in the current view, which are
coded as (*k*<sub>1</sub>, *k*<sub>2</sub>, ⋯, *k*<sub>*N*</sub>). The objective
is to select the four signals *Ind*<sub>*i*</sub> = (*k*<sub>*i*</sub>,
*k*<sub>*j*</sub>, *k*<sub>*m*</sub>, *k*<sub>*n*</sub>) that provide the
minimum GDOP value and that satisfy the following constraint: $$\left\{
\begin{array}{l} {k_{i} \leq k_{j} \leq k_{m} \leq k_{n}} \\ {1 \leq i \leq j
\leq m \leq n \leq N} \\ \end{array} \right.$$ where the size of the solution
space is $C_{N}^{4}$, *Ind*<sub>*i*</sub> represents an individual, and
*k*<sub>*i*</sub> is a gene on the chromosome. Without loss of generality, we
assume that the list of currently visible satellites is \[2, 5, 7, 14, 15, 20,
21, 25\] and possible solutions are \[2, 5, 7, 14\] or \[2, 5, 7, 15\]. A total
of 5 or 6 satellites must be selected when considering RAIM and fail detection
and exclusion (FDE), which means that the code’s dimension must increase to, for
example, \[2, 5, 7, 14, 15\] or \[2, 5, 7, 14, 15, 20\].
## The Fitness Function
The fitness function is a special type of objective function that indicates the
current number of individuals; a larger number is better in most cases. We
minimize the GDOP as follows to obtain a more accurate position:
$$\begin{array}{r} {Fit(t) = \frac{1}{\sqrt{tr\left( \left( G_{4}^{T}G_{4}
\right)^{- 1} \right)}}} \\ \end{array}$$ Note that according to, the fitness
function is monotonic and non-negative, and the number of calculations can be
reduced by omitting the square root, which yields $$\begin{array}{r}
{Fit^{\prime}(t) = \frac{1}{tr\left( \left( G_{4}^{T}G_{4} \right)^{- 1}
\right)}} \\ \end{array}$$
## Selection
During each generation, individuals are selected by the *Ps* probability to
produce the offspring, in which the individuals with higher fitness have
considerably higher probabilities of being selected.
We define the fitness selection operator as *T*<sub>*s*</sub> :
*S*<sup>*PopSize*</sup> → *S*, where *S* represents the solution space and
*PopSize* is the population size. Then, $$\begin{array}{r} {P\left\{
{T_{s}\left( {Pop} \right) = {Ind}_{i}} \right\} = {P\left\{ Ind_{i} \right\}} =
\frac{Fitness\left( {Ind}_{i} \right)}{\sum\limits_{k =
1}^{PopSize}{Fitness\left( {Ind}_{k} \right)}}} \\ \end{array}$$ where
*Fitness*(*Ind*<sub>*i*</sub>) is an individual’s fitness and the probability
distribution function *P*{*Ind*<sub>*i*</sub>} satisfies the following
condition: $$\left\{ \begin{array}{l} {P\left\{ {Ind}_{i} \right\} \geq 0} \\
{\sum\limits_{i = 1}^{PopSize}{P\left\{ {Ind}_{i} \right\} = 1}} \\ \end{array}
\right.$$
The role of the selection operator is to select the individuals that reproduce
stochastically; the roulette wheel selection method based on a proportional-
selection mechanism is the most widely used strategy in which a greater fitness
implies a higher selection probability.
To reduce the time required to find the optimal solution, the elitist
reservation strategy is used. Using a ranking based on the population’s fitness,
the best chromosomes are reserved for the next generation. This process
accelerates the algorithm’s convergence.
## Adaptive Crossover Operators
Crossover is a critical genetic operator that is also called recombination. In
this process, a couple of solutions are selected to create offspring that
inherit the characteristics from the “parents”. The process continues until a
new population is created.
Without loss of generality, *T*<sub>*c*</sub> : *S*<sup>2</sup> → *S* is a
stochastic map with cross probability *p*<sub>*c*</sub> for the single-point
crossover operator. ∀(*Ind*<sub>1</sub>, *Ind*<sub>2</sub>) ∈ *S*<sup>2</sup>,
*Y* ∈ *S*, there is $$P\left\{ {T_{c}\left( Ind_{1},Ind_{2} \right) = Y}
\right\} = \left\{ \begin{array}{lc} \frac{kp_{c}}{Len} & {Y \neq Ind_{1}} \\
{\left( 1 - p_{c} \right) + \frac{kp_{c}}{L}} & {Y = ind_{1}} \\ \end{array}
\right.$$ where *Len* is the length of the chromosome.
In general, the cross probability *P*<sub>*c*</sub> strongly influences the
performance of the MGA.
During the initial stage of the MGA’s evolution, if the best chromosome is
considerably more fit than the other individuals, the optimum chromosome has a
substantially higher probability of being selected, which causes the algorithm
to converge to a local minimum. Therefore, a gene with a high fitness should be
restricted from over-reproducing to maintain the gene diversity at the initial
stage of population evolution. In turn, the average fitness of all individuals
is approximately equal to the maximum value of the population. Consequently, the
possibility of selecting an individual with an average fitness is equal to that
of selecting the chromosome with the highest fitness, which eliminates
competition between excellent individuals. Then, the selection operation becomes
random, which results in the worst performance when searching for the best
chromosome during the post-evolutionary stage. is a demonstration of single-
point crossover operation for MGA-based satellite selection.
The following novel adaptive strategy for generating the cross probability for
different evolutionary stages that controls the gene diversity to a certain, but
not large, degree is proposed: $$P_{c} = \left\{ \begin{array}{lr} {P_{cmax} -
\frac{\left( {p_{cmax} - p_{cmin}} \right)\left( {Fitness^{\prime} -
Fitness_{avg}} \right)}{Fitness_{\max} - Fitness_{avg}}} & {Fitness^{\prime}
\geq {Fitness_{avg}}} \\ P_{cmax} & {Fitness^{\prime} \leq {Fitness_{avg}}} \\
\end{array} \right.$$ where
*Fitness*<sub>*max*</sub> and *Fitness*<sub>*avg*</sub> denote the maximum and
minimum fitness of the population, respectively. *Fitness*′ is the larger one
between the pair of parents. *p*<sub>*cmax*</sub> denotes the maximum
probability of crossover. *p*<sub>*cmin*</sub> denotes the minimum probability
of crossover.
## Adaptive Mutation Operators
The mutation operator brings a new gene into the population, keeping the
population’s diversity from converging prematurely. is an example of single-
point mutation operation for MGA-based satellite selection.
The mutation operator is used to alter an individual gene for the purpose of
population diversity; however, the MGA degenerates into a random search when the
mutation probability *P*<sub>*m*</sub> is high. In contrast, the probability of
producing certain useful genes is zero when *P*<sub>*m*</sub> is low. To improve
the algorithm’s ability to find a global optimum and to avoid converging
prematurely or becoming stuck at a local minimum, the adaptive mutation is
$$P_{m} = \left\{ \begin{array}{lr} {P_{mmax} - \frac{\left( {p_{mmax} -
p_{mmin}} \right)\left( {Fitness^{\prime} - {Fitness_{avg}}}
\right)}{{Fitness_{\max}} - {Fitness_{avg}}}} & {Fitness^{\prime} \geq
{Fitness_{avg}}} \\ P_{mmax} & {Fitness^{\prime} \leq {Fitness_{avg}}} \\
\end{array} \right.$$ where
*Fitness*<sub>*max*</sub> and *Fitness*<sub>*avg*</sub> denote the maximum and
minimum fitness of the population, respectively. *Fitness*′ is the larger one
between the pair of parents. *p*<sub>*mmax*</sub> is the maximum probability
mutation. *p*<sub>*mmin*</sub> is the minimum probability mutation.
## The Hybrid Genetic Algorithm
To utilize the benefits of the two classes of approaches, a hybrid genetic
algorithm that combines the advantages of an MGA and fast selection is proposed.
To allow the algorithm to quickly find the best solution, individuals may be
initially created in the areas with the higher probability for optimal
solutions. It is easy to obtain an equivalent formulation of when the receiver
position offset is described in the ECEF coordinate system.
$$\widetilde{G}\mspace{180mu}\begin{bmatrix} {\bigtriangleup e} \\
{\bigtriangleup n} \\ {\bigtriangleup u} \\ {\bigtriangleup\delta t_{u}} \\
\end{bmatrix} = b$$ where \[△*e* △*n* △*u* △*δt*<sub>*u*</sub>\]<sup>*T*</sup>
is the state variable to be determined; then, the geometry matrix
$\widetilde{G}$ becomes $$\widetilde{G} = \begin{bmatrix} {-
cos\theta^{(1)}sin\alpha^{(1)}} & {- cos\theta^{(1)}cos\alpha^{(1)}} & {-
sin\theta^{(1)}} & 1 \\ {- cos\theta^{(2)}sin\alpha^{(2)}} & {-
cos\theta^{(2)}cos\alpha^{(2)}} & {- sin\theta^{(2)}} & 1 \\ \vdots & \vdots &
\vdots & \vdots \\ {- cos\theta^{(N)}sin\alpha^{(N)}} & {-
cos\theta^{(N)}cos\alpha^{(N)}} & {- sin\theta^{(N)}} & 1 \\ \end{bmatrix}$$
where *α*<sup>(*n*)</sup> and *θ*<sup>(*n*)</sup> are the elevation and azimuth
angles of the *n*th satellite, respectively. The main idea behind this step is
to randomly generate all of the chromosome subsets with geometries similar to
that of the optimal subset of the population by grouping all of the satellites
and randomly selecting one satellite from each group to form a chromosome subset
according to their azimuths and elevations.
## Termination
This evolutionary process for MGA satellite selection arrives at an endpoint
when one of the following conditions has been reached:
1. A solution is found that satisfies the minimum criterion, *GDOP* \< 3.
2. The number of generations reaches 20.
# Results
To demonstrate the performance of the proposed MGA satellite selection
algorithm, the OEMStar, which is one of NovAtel’s OEM global navigation
satellite system receiver platforms, was used to collect broadcast ephemeris
data and to calculate all of the satellites’ positions in the ECEF coordinate
system every second for 12 hours. The OEMStar receiver was placed at the
author’s institute, which has ECEF coordinates of \[−2258692.95, 4405376.94,
4007987.94\]. At the beginning of the evolutionary process, four different
satellite signals were randomly selected to compute the GDOP from the geometry
matrix G, using. The following experiments were conducted using Matlab 2010 on a
personal computer with an Intel Core(TM)2 Duo CPU and 2 GB of memory.
## Simulation Parameters
The primary parameters of the proposed MGA satellite selection algorithm include
the population size, the crossover probability, the mutation probability and the
number of evolution iterations. These parameters influence the convergence speed
and accuracy of the algorithm. A high crossover probability accelerates the
creation of individuals and increases the possibility of an excellent gene being
destroyed, which negatively affects the process of evolution. In contrast, when
the crossover probability is low, the speed at which new individuals are created
decreases, and the search process stagnates. With regard to the mutation
probability, a high probability results in the MGA being closer to a random
search algorithm, and a low probability reduces the likelihood of producing new
individuals and of premature convergence. lists the values of all of the
parameters used in the simulations.
# Discussion
GPS satellites orbit the earth once approximately every 12 hours, and as shown
in, there are approximately 7–12 satellites visible from any point on the earth
at any given time. shows the cumulative probability distribution function for
the number of visible satellites. shows that the GDOP calculated using the all-
in-view (AIV) method is minimized, and the proposed MGA’s performance is
equivalent to that of the optimal selection algorithm. The GDOP of the optimal
selection algorithm is slightly better than that of the AIV method, whose
performance, however, is better than that of the neural network and the fast
selection algorithm, particularly in terms of accuracy and efficiency. A
comparison is shown in. A comparison of the residuals of the GDOPs of the
various algorithms is provided in, where it can be observed that the proposed
MGA is the most accurate and has the smallest residuals. shows the average
fitness function and the best individual fitness versus the number of
iterations. The average fitness function gradually decreases over time as the
process of evolution continues, which shows that the algorithm converges and
that the best chromosome is eventually obtained.
The proposed novel MGA satellite selection algorithm is not only suitable for
the common problem of selecting 4 satellites but can also be used to select 5 or
6 satellites under RAIM or FDE conditions without modification. presents the
GDOP performance curve computed by the MGA, which shows that the GDOP gradually
decreases as the number of satellites selected, namely, 4, 5 and 6, increases.
# Conclusion
A novel method for selecting satellites for GPS use was proposed and shown to
provide both parallel and global convergence. The MGA can select a subset of
satellites of any size that satisfies the position requirements using an elite
conservation strategy, adaptive mechanism, hybrid genetic algorithm and
reasonable designs of the fitness function, and the selection, crossover and
mutation operators. Comprehensive simulations were conducted and demonstrated
that the MGA-based satellite selection method can effectively select an optimal
subset of the available satellites in both conventional and RAIM modes. The
latter is more feasible and adaptable to the GPS receivers that are used in
handset equipment and mobile phones. An unexpected discovery was that this
method can be applied to not only single constellation systems such as GPS but
also to multi-constellation systems because of its ability to select more than 4
satellites.
# Supporting Information
The authors would like to thank the Associate Editor and anonymous reviewers for
their valuable comments, which improved the presentation of this paper.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JS. Performed the experiments:
GX YK. Analyzed the data: JS GX YK. Contributed reagents/materials/analysis
tools: GX. Wrote the paper: JS. Contributed to the use of GPS receiver: GX. |
# Introduction
Improvement in lifestyle and changes in consumption habits mean that livestock
production aims to provide sufficient meat of improved quality. Meat quality and
production are influenced by intramuscular fat (IMF) content and skeletal muscle
development. For example, the content of IMF and the myofiber type can affect
meat quality traits such as flavor, juiciness, water holding capacity and
tenderness\[–\]. In chickens, IMF is not visible and not anatomically separable,
which makes it difficult to investigate the mechanism of its deposition. Protein
profiles of whole muscle, therefore, are important in understanding the
mechanisms for both muscle development and IMF deposition.
The short lifespan of chickens makes them an excellent model for studying
various aspects of development. Some of the molecular markers of muscle
structure/metabolism in livestock have also been identified by genome scans, but
no studies using proteomics technology have linked muscle growth and IMF content
in chickens. It is desirable to analyze the expression profile of proteins in
chicken skeletal muscle at different ages. Many studies have characterized
proteins from 2-DE gels in pigs, cattle, and layers at different stages of
embryonic development to early growth after hatching. Doherty et al have
characterized the proteome of layer chicken breast muscle using two-dimensional
gel electrophoresis (2-DE) from 1 to 27 days after hatching. Fifty-one proteins
had mass spectra that matched existing chicken proteins in on-line databases.
For many of these proteins, there were dramatic changes in relative expression
levels during the 27 days of growth. Proteomic profiling of the breast muscle of
Thai indigenous chickens during the growth period were also analyzed using the
2-DE method. A total of 259, 161, 120 and 107 protein spots were found to be
expressed in the chicken breast muscles at 0, 3, 6 and 18 weeks of age,
respectively. From these proteins, five distinct spots were significantly
associated with chicken age. These were characterized and showed homology with
phosphoglycerate mutase 1 (PGAM1), apolipoprotein A1 (APOA1), triosephosphate
isomerase 1 (TPI1), heat shock protein 25 kDa (HSP25) and fatty acid binding
protein 3 (FABP3). In addition, by application of an isobaric tagging for
relative and absolute quantification (iTRAQ)-based approach, the proteomes of
bovine embryos at the zygote and 2-cell and 4-cell stage with MII oocytes as a
reference were quantitatively analyzed. Bioinformatic analysis of 87
proteins that differed significantly in abundance between the four stages
revealed proteins involved in the p53 pathway, lipid metabolism, and mitosis,
indicating that these processes may play pivotal roles in embryonic
development. All of these studies showed the utility of proteomics as a tool
for uncovering the molecular basis of physiological differences in muscle during
these growth periods. Compared with the methods previously used (1D and 2D
gels), the isobaric tags for relative and absolute quantitation (iTRAQ) analysis
in the present study is more accurate and has been widely applied to investigate
the proteome of different organisms at different developmental stages.
The present study used advanced proteomics methodology (iTRAQ) to identify
differentially expressed proteins in breast muscles of slow-growing chickens at
different post-hatching ages.
# Materials and Methods
## Ethics Statement
All of the animal experiments were conducted in accordance with the Guidelines
for Experimental Animals established by the Ministry of Science and Technology
(Beijing, China). Animal experiments were approved by the Science Research
Department (in charge of animal welfare issue) of the Institute of Animal
Sciences, CAAS (Beijing, China).
## Animals
Forty female Beijing-You chickens were obtained at day 1 from the Institute of
Animal Science, Chinese Academy of Agricultural Sciences (Beijing, China), and
were randomly assigned to four groups of 10. Individuals were reared in
stairstep caging under continuous lighting using standard conditions of
temperature, humidity and ventilation. The same diet was fed to all chickens and
a three-phase feeding system was used: the starter ration (d 1 to d 28) with
21.0% crude protein and 12.12 MJ/kg, the second phase (d 28 to d 56) with 19.0%
crude protein and 12.54 MJ/kg, and the last phase (after d 56) with 16.0% crude
protein and 12.96 MJ/kg. Feed and water were provided ad libitum during the
experiment. All birds were fasted for 12 h, and weighed before being killed by
stunning and exsanguination. The left breast muscles were collected from 10
chickens at day 1 (hatching), 56 (fast growth age), 98 (marketing age) and 140
(first egg age). All samples (200–300 mg) were snap-frozen and stored at −80°C
before analysis. The entire right breast and livers were collected, weighed and
stored at −20°C for phenotypic measurement.
## IMF measurement
IMF content of breast and fat content of the liver were determined by Soxhlet
extraction, as described previously, and expressed as percentages of the
dry weight of the breast muscle.
## Protein extraction
Frozen breast muscle tissues (\~200 mg) was homogenized in 1 mL lysis buffer
containing 7 M urea (Sigma, St Louis, MO, USA), 2 M thiourea (Sigma), 4% (w/v)
3–3 (cholamidopropyl) dimethylammonio-1-propanesulfonate (CHAPS; Sigma), 65 mM
dithiothreitol (DTT; Sigma), and 0.05% (w/v) protease inhibitor (Sigma). The
homogenates were held on ice for 30 min and centrifuged for 30 min at 12,000
*g*, to remove insoluble components. The total protein concentration of each
sample was determined with a 2-D Quant kit (GE Healthcare, Pittsburgh, PA, USA).
The ten samples of each age group were pooled using equal amounts of protein
then the four pools were diluted to the same concentration with Tris-buffered
saline (TBS) before iTRAQ labeling. The samples were stored at −80°C until
analysis. Each pool was tested twice.
## iTRAQ labeling
After precipitation with acetone, the protein (200 μg) of each pool was
dissolved with 1 M DTT for 1 h at 37°C and kept in the dark with 1 M
indole-3-acetic acid (IAA) for 1 h at room temperature. Samples were dissolved
and centrifuged twice with 120 μl UA (8 M urea in 0.1 M Tris.HCl, pH 8.5), and
then re-dissolved and centrifuged three times with 100 M lautyltrethylammonium
bromide (LTEAB) (1 M). The proteins (2–4 μg) were digested with trypsin
(trypsin: protein = 1:50; Sigma) and incubated at 37°C overnight. Each peptide
pool was then passed through a 0.2-μm centrifugal filter for 20 min at 10,000
*g* at 20°C. Labeling of each pooled sample was 2-plex, where two reporter tags
were used; hatchling samples were labeled with reporter tags 113 and 117; those
from day 56 with reporter tags 114 and 118; the pool at day 98 with reporter
tags 115 and 119; and the last pool from day 140 was labeled with reporter tags
116 and 121. The four 2-plex labeled samples were then combined into a single
8-plex sample mixture and dried by centrifugal evaporation.
## Strong cation exchange (SCX) separation and reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS)
The combined peptide mixture was analyzed by RPLC-MS/MS for simultaneous
identification and quantification. The sequence of a peptide is determined from
the products that are generated from proteolytic cleavage of the protein and the
relative quantity of a given peptide among the treated samples is determined
from the intensities of reporter ion signals also present in the MS/MS scan.
iTRAQ-8 plex labeling reagents (Applied Biosystems, Foster City, CA, USA) were
added to the peptide samples, which were incubated at room temperature for 2 h.
The digested protein samples were separated using multidimensional liquid
chromatography (LC). In the first dimension, the peptide mixtures were
fractionated using an Ultimate LC system (Shimadzu 20AD, Kyoto, Japan) connected
to an SCX column (Polysulfoethyl column, 2.1 mm × 100 mm, 5 u, 200 A; Nest
Group, Southborough, MA, USA). A linear binary gradient from solvent A (10 mM
KH<sub>2</sub>PO<sub>4</sub> (Sinopharm Chemical Reagent Co. Ltd, Shanghai,
China) and 25% acetonitrile (ACN, pH 2.6; Fisher Scientific, Fair lawn, NJ,
USA), to solvent B (10 mM KH<sub>2</sub>PO<sub>4</sub>, 0.35 M KCl (Sinopharm
Chemical Reagent Co. Ltd.), 25% ACN, pH 2.6 was applied: 0%–5% solvent B over 5
min, 5%–25% solvent B over 35 min, then 35%–100% solvent B over 10 min, with a
flow rate of 200μl/min and detection at 214/280 nm. The entire run lasted 1 h,
and 20 SCX fractions were collected. These fractions were vacuum dried (rotation
vacuum concentrators, Christ RVC 2–25; Christ, Germany) and re-dissolved in 0.1%
formic acid (Tedia, Fairfield, OH, USA) and 5% ACN.
Based on the SCX chromatograms, the 20 SCX fractions were combined into eight
pools then desalted by ZORBAX 300SB-C18 column (5 μm, 300 Å, 0.1 × 150 mm;
Microm, Miami, FL, USA). The pooled SCX fractions were automatically injected by
a Famos autosampler and separated by an UltiMate capillary LC system (Dionex/LC
Packings) and fractionated on a C18 PepMap main column (5μm, 300 Å, 0.1 × 150
mm; Microm, Miami, FL, USA) using a linear binary gradient (solvent A: 0.1%
formic acid, 5% ACN; solvent B: 0.1% formic acid, 95% ACN). High Performance
Liquid Chromatography (HPLC) linear gradients were from 0% solvent B (5 min) to
35% (70 min) and from 35% to 100% (120 min) at a flow rate of 0.3μl/min.
The peptides were eluted from the LC column and automatically deposited using a
Probot spotting device. Mass spectrometry (MS) was conducted with a QSTAR XL
instrument (Applied Biosystems).
## Data analysis
Peptide identification from the QSTAR XL data was carried out using the Paragon
algorithm in the ProteinPilot 4.2 software package (Applied Biosystems).
MS/MS was performed on the four most abundant ions and the proteins identified
by searching the SWISSPROT-vertebrate and National Center for Biotechnology
Information (NCBI) databases. The following parameters were used for searching:
trypsin as enzyme, fixed modification of methyl methanethiosulfate labeled
cysteine, iTRAQ as sample type, no special factors, biological modification,
thorough identification search, and fragmentation mass accuracy, which were
built-in functions of ProteinPilot software, and the Paragon method was adopted.
Then the name, function, International Protein Index (IPI), and similar
characteristics were obtained from the Uniprot database. For protein-abundance
ratios measured using iTRAQ, 1.5-fold up-regulation and 0.75-fold down-
regulation change and the p-value \< 0.05 (the p-value is generated from the
peptide ratios used for quantitation) were set as the threshold for significant
changes.
## Western blotting
The pools of proteins from each age group were mixed (4:1) with 5× sample buffer
\[0.5 ml 0.5 mM Tris.HCl (pH 6.8), 0.1 g SDS, 0.005 g bromophenol blue, 0.5 ml
glycerol, 0.078 g DTT\]. Proteins (40 μg) were boiled for 5 min and separated on
SDS-PAGE in running buffer (25 mM Tris.HCl, pH 8.3, 1.4% glycine, 1 g SDS; Mini-
PROTEAN Tetra Electrophoresis Cell, 0.75 mm thickness; Bio-Rad, Hercules, CA,
USA) in two stages (30 min at 80 V, and 60 min at 120 V). The gels were
transferred to Polyvinylidene Fluoride (PVDF) membranes (Millipore, Billerica,
MA, USA) in ice-cold transfer buffer (25 mM Tris.HCl, pH 8.3, 1.4% glycine, 20%
methanol; Mini Trans-Blot Electrophoretic Transfer Cell; Bio-Rad) at 200 mA for
1 h. Membranes were blocked with 5% non-fat milk (Becton, Dickinson and Company,
Franklin Lakes, NJ, USA) in TBST (10 mM Tris.HCl, pH 8.0, 150 mM NaCl, and 0.1%
Tween 20) for 90 min. Primary antibodies for Heat shock protein beta-1 (HSPB1)
(diluted 1:400; Abcam, Cambridge, MA, USA), Apolipoprotein (APO)A1 (diluted
1:300; Biorbyt, Cambridge, UK), aldehyde dehydrogenase (ALDH)1A1 (diluted 1:300;
Biorbyt), malate dehydrogenase (MDH)1 (diluted 1:300; Biorbyt), annexin (ANX)A6
(diluted 1:800; Sigma), and fast skeletal muscle troponin T isoform (TNNT3)
(diluted 1:800; Sigma) were incubated overnight at 4°C. Membranes were washed
twice for 10 min in TBST and once for 10 min in TBS. Horseradish peroxidase
(HRP)-labeled anti-goat and anti-mouse secondary antibodies (Thermo Scientific
Pierce, Waltham, MA, USA) were diluted 1:15,000 in TBST and incubated with the
membranes for 90 min. After washing twice for 10 min in TBST and once for 10 min
in TBS, immunoreactive proteins were visualized using a chemiluminescent HRP
substrate (Millipore) in a dark room. The exposed films were analyzed for their
gray-scale value using Image J.
# Results and Discussion
## Proteomic analysis of breast muscle
Traits related to breast muscle weight and IMF content were measured. The breast
muscle absolute weight was obviously increased, but the breast muscle weight,
relative to body weight, increased more slowly with age. Another study, using
Beijing-You and western-type broilers, also showed that the breast muscle
weights significantly increased with growth of the chickens. Saneyasu, et
al.also investigated the change of body and breast muscle weights at 7, 14,
28, and 49 days of age, and showed significant increases in both with age. The
relative weight of the breast muscle increased slowly, indicating a slight
favoring of its growth over that of the whole body. Chartrin et al.
investigated lipid deposition in breast muscle of mule ducks at days 1 to 98 and
found that there are two periods of IMF deposition. The first, from day 1 to 42,
is when lipids (mainly phospholipids and cholesterol provided by the egg yolk)
stored in the adipocytes during embryonic life were transferred to the muscle
fibers and used for growth and energy requirements and the second, after day 42,
is when muscle again stores lipids. The present result is consistent with that
finding as the content of IMF was highest at day 1, decreased dramatically by
day 56, then increased again from day 56 to 140.
The pooling strategy was adopted in this study, as it minimize the differences
due to subject-to-subject variation and better identifies characteristics of the
population. Clustering showed that the protein expression profile was
consistent for each of the repetitions. Over 5000 proteins were identified and
those accurately identified in breast muscle at days 1, 56, 98 and 140 were 494.
Details of all accurately identified proteins as well as those at each sampled
age are shown in. Cluster analysis of all proteins expressed at different ages
showed that the proteins in breast muscle at post-hatching ages (days 56, 98 and
140) were more similar than those at hatching, and the proteins at day 56 and
day 98 were similar.
Two hundred and four differentially expressed proteins were defined and analyzed
(P \< 0.05, indicating that the quantity in one pool was \> 1.5 or \< 0.7,
compared to the other pool, for each pairwise comparison). To gain insight into
the changes between each stage, four groups of proteins by Gene Ontology
analysis were compared: day 56 vs. day 1, day 98 vs. day 56, and day 140 vs. day
98. Proteins related to glucose and intermediary metabolism were abundant from
day 1 to 56; proteins involved in muscle development were abundant from day 56
to 98; and from day 98 to 140, translation and protein folding processes were
abundant. The protein expression profiles were similar at the three post-
hatching ages (days 56, 98 and 140) but differed from those at day 1 by
clustering analysis. The molecular mechanisms of muscle development and IMF
deposition are different at hatching and post-hatching stages.
## Highly upregulated or downregulated proteins in hatchlings compared to post-hatching chickens
The proteins were defined as age-specific, highly upregulated or downregulated
when the content differed \>10 fold compared to that at other ages. As shown in,
many proteins were highly upregulated at day 1, such as ADP-ribosylhydrolase
like 1 (ADPRHL1), alpha-2-HS-glycoprotein (AHSG), apolipoprotein A (APOA1,
APOAIV), histone family (H1, H2B-VII, HIST2H2AC, H4), thymocyte nuclear protein
1 (THYN1), myosin light polypeptide 6 (MYL6), isocitrate dehydrogenase
\[NADP\](IDH), peptidyl-prolyl cis-trans isomerase (PPIA), sarcalumenin (SRL),
tubulin beta-7 chain (TUB7) *inter alia*. Some proteins were highly
downregulated at day 1, which were mostly involved in energy metabolism and
muscle development; for example, adenylate kinase isoenzyme 1 (AK1), fructose-
bisphosphate (ALDOA.ALDOB.ALDOC), creatine kinase S-type, mitochondrial (CKMT2),
desmin (DES), fructose-1, 6-bisphosphatase 2 (FBP2), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), glycogen phosphorylase (GPH1), glucose-6-phosphate
isomerase (GPI), L-lactate dehydrogenase A chain (LDHA), phosphoglycerate kinase
1 (PGK1), malate dehydrogenase 1and 2 (MDH1, MDH2), malic enzyme 1 (ME1),
phosphoglycerate mutase 1(PGAM1), phosphorylase B and L chain (PYGB,PYGL),
acylphosphatase (ACYP), alpha-actinin-2 (ACTN2), troponin C, skeletal muscle
(TNNC2), troponin I, fast skeletal muscle (TNNI2), fast skeletal muscle troponin
T isoform (TNNT3), triosephosphate isomerase 1 (TPI1), tropomyosin alpha-1 chain
(TPM1) etc.
## Over-represented pathways in breast muscle from day 1 to 56
Between days 1 and 56, breast muscle weight increased about 100-fold and IMF
decreased about 100-fold. Thus, this is a critical stage for muscle growth and
depletion of IMF.
Comparing the protein profiles at days 1 and 56, 191 differing proteins were
identified (the fold difference was \> 1.5 or \< 0.7). The 19 metabolic pathways
were enriched during this fast growing stage based on a KEGG pathway analysis.
The protein–protein interaction network of the differentially expressed proteins
identified for this interval (days 56 vs. 1) was also analyzed by web-tool
STRING 10.0 (<http://string-db.org>); there were two functional modules. The
first related to metabolic pathways, including glycolysis/gluconeogenesis
pathway, insulin signaling pathway, and lipid metabolic pathway. This module
might relate to the significant changes in breast muscle, such as fast muscle
growth and IMF deposition. The second module involved the ribosomes.
## Active pathways and key differentially expressed proteins at day 1
Within the 19 metabolic pathways, some differentially expressed proteins that
were highly expressed at day 1 compared to day 56 were involved in the lipid
metabolic pathway, such as valine, leucine and isoleucine degradation, and fatty
acid degradation and elongation in mitochondria.
Of eight differentially expressed proteins involved in the fatty acid
degradation pathway, five (CPT-2, ACADL, HADHA, HADHB and ACAA2) were more
abundant at day 1 than at day 56, and ALDH1A1 and ALDH2 were significantly lower
at day 1 compared to day 56 (Abbreviations used in this paper are listed in
Tables). It was deduced that lipid oxidation was more active and fatty acid
synthesis was less active at day 1 compared to day 56.
The differentially expressed proteins, APOA1 and APOAIV, were identified , which
was consistent with previous studies. The reason may be that a large number
of lipoproteins take up cholesterol from the yolk sac membrane at hatching,
and the production of the APOs was stimulated by lipoproteins. Synthesis of
APOAI in the skeletal muscle of hatchling chicks acts as a local lipid
transporter in early post-hatching development. This is consistent with the
phenotype where the content of IMF at day 1 is about 8 times higher than that at
day 56.
Expression of histone family and ribosomal family proteins differed greatly
between hatching and post-hatching (56, 98 and 140 days). Histones play a
pivotal role in regulating gene expression by controlling the access of key
regulatory factors and complexes to chromatin, which is essential for
transcription, DNA replication, DNA repair and DNA recombination. The
organization of chromatin is considered to be regulated by post-translational
modification of histones, such as methylation, acetylation, phosphorylation and
ubiquitination. Many studies have shown that myogenesis is controlled
through sequential chromatin regulation by the selection of the histone variant
and the appropriate histone modification. For example, in mouse embryos, a
bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated
skeletal muscle genes before embryonic skeletal muscle differentiation.
Ribosomal proteins, in conjunction with rRNA, make up the ribosomal subunits
involved in the cellular process of translation and protein biosynthesis. It has
been demonstrated that differential mRNA translation controls protein expression
of specific subsets of genes during myogenesis, and one of a subset of
transcripts that is enriched for mRNAs encoding ribosomal proteins is regulated
at the translational level. Ribosomal proteins were also highly expressed during
mature adipogenesis. A large group of ribosomal proteins was identified in
chickens, which may partly explain why differentiation of myocytes and
preadipocytes within muscle occurs mainly before hatching.
## Active pathways and key differentially expressed proteins at day 56
Many metabolism-related proteins were more abundant at day 56 than at day 1.
There were 24 upregulated proteins, related to cytoskeleton and actin binding,
including actin, cofilin, desmin, actinin, myosin, calpain, calmodulin,
troponin, myomesin and myozenin.
These proteins were involved in muscle-development-related pathways, such as
glycolysis/gluconeogenesis, hypertrophic cardiomyopathy (HCM), insulin
signaling, cardiac muscle contraction and dilated cardiomyopathy (DCM). Sixteen
upregulated proteins related to the glycolysis/gluconeogenesis pathway were more
abundant at day 56, and eight proteins in the HCM pathway and six (LMNA, DES,
TTN, TPM1, TPM2, MYH6) were highly abundant at day 56.
The period from day 1 to 56 is a fast growing stage for skeletal muscle. The
identified proteins play a critical role in all skeletal and cardiac muscle in
the early stages of development. Specifically, the energy from
glycolysis/gluconeogenesis metabolism is needed for developing skeletal muscle.
Insulin signaling pathway proteins are involved in the proliferation and
differentiation of preadipocytes and myocytes\[–\], and are prominent for
coordinating myofiber growth, muscle hypertrophy and muscle regeneration\[–\].
calmodulin (CALM), connectin/titin (TTN) and troponin C (TnC) are activated by
the second messenger Ca<sup>2+</sup> and stimulate expression of troponin I
(TnI), troponin T (TnT), actin, myosin and muscle development\[–\]. In the
present study, the amount of TTN, TnC (TNNC2), TnI (TNNI2), TnT (TNNT3), actin
(ACTA1), myosin (MYBPC1, MYBPC2, MYBPH, MYLPF, MYH6), M line (MYOM2) and Z line
(MYOZ1, MYOZ3) was enhanced, related to muscle development at day 56. Muscle
contraction and hypertrophy are the main mechanisms influencing muscle
development, along with the insulin signaling and HCM pathways at day 56.
## Over-represented pathways in breast muscle from day 56 to 98 (market age)
From day 56 to 98, breast muscle weight and IMF about doubled and thus fast
growth of muscle and IMF deposition continues. For the local slow-growing
chickens used here, market age is around day 98.
There were 44 differentially expressed proteins identified when comparing days
56 and 98. Seven significant enrichment pathways were identified by KEGG
analysis, of which six are involved in muscle development. The only significant
functional module in the network analysis of protein interaction, however,
related to the ribosomes.
There were 12 differentially expressed proteins related to cytoskeleton and
actin binding, which was consistent with the high muscle growth rate at this
stage. Four more abundant proteins at day 98 than at day 56 related to HCM and
three related to focal adhesion. This suggests that the muscle development at
this stage was closely connected with muscle growth, and these proteins may play
important roles in focal adhesion and HCM during skeletal muscle development.
The proteins included ACTN4, extracellular matrix proteins (LAMA2 and LAMB1),
MYH6, TTN, DES and TPM2. Previous studies have made inroads into understanding
the mechanism underlying muscle development. The enzymes related to cytoskeletal
protein binding, including MYBPC2, MYBPC1, PDLIM3, ANXA2, SPTAN1, KBTBD10 and
TUB7 were highly abundant in breast muscle at day 98 compared to day 56. ANXA2
is a Ca<sup>2+</sup>-binding protein implicated in several biochemical
processes, including cell proliferation, ion-channel activation, cytoskeleton
rearrangement, cell–cell interactions and the bridging of membranes. ANXA2 forms
junctions between lipid bilayer structures through molecular bridging of their
external leaflets. From day 56 to 98, focal adhesion, tight junctions and
HCM play an important role in muscle development. Extracellular matrix proteins
had a key role in muscle growth at day 98, which was different from day 56.
ANXA2 may play an important role in lipid metabolism.
## Over-represented pathways in breast muscle from day 98 to 140
From day 98 to 140, breast muscle weight and IMF increased about 1.3-fold, so
muscle growth and IMF deposition are slowing from that occurring in the previous
phase. For the local slow-growing chickens used here, day 140 is near sexual
maturity.
Comparing the proteins at days 98 and 140, 58 were identified as being
differentially expressed with cytoskeletal and ribosomal proteins being less
abundant at day 140 than at day 98. The cytoskeleton is present in all cells,
and plays important roles in cellular processes such as differentiation and
apoptosis. As key regulators of cellular architecture, cytoskeletal components
contribute to physical processes such as adhesion and migration\[–\]. Ribosomal
proteins relate to the cellular processes of translation and protein
biosynthesis. This decrease in abundance suggests that the rate of growth and
the metabolic processes were slower at 140 than at 98 days. The only functional
module clustering in the protein–protein interaction network involved the
ribosomes.
Three pathways were identified by KEGG analysis, with 58 differentially
expressed proteins, including those of fatty acid metabolism and the proteins
related to lipid metabolism, ACADL, APOA1 and HADH, were more abundant at day
140 compared to day 98. So, the capacity for lipid production was increased and
that of oxidation decreased, resulting in lipid accumulation, perhaps explaining
the higher IMF content in mature birds compared to younger birds.
## Verification of content of key proteins by Western blotting
To validate the results of the iTRAQ testing, Western blotting was used to
examine the relative contents of six key functional proteins at the different
ages. Two proteins (MDH1 and TNNT3) related to muscle development, and four
proteins (ALDH1A1, ANXA6, APOA1, HSPB1) related to lipid metabolism. There was
acceptable consistency between the results of Western blotting and the fold-
change of differentially expressed proteins from iTRAQ analysis. No internal
reference was used here in the Western blotting because the abundance of
β-actin, β-tubulin, histone and GAPDH all differed significantly at hatching and
the post-hatching ages. The control sample used for Western blotting was a
composite of breast muscle proteins made by pooling the different ages. MDH1
plays an important role in transporting NADH equivalents across the
mitochondrial membrane, controlling tricarboxylic acid (TCA) cycle pool size and
providing contractile function, so the increased expression of MDHs is
required for the high demands of energy metabolism in developing tissues,
especially in those with high metabolic rate such as heart, skeletal muscle, and
brain. Troponin-mediated Ca<sup>2+</sup>-regulation governs the actin-activated
myosin motor function which plays a key role in the regulation of striated
muscle contraction in vertebrates. Point mutations in the *cTnT* gene have
been found in human familial hypertrophic cardiomyopathy, and the expression
of TnT isoform is regulated during heart and muscle development and
adaptation, suggesting that TnT plays an important role in muscle growth and
function. ALDHs are known to participate in oxidizing a plethora of endogenous
and exogenous aldehydes. ALDH1A1 was up-regulated in omental and
intramuscular preadipocytes during differentiation, and the increased levels
of ALDH1A1 in the obese omental fat might be involved in fat accumulation.
Annexin A6 (AnxA6) is a Ca<sup>2+</sup> and phospholipid binding protein that
acts as a scaffolding protein and regulates cholesterol transport along endo-
and exocytic pathways. Loss of AnxA6 alters both lipid and glucose homeostasis,
resulting in increased lipolysis and high density lipoprotein increased in
*AnxA6* KO mice. Apolipoprotein A1 (apoA1) is the major apolipoprotein
constituent of the high-density lipoprotein (HDL) and is involved in reverse
cholesterol transport. Variants in the apolipoprotein A1 (*APOA1*) gene play
an important role in the regulation of lipid transport\[–\]. Synthesis of APOAI
in the skeletal muscle of hatchling chicks acts as a local lipid transporter for
early post-hatching development. Heat shock protein beta 1 (HSPB1), a member
of the heat-shock family of proteins, is a relatively small (27 kDa) molecular
chaperone protein associated with cellular development, differentiation, and
signal transduction. HspB1 and its regulator genes (*FAS*, and *AGT*) were
shown to be good candidate genes associated with intramuscular fat content in
the longissimus muscle of Korean cattle.
# Conclusion
In summary, the present study provides a useful resource for further
investigating the roles of proteins expressed differentially in skeletal muscle
at different developmental stages. Such efforts will enable better understanding
of the molecular mechanisms of muscle development in chickens. The changes in
protein abundance with age have not been documented previously, and the extent
of the changes found here was unexpected. This study is the first step in
understanding post-hatching development on a proteome-wide scale, and indicates
the complexity of such an analysis. In addition, the present results suggest
that APOA1 and HSPB1 may be useful as molecular markers of IMF deposition in
chickens.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceived and designed the experiments:** RRL GPZ JWe. **Performed
the experiments:** RQF JL. **Analyzed the data:** JWa MQZ. **Contributed
reagents/materials/analysis tools:** HXC QHL JS. **Wrote the paper:** JL
RQF. |
# Introduction
Rheumatoid arthritis (RA) causes significant costs for society due to the
increased use of healthcare resources, sick leaves and early retirements.
Consequently, effective anti-rheumatic therapies have the potential to reduce
societal costs while also improving the patients’ quality of life. According to
current Finnish Care Guideline, treatment of RA should be initially treated with
a combination of methotrexate (MTX), hydroxychloroquine (HCQ), sulfasalazine
(SSZ) and a low-dose glucocorticoid. In case of an insufficient response or
intolerance, biological disease modifying anti-rheumatic drugs (bDMARDs), i.e.
abatacept (ABA), tocilizumab (TCZ), rituximab (RTX), sarilumab (SAR), and tumour
necrosis factor (TNF) inhibitors including etanercept (ETN), adalimumab (ADA),
infliximab (IFX), certolizumab pegol (CTZ), and golimumab (GOL) are prescribed.
The bDMARDs have comparable efficacy and not significantly differing safety
profiles. bDMARDs are recommended to be used in combination with methotrexate
rather than as monotherapy due to better efficacy and reduced immunogenicity.
However, ABA and TCZ as monotherapy have been shown to have similar efficacy as
in combination with MTX.
A previously published systematic review indicated RTX as the most cost-
effective bDMARD among patients with an insufficient response to bDMARD
treatment. Previous cost-effectiveness analyses were mostly based on the
efficacy measured in randomized controlled trials (RCT), and might therefore,
have limited generalizability to routine healthcare owing to stringent inclusion
criteria and brief follow-up. Nevertheless, a previous study based on
observational data showed RTX to provide small savings and quality-adjusted life
year (QALY) gains as a second line treatment as compared with TNF inhibitors.
Whether these findings are generalizable to Finnish healthcare is unknown. Also,
introduction of biosimilars has lowered the treatment costs of some TNF-
inhibitors as well as rituximab. The treatment of RA, especially in the field of
biological drugs, has changed a lot during previous years, and therefore, there
is a need for cost-effectiveness analyses based on real-world data reflecting
current treatment practice and providing valuable information for health-care
decision making. The objective of this study was to evaluate the cost-
effectiveness of ABA, TCZ, and TNF inhibitors as compared with RTX in RA
patients, who have previously been treated with TNF inhibitor using Finnish
patient-level registry data.
# Materials and methods
## Model structure
We developed a patient-level simulation model using R statistical programming
language 3.2.2 to estimate costs and outcomes associated with different bDMARDs
in the treatment of RA. The population consisted of RA patients who had
previously used a TNF inhibitor as their first bDMARD and were about to begin
their second bDMARD. The model simulated four alternative treatment regimens:
ABA, RTX, TCZ and a second TNF inhibitor. In this simulation TNF inhibitors were
considered together as a single group rather than as individual drugs due to the
same mechanism of action and similar effectiveness. Different routes of
administration for ABA and TCZ were also pooled as single groups. In the base
case analysis, the choice of admin route of ABA and TCZ was based on the
National Register for Biologic Treatment in Finland (ROB-FIN). Every treatment
regimen was simulated by identical cohorts of 1,000 patients. In case the
simulated bDMARD treatment was discontinued either due to lack of efficacy or
adverse events, patients were switched to another bDMARD in the beginning of the
next cycle until the patient had exhausted all four treatment options. After
that, patients were assumed to be treated with a 6<sup>th</sup> line treatment
until death. The outline of the model is presented in.
Patients were assumed to remain on any given treatment for at least 6 months. At
the end of each six-month period, the model individually evaluated for each
person whether he or she would continue treatment, discontinue it or die. The
length of the time period was set to six months as this was the average time
interval for routine care visits to rheumatologists in the (ROB-FIN).
The simulation used regression models to predict outcomes and costs for each
patient individually in each period. Each patients’ characteristics, history of
drug use and past treatment responses were recorded in the model and utilized as
predictors for future outcomes and costs. The analysis was conducted from a
societal perspective as the study included both direct and indirect costs. Half-
cycle correction was applied to both outcomes and costs. We analyzed direct
costs and both direct and indirect costs separately. Health outcomes also were
expressed as QALYs. Based on the Finnish recommendations for health economic
evaluations, all costs and benefits were discounted at 3.0% annually. Primary
outcome of the simulation was incremental cost-effectiveness ratio (ICER) per
QALY including only direct costs.
## Data sources
Primary data source for the model was the ROB-FIN and all assumptions were based
on observed data unless otherwise mentioned. ROB-FIN has been described in
previous publications. In brief, ROB-FIN is a longitudinal observational cohort
study established in 1999 to monitor effectiveness and safety of biologic drugs
in treatment of RA and was originally based on structured data collection forms
submitted by rheumatologists on patients´ routine care visits to outpatient
specialized healthcare. Starting in 2007, most of the data have been retrieved
from electronic patient monitoring systems. Additional data on the patients’
hospitalization and outpatient visits as well as sick leaves and disability
pensions were acquired from national healthcare registers, which were linked to
ROB-FIN using social security numbers.
Observed ROB-FIN data from baseline visit as well as subsequent follow-up visits
were used to construct the regression models later used to predict patients’
treatment response, utility and costs in the simulation. Independent variable
selection for the regression models was based on Akaike Information Criteria
although the choice of bDMARD therapy and its interactions with other variables
were always included where appropriate. Missing data among disease activity
parameters were imputed by multiple imputation whereas information on treatments
and use of healthcare resources were considered complete. Patients lost to
follow-up were treated as uninformative censoring while information on treatment
discontinuations were utilized in the modeling.
## Model inputs
### Baseline characteristics
The model population were sampled with replacement among the patients included
in ROB-FIN about to start their second bDMARD therapy thus preserving any
potential correlation between the variables. The baseline variables included in
the model comprised age, sex, weight, Body Mass Index (BMI), Health Assessment
Questionnaire (HAQ), DAS28, time from diagnosis of rheumatic disease, Rheumatoid
Factor (RF) status, and concomitant use of MTX, SSZ and HCQ along with the
patients’ healthcare costs during the past 12 months. The patients´ weight, BMI
and RF status were fixed at the baseline values.
### Clinical effectiveness
Treatment effectiveness was defined as an achievement of at least American
College of Rheumatology (ACR) 20% improvement, a moderate European League
Against Rheumatism (EULAR) response, or a Disease Activity Score 28 (DAS28)
value of less than 3.2. The actual observed data were used to construct
regression models, which in turn were used to predict the responses in the
simulation. Based on expert opinion, the treatment response to the
6<sup>th</sup> line treatment options was assumed to be the same as averaged
treatment response to all biological treatments in the model.
The patients’ underlying HAQ score was assumed to remain fixed at the baseline
value. The change in HAQ score in comparison to baseline was modeled and
subsequently predicted using a linear regression model. Any effect the treatment
could have on these disease activity parameters was assumed to be temporary and
re-evaluated in the next period.
### Clinical safety
In addition to lack of efficacy, treatment might be discontinued due to adverse
events and other reasons. The risk of discontinuation due to adverse events and
other reasons besides lack of efficacy was observed to be 0.068 across all
bDMARDs in average based on the available data in ROB-FIN. After every six-month
time period 0.017, 0.010 and 0.009 per cent of MTX, SSZ and HCQ users
discontinued the use of the said co-treatment, respectively.
### Mortality
Mortality rate adjusted by age and sex for patient-level data was based on the
life table in 2017 published by Statistics Finland. In this model, RA was
associated with mortality of general population. Despite using lifetime horizon
in the model, we assumed that patients would die at the latest at the age of 100
due to lack of mortality data beyond that age.
### Utility
Quality-adjusted life years (QALY) were calculated corresponding to EuroQol five
dimensions questionnaire (EQ-5D-3L) utilities predicted from the HAQ scores
utilizing a multinomial logistic regression model. The regression model was
based on data from a survey including both HAQ and EQ-5D-3L conducted in Finland
in 2009. Obtained EQ-5D-3L health stages were valued with the Finnish tariff.
### Costs
Direct costs comprised drug costs, administration costs of infusions, costs of
switching, outpatient and inpatient care, while indirect costs included early
retirement due to RA and sick leave. Costs of drugs were based on the Finnish
price list including the retail price without value added tax of drugs and the
dose in the label. The costs for infusion drugs were the wholesale prices of
Helsinki and Uusimaa Hospital District (Aaltonen T, personal communication,
April 20, 2016). The prices of biosimilar IFX, ETN, and ADA were used in the
base case analysis, whereas the price of biosimilar of RTX was taken into
account in sensitivity analysis. Administration costs of infusions including
intravenous treatment cost prices of bDMARDs for RA at Finnish hospitals were
derived from the Finnish study by Soini et al. The price for TNF inhibitor group
was an average for individual TNF inhibitors weighed by their actual usage.
Similarly, the costs for ABA and TCZ were weighed averages based on the prices
of subcutaneously and intravenously administered products. In the base case
analysis, the bDMARD was subcutaneously administered for 15 and 7 per cent of
ABA and TCZ users, respectively. Drug costs, administration costs of infusions
and dosages are presented in. Cost of switching was assumed to be equal to the
cost of one healthcare visit to internal medicine specialist in specialized
outpatient healthcare. Based on the expert opinion and in line with their
effectiveness, the costs of the 6<sup>th</sup> line treatment option were
considered to be same as the averaged costs of currently available biological
treatments.
Patients’ biannual healthcare and indirect costs were modeled using a linear
regression model based on actual observed data and later predicted in the
simulation using this model. Drugs in outpatient care were in 2019 euros and all
other costs were converted to 2017 euros using the price indices of Statistics
Finland.
## Sensitivity analyses
The model was stochastic in nature and the 1000 unique model runs with different
seeds for random number generator quantified the variability in the results.
Several subgroup and deterministic sensitivity analyses were carried out based
on 300 model runs to explore uncertainty and heterogeneity of the model results.
As ABA and TCZ can be administered either subcutaneously or intravenously, we
evaluated the influence of the choice of the administration route on the results
in the deterministic sensitivity analyses. Even though HAQ progression was
associated with age and the number of biologic treatment in the base case
analysis, we used annual HAQ progression rates of 0.03 and 0.06 in the
sensitivity analyses. Also, we included a scenario where we employed a British
tariff to value EQ-5D health stages. Although the patent of RTX has expired, RTX
biosimilar approved for the treatment of RA is not on the market in Finland.
Therefore, we considered RTX biosimilar with a price discount of 30% in
comparison to the reference medicinal product in sensitivity analysis. The time
horizon of 10 years and the discounting rate of 0% and 6% were explored in the
sensitivity analyses. In subgroup analyses we evaluated the heterogeneity of the
results related to the use of the concomitant csDMARD therapy, age, body mass
index (BMI), gender, presence of RF, prior use of MTX, and primary response to
the first TNF inhibitor.
## Ethical approval
Ethical approval for this study was granted by the Helsinki University Central
Hospital ethical committee (73/13/03/00/2014). The study permit to use the
patient records and cost data was granted by the Finnish National Institute for
Health and Welfare (THL/1497/.5.05.00/2013), Finnish Population Registry
(262/410/16) and the Social Insurance Institution of Finland (Kela 7/522/2016).
Written informed patient consents were acquired from patients who had been
included in ROB-FIN prior to the introduction of the electronic patient
monitoring systems.
# Results
## Baseline characteristics
At baseline, median age of patients included in the study was 56 years. Most of
the patients were female. Median HAQ score was 1.1, whereas median DAS28 was
4.6. More than half of the patients had a treatment response to the first TNF
inhibitor. The characteristics of the patients at the baseline are presented in.
## Base-case analysis
Lifetime drug costs without administration costs and costs of switching were the
lowest for RTX, but when administration costs and costs of switching were
included, drug costs were the lowest for TNF inhibitors. ABA had the highest
drug costs. However, ABA had the lowest healthcare costs, while RTX had the
highest healthcare costs. In total, the lowest and highest direct costs were
associated with the TNF inhibitors and RTX, respectively. Indirect costs ranged
from 148,718 € for TNF inhibitors to 165,300 € for RTX. Drug costs including
administration costs and costs of switching represented over half of the total
costs. QALYs ranged from 9.405 of RTX to 9.661 of TNF inhibitors. In our model,
patients died in average at the age of 85.59 (standard deviation 9.96).
TNF inhibitors, ABA, and TCZ had lower costs and higher QALYs than RTX, and
therefore, they were dominant in comparison to RTX. TNF inhibitors are the most
cost-effective treatment option, as they have the lowest costs and the highest
lifetime QALYs.
## Sensitivity analysis
The results regarding costs and QALYs from the subgroup and deterministic
sensitivity analysis are presented in and Tables. Instead of intravenous
administration, the use of self-administered ABA and TCZ decreased the
administration costs of drugs, but the choice of route of administration had no
effect on other costs. Similarly to base case results, RTX had the highest costs
and lowest QALYs when they were discounted at 0%, but when the discounting rate
of 6% was used, TCZ had the lowest QALYs. When the time horizon of 10 years was
used, RTX had the lowest drug costs including the costs of switching and the
administration costs, while TCZ was associated with the lowest QALYs. As
compared with RTX, TNF inhibitors were dominant, whereas the incremental cost-
effectiveness ratio for ABA was 144,213 €/QALY when the time horizon of 10 years
was used. RTX as a second-line treatment option had the highest direct costs
even when the price discount of 30% for RTX biosimilar was used, owing to the
highest outpatient and inpatient costs for RTX. Indirect costs increased and
QALYs decreased in case the bi-annual HAQ progression was fixed at 0.03 or 0.06.
Removing half-cycle correction had little effect on the results. QALYs decreased
when the health stages were valued with the British tariff. The costs were
higher and QALYs were lower for women than for men. As compared with patients
with no response to the first TNF inhibitor, primary responders had slightly
higher costs and QALYs. The QALYs ranged from 11.960 to 12.370 for patients with
negative RF status, while QALYs ranged from 9.159 to 9.415 for patients with
positive RF status. Also the costs were higher among patients with negative RF
status as compared to RF-positive patients. Furthermore, concomitant use of MTX
or other non-biologic therapies lead to increased QALYs in comparison to non-use
of MTX or biologic monotherapy, respectively.
# Discussion
Based on our patient-level simulation model using real-world data from Finland,
TNF inhibitors, ABA, and TCZ as a second-line biologic treatments for RA were
dominant as compared with RTX. Even though a commonly referred threshold for
cost-effectiveness has not been published in Finland, we have used the
willingness to pay threshold of 40,600€, representing the Finland´s gross
domestic product per capita in 2017 per QALY gained in this study. According to
our model, RTX was associated with the lowest drug costs, but when
administration costs and costs of switching were included, TNF inhibitors had
the lowest drug costs. RTX had the highest healthcare costs, and in total, RTX
was associated with the highest drug costs. TCZ was associated with the lowest
effectiveness. TNF inhibitors had the lowest costs and the highest QALYs, and
therefore, they were the most cost-effective treatment option.
Consistent with our results, previous studies showed similar effectiveness
between bDMARDs in patients with RA failing on TNF inhibitor, whereas other
studies suggest that changing to RTX is more effective than switching to an
alternative TNF inhibitor. Unlike our results, previously published results by
Lindgren et al. found that RTX treatment was associated with the lowest overall
costs and was the most effective option. Similarly, in a head-to-head RCT
between RTX, TNF-inhibitors, ABA and TCZ, RTX was associated with both the
lowest costs and the highest QALY gain. The Finnish study reported also RTX to
be most cost-effective treatment alternative for patients with RA who have
failed TNF inhibitor treatment. According to this study, life years were highly
similar between all bDMARDs, but they were the highest for RTX.
The main advantage of this study was that real-world data on costs and
effectiveness were based on registry data from the same population. Baseline
characteristics of the model and population and the assumptions used in the
model were mainly based on ROB-FIN data representing routine clinical practice.
Compared to the previous studies mostly based on efficacy derived from RCTs, our
simulation was based on observational data and therefore results are likely to
be more generalizable to the Finnish healthcare setting. In addition, cost data
derived from comprehensive Finnish national registers were employed whenever
possible. To eliminate the bias caused by confounders in the observational data,
we used several regression models to predict the patients’ treatment response,
utility and costs in the simulation.
Our model memorized changes in patient characteristics over time and enabled
simulation based on individual patients’ history. The history of what had
happened was an important aspect of the model because it affected the occurrence
of future events, their consequences and valuations, and many other aspects of
the simulation. The simulation followed up patients for their complete lifetime,
because RA is a chronic disorder that progresses over time. As such, long-term
consequences of any differences in disease progression, effect on life
expectancy, or drug discontinuation rates were assessed. The first TNF inhibitor
was not included in this model as it was assumed to be identical between the
comparators and would not affect the results. Reasons for discontinuation of RTX
treatment were severely underreported in ROB-FIN data and therefore, the risk of
adverse events was assumed to be equal across all treatment regimens. Also, the
discontinuation probabilities for non-biologic co-therapies were assumed to be
similar between the users of different biologics.
Because of high costs of original bDMARDs, interest has grown in biosimilars
that are comparable to the reference medicinal product in terms of efficacy and
safety. Many original biologic drugs have reached, or are approaching, patent
expiry. This will lead to increasing development and use of biosimilar drugs in
the future, offering considerable savings in comparison with the reference
medicinal product. Price competition after patent expiry may also reduce the
price of reference medicinal product, leading to remarkable cost savings.
Biosimilars of ETN, IFX, and ADA were considered in this analysis. The price of
RTX biosimilar used for the treatment of RA was not available, but we used price
discount of 30% for RTX biosimilar in the sensitivity analysis based on the
Finnish legislation concerning the confirming a reasonable wholesale price for
medicinal products. However, the price discount may be even bigger in the
future.
A potential limitation of this study is related to the dosing interval of RTX.
As compared with other biologics, RTX has a unique mode of action and long
dosing interval. In maintenance treatment, RTX infusions were often administered
on demand in Finland. Consequently, the patients treated with RTX came to visit
the rheumatologist only after the effect of the previous infusion began to wear
off, which is very likely to lead to an underestimation of the effectiveness of
RTX in our data, and therefore, leading to overestimation of healthcare costs.
Based on the registry data used in this study, RTX was administered every 7.98
months in maintenance treatment. Similar dosing interval of RTX was reported by
Keystone et al, whereas mean dosing interval of RTX varied between 11 and 13
months in daily clinical practice in Finland according to the study by Valleala
et al. Therefore, a relatively short dosing interval used in this study might
also be one reason to cause high costs of RTX treatment. In this model RA was
associated with mortality of general population, which is another limitation of
this study. According to the study by Kroot et al, mortality of RA patients was
comparable with the expected mortality of the general population of the
Netherland up to 10 years of RA. This finding was in line with the study by
Lindgqvist and Eberhardt et al.. Furthermore, Lacaille et al found that
mortality gap between RA and the general population in the first five years was
not observed in people with RA onset after year 2000. However, it is notable
that the patient populations in these studies comprised of patients with recent
onset of RA. Although mortality has decreased among RA patients over the past
decades, the general belief is that patients with RA, especially the more severe
cases, have a shortened life expectancy compared with the general population. We
assume that differences in QALYs between bDMARDS would have been bigger, but the
order of these results would have been the same, if the association between
disease activity and mortality in RA patients had been considered in the model.
We did not however, have sufficient data to create a prediction model for
mortality, which can be considered a limitation. Furthermore, patients’ erosive
progression could not be taken into account as data on this subject was not
available in ROB-FIN. The effect could be mitigated by inclusion of HAQ scores,
which have been shown to be correlated with the presence of joint erosions. Some
bias could also be caused by the lack of a generic health related quality of
life instrument. When we employed another valuation method in sensitivity
analysis, the QALYs decreased when health stages were valued with the British
tariff.
# Conclusion
Our patient-level simulation based on observational data showed that TNF
inhibitors were associated with the lowest costs and highest QALYs, whereas RTX
had the highest costs and lowest QALYs. TNF inhibitors, ABA, and TCZ were
dominant in comparison to RTX. As TNF inhibitors had the lowest costs and
highest QALYs, they were the most cost-effective treatment option.
# Supporting information
We acknowledge the support given to the National Register for Biologic Treatment
in Finland (ROB-FIN) by the Finnish Society for Rheumatology and all the
rheumatologists who have contributed to data collection.
[^1]: HR has received a fee for speaking from Pfizer. KT has received fees
for consultancy from Novartis and Pfizer. KP has received fees for speaking
and consultancy from Pfizer, MSD, Abbvie, BMS, Roche, Lilly, Novartis,
Sandoz, and Sanofi. DN has received fees for consultancy from AbbVie, BMS,
Lilly, MSD, Novartis, Pfizer, Roche, and UCB. SH works at ESiOR Oy, which
carries out studies, consultancy, education, reporting and health economic
evaluations for several pharmaceutical (including companies producing
biologic RA drugs), food industry, diagnostics and device companies,
hospitals, and academic institutions. ESiOR Oy did not play any role in
study design, data collection and analysis, decision to publish and did not
provide any financial support to work or author. All other authors have
declared no conflicts of interest. This does not alter our adherence to PLOS
ONE policies on sharing data and materials. |
# 1 Introduction
Over the past few decades, functional MRI has widened our understanding of the
functional organization of intrinsic brain networks and their role in cognition
and behavior. Classical univariate (i.e., voxel-wise) analyses of fMRI signal
(i.e., blood-oxygenation level-dependent, or BOLD) have been instrumental in
probing the specialized function of brain regions. More recent approaches using
functional connectivity and network neuroscience portray a complex and multi-
scale set of interactions between brain structures. Following this view, a wide
array of graph theoretical and complex systems tools have been used to describe
BOLD dynamics.
Despite these efforts, we still lack a unified mechanistic framework that
overcomes three key limitations. First, the features of the BOLD signal that are
important for neural activity are unclear. Several prior studies demonstrate a
relation between BOLD and slow amplitude features of cortical activity, and
between BOLD and the hemodynamic response function (HRF). These studies imply
that the low frequency component of the BOLD signal contains information
relevant to underlying neural dynamics, although it is also clear that the
signal contains artifact. Due to the mixture of signal and artifact in the BOLD
time series, it is possible that the common practice of band-pass filtering the
BOLD signal at low frequencies may exclude functionally relevant signal. Second,
many graph theoretic and network analyses are inherently descriptive in nature,
and lack the power to give a generative understanding of the relationship
between model inputs and outputs (for extensions of these approaches that move
beyond description into explanation and prediction, see). Finally, model-based
approaches often treat the brain as an isolated system by ignoring external
input, or assuming an artificial profile of internal and external noise.
To address these three limitations, we develop a generative framework that
explicitly includes exogenous input (e.g., external sensory or subcortical
structures’ inputs), and provide evidence that the brain’s activity can be
fruitfully understood in the context of its natural drivers. Specifically, we
use a multivariate autoregressive model with unknown inputs to capture the
spatiotemporal evolution of the BOLD signal driven by extra-cortical inputs.
These models have been used to characterize and predict the evolution of several
synthetic and biological systems. For instance, Chang and colleagues (2012)
leveraged a multivariate linear dynamical system’s framework and the patients’
intracranial EEG to model the cortical impulse response to the direct electrical
stimulation. Many prior studies use this and similar methods such as Granger
causality and dynamic causal modeling (DCM) for understanding the directed
functional connectivity of BOLD \[, –\]. While some prior studies account for
the effect of exogenous input, they typically assume a simple known and abstract
form of the input function. Moreover, the inability of models such as DCM to
capture signal variations beyond those caused by the external inputs makes the
connectivity estimation highly dependent on the assumed number and form of the
inputs.
In this work, we treat the exogenous inputs to the cortex as *unknown*
parameters of a linear time-invariant (LTI) system, which we estimate following
recent developments in linear systems theory. We use these developments to
provide new insights into how the brain responds to ongoing task requirements,
and to shine a light on factors that contribute to the dynamics of cortical
functional connectivity. To demonstrate our approach’s utility, we begin with a
proof-of-concept where we consider synthetic examples for which we retrieve the
external inputs’ spatiotemporal profiles of a known LTI system. We demonstrate
that unknown external inputs result in apparent changes in internal system
parameters, and consequently, in estimated external inputs’ error. Also, we show
that using internal system parameters estimated from time windows without
external stimulation significantly improves our ability to extract external
inputs’ profile from periods with external stimulation, expect for simulations
with relatively low external inputs and signal-to-noise.
Next, we test the hypothesis that variations in cortical dynamics during
different tasks or cognitive states can be accurately modeled as external
excitations on fairly stable interactions between cortical regions.
Specifically, we recover the unknown external cortical inputs during resting-
state and task scans for 96 subjects with the lowest motion artifact from the
Human Connectome Project (HCP). Our results demonstrate that using system
parameters estimated from resting-state scans enables uncovering the expected
spatiotemporal profiles of external sensory (i.e., visual cues) and task-related
extra-cortical inputs, while system parameters estimated from task scans result
in highly inaccurate input estimations. In addition, an in-depth examination of
estimated inputs during task scans reveals the spatiotemporal patterns of other
task-related inputs that were not captured by the abstract task regressors.
Lastly, we measure the non-stationarity of estimated external inputs over
resting-state scans to examine the assumption of the system’s time-invariance
and to identify exogenous determinants of the BOLD signal’s non-stationarity.
Recently, the nature of non-stationarity of BOLD signal and dynamic functional
connectivity has been a topic of scientific debate, as several recent
publications paint seemingly contrasting portraits of the processes’
stationarity underlying the brain’s functional dynamics. However, to the best of
authors’ knowledge, no study examines the BOLD signal’s stationarity in the
context of time-varying external inputs and their effects. Our results show that
the inputs to several brain regions, most notably over default mode network,
estimated from the resting-state scans display significantly high non-
stationarity compared to other brain regions. Together, we demonstrate that our
framework allows us to uncover spatiotemporal patterns and dimensionality of
unknown cortical drivers. These findings offer insight into how a relatively
static relation between brain regions and exogenous drivers can give rise to
complex cortical dynamics and contribute to their non-stationarity.
# 2 Materials and methods
## 2.1 Linear time-invariant (LTI) dynamical systems with external inputs
Each region *i* of interest (ROI) from which the BOLD signal is collected
provided us with a time series described by *x*<sub>*i*</sub>\[*k*\] at sampling
point *k* = 0, …, *T*. A total of *n* = 100 regions are considered and the
collection of these signals is captured by the vector $x\lbrack k\rbrack =
\left\lbrack x_{1}\lbrack k\rbrack\mspace{720mu}\ldots\mspace{720mu}
x_{n}\lbrack k\rbrack \right\rbrack^{\intercal}$, with *k* = 0, …, *T*, which we
refer to as the *state of the system* (i.e., it describes the evolution of the
BOLD signal across different regions). The evolution of the system’s state is
mainly driven by (*i*) the cross-dependencies of the signals in different
regions (not necessarily adjacent), and (*ii*) the external inputs that are
either excitation noise or inputs arriving from the environment surrounding the
regions captured by the state of the system (e.g., stimulus arriving from
subcortical structures not accounted for during BOLD signal collection).
Subsequently, a first step towards modeling the evolution of the system’s state
is: $$\begin{array}{r} {x\lbrack k + 1\rbrack = Ax\lbrack k\rbrack + Bu\lbrack
k\rbrack + \omega_{k},\quad k = 0,\ldots,T,} \\ \end{array}$$ where $A \in
\mathbb{R}^{n \times n}$ described the autonomous dynamics, $B \in \mathbb{R}^{n
\times p}$ is the input matrix that describes the impact of inputs (i.e.,
external drivers) $u\lbrack k\rbrack \in \mathbb{R}^{p \times 1}$ on the system
state’s evolution, and $\omega_{k} \in \mathbb{R}^{n}$ is the internal dynamics
noise (i.e., internal drivers) at sampling point *k*. Notice that $\left\{
x\lbrack k\rbrack \right\}_{k = 0}^{T}$ is the BOLD signal at the different ROIs
and is the only known. However, the state of the underlying neural activity is
unknown since we did not account for the hemodynamic response function (HRF) in
our reduced model. Therefore, the input in the model captures the external
drivers of regional BOLD and only indirectly, the underlying neural activity. In
order to determine the *parameters of the system*, i.e., (*A*, *B*, $\left\{
u\lbrack k\rbrack \right\}_{k = 0}^{T}$), we need to solve an optimization
problem that minimizes the distance between the system’s state *x*\[*k*\] and
the estimate of that state given by $\hat{x}\lbrack k\rbrack$ driven by the
unknown quantities. Specifically, we have the following optimization problem:
$$\begin{array}{rc} {\left\{ \hat{x}\lbrack k\rbrack \right\}_{k = 0}^{T} \in
\text{arg}\underset{z\lbrack 0\rbrack,\ldots,z\lbrack T\rbrack}{\text{min}}} &
{\quad \parallel z\lbrack k\rbrack - x\lbrack k\rbrack \parallel}_{2}^{2} \\
{\mspace{720mu}\text{s.t.}} & {z\lbrack k + 1\rbrack = Az\lbrack k\rbrack +
Bu\lbrack k\rbrack.} \\ \end{array}$$ Notice that this problem is more
challenging than the usual least squares problem considered when the parameters
of the system are known. Thus, similar to the method develop by, we perform the
following steps: (i) we assume that the state *z*\[0\] = *x*\[0\], and $\left\{
u\lbrack k\rbrack \right\}_{k = 0}^{T}$ is identically zero, to find an
approximation to *A*;(ii) assuming *A* is given by the initial approximation, we
provide a sparse low-rank structure to matrix *B* and we find an approximation
to both *z*\[0\] and $\left\{ u\lbrack k\rbrack \right\}_{k = 0}^{T}$, which
suffices to obtain *z*\[0\], …, *z*\[*T*\] subsequently, $\left\{ \hat{x}\lbrack
k\rbrack \right\}_{k = 0}^{T}$; and (iii) assume $\left\{ z\lbrack k\rbrack
\right\}_{k = 0}^{T}$ and $\left\{ u\lbrack k\rbrack \right\}_{k = 0}^{T}$ are
as approximated in step (ii) and determine an approximation to *B*. The process
consists of executing step (ii) and (iii) iteratively. Our experiments reveal
that the estimated parameters converge after a few iterations in both synthetic
and fMRI time series (S18 Fig). Additionally, to force the inputs to be used as
little as possible, since otherwise they could contain all the required
information to obtain the sequence $\left\{ z\lbrack k\rbrack \right\}_{k =
0}^{T}$ (e.g., consider *A* to be zero and *B* to be the identity matrix), the
optimization objective is rather given by ${\parallel z\lbrack k\rbrack -
x\lbrack k\rbrack \parallel}_{2}^{2} + {\lambda \parallel u \parallel}_{1} +
\lambda{\parallel B \parallel}_{1}^{2}$, which penalizes the use of the input
with a weight *λ* \> 0.—See section SI1 in for algorithm details.
We will demonstrate in the following results section that unaccounted external
inputs result in error in estimation of system matrix *A*. Therefore, in a
modified version of this algorithm, in step (i) we estimate *A* from *x*′\[*k*\]
measured during an extended window without external stimulation (e.g., resting-
state). Next, we repeat steps (ii) and (iii) iteratively—as detailed above.
Since we did not know the true dimensionality of the external inputs, we
approximated the dimensions of the input matrix *B* by performing principal
component analysis on the residuals of the models. As seen in S19 Fig in,
principal components 1–25 capture more than 80% of variance in the average
residuals and more than average 60% of subject-level residuals’ variance across
all tasks. In addition, we compared the goodness-of-fit of the LTI model with
and without external inputs using Akaike information criterion (AIC). Our
results demonstrate that incorporating external inputs does not results in
overfitting and improves the model’s fit—an effect most pronounced in higher
dimensional input matrices (S20 Fig). Finally, we demonstrate that we identify
the external inputs during the motor task similarly at high-dimensional input
matrices (S6 Fig), as indicated by the high correlation (\>0.8) of inputs
estimated using input matrix dimensions higher than 25 (S6I Fig). Therefore, we
select *p* = 25 for input matrix *B* to estimate the inputs from task fMRI time
series.
## 2.2 Spectral analysis of an LTI system
Provided an LTI description of the system dynamics, the autonomous evolution of
the dynamical system can be decomposed in a so-called *eigenmode decomposition*.
Briefly, consider the *n* eigenmodes (i.e., eigenvalues and the corresponding
eigenvectors) associated with *A*. Each eigenmode corresponds to an eigenvalue-
eigenvector pair (*λ*<sub>*i*</sub>, *v*<sub>*i*</sub>) satisfying
*Av*<sub>*i*</sub> = *λ*<sub>*i*</sub> *v*<sub>*i*</sub>, and it describes the
oscillatory behavior for a specific direction *v*<sub>*i*</sub>.
Specifically, for any given eigenvalue *λ*<sub>*i*</sub> represented in polar
coordinates (*θ*<sub>*i*</sub>, \|*λ*<sub>*i*</sub>\|), we have that it captures
the *frequency* characterized as $$f_{i} = \frac{\theta_{i}}{2\pi}\delta t,$$
where *δt* corresponds to the sampling frequency, and the *time scale* given by
$$\rho_{i} = \frac{\text{log}\left( \middle| \lambda_{i} \middle|
\right)}{\delta t},$$ which can be interpreted as the *damping rate*.
In particular, we can re-write $A = V\lambda V^{\intercal}$, where *V* =
\[*v*<sub>1</sub>, …, *v*<sub>*n*</sub>\] and *λ* = diag(*λ*<sub>1</sub>, …,
*λ*<sub>*n*</sub>) are the matrices of eigenvectors and eigenvalues.
Subsequently, we can apply a change of variable as *z*\[*k*\] = *V*\*
*x*\[*k*\], where *V*\* is the transpose conjugate, which implies that
$z_{i}\lbrack k\rbrack = v_{i}^{\intercal}x\lbrack k\rbrack$ is a weighted
combination described by the *i*<sub>*th*</sub> eigenvector associated with the
*i*<sub>*th*</sub> eigenvalue. Hence, this can be understood as the spatial
contributions of the *n* ROIs at a given (spatiotemporal) frequency
*f*<sub>*i*</sub>. Additionally, we can revisit the damping rate of the process
in such direction *v*<sub>*i*</sub> by reasoning as follows: first, we can
recursively obtain \|*z*<sub>*i*</sub>\[*k*\]\| =
\|*λ*<sub>*i*</sub>\|<sup>*t*</sup>\|*z*<sub>*i*</sub>\[0\]\|. Therefore, we
have the following three scenarios: (*i*) \|*λ*<sub>*i*</sub>\|\<1; (*ii*)
\|*λ*<sub>*i*</sub>\|\>1; and (*iii*) \|*λ*<sub>*i*</sub>\| = 1. In case (i) and
(ii), we can readily see that \|*z*<sub>*i*</sub>\[*k*\]\| → 0 and
\|*z*<sub>*i*</sub>\[*k*\]\| → ∞ as *k* → ∞, respectively. Lastly, in scenario
(iii), or practically, when \|*λ*<sub>*i*</sub>\|≈1, we have that the process
oscillates between *stability* and *instability*, and therefore these dynamics
are refer to as *meta-stable*.
In summary, the dynamical process *z*(*k*) describes the spatiotemporal brain
BOLD signal evolution. Specifically, the timescales are encoded in the
eigenvalues and the spatial contributions of the different ROIs are described by
the eigenvectors with a spatiotemporal timescale described by the associated
eigenvalues.
## 2.3 Dataset and preprocessing
We used data from the Human Connectome Project (HCP). As part of the HCP
protocol, subjects underwent two separate resting-state scans along with seven
task fMRI scans, both of which included two sessions. All data analyzed here
came from these scans and was part of the HCP S1200 release. The fMRI protocol
(both resting-state and task) includes a multi-band factor of 8, spatial
resolution of 2 mm isotropic voxels, and a TR of 0.72 sec (for more details
see). Subjects that completed both resting-state scans and all task scans were
analyzed. Each of the scanning sessions included both resting-state and task
fMRI. First, two 15-minute resting-state scans (eyes open and fixation on a
cross-hair) are acquired, for a total of 1 hour of resting-state data over the
two-day visit. Second, approximately 30 min of task-fMRI is acquired in each
session, including 7 tasks split between the two sessions, for a total of 1 hour
of task fMRI (for details see).
Head-motion artifacts result in significant error in the functional connectivity
estimates. Therefore, to minimize head-motion artifacts, we selected 100
subjects with the lowest mean frame-wise displacement in our study, where we
utilized a cortical parcellation with *N* = 100 parcels that maximizes the
similarity of functional connectivity within each parcel. Next to keep the same
subjects across the resting state and task scans, we removed the four patients
with missing either task or resting state scans. We preprocessed resting-state
and task data using similar pipelines. For resting-state, the ICA-FIX resting-
state data provided by the Human Connectome Project were utilized, which used
ICA to remove nuisance and motion signals. For task data, CompCor, with five
components from the ventricles and white matter masks, was used to regress out
nuisance signals from the time series. In addition, for the task data, the 12
detrended motion estimates provided by the Human Connectome Project were
regressed out from the time series. For both task and resting-state, the mean
global signal was also removed in an effort to remove the auto-correlated non-
physiological noise and reduce the model estimation error.
## 2.4 Statistics
We performed student’s *t*-test and Welch’s *t*-test to test the statistical
significance of the differences between the distributions of interest. Non-
parametric Wilcoxon rank-sum test were utilized for comparisons of distributions
with non-normal profiles. We corrected calculated test statistics for multiple
comparisons using false discovery rate (FDR) method, as well as the more
conservative Bonferroni method. To identify the task-specific fluctuations in
the average estimated inputs, for each brain regions we compared task-related
inputs to those estimated from resting-state time series (paired *t*−test, *p*
\< 0.05, FDR). In addition we also generated phase-randomized null time series
from each subjects’ BOLD times series for the task time series. We select the
phase-randomized null model since it maintains most of the statistical
properties of multivariate time series (e.g., autocorrelation, covariance).
Next, for each brain region, we compared the average empirical and null
estimated inputs for each time point (paired *t*−test, *p* \< 0.05, FDR).
To identify estimated inputs that display changes that correspond to different
task conditions in the motor paradigm, we first performed a principal component
analysis (PCA) on all estimated inputs (*U*) concatenated over all subjects.
Next, we identified a single input with the highest absolute principal component
(PC) loading for every component. We then multiplied the selected inputs with
negative PC loadings by −1. Next, we separately fitted a multiple linear
regression model for each PC’s inputs (*U*) using the known task-regressors. We
created task-regressors for different conditions by assigning every sample to
baseline (0) or one of six events (i.e., visual cue, left hand, right hand, left
foot, and right foot movements) based on their temporal proximity to events’
onsets and offsets. We repeated this analysis by shifting task-regressors by
different lags (0–12 TRs) to identify the lag that produces the best fit (i.e.,
highest *R*<sup>2</sup> values) for each region. Finally, we performed *t*−tests
on estimated coefficients at the group-level to identify task conditions
similarly echoed in estimated inputs associated with each PC across
participants. We also identified brain regions that correspond to the identified
inputs by performing group-level region-wise *t*−tests on input matrix *B*
elements that correspond to inputs *U* identified by PCs.
We examined the estimated inputs’ non-stationarity using two methods. First, we
used a sliding window approach to examine temporal fluctuations of estimated
inputs’ means over resting-state scans for all brain regions, measured from the
windowed-means’ standard deviation. Second, we used the nonlinear non-
stationarity index introduced by, with *α* = 0.9 and *β* = 1 exponent parameters
following their study, where *α* and *β* parameters control the relative
weighting between the importance of long versus large excursions in time series.
Therefore, non-stationarity indexes with our selected parameters give marginally
greater weighting to excursions’ height. Finally, to test the group-level
significance of both non-stationarity metrics, we first normalized the values
across all brain regions. Next, we used the *t*−test (FDR corrected for multiple
comparisons across all brain regions) to establish the statistical significance
of the measured non-stationarities across patients. Traditionally, researchers
have commonly used the 0.05 as the statistical significance level, though the
choice is largely subjective. Therefore to convey the probabilistic nature of
the statistical analysis and the proper interpretation of statistical test
results, in the manuscript, we refer to results of the commonly accepted
statistical threshold of 0.05 as “significant” and the more conservative
thresholds of 0.0005 or lower as “highly significant”.
## 2.5 Ethics statement
All subject recruitment procedures and written informed consents were approved
by the Washington University Institutional Review Board (IRB). For more details
see.
## 2.6 Retrieving the external inputs to a synthetic LTI system
We use the proposed method to explicitly model the contributions of internal
system dynamics and external inputs on the BOLD signal during rest and task. To
build intuition, we begin by estimating the internal system parameters and
unknown inputs using data simulated from a synthetic LTI model with four states
representing four brain regions. We first simulate the dynamics of our model,
where each region is driven by random internal noise, and only one region is
driven by an additional square pulse train. For details regarding the simulation
see section SI2 in. Next, we estimate internal system parameters (4 × 4 matrix
of interactions) and unknown inputs from the simulated time series, and to
recover the spatial and temporal profiles of the pulse train input. Although the
estimated inputs (green line) fluctuate time-locked to the ground-truth input,
their temporal profiles notably differ. We hypothesize that this divergence
arises from the error in system matrices estimated during periods with external
stimulations. In , we show that the LTI system parameters receiving time-varying
external inputs can falsely appear to change and diverge farther from the
ground-truth when examined over periods with external stimulation.
Consequently, we hypothesize that system matrices estimated from periods without
external inputs would improve our ability to capture the unknown inputs’ profile
accurately. shows that using a fixed system matrix estimated from periods
without external inputs significantly increases the similarity (correlation) to
the ground-truth inputs. We also demonstrate that although estimated inputs
contain noise, averaging inputs estimated over 100 simulations results in highly
accurate estimations (correlation = 0.99). The significant (Wilcoxon rank-sum
test, Bonferroni *p* \< 0.0001) changes in the input matrix *B*’s loading for
estimation windows overlapping the external stimulation periods, reveals the
unknown external inputs’ spatial profile (i.e., the blue input node). Together,
these results demonstrate that external inputs can increase estimation error in
system matrices, and consequently, input parameters. More importantly, these
results also show that identifying system matrices from periods without external
stimulation allow an accurate estimation of unknown external inputs’
spatiotemporal profiles.
Next, we generate synthetic time series by stimulating LTI systems, parameters
of which were estimated from subjects’ resting-state BOLD time series. We set
external inputs’ magnitude such that the global average stimulus-induced changes
in normalized simulated outputs match the largest average task-related changes
in a sample (social) task. We confirm that similar to the low-dimensional
example in, our approach is able to extract synthetic external inputs to high-
dimensional LTI models of BOLD signal dynamics. Likewise, employing system
parameters estimated from periods without external stimulation results in a
significant (*t*−test, *p* \< 0.05, *p* = 6.6 × 10<sup>−65</sup> and *p* = 2.9 ×
10<sup>−66</sup> for 1000 TR- and 250 TR-long estimation windows, respectively)
increase in the similarity between the ground-truth and estimated inputs. The
notably higher similarity between the average estimated to ground-truth inputs
than that of subject-level estimated inputs suggests that profiles of external
inputs are correctly approximated although with noise. Together these results
demonstrate the utility of our framework in identifying external inputs to LTI
systems, and highlight the importance of accurate estimation of model
parameters.
So far, we have examined the LTI system’s response in a low recording noise
level (signal-to-recording noise = 1000). Next, we examine the accuracy of the
retrieved model and input parameters at different recording and internal noise
levels. The contributions of the recording and internal noise to the BOLD
signal, for the most part, are unknown quantities. However, they play an
essential role in our ability to capture external inputs accurately. Simulating
the system’s response magnitude and variance (i.e., *t*-values) at various
recording and internal noise levels show how different noise levels can lead to
seemingly similar outputs.
Moreover, at high noise levels, the error increases notably in the system
parameters estimated from periods without external inputs, and consequently, in
the estimated input parameters during stimulation periods. Interestingly, at
such high noise levels, the system matrices estimated during stimulation periods
more accurately recover external inputs than those estimated during periods
without stimulation (S1 Fig in). These observations suggest that the choice of
system matrices and the goodness-of-fit of the estimated inputs can further
provide insight into the empirical noise levels. In the following, we consider
the proposed methodology in the context of quantifying important spatial and
temporal features of the internal system dynamics and external inputs estimated
from the HCP resting-state and task fMRI scans.
## 2.7 Capturing external drivers of BOLD signal
### 2.7.1 Brain’s large-scale oscillatory modes display heterogeneous spatiotemporal profiles
We begin by showing that the estimated system parameters during resting-state
reliably capture and reproduce known brain functional organization. Further,
because these parameters reside within a quantitative dynamical model, we
simultaneously capture both spatial (regions that are co-active) and temporal
(oscillation frequency) information through the *eigenmodes* of our estimated
system. Specifically, each eigenvector indicates an independent pattern of co-
active regions, and its corresponding eigenvalue determines both the oscillation
frequency and the change in amplitude of the activation patterns. Intuitively,
if we initialize our estimated system state to a pattern of activity
corresponding to an eigenvector, then the system states would oscillate and
dampen according to the associated eigenvalue’s characteristics (see more
details in Materials and Methods section).
To capture the spatial and temporal patterns of activity, we use our method to
estimate the internal system parameters from the resting-state time series (1200
TR ≈14.5 min). The high stability of the (i.e., slow damping rate) low-frequency
eigenvalues as seen in indicates that the system’s outputs are dominated by
lower frequency oscillations. To identify the eigenmodes with similar spatial
patterns across subjects, we aggregate all subjects’ eigenvectors and perform
*k*-means clustering analysis. We used the elbow method (optimal *k* ≈ 4),
Calinski-Harabasz, Davies-Bouldin, and Silhouette criteria (optimal *k* = 2) to
identify the optimal clustering resolution—for details, see SI5 and S3 Fig in.
The non-converging results across the different criteria suggest that the
community organization of eigenvector clusters does not display a distinct
optimal topological scale. We provide the course (*k* = 2) and finer scale (*k*
= 4) clusters in S2A Fig and, respectively. To ensure that the image acquisition
type (i.e., phase-encoding direction) or the scanning session does not affect
these results, we provide statistical comparisons between the coarse-scale
cluster’s stability and frequency in S4 Fig in. These results show that very
similar distributions and clusters are identified regardless of phase-encoding
direction or day of scans. Specifically, statistical comparisons (bootstrapping
*n* = 50, 000, *p* \< 0.05) fail to find any difference between cluster’s means.
To test the spatial inhomogeneity in the frequency and damping of these
clustered eigenvectors, we performed a pairwise comparison between the
distribution of eigenvalues corresponding to the eigenvectors in each of the
clusters (bootstrap *n* = 50, 000, Bonferroni corrected *p* \< 0.05). We found
significant differences in the frequencies and damping rates between all cluster
pairs, except for the comparison between the frequencies in clusters 3 and 4).
Together, these findings highlight the spatial heterogeneity in the frequency
and damping profiles of brain oscillations.
### 2.7.2 Task-specific increases in the extra-cortical input’s power
Up to now, we provided evidence that the system dynamics can capture the spatial
and temporal behavior of resting-state brain networks. Next, we try to assess if
the task-induced dynamics are driven by the external inputs, retrieved by the
proposed method. The sensory inputs to the brain are some of the major drivers
of cortical dynamics. Therefore, we hypothesize that the external inputs to the
subjects’ brains, as estimated by the proposed method, will mirror real-time
changes present in these task regressors (see S5 Fig in for details regarding
the task regressors).
To test this hypothesis, we apply our method to the fMRI activity to estimate
the internal system parameters and external inputs for each subject during task
performance (i.e., social, gambling, motor, working memory, language, and
relational). Then, we compare the average estimated inputs’ frequency spectrum
for each task. Statistical tests (Wilcoxon rank-sum test, FDR corrected, *p* \<
0.0005) reveal highly significant unique peaks, matching the expected external
task-specific frequencies. Note that the distinct task-induced peaks are
identified at low (\< 0.1 Hz) and high (\> 0.1 Hz) frequencies, even as high as
0.3–0.4 Hz.
### 2.7.3 Task-specific profiles of extra-cortical inputs
Next, we consider an LTI framework to quantify spatial and temporal features of
external inputs to the brain using HCP’s motor task dataset. The motor task
comprises 3-second long visual cues, where participants are asked to either tap
left or right fingers, squeeze left or right toes, or move their tongue over
12-second long periods following the visual cue’s offset. We select the motor
task since the high dimensionality of input and various task conditions in this
paradigm allows us to evaluate our framework’s ability to estimate external
inputs’ complex spatiotemporal structure. We aim to assess if we can retrieve
the external inputs that drive task-induced dynamics. We hypothesize that
subjects’ estimated external inputs will mirror real-time changes present in
known task regressors. Moreover, due to relatively lower levels of structured
external stimulations during resting-state scans, we hypothesize that the system
parameters estimated from subjects’ full-length resting-state time series will
increase the accuracy of external inputs estimated from motor task datasets.
demonstrates estimated inputs (input matrix *B* dimensions = 25, regularization
factor = 0.5) to all brain regions (i.e., *B* × *U*) averaged across all
subjects during the motor task. These results highlight the brain-wide
significant task-specific changes in the estimated inputs when system parameters
are estimated from the resting-state time series. We provide evidence of the
robustness of these results to changes in the input matrix *B*’s dimension (S6
Fig in). Conversely, the identified inputs using the system parameters
estimated from the subjects’ motor task time series notably reduces our ability
to capture the task-related changes.
We establish these observations’ statistical significance by comparing the
external inputs estimated from task datasets against those from subjects’
resting-state scans (paired *t*−test, *p* \< 0.05, FDR corrected for multiple
comparisons). Comparisons against the phase-randomized null time series also
provide converging observations (S7 Fig). We also use multiple linear regression
analyses to assess the estimated inputs’ similarity to the known temporal
profile of the task regressors. Our results demonstrate that external inputs
estimated using the full-length resting-state system parameters result in
significantly (paired *t*−test, *p* \< 0.05, Bonferroni corrected for multiple
comparisons) improved fit (measured by *R*<sup>2</sup> values), compared to
system parameters estimated from the motor task (S8 Fig in). We also find
similar results when resting-state system parameters were estimated from a short
(250 sample) window that match task scans’ length (S8B Fig). Together these
results highlight the importance of the modeled system’s accuracy in capturing a
reliable picture of the brain’s external inputs.
Next, we examine the temporal (i.e., *U* matrix) and the spatial (i.e., input
matrix *B*) profiles of the external inputs (estimated using resting-state
system parameters), to demonstrate how the estimated inputs reveal the
dimensionality and the spatiotemporal dependencies of the task-related inputs.
Prior works using univariate and multivariate analyses of HCP task datasets have
demonstrated that activation induced by the hand, foot, and tongue movements can
be localized over the somatomotor network. Therefore, we expect the
dimensionality of the external inputs to roughly match or exceed those of task
conditions (i.e., six dimensions). As mentioned in the Materials and Methods
section, the principal component analysis reveals that in all HCP task
conditions, principal components (PCs) 1–25 explain more than 80% of the
variance in the model’s average residuals. Therefore, we choose *p* = 25 as the
input matrix *B* dimension in.
We performed principal component analysis on external inputs estimated temporal
profiles (i.e., *U*) concatenated across all subjects to identify the input
patterns similarly identified over the group. shows the temporal profile of the
concatenated inputs’ PCs 1–15. As seen in, the first few PCs (≈ 9) explain a
relatively larger portion of the variance. shows the high similarity between
known task regressors and PCs’ temporal profiles. We quantify this similarity
using subject-level multiple linear regression analysis of the estimated inputs
using the known task (motor) regressors. We note apparent time lags between the
known and estimated inputs. Therefore, we perform the multiple linear regression
analysis using various lags. shows distributions of lags (samples) that yield
the highest *R*<sup>2</sup> values for PCs 1–9. shows the group average
coefficients estimated from external inputs associated with each PC (i.e.,
external inputs with highest PC weights). We used the group average optimal lag
(based on *R*<sup>2</sup> values) identified in in. Estimated coefficients have
significant values, only in PCs 1–9. These results demonstrate that the
estimated inputs provide insight into the extra-cortical drivers’
dimensionality.
Next, we examine spatiotemporal profiles of subject-level estimated inputs
associated with these components to understand their relationship to the
external stimuli. demonstrate that compared to other PCs, the inputs associated
with PCs 1–4 and 6 fit task regressors relatively better, indicated by
significantly (Wilcoxon rank-sum test, *p* \< 0.05, FDR corrected for multiple
comparisons) higher *R*<sup>2</sup> values. reveals that PCs 1–4 and 6 are
associated with the visual cue, hand and feet movements (maximum coefficient in
left hand), all movements (maximum coefficient in right hand), feet movements
(maximum coefficient in left foot), and tongue movements, respectively. S9 Fig
in shows that the brain regions with the highest average absolute input matrix
*B* values corresponding to PCs 2, 4, and 6 reveal the same regions identified
in the somatomotor cortices using general linear model analysis of BOLD time
series for hand, foot, and tongue movements.
The input matrix *B* also captures the spatiotemporal relationship between the
inputs across different conditions. For instance, S9A Fig in shows that hand or
feet movements are associated with simultaneous positive and negative (e.g.,
inhibition or deactivation) inputs to the contra- and ipsilateral somatomotor
cortices, respectively. also shows that PCs 5, 1, and 3 reveal the temporal
order of inputs to visual, dorsal attention, and finally, somatomotor cortices
following the onset of visual cue. Note that the spatial and temporal profile of
PC 5 demonstrates the inverse relationship between inputs to visual and
somatomotor cortices. This unexpected temporal profile contributes to the low
similarity of PC 5 to task regressors in. We show that changing the delay
between estimated inputs and task regressors changes the coefficient patterns
with significant loading (S10 Fig). These results demonstrate an early positive
relationship of PC 5 input with visual cue blocks, followed by a later positive
(negative) relationship with left-hand movements (visual) blocks.
Finally, in we demonstrate that PCs 7, 8, and 9 are primarily associated with
the right foot movement blocks. However, the significantly smaller
*R*<sup>2</sup> values of these PCs than other PCs in indicates the lower
similarity of corresponding estimated inputs’ temporal profiles to those of task
regressors. Closer examination of these inputs’ spatiotemporal profiles reveals
that in addition to changes related to left-hand movements, these PCs capture
the rapid sequence of inputs to frontal and somatomotor cortices following the
motor task block’s offset and the baseline (i.e., no task) onset (S11 Fig).
Together, these results suggest that an LTI model of cortical dynamics can
reveal the unknown spatiotemporal profiles of the BOLD signal’s external task-
related drivers.
We provide additional analysis and discussion on model parameters and their
effect on the reported results in the in document. We explored sparsity
constraints on the system and input parameters in SI5. S12 Fig in demonstrates
that increasing the system matrices’ sparsity reduces the model’s goodness-of-
fit (measured using the AIC criterion). In the same vein, the increased
spatiotemporal sparsity of the inputs overall reduces the accuracy (measured
using the *R*<sup>2</sup> value of the linear regression) of the estimated
inputs (S13 Fig). Nevertheless, estimated inputs’ group-level PCA reveals that
the higher sparsity constraints can improve the accuracy of specific empirically
identified input patterns (S14 Fig). In addition, we examined the effect of the
estimation window’s size on the input’s accuracy in SI6. These results show that
a smaller estimation window (3 min) provide comparable results to the full-
length window, however overall it increases the accuracy of mean inputs to many
brain regions (S8 Fig) and several main input patterns (S15 Fig). Finally, we
explored the sensitivity of the identified input patterns to the factorizations
method in the SI7. These results demonstrate that PCA decomposition of the
model’s residuals reveals the analogous primary input patterns (S16 Fig)
uncovered by our spatiotemporal regularization scheme.
### 2.7.4 Non-stationarity of inputs to resting-state networks
So far, we showed that adopting a time-invariant model of the intrinsic
relationship between large-scale brain regions allows us to extract the unknown
external drivers of cortical dynamics. Our results demonstrate that the resting-
state paradigm serves as a viable option for a more accurate estimation of
internal system parameters. However, sensory and other extra-cortical inputs are
still present during resting-state scans, resulting in system parameters and
input estimation errors. Despite the estimation error in the external inputs’
profile, we hypothesize that quantifying the non-stationarity of the estimated
resting-state inputs provides information on the external factors that
contribute to resting-state BOLD signal non-stationarities.
We quantify estimated inputs’ non-stationarity for every brain region (i.e., *B*
× *U*) from the temporal fluctuations (i.e., standard deviation) of external
inputs’ means, measured using a sliding window. shows brain regions that exhibit
significantly high input means’ fluctuation across different sliding window
sizes (see methods for details). We demonstrate the results for sliding windows
of 6, 24, and 50 samples (TR = 0.72 sec) lengths and half window-length shifts.
We also measure the non-stationary of external inputs during resting-state scans
using the nonlinear measure developed by and find converging results. We find
several brain regions within DMN consistently display high non-stationarity
values. Statistical comparisons between the quantified non-stationarity of
estimated inputs to identified brain regions in reveal the significantly
(Welch’s *t*-test, *p* \< 0.05, Bonferroni corrected for multiple comparisons)
higher non-stationarity of external inputs to identified DMN regions relative to
several other resting-state networks (S17 Fig). Together, these results reveal
that time-varying external inputs may partly contribute to the previously
reported resting-state BOLD signal’s non-stationary, and the LTI model offers an
avenue to determine the spatiotemporal profiles of these unknown external
sources.
# 3 Discussion
Based on the theory of embodied cognition, the evolution and emergent function
of the brain can be best understood in the context of the body and its
interactions with the environment. In this view, the information does not exist
in an abstract form outside the agent, instead, it is actively created through
the agent’s physical interaction with the environment. Therefore, understanding
the native structure of the external inputs to the brain, as well as the
interaction between the brain and its exogenous drivers, is germane to
understanding the functional dynamics of the embodied brain.
What are the external drivers of BOLD signal? Current theories suggest that
cortical outputs reflect changes in the balance between the strong recurrent
local excitation and inhibition connectivity, rather than a feedforward
integration of weak subcortical inputs. Changes in this balance heavily affects
the local metabolic energy demands and consequently the regulation of cerebral
blood flow and the BOLD signal, despite the net excitatory or inhibitory output
of the circuits. Inhibition in principle can lead to both increases and
decreases in metabolic demands. Moreover, cortical afferents and microcircuits
can function as *drivers* by transmitting information about the stimuli, or
alternatively as *modulators* by modulating the sensitivity and context-
specificity of the response. Excitatory sensory information, transmitted mostly
via glutamatergic or aspartergic drivers, combined with the strong evoked
recurrent GABAergic interneurons are a major part of neurotransmission dynamics,
which in turn affect the local cerebral blood flow (CBF). Likewise, regulation
of cortical excitability mediated by neuromodulatory neurotransmitters including
acetylcholine, norepinephrine, serotonin, and dopamine can also significantly
effect CBF and the BOLD signal.
What do input parameters of an LTI model capture in BOLD fMRI? We show that an
LTI system acts predominantly as a high-pass filter and highlights the rapid
transient fluctuations in the BOLD signal. We provide evidence that the
influence of sensory inputs is identifiable in the estimated inputs to sensory
cortices. More importantly, the task-related changes that are temporally
decoupled from the sensory stimuli, such as the motor cortex’s activation
following the offset of visual cues and onset of behavioral outputs, are also
captured as external inputs to the LTI system.
Prior research has reported brain-wide and heterogeneous task-related changes in
the BOLD signal power spectrum and estimated system parameters. However, we
provide evidence that the time-varying unknown exogenous (i.e., extra-cortical)
inputs also likely contribute to non-stationarities in the cortical dynamics.
Specifically, we demonstrate in silico that determining the LTI system’s
parameters from periods with unknown stimuli can lead to high estimation errors
in system and input parameters. We verify these observations empirically by
showing that LTI system parameters identified from resting-state, instead of
task BOLD time series, result in notably more accurate identification of unknown
extra-cortical inputs’ spatiotemporal profiles in task scans. Our results have
implications for the common interpretation of correlation-based functional
connectivity changes as altered intrinsic relationships between regions. More
importantly, our findings highlight the importance of modeling and interpreting
the brain’s dynamic functional connectivity and non-stationarity as an open
system.
Can the brain during resting-state scans be fully described as a *linear* and
*time-invariant* system? Prior studies demonstrate that temporal fluctuations in
the BOLD signal (\< 0.1 Hz) cannot be fully attributed to linear stochastic
processes, and suggest that the nonlinearities in the BOLD signal could be
attributed to the presence of a strange attractor. Additionally, other
neuroimaging studies using paradigms such as “temporal summation” have more
directly probed the *system* and provide evidence of system nonlinearities.
Model-based approaches such as work by have concluded that nonlinear
transduction of rCBF to BOLD is sufficient to account for the nonlinear
behaviors observed in the BOLD signal. However, care should be taken in the
interpretation of these results as in the temporal summation framework, where
the profile of input is assumed to be known and is approximated by an abstract
stimulus representation. We believe our framework provides a novel avenue for
testing the system linearities through the examination of the estimated unknown
inputs in summation paradigms. Specifically, the delay between estimated and
known external inputs can be further leveraged to tease out the nonlinear
components of hemodynamic response function (e.g., vascular) from the neural
impulse response function.
Stationary signals are characterized by time-invariant statistical properties,
such as mean and variance. To date, several tests have been proposed to examine
the non-stationarity of BOLD time series and the presence of dynamic functional
connectivity, including test statistics based on the variance of the FC time
series, the FC time series’ Fourier transform, multivariate kurtosis of time
series, non-linear test statistics, and wavelet-based methods, among others.
These methods commonly compare measured properties between the time series of
empirical data and a suitable surrogate or null time series that is designed to
lack time-varying properties through non-parametric resampling, phase-
randomization, or generative models, and the choice of measured properties and
null models profoundly impact on the outcomes of stationarity tests in
conflicting reports on BOLD signal.
Notably, the presence of non-stationarity in the outputs does not directly imply
the underlying system’s non-stationarity. An LTI system’s outputs, for instance,
while receiving non-stationary external inputs, can also display time-varying
properties. As mentioned earlier, using internal system parameters of an LTI
system estimated over resting-state scans enables more accurate identification
of exogenous inputs’ spatiotemporal profile task scans. These results suggest
that a large-scale stationarity model of the brain with time-varying external
inputs can, in theory, account for a large portion of the observed task-related
changes in cortical dynamics. It is worth noting that any possible task-related
changes in the underlying system parameters are also captured as external inputs
in an LTI framework. Therefore, from the system identification and model-fitting
perspective, it is likely that a linear switching system with higher degrees of
freedom would improve the fit. Beyond the goodness-of-fit of the model, care
should be taken in interpreting the epiphenomenal large-scale models’ parameters
and their changes at the micro-scale biophysical level.
However, the impetus for this work is to highlight the estimates’ notable
sensitivity to the unknown, and thus, unaccounted external inputs. More
practically, when simulated with a wideband unknown external inputs, our results
suggest that an open LTI model estimated during resting-state allows us to
uncover the influence of these unknown drivers of BOLD dynamics. Nevertheless,
participants’ cortices receive external stimulation even during resting-state
scans, contributing to estimation inaccuracy and the system’s outputs’ non-
stationarity. In this work, we aim to disentangle the non-stationarity of the
*system* from its *outputs* over resting-state by examining estimated inputs’
non-stationarity. Our results show that external inputs’ non-stationarity over
resting-state scans are spatially inhomogeneous, with identified DMN regions
showing the highest levels consistently across different analyses. These
observations are in line with prior reports of higher dynamic functional
connectivity of these brain structures over rest. The identified non-stationary
inputs during resting-state scans also imply that we should expect more error in
the estimated spectral profile of the aforementioned regions. Therefore, future
work should explore leveraging other states of consciousness, such as sleep with
lower global cortical activity, to address this limitation. Despite the presence
of possible confounding factors such as unaccounted nonlinearities and non-
stationarities in the recording noise, our framework and observations provide
new insight into the external drivers of cortical dynamics and factors that
contribute to their non-stationarity. Recent system-identification and control-
theoretic work have also demonstrated the utility of a stationary system in
explaining BOLD dynamics. Together these findings pave the way for principled
model-based control of pathological brain dynamics, such as depression and
schizophrenia, using open-loop external or closed-loop neurofeedback
stimulation.
Historically, a narrow band of slow frequencies between 0.01 to 0.1 Hz was
thought to contain information relevant to underlying neural activity, and that
the higher frequency (\> 0.1 Hz) BOLD activity considered mainly as an artifact.
Our results also demonstrate that the primary oscillatory modes of the LTI model
of the resting-state BOLD display similar slow frequencies heterogeneously over
the brain. In addition, the hemodynamic response function (HRF) is also expected
to dampen the higher frequency neural activity significantly. More recent
evidence, however, portrays a broadband picture of BOLD signal fluctuations with
frequencies up to 0.25 Hz and even higher. We also provide converging evidence
that despite the expected low-pass filtering of HRF, information about the
stimulus-related activity can still be extracted from the BOLD signal even as
high as ≈ 0.4 Hz. Future work can leverage acquisition protocol with higher
sampling rates than HCP and rapid stimuli capable of inducing brain-wide
activations to accurately delineate the inputs’ attenuation profile by HRF at
higher frequencies. In line with previous reports of intrinsic functional
connectivity networks, our clustering analysis reveals the low dimensionality of
the system eigenmodes as the eigenvectors can be roughly grouped in a small
number of spatial patterns. However, we show that depending on the task, the
dimensionality of inputs can be high; for example, in the motor task with
multiple conditions, we identified task-specific inputs to different ROIs across
motor cortices. Future work should use our proposed framework to identify the
highest bound of input dimensionality using higher resolution parcellations or
voxel-wise modeling of the BOLD signal.
However, the HRF plays another critical role in biophysical models where it
enables the approximation of the latent neural states from the BOLD signal. This
is one of the main limitations of our simplified model, as it incorrectly
assumes that the BOLD signal in one region (instead of the underlying neural
activity) can cause changes in the BOLD signal in the connected regions. This
assumption for spatially inhomogeneous HRF functions can, in theory, lead to
incorrect identification of the external inputs’ focus and error in the
direction and speed of the interactions within functional networks. We believe
the overlapping patterns of inputs and the task activation maps identified using
the conventional univariate general linear model analysis suggest that the
above-mentioned error is likely tolerable. To improve the estimated unknown
inputs’ accuracy, future work should leverage the formulated quantitative
spatiotemporal models, or the more recent models informed by the precise
mechanisms of neurovascular coupling. Nevertheless, care should be taken in
these or other related deconvolution-based inferences, since as mentioned
earlier, they rely on the assumption of a known profile of HRF or inputs. Future
work can also leverage neural adaptation paradigms to influence the neural
response timing and help tease out the neural and vascular components’
contributions to the modeled inputs. Comparing our identified inputs with those
extracted from other neuroimaging modalities such as Magnetoencephalography
(MEG) that are more direct measurements of the underlying neural activity will
also us to further decouple the aforementioned mechanisms.
Structured recording noise such as autocorrelated noise can negatively impact
the modeled system, and the estimated input. Although we have included global
mean signal regression (GSR) as a preprocessing step to account for the shared
global noise that is present in many of the functional networks, our model is
unable to account for other unknown structured (e.g., autocorrelated) and time-
varying recording noise. Moreover, GSR may also introduce artifact, as in
addition to the shared noise, it also removes any global activation patterns
(e.g., vigilance or arousal) and can alter the correlation structure. These
limitations are the source of ongoing controversy around this noise reduction
method. Having weighed the potential drawbacks of GSR against the major concerns
regarding the significant global artifacts such as the cardiac and respiratory
noise, we adopted this preprocessing step. Nevertheless, it would be beneficial
to investigate the spectral profile of the global signal and the impact of GSR
on the estimated system and inputs’ spectral characteristics.
One of the current limitations of our proposed framework is that the estimated
inputs’ accuracy depends on the internal and recording noise levels. We show
that group-level analysis and repeated measurement designs are effective
strategies to increase signal-to-recording noise and to increase the estimated
inputs’ accuracy. In addition, although we can not accurately tease out the
contributions of internal noise from other sources of noise, our simulations and
experimental results suggest lower levels of internal noise relative to external
drivers in task fMRI. We draw this conclusion based on the relatively large
input estimation errors associated with system parameters identified during
external stimulation.
We used individual subjects’ resting-state datasets to identify the system
parameters for uncovering the unknown inputs from the BOLD signal. Although
beyond the scope of our current work, it is critical to comprehensively examine
the identifiability of the estimated system parameters across different scanning
sessions, types, and individuals. Subsequently, it would be interesting to
explore further the extent to which our proposed system identification framework
can highlight the shared features across subjects or increase the accuracy of
the subject-specific mapping of spatiotemporal dynamics.
Finally, it is worth highlighting that model-based *data-driven* methods such as
our proposed framework and the *hypothesis-driven* methods such as DCM are
complementary approaches, suited for interrogation of different aspects of
system and output dynamics. For instance, DCM can also be leveraged fruitfully
for a more accurate estimation of the system, and consequently, external input
parameters using highly controlled experimental designs with known external
input profiles. Though, as mentioned before, care should be taken in the
interpretation of the results produced by methods that incorporate priors, as
the boxcar regressors commonly used to model the profile of external inputs are
merely abstractions and do not account for other possible factors such as
anticipatory responses, adaptation, or other unknown drivers that shape the
profile of external inputs. However, data-driven approaches are particularly
advantageous when the brain is driven by extensive complex inputs, for instance,
during naturalistic stimuli (e.g., watching a movie), or in general, if we lack
a priori information or hypothesis on the structure of external inputs—for
instance, during the healthy resting-state or pathological brain activity such
as epileptic discharges.
# 4 Conclusion
We show that the proposed framework provides an avenue to uncover the structure
of the unknown drivers of BOLD signal fluctuations and shines light on factors
that contribute to its apparent non-stationarities. However, more significantly,
our results highlight the importance of modeling and interpreting the brain’s
dynamic functional connectivity as an *open* system. Broadly, our approach
provides a framework for understanding the brain’s large-scale functional
dynamics and non-stationarities, mechanistically via the modeled system and its
time-varying drivers.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
The prevalence of mouth breathing among children remains controversial but is at
most reported to be 50–56%. Mouth breathing is defined as using the mouth alone
or the mouth and nose instead of the nose alone for respiration for longer than
6 months. Mouth breathing is thought to be caused by mechanical factors such as
septal deviation and adenotonsillar hyperplasia, inflammatory diseases such as
allergic rhinitis, congenital malformation, and behavioral mouth breathing \[,
\].
The functions of the nasal cavity are air-conditioning, olfaction, and defense,
but mouth breathing causes environmental air to bypass these nasal functions,
allowing air to directly enter the lower respiratory tract, which can cause
airway hyperreactivity and chronic bronchial inflammation. Two case–control
studies showed that children suffering from asthma exhibit more mouth breathing
behaviors than controls. Meanwhile, a cohort study revealed that the risk of
otitis media with effusion is 2.4 times higher in mouth breathers than nose
breathers.
In addition, mouth breathing might be associated with skin diseases, given its
previously demonstrated relationships with periodontal disease and enlarged
tonsils. Periodontal disease is associated with chronic skin diseases such as
chronic urticaria, chronic pigmented purpura, and chronic nodular prurigo. Mouth
breathers had an increased risk of gingivitis in a case-control study, while
patients suffering from adenotonsillar hypertrophy, which is a cause of mouth
breathing, have an increased risk of periodontal disease, which improves after
adenoidectomy. In addition, Valera et al. reported that children aged 3–6 years
with enlarged tonsils had a significantly increased risk of mouth breathing.
Streptococcal tonsillitis is associated with psoriasis, while some reports
indicate tonsillectomy improves psoriasis.
However, no population-based studies have investigated the relationship between
mouth breathing and the prevalences of pediatric diseases, including atopic
dermatitis, which is a highly prevalent skin disease in children.
Accordingly, this study investigated the relationship of mouth breathing with
the prevalences of various diseases including atopic dermatitis by using a
questionnaire targeting preschool children in day nurseries.
# Materials and Methods
## Design, setting, and participants
Aimed to have a total of 600 respondents, we targeted preschool children aged
2–6 years who attended day nurseries in Tokushima City. The questionnaire was
distributed at 13 randomly selected day nurseries in Tokushima City. We
distributed anonymous questionnaires to the parents or guardians from November
27 to December 16, 2013. The questionnaires were submitted through collection
boxes in each day nursery.
This was a population-based cross-sectional study performed with the permission
of the Ethics Committee of the Tokushima University Hospital. After obtaining
written informed consent from the head of each day nursery, we distributed the
questionnaires to parents/guardians attached with an explanatory leaflet
specifying that their submission of the questionnaire was considered consent.
Within the leaflet, we also informed the parents/guardians that some, as yet
unknown, behavioral habits might contribute to the development of some diseases;
however, we did not provide information regarding the specific potential
associations.
## Questionnaire
The questionnaire included questions on the following: age, sex, smoking habits
of family members, behavioral habits, present and previous diseases, and present
and previous diseases of parents. A multiple choice question was used to collect
information about present diseases, including allergic rhinitis, chronic
sinusitis, asthma, chronic bronchitis, pollinosis, atopic dermatitis,
tonsillitis, otitis media, chronic headache, proteinuria, and hematuria. In
addition to the present diseases, previous diseases included acute sinusitis,
acute otitis media, chronic otitis media, pneumonia, and meningitis. As some
cases of allergic rhinitis are pollinosis, we treated pollinosis as allergic
rhinitis. Excluding mouth breathing, which is described in the next section, we
collected information about the following behavioral habits: regular bedtime and
rising time, sleeping hours, sleeping posture, pacifier use, and dietary habits
including mastication and food and drink preferences.
## Mouth breathing criteria
Although there is no widely adopted questionnaire for evaluating mouth
breathing, we prepared the following 3 items for detecting mouth breathers in
daytime (MBD): “breathes with mouth ordinarily,” “mouth is open ordinarily,” and
“mouth is open when chewing.” These items were developed with reference to
generally used methods including the Glatzel mirror, lip closure, and the water
test. “Breathes with mouth ordinarily” corresponds to the Glatzel mirror, which
judges mouth breathing by vapor emanating from the mouth using a mirror placed
below the child’s nose. “Mouth is open ordinarily” corresponds to lip closure,
which is determined according to soft contact between the upper and lower lips.
“Mouth is open when chewing” corresponds to the water test, in which children
hold some water in their mouth while keeping their lips closed without
swallowing for 3 minutes. The necessity of opening the mouth during mastication
suggests the child is not in the habit of complete nose breathing. Children who
met 0–1 and 2–3 of the above criteria were considered nasal breathers in daytime
(NBD) and MBD, respectively.
We adopted the following 3 items to detect mouth breathers during sleep (MBS):
“snoring,” “mouth is open during sleep,” and “mouth is dry when your child gets
up.” Snoring is significantly associated with MBD and MBS. Dry mouth is caused
by dry air passing through the mouth unless a loss of saliva occurs. Children
who met 0–1 and 2–3 criteria were considered nasal breathers during sleep (NBS)
and MBS, respectively.
For each item, we defined positive or negative choices for mouth breathing
before distributing the questionnaire. As shown in, the underlined and non-
underlined choices were defined as positive and negative for mouth breathing,
respectively. We did not indicate in the questionnaire which questions were used
to analyze mouth breathing or which choices were used to determine mouth
breathing.
Complete mouth breathers (CMB) were defined as children who met the criteria for
both MBD and MBS, partial mouth breathers (PMB) met the criteria for either MBD
or MBS, and complete nasal breathers (CNB) met the criteria for both NBD and
NBS.
Because nasal congestion, which is the main symptom of rhinitis and sinusitis,
is an important cause of mouth breathing, we included an item for “nasal
congestion”; it was assessed as negative only if “not blocked unless cold” was
selected and assessed as positive if “often blocked” or “always blocked” was
selected.
Genetic factors were considered positive if the parents selected the
corresponding disease from among present and/or previous diseases, because
atopic dermatitis and asthma improve naturally with age and allergic rhinitis is
classified as intermittent, seasonal, or persistent according to the nature of
the allergen. Smoking was categorized according to whether family member(s)
smoked around the children or not.
## Statistical analysis
Statistical analyses were performed by using SPSS version 21 (IBM Corp, Armonk,
NY, USA). Continuous variables are presented as mean ± standard deviation (SD)
or median and interquartile range (IQR).
For univariate analysis, categorical variables such as habits and disease
presence were analyzed by Pearson’s *χ*<sup>2</sup> tests or Fisher’s exact
tests where appropriate. The results are presented as odds ratios (ORs) and 95%
confidence intervals (CIs). To adjust the ORs of MBD and MBS with respect to
disease prevalence, the Mantel–Haenszel test was performed using variables with
*p* \< 0.25 in univariate analysis as potential confounders.
For multivariate analysis, forward stepwise multiple logistic regression was
performed by using breathing pattern (i.e., MBD or MBS) as the dependent
variable and categorical variables showing *p* \< 0.25 in univariate analysis as
independent variables. Bonferroni corrections for multiple comparisons were made
as a *post hoc* analysis. The level of significance was set at *p* \< 0.05
except in cases of multiple comparisons, in which *p* \< 0.016 was used.
We used the phi coefficient to indicate the effect size in the Pearson’s
*χ*<sup>2</sup> test. For statistical power, we performed *post hoc* power
analysis using G\*power (ver. 3.1.9.2, Erdfelder, Faul and Buchuner, Germany).
# Results
We distributed questionnaires to 1036 subjects from November 27 to December 16,
2013 and collected 552 responses for a response rate of 53.3%. No reminder
letters were distributed. Thirteen responses were excluded because of age: 11
were outside the target age range and 2 had no age given. A further 71 responses
were excluded because of at least one unanswered question. Therefore, a total of
468 valid responses were collected for a response rate of 45.2%.
The subjects’ characteristics are shown in. Mean age was 4.5 ± 1.2 years with a
median of 4.5 years (IQR: 3.4–5.5 years). The subjects were divided into 3
groups according to the main diseases reported: atopic dermatitis, asthma, and
allergic rhinitis. Children with atopic dermatitis were significantly associated
with a history of asthma and/or allergic rhinitis. In contrast, asthmatic
children were significantly associated with a history of allergic rhinitis,
atopic dermatitis, and/or pneumonia. The most afflicted children were those with
allergic rhinitis, who had a history of all documented conditions except
tonsillitis. Each group had a family history of their condition, and the atopic
dermatitis group was also significantly associated with a family history of
allergic rhinitis.
The results show that 57.5% of the children had substantial nasal breathing
difficulties, including CMB (23.9%) and PMB (33.5%). The symptoms appeared to be
more frequent at night; 45.9% were MBS compared to 35.5% who were MBD.
Atopic dermatitis was significantly associated with both MBD and MBS (*p* =
0.001 and *p* = 0.002, respectively). Asthma was significantly associated with
only MBD (*p* = 0.013), while allergic rhinitis was significantly associated
with both MBD and MBS (*p* = 0.035 and 0.006, respectively). Nasal congestion
was significantly associated with a risk of all 3 diseases, which was considered
a confounder. Also, nasal congestion, which was present with \[4/97 (4.1%)\] and
without \[1/371 (0.3%)\] chronic sinusitis, was associated with chronic
sinusitis (OR: 15.9, 95% CI: 1.8–144.1, *p* = 0.007, Fisher’s exact test).
According to the results of the univariate analysis, we adopted previous disease
(i.e., asthma and allergic rhinitis), family history of disease (i.e., atopic
dermatitis, asthma, and allergic rhinitis), and nasal congestion as confounding
factors for atopic dermatitis. Meanwhile, previous disease (i.e., atopic
dermatitis, allergic rhinitis, and pneumonia), family history of disease (i.e.,
asthma), and nasal congestion were adopted as confounding factors for asthma.
Because nasal congestion can induce mouth breathing and is one of the main
symptoms of allergic rhinitis, we excluded allergic rhinitis from subsequent
analyses. After adjusting for confounders, atopic dermatitis was significantly
associated with risks of both MBD (OR: 2.6, 95% CI: 1.3–5.4, *p* = 0.010) and
MBS (OR: 4.1, 95% CI: 1.8–9.2, *p* = 0.001), although asthma was not
significantly associated with a risk of MBD (OR: 1.3, 95% CI: 0.7–2.7, *p* =
0.508). Multiple logistic regression for atopic dermatitis was subsequently
performed by including the categorical variables listed above. The ORs of MBD
and MBS for atopic dermatitis were 2.2 (95% CI: 1.2–4.2) and 2.7 (95% CI:
1.4–5.3), respectively; these values were lower than those for family history of
atopic dermatitis and history of asthma and allergic rhinitis. Nasal congestion
and parental history of asthma and allergic rhinitis were not significant risk
factors.
The effect sizes of MBD and MBS for atopic dermatitis were 0.149 and 0.141,
respectively, and 0.897 and 0.862, respectively, represented the statistical
power in the *post hoc* analysis, indicating sufficient power.
Atopic dermatitis was present in 7.0% of children with CNB (n = 199), 12.7% of
children with PMB (n = 157), and 22.3% of children with CMB (n = 112) (*p* \<
0.001). The prevalence of atopic dermatitis in CMBs was higher than that in PMBs
(*p* = 0.038), but the difference was not significant after Bonferroni
correction.
# Discussion
The results of the present study indicate mouth breathing is significantly
associated with atopic dermatitis and asthma. In particular, atopic dermatitis
was significantly associated with both MBD and MBS after adjusting for
confounding factors. Moreover, the prevalences of these diseases showed an
increasing trend with an increasing extent of mouth breathing. To our knowledge,
this is the first study to evaluate the impact of breathing patterns on the
prevalence of atopic dermatitis. Although two studies report that mouth-
breathing children with positive skin-prick test results have higher prevalences
of asthma and sleep apnea than those with negative results, they did not compare
mouth breathers with nasal breathers.
No previous study has analyzed the prevalence of mouth breathing among Japanese
children. In the present study, the prevalences of MBD and MBS were 35.5% and
45.9%, respectively; however, these results were based on a questionnaire that
has not been assessed for validity. The studies from other countries report a
wide range of the prevalence of mouth breathing, 4–56%. Brazil has the highest
prevalence, which exceeds 50% based on clinical assessment. On the other hand,
the lowest prevalence is in India, at 4–7% based on clinical assessment. The
prevalences of mouth breathing in England and New Zealand are 23% and 19%,
respectively. However, direct comparison among studies is difficult owing to
varying criteria. Standardized criteria for mouth breathing using clinical
assessment and questionnaires are required to more precisely investigate
differences in mouth breathing prevalence.
Atopic dermatitis is a chronic skin disorder characterized by pruritus and
inflammation that mostly develops during childhood and is strongly associated
with the allergic history of patients and relatives. Filaggrin, which is encoded
by the *FLG* gene, is a crucial protein for skin barrier function. Two meta-
analyses show that children with an abnormal *FLG* gene are 3.12–4.78 times more
likely to have atopic dermatitis than normal subjects. On the other hand, the
prevalences of chronic pediatric diseases, including atopic dermatitis as well
as asthma and allergic rhinitis, vary greatly worldwide. The prevalences of
these diseases have increased in the late 20<sup>th</sup> century, although this
trend has not held true in the last 10 years. These findings suggest that, in
addition to genetic factors, environmental factors play important roles in these
diseases.
This was a population-based cross-sectional study; therefore, causal
relationships cannot be determined. However, if mouth breathing is shown to
contribute to atopic dermatitis in future cohort studies, guidance to avoid
mouth breathing should be provided to children and parents/guardians to prevent
atopic dermatitis. Furthermore, otolaryngologist help should be considered, as
necessary, if children find it difficult to quit mouth breathing.
It is possible that periodontal disease and/or tonsillitis might mediate the
mechanism underlying any association between mouth breathing, as an
environmental factor, and atopic dermatitis. Satoh et al. suggested that immune
reactions mediated by bacterial–immune complexes, superantigens, or toll-like
receptors might induce skin diseases. However, the present questionnaire did not
collect periodontal information. Furthermore, although the present results
showed no significant relationship between history of tonsillitis and the
prevalence of atopic dermatitis, inquiring about the number of repeated
tonsillitis episodes might have revealed an association. On the other hand, it
is possible that children could have mild nasal congestion unnoticed by their
guardians. Thus, case–control studies with otolaryngological diagnostic tests
are required to confirm the relationship between mouth breathing and atopic
dermatitis. Furthermore, sleep disturbance due to the intense pruritus of atopic
dermatitis can cause daytime sleepiness, which might in turn cause behavioral
mouth breathing.
According to the Japanese Ministry of Health, Labour, and Welfare, the
prevalences of atopic dermatitis in 3- and 6–7-year-old children are 13.2% and
11.8%, respectively. Meanwhile, the prevalence of asthma in a study of 34,699
children aged 4–5 years at randomly selected nurseries in Japan was 11.2%, and
the lifetime prevalences of atopic dermatitis, asthma, and allergic rhinitis
were 16.0%, 16.1%, and 17.6%, respectively. In the present study, the
prevalences of atopic dermatitis, asthma, and allergic rhinitis were 12.6%,
9.8%, and 13.0%, respectively; the lifetime prevalences were 15.4%, 14.5%, and
18.2%, respectively. Thus, the present results are similar to those of previous
studies, suggesting the subject group is representative of Japanese children
aged 2–6 years.
In this study, 45.9% of children had family members who smoked. This is similar
to the prevalence of smokers among Japanese men and women 20–40 years old: 40%
and 12%, respectively. However, having a family member who smoked was not
associated with disease prevalence. Possible reasons for this are the low rate
of smokers who smoke around their children (7.1%) and parents who stop smoking
when their child develops a disease.
There are some limitations in this study, as already mentioned. First, the
diagnoses of atopic dermatitis and mouth breathing were dependent on the
questionnaire results. Because we created the questions and criteria for mouth
breathing, the lack of experimental data and questionnaire validity limits the
strength of our findings. Second, the valid response rate was comparatively low
at 45.2%. Third, as this was a cross-sectional study, causal relationships
cannot be determined. Although we cannot exclude facial injuries affecting nasal
breathing, such cases would be rare and are unlikely to affect the results. The
effects of common cold and flu appear quite small, because we inquired about the
normal state of the children in the questionnaire. Finally, regarding genetic
factors, only the parents’ information was collected and not that of sibling or
grandparents. Therefore, genetic factors may have been underestimated.
# Conclusion
Mouth breathing is significantly associated with atopic dermatitis in Japanese
preschool children aged 2–6 years. Additional case–control and cohort studies
are required to confirm this relationship. Furthermore, studies targeting school
children and adults would also help clarify this association.
# Supporting Information
We thank the heads and staff members of the day nurseries for their cooperation.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: HY KT. Performed the
experiments: HY ST YN SK RT SY NS MK. Analyzed the data: HY SK RT SY NS MK
KT AT. Contributed reagents/materials/analysis tools: KT. Wrote the paper:
HY TS KT. |
# Introduction
Theoretical work in social psychology frequently seeks to integrate phenomena
that are otherwise studied separately. One recent effort in this regard, the
Balanced Identity framework proposed by Greenwald and colleagues, builds on
classic theories of cognitive consistency, by arguing that constructs such as
attitudes, identification, and self-esteem reflect a coordinated set of
associative relationships in semantic space. The framework makes formal
predictions regarding how such constructs should relate, making it
straightforward to test and extend in new directions. In particular, it provides
a means of testing the ideas that intergroup bias emerges as a natural outgrowth
of self-related positivity interacting with group identification.
More specifically, Balanced Identity entails the claim that ingroup preference
must be understood in the context of several interrelated cognitive constructs,
namely self-esteem and ingroup identification. These three constructs are
conceptualized as occupying a triangular constellation of mutual associative
influence (depicted). This cognitive structure allows a specific set of
predictions to be made regarding the expected relationships among the
constructs: any one construct can be predicted from the product of the other
two. That is, one can predict the strength of a *group attitude* from the
product of *self-esteem* and *identification* with that group, *self-esteem*
from the interactive effect of *identification* and *attitude*, and so on.
Because the model purports to describe patterns based on an associative memory
system, it is suggested that these relationships will be most prevalent when
constructs are themselves measured at the implicit or associative level, and
will not necessarily apply to more explicitly held, propositional forms of
information. Recent meta-analytic evidence provides strong support for the
presence of these relationships across multiple attitude and stereotype domains.
So far, the Balance framework has been used to examine attitudes and stereotypes
towards a range of groups, but primarily ones that are enduring and central to
mature identity, such as gender and race/ethnicity. Capturing attitudes and
identifications in these domains is a crucial test of model fitness, but because
individuals have usually been members of these groups for a long time, we know
very little about how the consistency processes postulated by Balanced Identity
initially form. Is consistency the result of a protracted process of dissonance
reduction in which inconsistencies are gradually reduced as related concepts are
repeatedly co-activated? Alternatively, when a new attitude object (such as a
social group) is encountered and evaluated, is the form of that evaluation
immediately constrained by the strength and direction of existing cognitions
(such as self-esteem), such that the new cognition is, from its genesis, in
balance with its neighbors? Long-standing social affiliations cannot be used to
address this question, because consistency observed long after their acquisition
could be the result of either process. At present, the shortest time scale that
has been investigated is about a week, in the specific case of attitudes towards
residential colleges at a university.
The current research seeks to explore this problem space by asking whether
attitudes and identifications learned just minutes before, within the context of
the experimental setting, conform to the predictions of the Balanced Identity
model. Participants are assigned to previously unfamiliar ‘minimal’ social
groups, and self-esteem, (minimal) group attitude, and (minimal) group
identification are assessed. If these cognitions conform to the predictions of
Balanced Identity, it would favor the possibility that cognitive consistency
emerges immediately, without requiring a gradual period of dissonance reduction.
At a broader level, this inquiry can be considered a test of the generality of
the Balance framework: Does it appear only with respect to highly familiar,
personally important and culturally salient social groups, or can we observe it
from the earliest moments of social affiliation, even with previously unfamiliar
and minimally meaningful social groups?
The present work also addresses a second question regarding how the Balance
Framework should be interpreted. All current investigations have been
correlational designs, demonstrating that the posited pattern of relationships
does exist with respect to real-world social groups. But the theory presumes to
go beyond description; it posits that, for a given association, surrounding
associations are, at least in part, *causally constitutive* of it. Testing this
causal assumption requires moving beyond the correlational framework and
directly manipulating constructs within the Balance framework to see if that
manipulation affects neighboring constructs in the predicted directions. Thus,
in addition to testing whether cognitive balance appears with newly encountered
“minimal” social affiliations, in a follow-up series of experiments, each of the
three “legs” of the Balance triangle is manipulated independently in either the
positive or negative direction, and the effect of that manipulation on related
constructs is measured. This design allows us to test two corollary questions.
First, does cognitive balance survive the direct manipulation of one of the
cognitive constructs? An affirmative answer would again suggest that the balance
relationships are rapidly emergent. Second and relatedly, several studies have
suggested that balance does not emerge when the ingroup in question is less
positively evaluated overall, for example when the ingroup is stigmatized, lower
status, or more ambivalently viewed with respect to valence. That raises the
possibility that balance will be particularly disrupted when we manipulate
constructs in the negative direction, e.g. by artificially reducing ingroup
preference. Observing this in the present context would help to establish the
generality of that previously observed phenomenon.
The Minimal Groups Paradigm (hereafter MGP) has established that participants
consistently show preferences for previously unfamiliar, randomly assigned
ingroups, and that these preferences occur across substantial methodological
variation and on both self-report and implicit measures. Because prior knowledge
is controlled for through the novelty of the grouping dimension, the
psychological consequences of “mere membership” can be directly assessed,
providing a window into the generalized cognitive processes that underlie
responses to group boundaries. This strategy has already proven valuable,
showing, for example, that the tendency to show better recall for ingroup faces,
to associate anger with outgroups, and to show signature neural responses to
outgroup faces are all general intergroup responses that emerge in similar
fashion for highly familiar groups as well as minimal groups and that appear
early in development, suggesting that it is a reflection of core intergroup
processes. In the current context, the question is whether the patterns of
relationships described by Balanced Identity emerge outside the context of well-
established and socially meaningful social collectives, i.e. in the minimal
groups setting.
## Overview of the present research
In Experiment 1, participants are randomly assigned to a minimal social group,
implicit and explicit group attitudes, group identification, and self-esteem are
assessed, and the predictions of Balanced Identity regarding the relationships
between the three constructs are tested. I predicted that cognitive balance
would be present, supporting the possibility that Balanced Identity can serve as
a general explanatory theory of intergroup bias, and that the relationships it
describes emerge immediately and automatically in newly formed cognitions.
In Experiments 2–4, each of the three “legs” of the balance triad are
manipulated prior to measurement. That is, each study experimentally manipulates
one of group identification, group attitude, or self-esteem in either a positive
(increasing strength) or negative (decreasing strength) direction. Manipulating
all three constructs provides for an examination of the possibility that some of
the links are more or less susceptible to manipulation than others. Most
notably, at least within a minimal groups setting, self-esteem has a different
status than identification with or attitudes towards a minimal group in that it
is precedes the experimental setting. Indeed, positive implicit self-esteem is
enduring, strong, and broadly present across persons and cultures. By contrast,
within the minimal groups setting, identification and attitude are emergent
phenomena not supported by a long history of activation and/or reinforcement.
This might make self-esteem more resistant to influence via manipulations of the
others constructs, or it could make self-esteem more difficult to manipulate
more generally and thus a less reliable inroad into affecting the other elements
of the balance constellation. Thus, manipulating the identification with a
social ingroup, for example, might be more likely to affect attitudes towards
that group than self-esteem itself. Across these three experiments, I predicted
that manipulations of one component of balance would affect the others,
providing support for a causal reading of Balanced Identity.
# Experiment 1: Balanced Identity in the Minimal Group Paradigm
## Materials and Methods
### Participants
All research reported in this paper was approved by the University of
California, Merced Institutional Review Board. Ninety-seven participants,
recruited from a university research study pool composed of undergraduate
students participating for credit towards a course requirement in one of several
psychology courses, participated in Experiment 1. Participants were highly
diverse in terms of self-reported race/ethnicity (Asian = 40%, Hispanic = 33%,
White = 19%, Black = 7%). Computer failure led to the elimination of data from
seven participants prior to any analyses.
### Procedure
Participants were greeted by a research assistant who secured written, informed
consent and then escorted them to a private lab room. The participants were
seated alone at a personal computer. The entire procedure took approximately 20
minutes, at which point they were debriefed and released.
### Minimal group induction procedure
The experiment always began with the minimal group induction procedure, modeled
after the recommendations provided by Pinter and Greenwald, who directly
compared several minimal groups induction procedures. Participants read a short
paragraph indicating that the study involved two groups (the “Copley group” and
the “Dawson group”), one of which they belonged to. They would next be
introduced to the names of the members of their ingroup, which they had to learn
in order to complete the later portions of the experiment. Participants then
observed a static display listing the names of six members of their ingroup for
45 seconds. Following that, participants completed a name assignment task in
which names appeared in the middle of the screen and had to be assigned to
either the Copley or Dawson group by pressing one of two keys (the ‘e’ or the
‘i’ key), which were indicated on the screen next to the appropriate group
label. For the initial 40 trials, participants were provided with a color cue
indicating the group membership of each name, making categorization
straightforward, but in a subsequent 30 trials, the color cue was removed so
that participants would be encouraged to remember the names. Error feedback was
presented when a name was miscategorized, and participants had to correctly
categorize the name before continuing to the next trial. This procedure produced
acceptable accuracy at group categorization (mean accuracy across the last 30
trials in all studies was greater than 93%). While participants always learned
ingroup names first, the ingroup/outgroup name and the pairing of names to
groups were counterbalanced across participants. This completed the minimal
group induction procedure.
### Measures
The primary dependent measures were three Implicit Association Tests. The IAT is
a dual categorization task in which, in the critical blocks, participants
alternate between two categorization tasks involving four total target
categories, but respond using only two response keys. The logic of the task is
that if cognitively associated categories share a response key, responses will
be facilitated, resulting in faster responding and less errors. For example,
when categorizing positive words and words relating to the self with one key,
and negative words and words relating to others with the other key, responding
will generally be faster because of the cognitive congruence between positive
words and a positively evaluated self. When the opposite pairings needs to be
made (i.e., self paired with negative words, other paired with positive words),
responding will generally slow down and the error rate will generally increase.
The IAT is now the most well-validated measure of implicit attitude.
The three IATs employed here were a group attitude IAT (contrasting Copley names
and Dawson names as well as positive and negative adjectives), a group
identification IAT (contrasting Copley and Dawson names as well as words
relating to self and other, such as “me”, “my”, “I” versus “they”, “their”, and
“them”), and a self-esteem IAT (contrasting self and other words as well as
positive and negative adjectives). Because the strength of observed associations
might depend on the temporal relationship between the IATs and the induction
procedure, the two group-relevant attributes (attitude and identification) were
measured prior to self-esteem. Thus, participants completed the attitude and
identification IATs first (with task order counter-balanced across participants)
followed by the self-esteem IAT. After the implicit measures, participants
completed a short battery of self-report measures corresponding to the same
three constructs of group attitude, group identification, and self-esteem.
Following Pinter & Greenwald, these questions involved reporting on their liking
for each of the two groups (e.g., “I like the Copley group”), their
identification with each group (e.g. “I identify with the Dawson group”), and
their liking for the self versus others (e.g. “I like myself”). To facilitate
comparison with the IAT, difference scores were produced such that positive
numbers indicate greater liking for and identification with the ingroup and
greater liking for the self. The order of these measures was matched to the
order of the IATs described above.
## Results
All data reported in these experiments are available online via the Open Science
Framework, hosted at <https://openscienceframework.org/project/Vvo9A>.
### Descriptive statistics
Following prior work with the IAT, response latencies greater than 10,000
milliseconds (ms) or less than 400 ms were dropped, and participants with
excessive extremely fast trials (\>10% of trials \<300 ms) were dropped
entirely; such participants are generally rapidly pressing buttons without
responding to task instructions. These criteria led to the exclusion of four
participants from the attitude and identity IATs and eight participants from the
self-esteem IAT. Response time data were then used to calculate an effect size,
the IAT *D*, reflecting the relative speed advantage in one condition. *D* was
used in all analyses reported below, but for ease of interpretation we also
include Cohen's *d* of the simple effects when reporting descriptive statistics.
There were no main effects of group (Copley or Dawson), name-group pairing, or
explicit group assignment on any dependent measures, so these factors were
dropped from preliminary analyses. Beginning with the implicit measures,
preference for the ingroup was robust, *D* = .29 (.36), *t*(85) = 7.37,
*p*\<.001, *d* = .83, as was identification with the ingroup, *D* = .47 (.41),
*t*(85) = 10.50, *p*\<.001, *d* = .93. Implicit self-esteem was strong and
positive, *D* = .53 (.28), *t*(81) = 17.23, *p*\<.001, *d* = 1.18. Consistent
with the notion that the MGP is essentially a manipulation of ingroup
identification, identification as measured by the IAT was stronger than attitude
as measured by the IAT, paired *t*(81) = 3.56, *p*\<.001. Preference for and
identification with the ingroup were modestly correlated, *r*(80) = .35,
*p* = .001, but self-esteem correlated with neither identification, *r*(78)
= −.15, *p* = .18, nor preference, *r*(79) = .05, *p* = .65.
Evidence for an effect of group membership was also present at the self-report
level. Participants expressed more liking for the ingroup, *M* = .88 (1.7),
*t*(89) = 4.92, *p*\<.001, *d* = .52, as well as greater identification with
the ingroup, *M* = 1.56 (2.1), *t*(89) = 6.95, *p*\<.001, *d* = .74
Participants also reported somewhat greater liking for the self as compared with
others, *M* = .28 (1.2), *t*(89) = 2.17, *p* = .03, *d* = .23. Correlations
among self-report measures were consistently present, all *r*(88)\>.28,
*p*\<.01. Strongest was the correlation between attitude and identification,
*r*(88) = .66, *p*\<.001. Implicit and explicit measures did not correlate, all
\|*r*\|\<.18, *p*\>.12.
### Balanced Identity Analyses
Following the original formulation of Balanced Identity, evidence of balance is
assessed for each of the three possible regression models created by predicting
each construct from the other two. This involves first modeling each criterion's
association strength from the product of the other two, and then in a second
step entering the two predictors as main effects. The prediction is that the
addition of these two main effect terms will not increase the predictive power
of the model because the relationship is wholly accounted for by the product.
This produces four tests that each model can be assessed against:
1. The regression coefficient associated with the interaction should be
numerically positive and statistically significant at Step 1. A failure at
this step indicates that the primary prediction of the theory has not been
confirmed, and testing often ceases at this point. However, if this
prediction is upheld then:
2. the coefficient associated with the interaction should remain
numerically positive at Step 2 (after the main effect terms have been
added);
3. neither regression coefficient associated with the main effect terms
should statistically differ from zero at step 2; and
4. the increase in criterion variance (*R<sup>2</sup>*) at Step 2 should
not be statistically significant.
Thus, these four tests can be used for each of the three regression models,
providing 12 total tests that summarize the extent to which the predictions of
cognitive balance are met in a given data set (independently for self-report and
implicit data).
Results of these analyses are presented in for both implicit and explicit data.
For implicit data, evidence of balance was uniformly high, with all three models
passing all four tests described above, except for one partial failure at Step 2
in which, for one model, one of the two main effect terms was statistically
significant despite the presence (and continued statistical significance) of the
interaction term. Nonetheless, 11 of 12 possible tests were passed, providing
strong evidence of cognitive balance. For explicit results, evidence of balance
was mixed, with all three models failing at least one test at Step 2 (in total,
eight of 12 tests were passed). Thus, even though explicit measures were
strongly correlated at the bivariate level (considerably more so than their
implicit counterparts), they did not conform as well to the more complex pattern
of relationships specified by the Balanced Identity model, replicating much
prior work with this design.
## Discussion
Upon inducing associations between participants and one of two novel groups,
robust preference for and identification with the ingroup emerged, especially
when measured at the implicit level. These findings add another replication to
the large body of work employing the MGP. The novel contribution, however, is
the demonstration that the predictions of Balanced Identity are satisfied by
emergent cognitions in the MGP and therefore need not rely on an iterative or
otherwise protracted period of enculturation or experience.
The fact that new group-related attitudes and identifications formed in such a
way as to be in balance with pre-existing associations (i.e., self-esteem)
suggests that the Balanced Identity relationships can thought of as causal
determinants of the strength and direction of newly formed associations such as
group attitudes. The next set of studies directly tests causality by
manipulating each construct and measuring the effect of this manipulation on
related constructs.
# Experiment 2: Manipulating Group Identification
This experiment follows a nearly identical procedure to that described in
Experiment 1, except that, following group assignment, the strength of the
associative self-group relationship is manipulated. Because Balanced Identity
emerges so much more consistently at the implicit level, a manipulation of group
identification that also targeted associations (as opposed to the explicit
reporting of a self-group relationship) was employed. The central question,
then, was whether manipulating identification will affect group attitude and/or
self-esteem, and what effect, if any, this will have on the emergence of
cognitive balance. This experiment also begins to explore a secondary question.
Some prior work has suggested that balance does not emerge as reliably for
members of lower status groups, such as members of less prestigious residential
colleges or ethnic minorities. This pattern can be formalized as the prediction
that balance will not be as robust for groups that are less positively
evaluated. The current manipulation involved artificially increasing group
identification in half the participants, and artificially decreasing it in the
other half. If the phenomenon described above is general, we could see a
disruption of balance specifically in individuals in the latter condition.
## Methods
### Participants
One hundred twenty-two participants were recruited from the same population and
following the same procedure described in Experiment 1.
### Procedure
The procedure was closely modeled after that employed in Experiment 1;
participants were assigned to groups in the same way as described there. They
completed a manipulation of group identification followed by the same dependent
measures as in Experiment 1.
### Manipulation of group identification
The self-group association was manipulated using a partial-IAT procedure.
Participants were told that they were to imagine that they had the opportunity
to spend a large amount of time with one of the two groups (the ingroup or the
outgroup, as a between-participants factor). To simulate this experience, they
would perform a categorization task in which they would categorize words related
to the self with members of that group and words related to others with members
of the other group. They completed two blocks of 60 trials in which they either
repeatedly responded to self and ingroup names using one key and other and
outgroup names using another key, or repeatedly responded to self and outgroup
words using one key and other and ingroup words using another key. Thus, as a
between-participants factor, the manipulation was designed to either bolster the
self-ingroup relationship or weaken that relationship while bolstering the self-
outgroup relationship. To avoid associating any response with a particular side
of the screen, the pairing of group and self was counterbalanced across the two
blocks of trials.
### Measures
The same measures used in Experiment 1 were used here, namely implicit and
explicit measures of group attitude, group identification, and self-esteem.
## Results
### Descriptive statistics
Standard exclusion criteria for IAT results led to the elimination of data from
10 participants from the attitude and identity IATs and 16 participants from the
self-esteem IAT. There was one significant effect of ingroup name (Copley or
Dawson), with stronger ingroup preference for participants assigned to the
Copley group, *t*(109) = 2.84, *p* = .005. However, because this was the only
effect of group name across the four experiments reported here and because it
did not interact with other reported findings, it is not interpreted further.
Overall patterns of results were highly similar to Experiment 1, with
participants exhibiting robust preference for the ingroup, *D* = .30 (.37),
*t*(110) = 8.34, *p*\<.001, *d* = .78, robust identification with the ingroup,
*D* = .49 (.36), *t*(111) = 14.42, *p*\<.001, *d* = 1.06, and strong implicit
positive self-esteem, *D* = .47 (.30), *t*(103) = 15.91, *p*\<.001, *d* = 1.18.
As in Experiment 1, identification was stronger than attitude, paired *t*(108)
= 4.65, *p*\<.001. Preference for and identification with the ingroup were
modestly correlated, *r*(107) = .35, *p*\<.001, but again self-esteem as not
correlated with attitude, *r*(101) = .13, *p* = .21, or identification,
*r*(102) = .13, *p* = .20. Evidence for an effect of group membership was also
present at the self-report level. Participants expressed more liking for the
ingroup, M = .70 (1.7), *t*(121) = 4.50, *p*\<.001, *d* = .41, as well as
greater identification with the ingroup, M = 1.3 (2.4), *t*(121) = 5.95,
*p*\<.001, *d* = .54. Participants also reported somewhat greater liking for the
self as compared with others, M = .47 (1.2), *t*(121) = 4.39, *p*\<.001,
*d* = .39. Correlations among self-report measures were moderate, all *r*(120)
\>.39, *p*\<.001. Strongest was the correlation between attitude and
identification, *r*(120) = .59, *p*\<.001. Implicit and explicit measures did
not correlate, all \|*r*\|\<.11, *p*\>.25.
### Effect of identification manipulation
As noted above, half of participants completed a manipulation designed to
strengthen the self-group relationship (the “match” condition), while the other
half completed a manipulation designed to weaken it (the “mismatch” condition).
The manipulation was effective; participants in the match condition exhibited
markedly stronger ingroup identification as measured by the IAT,
*M*<sub>MATCH</sub> = .70 (.27), *M<sub>MISMATCH</sub>* = .29 (.32), *t*(110)
= 7.38, *p*\<.001, *d* = 1.4, though identification remained positive and
significant even in the mismatch condition, *t*(57) = 6.98, *p*\<.001. This
manipulation also affected ingroup attitude, *M*<sub>MATCH</sub> = .37 (.37),
*M<sub>MISMATCH</sub>* = .22 (.39), *t*(109) = 2.20, *p* = .03, *d* = .41, but
did not affect self-esteem, *M*<sub>MATCH</sub> = .49 (.27),
*M<sub>MISMATCH</sub>* = .45 (.33), *t*(102) = .61, *p* = .54, *d* = .13. The
manipulation had no effect on any of the explicit measures, all *t*\<.72,
*p*\>.47.
### Balanced Identity Analyses
provides a summary of results of the Balanced Identity analyses for implicit and
explicit data, collapsing across condition. Overall, evidence for balance was
strong at the implicit level but weak at the explicit level, with 12 and 7 tests
passed, respectively. To examine whether the relative degree of balance differed
depending on experimental condition, and in particular whether balance was less
robust in the mismatch condition in which identification was reduced, these
analyses were also conducted separately for each condition. However, one
potential limitation of such an approach must be noted. By intervening on group
identification in either the positive or negative direction, we may have
artificially reduced the variability in that measure, which would work against
finding evidence of balance on statistical rather than conceptual grounds. Thus,
the following analyses should be interpreted with this in mind and in the
context of supplementary analyses presented in the next section.
provides these analyses sub-divided by condition (match or mismatch). However,
when broken down by condition the results are more nuanced. Beginning with the
implicit data, evidence for balance was once again strong in the match
condition, with 12 out of 12 tests successfully passed. The results were quite
different in the mismatch condition. All three models failed at Step 1,
indicating that the interaction did not predict the criterion, nor did it do so
at Step 2 when main effects were included. Thus, there was no evidence of
balance when group identification was manipulated in the negative direction. The
results from explicit data, while not providing strong evidence of balance, were
relatively consistent across the two conditions, with 8 of 12 total tests passed
for data from the match condition and 6 of 12 from the mismatch condition.
### Supplementary analysis: Comparison with Experiment 1
Because our design did not feature a control group for whom attitudes were not
manipulated, the relative degree of change from baseline in the match versus
mismatch condition is difficult to ascertain. However, Experiment 1 resembles
such a control group, in that the design was identical to Experiment 2 except
there was no manipulation of the identification construct. Indeed, the means
across these two experiments (collapsing cross condition in Experiment 2) were
highly similar, and did not differ significantly, all *t*\<1.37, *p*\>.17. Thus,
under the assumption that the means in Experiment 1 are a reliable estimate of
what we would have observed in Experiment 2 if there had been no manipulation,
we can gain some insight into whether one condition was a more powerful elicitor
of change by comparing Experiment 1 to the two conditions of Experiment 2. We
can also use this analysis to explore the issue of reduced variability described
above by comparing variances directly to see if variation was reduced with
respect to any of the constructs. Results are presented in, showing the average
change and associated standard errors for the comparisons between Experiment 1
and the match and mismatch conditions of Experiment 2. The results suggest
equivalence in the strength of the positive and negative manipulation; i.e., it
does not appear to have been easier to increase or decrease identification and
attitudes.
Supporting the worry that the manipulation might have reduced variance, the null
hypothesis of equal variance was rejected in only the two cases in which ingroup
identification in Experiment 1 was compared with ingroup identification in the
match and mismatch conditions of Experiment 2. As expected, in both cases
variability was lower in the Experiment 2 conditions. However, this finding does
not appear able to explain the lack of balance found in the mismatch condition
as compared to the match condition. This is because while variance was reduced
relative to Experiment 1, it was reduced equivalently in the match and mismatch
conditions, in which the null hypothesis of equal variance could not be
rejected, *F*(57, 53) = 1.39, *p* = .23. Thus, the differential degree of
balance cannot be attributed to reduced variability in one of the criterion
constructs.
## Discussion
In Experiment 2, identification with a minimal ingroup was manipulated by either
weakening or strengthening the associative relationship between self and group
in a partial-IAT procedure. This manipulation was successful and constituted a
large effect; participants in the “mismatch” condition were much less implicitly
identified with their ingroup. This manipulation did not, however, affect self-
reported identification, and participants continued to explicitly identify with
their ingroup. This demonstrates that the manipulation did not simply *confuse*
participants about their membership, but rather affected the associative
relationship between self and group. Importantly, the manipulation of
identification also affected implicit attitude, with participants in the
mismatch condition showing weaker implicit preference for their ingroup. Thus,
manipulating one leg of the “Balance triangle” exerts causal influence on at
least one of its neighbors, lending support to a causal rather than merely
descriptive interpretation of these relationships. It is interesting that
implicit self-esteem was not similarly affected. In general, a causal
interpretation of Balanced Identity merely predicts that changing one construct
will change related constructs so as to preserve balance; it is agnostic with
respect to exactly *how* the equation is balanced. That is, when group
identification changes, the cognitive system could respond by adjusting self-
esteem, group attitude, or both. In this case, the predominant operation was to
decrease the strength of group attitude. A plausible interpretation of this
pattern of results is that because the self-positive association precedes the
experimental setting and generalizes far beyond it, it is more resistant to
minor perturbations via associated constructs of the sort created here. More
leverage will be gained on this issue after manipulating the other constructs
and describing their downstream effects.
Interestingly, the mismatch condition, which reduced group identification (and
consequently group attitude), also disrupted balance; tests of balance in this
condition all failed at the first step. By contrast, participants who had their
group identification increased through the match manipulation continued to show
robust balance, passing all 12 tests. This finding supports past work contending
that belonging to a low status or otherwise non-liked group prevents cognitive
balance, though the present findings allow us to generalize this considerably
because “low status” in this case in no way reflects richer conceptions of
enculturation but rather the results of a simple associative manipulation
designed to reduce the self-group association. Supplementary analyses also
suggested that the failure to find balance in the mismatch condition cannot
simply be attributed to reduced variance in one of the constructs. The
implications of this finding will be discussed further as data from the other
experiments is presented. More broadly, however, we again saw clear evidence of
balance on implicit but not on explicit measures.
# Experiment 3: Manipulating Group Attitude
This experiment followed a nearly identical procedure to that described in
Experiment 2, except that group attitude was manipulated instead of group
identification. In one direct sense, the MGP is in fact a manipulation of group
identification; that is, it attaches participants to a group. By contrast, it
does not provide evidence directly relevant to attitude or self-esteem. Thus,
the manipulation in Experiment 2, above, could have been effective because it
directly targeted the same factor implicated in the minimal group paradigm
itself. If so, the causal effects found in that experiment could be thought of
as simply strengthening or weakening the minimal group induction itself, rather
than a consequence of causal relationships specified by the Balanced Identity
model. However, if those effects were in fact the result of the consistency-
generating processes described by Balanced Identity, results should be similar
if another leg of the Balance triangle (in this case, group attitude) is
manipulated. The central question, then, is whether manipulating attitude
towards the newly assigned minimal group will affect the other two related
constructs. A secondary question is whether artificially decreasing group
attitude through our manipulation will disrupt the formation of cognitive
balance.
## Methods
### Participants
One hundred thirty-five participants were recruited from the same population and
following the same procedure described in Experiments 1 and 2.
### Procedure
The procedure was closely modeled after that employed in Experiment 2, except
that the manipulation targeted attitude instead of identity.
### Manipulation of group attitude
As in Experiment 2, a partial-IAT procedure was employed. Participants again
completed two blocks of 60 trials in which, as a between-participants factor,
they either repeatedly responded to ingroup and positive words using one key and
outgroup and negative words using another key, or repeatedly responded to
outgroup and positive words using one key and ingroup and negative words using
another key. Thus, as a between-participants factor, the manipulation was
designed to either increase or decrease the positive association with the
ingroup (and affect the positive association with the outgroup in the opposite
direction).
### Measures
The same measures used in Experiment 1 were used here.
## Results and Discussion
### Descriptive statistics
Standard exclusion criteria for IAT results led to the elimination of data from
nine participants from the identification IAT and 11 participants from each of
the other IATs. There were no effects of ingroup name or task order, and so
these factors were dropped from subsequent analysis. Overall results again
mirrored those described in the prior experiments, with participants exhibiting
robust preference for the ingroup, *D* = .28 (.42), *t*(123) = 7.60, *p*\<.001,
*d* = .68, robust identification with the ingroup, *D* = .48 (.29), *t*(125)
= 18.45, *p*\<.001, *d* = 1.26, and strong implicit positive self-esteem,
*D* = .51 (.31), *t*(123) = 18.33, *p*\<.001, *d* = 1.37. Identification was
again stronger than attitude, paired *t*(123) = 18.33, *p*\<.001. As in prior
studies, implicit identification with the ingroup was stronger than implicit
preference for the ingroup, paired *t*(122) = 5.21, *p*\<.001. Turning to
correlations between constructs, preference for and identification with the
ingroup were modestly correlated, *r*(121) = .38, *p*\<.001, and weaker
correlations were observed between attitude and self-esteem, *r*(120) = .23,
*p* = .01, and identification and self-esteem, *r*(121) = .19, *p* = .04.
Participants self-reports also revealed more liking for the ingroup, M = 1.0
(1.8), *t*(134) = 6.43, *p*\<.001, *d* = .56, as well as greater identification
with the ingroup, M = 1.8 (2.2), *t*(134) = 9.29, *p*\<.001, *d* = .82.
Participants also reported somewhat greater liking for the self as compared with
others, M = .50 (1.1), *t*(134) = 5.18, *p*\<.001, *d* = .45. Correlations
among self-report measures varied from weak in the case of self-esteem and
attitude, *r*(133) = .17, *p* = .051, moderate for self-esteem and
identification, *r*(133) = .30, *p*\<.001, and strong in the case of attitude
and identification, *r*(133) = .61, *p*\<.001. Implicit and explicit measures
did not correlate, all \|*r*\|\<.15, *p*\>.10.
### Effect of attitude manipulation
Half of the participants completed a manipulation designed to strengthen
preference for the ingroup (“match” condition) while the other half completed a
manipulation designed to weaken it (“mismatch” condition). This manipulation was
effective; participants in the match condition exhibited markedly stronger
ingroup attitude as measured by the IAT, *M*<sub>MATCH</sub> = .56 (.31),
*M<sub>MISMATCH</sub>* = .03 (.32), *t*(122) = 9.29, *p*\<.001, *d* = 1.66.
Indeed, participants in the mismatch condition no longer preferred their ingroup
following this manipulation, *t*(64) = .74, *p* = .45. This manipulation also
affected ingroup identification, *M*<sub>MATCH</sub> = .58 (.29),
*M<sub>MISMATCH</sub>* = .39 (.27), *t*(124) = 3.93, *p*\<.001, *d* = .68,
though identification remained positive and significant in the mismatch
condition, *t*(64) = 11.61, *p*\<.001. This time the manipulation also affected
implicit self-esteem, *M*<sub>MATCH</sub> = .59 (.30),
*M<sub>MISMATCH</sub>* = .44 (.31), *t*(122) = 2.91, *p* = .004, *d* = .49. The
manipulation had no effect on explicit measures, all *t*\<1.32, *p*\>.19.
### Balanced Identity Analyses
provides a summary of results of the Balanced Identity analyses for implicit and
explicit data, collapsing across condition; again, balance was clearly observed
in the implicit but not explicit data. provides these analyses sub-divided by
condition (match or mismatch). Again results suggest that balance was disrupted
in the mismatch condition. For implicit data in the match condition, evidence
for balance was strong, with 10 out of 12 tests successfully passed, though it
is important to note that one model (with self-esteem as the criterion) failed
to show a significant interaction at Step 1, an important deviation from
predictions. At the explicit level, evidence of balance was completely absent,
with all models failing all tests. For participants in the mismatch condition
all three models focusing on implicit data failed at Step 1, indicating little
support for balance. For explicit data, there were some hints of balance, though
not definitive, with 8 of 12 tests passed successfully.
### Supplementary analysis: Comparison with Experiment 1
As described in Experiment 2, we can estimate the relative degree of change
affected by the match versus mismatch condition by comparing to the means from
Experiment 1 (no manipulation). As in Experiment 2, the overall means from
Experiment 3 (collapsing across conditions) did not differ significantly from
those observed in Experiment 1, all *t*\<.34, *p*\>.74. Results of the
comparisons between Experiment 1 means and the two conditions in Experiment 3
are presented in. The results again sugest equivalence in the strength of the
positive and negative manipulation.
Interestingly, we again observed some cases in which variance appeared to have
been reduced by the manipulation, but instead of occurring with respect to the
manipulated construct of group attitude, it again appeared only with respect to
group identification (in both the match and mismatch conditions). However, as in
Experiment 2, the variances between match and mismatch conditions themselves did
not differ, suggesting that while manipulation appears to reduce variance, that
reduced variance cannot account for the varying degrees of balance observed
between match and mismatch conditions.
The results of Experiment 3 lend further support to a causal interpretation of
Balanced Identity by showing that intervening on implicit attitudes robustly
affected both implicit group identification and self-esteem. Participants whose
implicit preference for their ingroup was reduced also implicitly identified
with that group less, and even showed less implicit self-related positivity. In
addition, and as in Experiment 2, group identity, attitude, and self-esteem
remained closely related in the manner predicted by Balanced Identity when group
attitude was reinforced, but not when it was disrupted, suggesting that
depressing ingroup preference prevents cognitive balance.
# Experiment 4: Manipulating Self-Esteem
A nearly identical procedure to that described in the previous two experiments
was employed, except that this time the self-positive association was
manipulated. This association differs in one profound respect from the group
attitude and group identification associations, in that it is an enduring
construct (i.e., implicit self-esteem) that exists before and endures after the
experimental paradigm in which participants find themselves. That is, the
minimal group paradigm that participants take part in here *produces* group
attitude and group identification; it does not *produce* self-esteem in the same
way, because the self-valence link is an enduring representation in semantic
memory. Does this make it more resistant to the form of manipulation used above?
And does it affect the influence it exerts on associated constructs?
## Methods
### Participants
One hundred sixteen participants were recruited from the same population and
following the same procedure described in prior experiments.
### Procedure
The procedure was closely modeled after that employed in Experiments 2 and 3,
except that the manipulation targeted the self-valence association.
### Manipulation of implicit self-esteem
The same partial-IAT procedure used in prior experiments was used here. That is,
as a between-participants factor, participants either repeatedly responded to
self and positive words using one key and other and negative words using another
key, or repeatedly responded to other and positive words using one key and self
and negative words using another key. Thus, the manipulation was designed to
either increase or decrease the positive association with the self while
affecting the association with other-related concepts in the opposite direction.
### Measures
The same measures used in Experiment 1 were used here.
## Results and Discussion
### Descriptive statistics
Standard exclusion criteria for IAT results led to the elimination of data from
three participants from the attitude IAT, seven from the self-esteem IAT, and 11
from the identification IAT. There were no effects of ingroup name or task
order, and so these factors were dropped from subsequent analysis. Overall
results again mirrored those described in the prior experiments, with
participants exhibiting robust preference for the ingroup, *D* = .27 (.35),
*t*(112) = 8.27, *p*\<.001, *d* = .64, robust identification with the ingroup,
*D* = .41 (.36), *t*(104) = 11.69, *p*\<.001, *d* = 1.2, and strong implicit
positive self-esteem, *D* = .47 (.35), *t*(109) = 14.05, *p*\<.001, *d* = 1.0.
Identification was again stronger than attitude, paired *t*(102) = 3.05,
*p* = .003. Unlike in prior studies, preference for and identification with the
ingroup were not correlated, *r*(101) = .06, *p* = .56, though weak
correlations were observed between attitude and self-esteem, *r*(107) = .20,
*p* = .03, and identification and self-esteem, *r*(100) = .23, *p* = .02.
Participants self-reports also revealed more liking for the ingroup, M = .78
(1.4), *t*(114) = 6.08, *p*\<.001, *d* = .56, as well as greater identification
with the ingroup, M = 2.0 (2.1), *t*(114) = 10.38, *p*\<.001, *d* = .95.
Participants also reported somewhat greater liking for the self as compared with
others, M = .46 (1.2), *t*(115) = 4.19, *p*\<.001, *d* = .38. Preference and
identification were highly correlated, *r*(113) = .59, *p*\<.001, but no other
bivariate correlations reached significance, both *r*(113) \<.12, *p*\>.23.
Implicit and explicit identity were moderately correlated, *r*(102) = .28,
*p* = .004, but no other implicit-explicit correlations achieved significance,
both \|*r*\|\<.10, *p*\>.33.
### Effect of manipulation
Half of participants completed a manipulation designed to strengthen association
between the self and positivity (“match” condition), while the other half
completed a manipulation designed to weaken it (“mismatch” condition). This
manipulation was effective; participants in the match condition exhibited
stronger implicit self-esteem as measured by the IAT, *M<sub>MATCH</sub>* = .56
(.34), *M<sub>MISMATCH</sub>* = .40 (.34), *t*(108) = 2.53, *p* = .01,
*d* = .47, though the size of this effect is considerably smaller than in the
previous two experiments, suggesting that self-esteem is indeed more resistant
to manipulation in this fashion. The manipulation also affected ingroup
preference, *M*<sub>MATCH</sub> = .36 (.33), *M<sub>MISMATCH</sub>* = .20 (.35),
*t*(111) = 2.39, *p* = .02, *d* = .47, but did not affect ingroup
identification, *M*<sub>MATCH</sub> = .46 (.32), *M<sub>MISMATCH</sub>* = .37
(.39), *t*(103) = 1.38, *p* = .17, *d* = .25. Positive self-esteem, ingroup
identification, and ingroup preference remained statistically significant in
both match and mismatch participants, all *t*s\>4.5, *p*s\<.001. The
manipulation had no effect on explicit measures, all *t*\<.43, *p*\>.67.
### Balanced Identity Analyses
provides a summary of results of the Balanced Identity analyses for implicit and
explicit data, collapsing across condition; overall, evidence for balance was
strong at the implicit level but weak at the explicit level, with 12 and 6 tests
passed, respectively. provides these analyses sub-divided by condition (match or
mismatch). When broken down by condition the results of this study were somewhat
less definitive than in the prior two investigations. For participants in the
match condition, implicit data led to 9 of 12 tests being passed, but two of the
failures were at Step 1, where for models with identification and self-esteem as
criterion, the interaction was directionally consistent but did not reach
significance. For explicit data, only 3 of 12 tests were passed, suggesting no
balance at this level. In the mismatch condition, there was for the first time
some evidence of balance with 10 of 12 tests passed, though again the two
failures were at Step 1. For explicit data in the mismatch condition, 6 of 12
tests were passed. In one sense, these data are broadly consistent with prior
experiments, in that there was better evidence of balance at the implicit than
explicit level. However, evidence of balance was this time as good in the
mismatch condition as in the match condition. Additionally, both match and
mismatch analyses revealed failures at Step 1, which represent particularly
dramatic divergences from predictions.
One plausible interpretation of the maintenance of balance in the mismatch
condition concerns the strength of our manipulation. The manipulation of self-
esteem, while statistically significant, was considerably weaker than the
manipulation of the other constructs in the previous studies (*d<sub>self-
esteem</sub>* = .44, *d<sub>attitude</sub>* = 1.77,
*d<sub>identification</sub>* = 1.40; see also, introduced below). Thus, the
manipulation might not have created a powerful enough perturbation to effect
balance as dramatically, perhaps because self-esteem, an enduring and central
aspect of personhood, is particularly resistant to negative perturbations.
### Supplementary analysis: Comparison with Experiment 1
As described in Experiment 2 and 3, above, we again compared results to the
means from Experiment 1, which can serve as a no-manipulation baseline. As in
the prior two experiments, collapsing cross condition in Experiment 4 reveals
means that do not differ significantly from those in Experiment 1, all *t*\<1.3,
*p*\>.20. Results for the comparisons between Experiment 1 and the match and
mismatch conditions of Experiment 4 are presented in, showing the average change
and associated standard errors. Unlike in the previous two experiments, visual
inspection of the figure suggests that the manipulation of self-esteem was not
particularly effective in the match condition (i.e., the mismatch bars are the
only ones that are reliably displaced from 0). Thus, it appears that the
manipulation was not powerful enough to inflate already robust implicit self-
esteem but was powerful enough to (presumably temporarily) decrease it. As in
Experiment 2, results again suggested a (in this case marginal) reduction of
variance for the intervened upon construct (self-esteem). But as in the prior
experiments, the variances between match and mismatch conditions in Experiment 4
did not differ.
# General Discussion
The current study investigated whether consistency between group attitude, group
identification, and self-esteem—which has been widely demonstrated with respect
to familiar and salient real-world groups—would emerge immediately following
random assignment to minimal social groups. Four experiments provided a strong
affirmative answer: cognitive balance was robustly present in every case, but
(as has been noted with real-world groups) only at the implicit level. These
findings strongly suggest that cognitive balance need not stem from a history of
gradual revision or iterative dissonance reduction. Rather, the initial form
that intergroup cognitions take is well described by the predictions of Balanced
Identity, suggesting that new cognitions are constrained by existing ones as
described by the Balance model.
Relatedly, these studies provide direct evidence for a causal (as opposed to
merely descriptive) reading of Balanced Identity by showing that manipulating
any one leg of the balance triad (e.g., group attitude, group identification,
self-esteem) affected related constructs while preserving the broader pattern of
cognitive balance. Establishing these causal connections within the framework of
the minimal group paradigm supports a reading of Balanced Identity as a
generalized causal account of intergroup bias; intergroup bias can be
conceptualized as the interactive effects of self-esteem and group
identification. What's more, it suggests new pathways to intervention.
Intergroup bias can be intervened with not only directly but also by
manipulating the two related constructs of group identification and self-esteem.
Finally, the present results bolster the prior observation that belonging to a
group that is not positively evaluated inhibits balance for members of that
group. When we intervened on group attitude or identification, participants who
experienced the “negative” intervention that decreased the strength of that
construct did not show cognitive balance. This was not merely the result of any
artificial intervention, as participants who experience the “positive”
intervention increasing the strength of the same construct did show cognitive
balance. Nor was it the result of a reduction in variance in the manipulated
construct; while manipulation did appear to reduce variance (as compared to
Experiment 1, in which there was no manipulation), it reduced variance
equivalently across the match and mismatch conditions and so cannot uniquely
explain the different degrees of balance that were then observed. Results of a
final experiment manipulating self-esteem were somewhat more equivocal, however,
perhaps because the effort to change implicit self-esteem was less successful
than the other two manipulations, and the effects it did have may have been
asymmetrical, predominately deflecting implicit self-esteem in the negative
direction. In any case, one suggestive interpretation of this broader pattern
is that, when belonging to a group that is negatively evaluated, balance is
disrupted so as to prevent that negativity from bleeding over into self-esteem.
While broadly consistent with some motivational accounts of self-group
relationships such as Social Identity Theory, it is striking that this
phenomenon is seen most clearly at the implicit level, where we would expect
that motivational concerns would be at least somewhat attenuated. Indeed, a
question raised by these findings is how the implicit system “knows” to disrupt
balance in the face of a negative perturbation while retaining it in the face of
a positive perturbation. The microdynamics of these changes could be a fruitful
topic for future study.
Stepping back somewhat, some past research has postulated that intergroup bias
is the result of self-related positivity that associatively spreads from the
self to ingroups. However, support for this claim has been mixed. The present
research suggests that the reason for this mixed support may be that, in trying
to associate self-esteem with group bias, one crucial construct, group
identification, has been left out. In a sense, it is attempting to impose a
bivariate model (i.e., linkages between self-esteem and group attitude) on a
multivariate and interactive phenomenon. Looking solely at the link between
group attitude and self-esteem in the present studies, the correlations were a
modest *r* = .29, and the relationship was significant in some but not other
studies. In other words, the same mixed support found in prior investigations
appeared in these data. By contrast, when predicting each construct from the
product of the other two, implicit data yielded a statistically significant and
positive relationship in the great majority of tests across these four studies
(with the exception of the cases in which the constructs were artificially
reduced through intervention). As with prior work employing the Balanced
Identity framework, however, the predicted pattern appears to characterize
*implicit* constructs much better than *explicit* constructs. Indeed, as
compared with the 47/48 successful statistical tests for implicit data
(collapsing for the moment across condition), only 24/48 tests were passed for
self-report data. Indeed, it is possible that the relationships between self-
report constructs may be better captured by bivariate relationships, which were
generally sizable, as compared with the implicit data, which are better-
described by the multiplicative relationships specified by Balanced Identity.
One general pattern emerging from my manipulation of individual components of
the balance triad was that, while each construct is easily manipulated, the
influence of that manipulation on the more “downstream” constructs was not
perfectly predictable. That is, it did not always affect *both* theoretically
related constructs. Suggestively, the pattern was at least partially structured.
Specifically, group attitude and group identification mutually affected one
another, and group attitude and self-esteem mutually affected one another.
However, self-esteem and group identification did not affect one another in
either direction. Future work will be necessary to understand whether this
pattern is replicable and, if so, why it occurs.
One potential weakness of the present design is that the method used to
manipulate the constructs in question was closely related to the method used to
measure them following their manipulation. That is, a partial-IAT procedure was
used to manipulate the constructs, which were then measured with a full IAT.
While the left/right side pairing was balanced at the participant level such
that a simple side-bias is unlikely, it is nonetheless possible that the
partial-IAT procedure affected subsequent IATs to some extent. Future work could
make the method of manipulation more distinct, for example through a
conditioning paradigm, or could alter the method of construct measurement by
using a different implicit measure.
In closing, this study highlights the value of the minimal group paradigm as a
methodological tool in intergroup social cognition research. By abstracting away
from the variable knowledge and informational complexity that characterizes
real-world groups, we can gain a clear perspective on how group affiliations
emerge and change across contexts and can observe the emergence of such
cognitions from their earliest moments. Of course, it is always important to
verify that findings with minimal groups generalize to real groups. In the
present context, this has already been accomplished, as cognitive balance has
been widely observed with respect to real-world groups. But that observation
left open several questions regarding the causal status and informational time
course of such consistency. The minimal group paradigm became a powerful tool
for addressing these issues in a controlled manner. At a higher level, by
demonstrating that a phenomenon observed with respect to real-world groups also
occurs with respect to novel social groups, we are able to reduce a set of
specific observations about specific groups to a general principle governing
intergroup cognition more broadly. This opens the door to interpreting Balanced
Identity as a general account of intergroup attitudes (as well as, of course,
intergroup identifications and self-esteem), offering a model for both
prediction and intervention. Future work focusing on linkages between self-
esteem and attitudes will benefit by incorporating these insights and ensuring
attention is also paid to the closely related construct of group identification.
[^1]: The author has declared that no competing interests exist.
[^2]: Conceived and designed the experiments: YD. Performed the experiments:
YD. Analyzed the data: YD. Wrote the paper: YD. |
# Introduction
Animal behavior has the potential to contribute substantially to conservation
efforts, but the field of conservation behavior has been slow to develop. Only
recently have attempts been made to develop unifying principles and overarching
frameworks for the study of conservation behavior. Because behavior is one of
the most flexible traits an animal may possess, it represents a promising
mechanism by which species may cope with human-induced rapid environmental
change (HIREC).
Behavioral flexibility is the ability of an animal to modify its behavior under
different environmental conditions. Behavioral responses are often rapid and
reversible, and afford animals some degree of control over their external
stimuli by choosing their surrounding environment. Appropriate behavioral
responses to environmental change are vital for animals that live in naturally
heterogeneous environments, and will likely be critical for the persistence of
many species in the face of HIREC.
Both natural environmental heterogeneity and the effects of HIREC occur across
time and space, and this environmental variability affects species occurrence,
distribution, abundance, population stability, and individual behavior. Some
habitat types are relatively stable, while others are highly variable in their
natural state. For example, wetland biota is influenced by a variety of both
predictable (tides, seasonal flows) and stochastic (flood, drought) changes on a
variety of time scales. Anthropogenic changes to wetlands, such as modification
of tidal inundation regimes through the construction of earthen dikes/levees,
can greatly interfere with these natural patterns of heterogeneity. Because
coastal wetlands are both highly variable in their natural state and have been
heavily modified by human activities, organisms that live in coastal wetlands
present opportunities for the study of both behavioral flexibility in response
to natural heterogeneity and behavioral responses to HIREC.
We investigated the response of the endangered northern salt marsh harvest mouse
(*Reithrodontomys raviventris halicoetes*) to both natural and anthropogenic
environmental heterogeneity. The salt marsh harvest mouse is an ideal species in
which to study behavioral flexibility and responses to HIREC, because it is
endemic to changeable wetland habitats and currently occurs in both natural
tidal (full tidal influence) and anthropogenically-altered (diked) wetlands.
Features of salt marsh harvest mouse biology that are consistent with adaptation
to tidal environments become clear when comparisons are made to the sympatric
congener the Western harvest mouse (*R. megalotis*), which occurs in tidal
marshes but also in a variety of other habitat types. Compared to Western
harvest mice, the salt marsh harvest mouse is a stronger swimmer and is more
capable of consuming sea water. They are also capable of entering torpor, a
mechanism thought to aid small mammals in coping with osmotic stress, and are
more active during warmer daytime temperatures which allows for a lower resting
metabolic rate than activity during cool, damp nights. Finally, as a species
that naturally resides in a highly changeable environment, the salt marsh
harvest mouse may possess a high level of behavioral flexibility that
predisposes it to alter its behavior to utilize resources in novel habitat
types, such as diked wetlands.
Understanding how salt marsh harvest mice use their habitat during daily tidal
flooding is especially important because tidal restoration is the primary
conservation measure for preserving this species which is endangered due to
dramatic habitat loss (∼90%) and faces further loss due to predicted sea level
rise. As the only terrestrial mammals that is entirely restricted to coastal
marshes, the salt marsh harvest mouse must somehow avoid drowning during high
tides. It has long been believed that salt marsh harvest mice spend the majority
of their time in wetlands, and move upland only to escape tide and flood waters.
Since the 1950s, researchers have attempted to characterize salt marsh harvest
mouse behavior during tidal inundation to determine whether mice move vertically
into tall vegetation or horizontally upland to escape the tide,. In tidal
wetlands salt marsh harvest mice typically experience tidal flooding twice a
day. In contrast, diked wetlands are flooded continuously during the rainy
season (approximately October through March), and dried out for the remainder of
the year. Thus, the patchwork of modified and unmodified habitat in the San
Francisco Bay Estuary presents the opportunity to study habitat use of mice in
both highly changeable natural (tidal) habitat and highly modified (diked,
surrounded by levees with water control structures) habitat that is temporally
more stable.
Conclusions from previous studies of the behavior of the salt marsh harvest
mouse at high tide have been split on the question of how mice deal with daily
tidal inundation of their habitat. Some concluded based on trapping data or
visual observations that animals move out of tidal wetlands and into upland
areas or onto levees to escape rising waters, , and others concluded that they
remain in tall, dense vegetation over water where they can easily move about in
the thatch layer. However, although trapping methods allow researchers to
determine that a mouse had been at a location at some point during the night, it
is impossible to tell from these previous trapping studies where mice were
during the time that the habitat was actually flooded. The contrasting
conclusions of previous studies suggest that the only way to definitively
understand salt marsh harvest mouse habitat use during high tide is through the
use of radio telemetry.
The objective of this study was to determine how salt marsh harvest mice respond
to both natural environmental heterogeneity and HIREC, focusing on the effects
of regular tidal inundation. We approached this investigation from a
conservation behavior perspective which uses general principals of animal
behavior to address conservation issues. We applied two questions from a
recently developed conservation behavior framework. One focus of the Berger-Tal
et al. framework stresses performing behavior-based management, which considers
behavior in conservation decision making protocols. In our case, answering the
question *Do salt marsh harvest mice move vertically (into tall vegetation) or
horizontally (into upland areas) to escape the high tide*? will allow managers
develop restoration and enhancement priorities in the little habitat that
remains, and is a research priority explicitly identified in the recovery plan
for this species. A second focus of the framework addresses if and how HIREC
affects behavior, in the case of this endangered species, *Do mice in
anthropogenically altered diked wetlands behave differently than those subject
to tidal influence*?.
To answer these questions, we tracked mouse movements during high tide events in
natural tidal wetlands using radio telemetry to identify the type of refuge used
to escape incoming water. During the same periods, we tracked mice in adjacent
diked wetlands. This allowed us to determine space use and estimate movement
rates in response to high tides, as well as independent of tidal influence in
diked habitats. With regard to our first question, we predicted that during
tidal flooding, mice would remain in tall vegetation over water. Our predictions
for the second question depended on the results of the first. If the prediction
that mice remain in tall vegetation during the high tide was confirmed, we
expected that movement distances (and thus rates) in natural tidal and diked
wetlands would be similar. In contrast, if mice instead moved long distances
upland to escape the tide, then we expected that movement rates in natural tidal
wetlands would be greater than those in diked wetlands.
# Methods
## Ethical note
The research reported here was conducted under an approved IACUC protocol from
New Mexico State University (2011–013) and a Memorandum of Understanding between
the California Department of Fish and Wildlife and the United States Fish and
Wildlife Service for handing of endangered species, with direct involvement and
supervision by the California Department of Fish and Wildlife. All procedures
were consistent with the guidelines for the use of wild mammals in research from
both ASAB/ABS and the American Society of Mammalogists.
## Study Area
We conducted this study between May and August 2011 in the Suisun Marsh in
Solano County, CA, USA (38° 11′ 15.86″ N, 122° 3′ 52.67″ W), which is a large
wetland complex in the San Francisco Bay Estuary. We established six study areas
which were grouped into three blocks, each containing one natural tidal and one
diked wetland. Study areas were established on public lands with the permission
of the California Department of Fish and Wildlife and the Suisun Resource
Conservation District. Paired wetlands (one tidal and one diked) within each
block were 100 to 600 m apart and separated by levee roads or sloughs, and the
three blocks were 1 100 to 8 500 m apart. Although salt marsh harvest mice are
capable of moving distances similar to the distances between our study wetlands,
we did not observe movement between these wetlands during our study. All tidal
wetlands were subject to full natural tidal action. All diked wetlands were cut
off from water access for the summer and were essentially dry.
Vegetation on the study sites included pickleweed (*Salicornia virginica*, also
known as Virginia glasswort), Olney's threesquare bulrush (*Schoenoplectus
americanus*, also known as chairmaker's bulrush), cattail (*Typha* spp.),
saltgrass (*Distichlis spicata*), tule (*Sc. acutus*), common reed (*Phragmites
australis*), Baltic rush (*Juncus balticus*) and other halophytic species. Plant
community composition was broadly similar between natural tidal and diked areas,
although tidal areas tended to be dominated by reeds and rush (mean height ±1
*SE* = 138.13±27.84 cm), while the diked areas tended to be dominated by
pickleweed (mean height ±1 *SE* = 42.40±6.00 cm). Although the vegetation was
similar overall, diked wetlands had a higher proportion of bare ground, which
lead to a lower calculated mean height.
## Live Trapping
During more than 3 500 trap nights, we captured small mammals using collapsible
Sherman live traps (SFA, 7.62×8.89×23.50 cm; H.B. Sherman Traps Inc.,
Tallahassee, FL). We placed trap grids in vegetation types known to support
large salt marsh harvest mouse populations, including pickleweed and Olney's
threesquare bulrush. Traps were placed at 10 meter spacing within grids
consisting of 50 or 100 traps, depending upon wetland configuration and mouse
density. We placed traps flat on the ground in diked wetlands. In natural tidal
wetlands, we placed traps above the water line, nested securely in the
vegetation. We placed paperclips at the top of trap doors to leave a small space
between the door and the body of the trap to prevent the tails of salt marsh
harvest mice from getting caught when traps closed. We baited traps with ground
walnut and mixed birdseed, and added cotton batting to the traps for warmth.
Traps were opened at sunset, and checked and closed at sunrise. We trapped and
tracked mice simultaneously in paired natural tidal and diked wetlands within
each block during the same moon phases. At each block, trapping was initiated
4–5 days before the highest tide of the full and new moons. Tracking began 2–3
days before the highest high tide of the lunar phase, and typically continued
until 2–3 days after the highest tide.
## Radio Tracking
To maximize the potential effect of tidal inundation on movement, we tracked
mice during several days surrounding the full moon, which is typically when the
highest tides occur. To control for potential effects of bright lunar
illumination during the full moon, we also tracked during the new moon when
tides are also very high but lunar illumination is low. We tracked mice during
sequential full and new moon phases for each block, with a different set of mice
tracked during each lunar phase. This yielded a 2×2 factorial arrangement with
four “treatments”: tidal wetland during the full moon, tidal wetland during the
new moon, diked wetland during the full moon, and diked wetland during the new
moon.
To track movements of salt marsh harvest mice, we fitted individuals with BD-2NC
radiotransmitters (Holohil, Inc., Carp, Ontario, Canada). Transmitters were
equipped with the smallest battery possible, resulting in a total transmitter
package mass of approximately 0.5 g. Because of the small size of salt marsh
harvest mice (adult mass = 7.6–14.5 g), only the largest individuals could be
collared in order to meet the recommendation that collars not exceed 5% of body
mass. Because females of sufficient size were commonly pregnant or lactating, we
used only males weighing ≥10 g. We collared up to five of the largest
individuals per wetland per lunar cycle (up to ten individuals per block per
lunar cycle), yielding a total of 42 mice, 23 in the tidal wetlands and 19 in
the diked wetlands. We collared mice in the field without using anesthesia and
held them for observation for ten minutes following the collaring procedure;
none showed any adverse reactions. Following the observation period, we released
mice at their capture location and began tracking their movement during the
second high tide following release (approximately 12 hours later). We tracked
each individual for up to six days (mean; *SD* = 4.3; 0.99 days, min = 2 days,
max = 6 days), collecting location data approximately once an hour for 2 to 3
hours on either side of the peak of both diurnal and nocturnal high tides. When
tracking was complete, we re-trapped mice and removed their collars. Due to some
dropped collars and unexpectedly short battery lives, not all collared
individuals yielded sufficient data for analysis.
We used standard radiotracking methods to estimate individual locations, using
triangulation from known coordinates, with two bearings recorded per location by
two observers (a total of four bearings from different points were used to
triangulate each location). By allowing the location of animals from a distance,
triangulation minimized the influence of observer presence on mouse movement and
reduced damage to habitat. It also allowed us to track mice in flooded areas
that were inaccessible to humans on foot. Location estimates were generated
using Lenth's technique for maximum-likelihood estimates (MLE) in the computer
program LOCATE III. To ensure that mice retained their collars and were not
caught in vegetation, we homed in on each mouse following each high tide. We
then recorded location coordinates using a handheld GPS unit with submeter
accuracy (Trimble GeoExplorer 3; Trimble Navigation Limited, Sunnyvale, CA,
USA). Homing locations also allowed us to estimate the accuracy of triangulated
locations. When a homing location was recorded within 15 minutes of a
triangulated location, we calculated the distance between the homing location
and the triangulated location to estimate triangulation error. The distance
between triangulated and homed points was 15.51; 8.83 meters (mean; *SD*,
N = 37). However, this error distance is probably an overestimate, because mice
are capable of moving large distances in short periods of time, and because
animals often move away from researchers during the homing process, which would
increase the distance between triangulated and homed locations.
## Determining Refuge Use
Our first goal was to determine whether salt marsh harvest mice moved vertically
into emergent vegetation or horizontally into upland areas during the high tide.
We examined mouse locations that were recorded within one hour of the high tide
in relation to the tide height. We created maps of inundation for each tidal
site using water surface data. To measure water surface level, we placed tide
level markers throughout tidal areas within and around trap grids at 20 meter
spacing, and measured the maximum height reached by water following each high
tide. Tide markers were wooden dowels marked each centimeter and dusted with
colored chalk. Tide height was estimated by examining dowels following high tide
and noting the height to which chalk had been washed away. We recorded
coordinates of all tide markers using a handheld GPS unit with submeter accuracy
(Trimble GeoExplorer 3; Trimble Navigation Limited, Sunnyvale, CA, USA).
We input all tide measurements as points into ArcMap 10 and interpolated a
raster surface using an Inverse Distance Weight method (Inverse Distance Weight,
ArcGIS 10.0; Environmental Systems Research Institute, Redlands, CA, USA) which
overlaid well with aerial imagery and was consistent with observations in the
field. Over this raster surface, we overlaid all mouse locations recorded
“during” the high tide. Points occurring within one hour before or after the
time of the highest point of the tide were categorized as “during” the high
tide. If a mouse location fell in an area where water level was ≤1 cm we
considered it “upland”. If a mouse location fell in an area where water level
was \>1 cm it was considered “over water”. If a location was determined to be
upland, we assumed the mouse moved horizontally to escape the tide. If a
location was categorized as over water, we assumed that the mouse had remained
in emergent vegetation to escape the tide. Refuge use was determined using the
lowest high tide of both the full and new moon periods during which mice were
tracked, thus represented the most conservative categorization of locations as
over water. That is, locations were only considered to be over water if they
would have been over water during the lowest high tide that we observed at a
site. Locations that fell within 15 m of the boundary between inundated and
unindated areas were excluded from analysis of refuge use. Because these
“boundary” locations were within the average triangulation error distance, we
could not definitively determine whether they were over water or not.
We also performed an analysis using minimum convex polygons (MCP) of salt marsh
harvest mouse locations to test for differences in space use between mice in
tidal and diked wetlands. Individual locations were grouped as “before”,
occurring more than one hour before high tide; “during”, within one hour before
or after the high tide; and “after”, occurring more than one hour following the
high tide. Using the Minimum Bounding Geometry-Convex Hull tool in ArcMap we
created “before”, “during”, and “after” polygons for each mouse during each time
period for which we recorded ≥6 locations. Then using the Intersect tool in
ArcMap we calculated what percentage of the “before” and “after” MCPs overlapped
the “during” MCP.
## Estimating Movement Rates
Our second goal was to determine whether there were differences in movement
distances between mice in natural tidal wetlands and mice in diked wetlands.
However, because the time intervals between location estimates were not equal,
it was necessary to first standardize movement data as rates. Movement rate is
the Euclidean distance between two consecutive point locations divided by the
time elapsed between those points. We calculated movement rates using all
locations of an individual that were recorded less than three hours apart
before, during and after high tide events (mean ±1 *SE* = 83.94±1.40 minutes
between successive points, min = 21 minutes, max = 180 minutes). Based on
average times of sunrise and sunset during the study, we categorized movements
occurring between 0601 and 2000 hours as “diurnal” and movements between 2001
and 0600 as “nocturnal”, and calculated average movement rates separately for
day and night for each mouse. We omitted from analysis 18 movement rates that
spanned the diurnal/nocturnal time cut-offs. The number of movement rates per
mouse per time of day (diurnal vs. nocturnal) ranged from 1 to 17, with an
average of 7.28; 3.87 (mean; *SD*) rates per individual per time of day (total
movement rates = 411). Mice with \<5 movement rates were excluded from
analysis, leaving a total of 29 mice for which we analyzed movement rate data,
15 in tidal wetlands and 14 in diked wetlands.
## Analysis
Statistical analysis of the MCP data was performed using paired t-tests to
compare space use before and after the high tide to space use during the high
tide. Statistical analysis of movement rates was conducted using PROC MIXED in
SAS 9.3 (SAS Institute Inc., Cary, NC, USA). We used a replicated block design
with blocks and the combination of wetland type and moon phase as fixed effects
(four “treatments”: tidal full, tidal new, diked full, and diked new), and mouse
identification number as a nested random effect, with time of day (diurnal or
nocturnal) as a repeated factor. Tukey post-hoc tests were used to detect
differences among treatments using an alpha level of 0.05 for statistical
significance. Only locations from mice in natural tidal wetlands were used for
the refuge use analysis, while locations from all mice with sufficient data were
used in the space use and movement rate analysis.
# Results
## Refuge Use
For mice in natural tidal wetlands, we recorded a total of 167 locations within
1 hour of high tide from 16 mice (mean; *SD* = 11.48; 4.02 points per mouse).
Forty-two of these 167 locations (∼25%) were within 15 m of the boundary between
areas that we considered “upland” versus “over water”. Because these locations
were within our average triangulation error from the boundary, we could not
classify them to either category with confidence, and they were eliminated from
further analysis. Of the remaining 126 locations, 125 (\>99%) were definitively
in vegetation over water and 1 (\<1%) was upland. It is worth noting that even
if locations falling within 15 m of the boundary between inundated and unindated
areas were included, only 6% (10/167) would have been categorized as “upland”.
Because we used the most conservative definition of “over water” (e.g., that the
position would have been over water during the lowest high tide during which
mice were tracked), 6% is likely an overestimate of the frequency with which
mice used upland refuges.
When comparing the MCP of points for individuals before (t-test:
t<sub>15</sub> = 1.327, P = 0.20; diked: n = 8, mean ± SE = 0.61±0.09; tidal:
n = 9, mean ± SE = 0.44±0.09) and after (t-test: t<sub>18</sub> = −0.55,
P = 0.59; diked: n = 10, mean ± SE = 0.56±0.09; tidal: n = 10, mean ± SE
= 0.49±0.08) high tide to the MCP during high tides we saw no significant
difference between tidal and diked wetlands. We found no evidence that salt
marsh harvest mice in tidal wetlands shift their space use more or less than
mice in diked wetlands during the same high tide periods.
## Movement rates
Because mice remained over water instead of moving large distances to use upland
refuges to escape high tides in natural tidal wetlands, it was not surprising
that we found no significant difference in movement rates among treatments
(combinations of wetland type and moon phase; \[repeated-measures ANOVA:
F<sub>3,24</sub> = 0.92, P = 0.44\]). As expected for a primarily nocturnal
species, we did find an effect of time: diurnal movement rates were lower than
nocturnal movement rates (repeated-measures ANOVA: F<sub>1, 23</sub> = 7.46,
P = 0.01). Post-hoc comparisons of diurnal and nocturnal movement rates within
treatments suggested that this difference was primarily due to diked habitats,
in which diurnal movement rates during the new moon were considerably lower than
nocturnal movement rates (Tukey post-hoc test: P = 0.012). In contrast, in
natural tidal wetlands, diurnal and nocturnal movement rates were very similar.
These data indicate that in natural tidal wetlands, mice are moving almost as
much during the day as at night.
# Discussion
Understanding how animals cope with both naturally-occurring heterogeneity and
HIREC in their environment can be crucial to the conservation of threatened
species such as the salt marsh harvest mouse. Behavioral flexibility represents
a coping mechanism likely to be crucial for many animals in the face of
anthropogenic change in their environment. The purpose of this study was to
determine how salt marsh harvest mice respond to both natural heterogeneity and
HIREC. Following the framework of Berger-Tal et al., we answered two questions.
First, we considered behavior-based management and asked *do salt marsh harvest
mice move vertically (into tall vegetation) or horizontally (into upland areas)
to escape the high tide*? Determining high tide refuge use by the salt marsh
harvest mouse is a high conservation priority for this endangered species. We
used radio telemetry to show that the vast majority of the time, male salt marsh
harvest mice in the Suisun Marsh used emergent vegetation to escape tidal
inundation, suggesting that maintenance of healthy intertidal vegetation is
important in the conservation of small mammals and protection of biodiversity in
this system. This conclusion is supported by extensive live-trapping data from a
previous study that indicated that salt marsh harvest mice are only rarely
trapped in upland areas, and is not surprising in the Suisun Marsh where
vegetation is generally tall and thick providing extensive structure and cover.
Our behavioral results will allow for improved habitat management, allowing
managers to concentrate conservation efforts on maintaining intertidal habitat
rather than expending efforts enhancing upland habitats that salt marsh harvest
mice rarely use.
Secondly, we addressed whether and how HIREC causes changes in behavior ;
specifically, do salt marsh harvest mice in anthropogenically altered diked
wetlands behave differently than those subject to tidal influence? The lack of a
significant difference in nocturnal movement rates between natural tidal and
diked wetlands would suggest that this anthropogenic change has not affected the
behavior of this species. However, diurnal movement rates were consistently
lower than nocturnal movement rates in diked wetlands, a pattern not observed in
tidal wetlands. This result indicates that mice in human-influenced diked
wetlands may have altered their diurnal movement behaviors in anthropogenically-
modified diked wetlands, moving less during the day. Current telemetry efforts
support these results and are revealing similar trends throughout the day, not
just during high tides (K.R. Smith, unpublished data).
There are two potential explanations for the observed decrease in movement rate
in diked wetlands during the day. First, anthropogenic habitat modification may
have had an adverse effect on salt marsh harvest mice. For example, differences
in habitat structure or a lack of inundation may either allow for increased
access by diurnal predators or decreased cover for mice, potentially leading to
reduced diurnal movement rates in diked wetlands (mice are *forced* to seek
refuge during the day). A second potential explanation is that removal of tidal
influence has instead had a positive effect on salt marsh harvest mice. For
example, mice in diked wetlands may have increased availability and/or
accessibility to resources, or an increased ability to cache resources, which
could allow mice to restrict their foraging to night-time hours, remaining in
refuges during the day when predation pressure is high (mice are *choosing* to
seek refuge during the day). This possibility is supported by our observations
of salt marsh harvest mice using underground refuges, and taking cover in voids
below a hummocks of cattails during the day in three separate diked wetlands,
behavior that would not be possible in a tidal wetland.
Habitat loss has been the primary threat to the salt marsh harvest mouse, and
with the level of existing and continuing development in the San Francisco Bay
Estuary, subsidence, and sea level rise, the marshes utilized by the salt marsh
harvest mouse are vulnerable to additional habitat loss. Every remaining hectare
of salt marsh harvest mouse habitat is crucial, and maximizing the value of this
habitat through restoration and enhancement will be key to the persistence and
recovery of the species. There are currently at least three plans that call for
restoration of diked wetlands to tidal influence in the Suisun Marsh in the
immediate future: Suisun Marsh Habitat Management, Preservation, and Restoration
Plan, the Bay Delta Conservation Plan, and the Fish Restoration Program
Agreement. Refuge use by salt marsh harvest mice is a behavior that must be
taken into account when planning and implementing these tidal restorations.
There is currently little research investigating the differences in value of
various habitat types to the salt marsh harvest mouse. Our trapping was not
designed to assess effects of wetland type on populations or densities of salt
marsh harvest mice, but another study suggests that diked wetlands may be at
least as valuable to conservation efforts as tidal wetlands, by supporting
higher densities of mice. Additionally, large populations of salt marsh harvest
mice have recently been found in diked wetlands, even during flooded winter
months (K.R. Smith, unpublished data). This suggests that this species is
capable of thriving in flooded diked wetlands by remaining in tall vegetation
over standing water. Given that much of the remaining salt marsh harvest mouse
habitat in the Suisun Marsh exists as diked wetlands, the behavior and ecology
of individuals living in these modified habitats warrants further exploration.
It is important to note that our findings may not necessarily apply to the
entire range of the salt marsh harvest mouse. In the Suisun Marsh tidal
vegetation is tall and marshes are wide, and there is an abundance of diked (21
044 hectares) and upland (11 210 hectares) habitat available, while only 21% of
historic tidal habitat remains (2 550 hectares). The two other major strongholds
for this species differ in marsh width and vegetation type: in San Pablo Bay
many marshes are wide, but vegetation is short, and in the South San Francisco
Bay marshes are narrow and vegetation is short. In these areas, mice may employ
alternate strategies when escaping the tide; for example, if vegetation is
shorter than the water height at high tide, mice may have no choice but to move
upland where they may be exposed to predation.
Finally, due to a number of limiting factors including minimum transmitter size
and logistical constraints, we were unable to track female or young salt marsh
harvest mice, so any speculation about their behavior should be made with care.
Indeed, a recent study of another endangered wetland rodent, the New Mexico
jumping mouse (*Zapus hudsonius luteus*), found different patterns of habitat
use by males and females. As technology advances and radio collars become
smaller, it would be prudent to repeat this study using both male and female
mice, as well as sub-adults and juveniles.
The results of this study provide insight to a question that researchers have
been investigating for over 55 years: where do salt marsh harvest mice go during
the high tide ? This study increases our understanding of the behavior of
endangered salt marsh harvest mice in tidal and diked wetlands, suggesting that
these small mammals may be capable of coping with the challenge of living in
tidal wetlands with little to no emergent land as long as vegetation is
sufficiently thick and tall. They also indicate that small mammals may exhibit
different behaviors in diked wetlands than in tidal wetlands, highlighting the
need to consider behavior when planning for conservation or management projects
and shedding light on the need for comprehensive management strategies that
account for potential behavioral differences in populations. Behavioral
flexibility may be critical for threatened species in the face of HIREC. As we
cause further changes to the environment both directly, through processes such
as urbanization, and indirectly, though processes such as climate change, the
work of behaviorists will be critical in understanding behavioral responses of
threatened species, like the salt marsh harvest mouse, to HIREC.
For invaluable field assistance we thank S. Estrella, D. Fidler, M. Riley, R.
Pardee, L. Preteska, L. Patterson. For logistical support we thank S. Chappell
and B. Wickland and the Suisun Resource Conservation District, and the
California Department of Water Resources.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: KRS LBT KEM. Performed the
experiments: KRS LBT. Analyzed the data: KRS WG KEM. Contributed
reagents/materials/analysis tools: KRS LBT WG KEM. Wrote the paper: KRS LBT
WG KEM.
[^3]: Current address: Wildlife, Fish, and Conservation Biology Department,
University of California at Davis, Davis, California, United States of
America |
# Introduction
Miniature legged robots are transportable, inconspicuous, and can pass through
tiny openings and narrow corridors, which makes them excellent navigators for
search and rescue missions at disaster sites. However, even state-of-the-art
miniature legged robots have not yet matched the walking system of insects
(ideal miniature legged systems in nature) in terms of power efficiency and
motion controllability, despite many of them being inspired by the locomotion
systems of living insects.
What if a living insect itself could be controlled using an electrical
stimulator? That is, what if we could create an insect–machine hybrid robot—a
fusion of a living insect and a man-made device (electrical stimulator)—to
elicit our desired motions and behaviors? Compared with the power consumption in
entirely man-made miniature legged robots, which is on the order of 100–1000 mW,
that of an insect–machine hybrid robot can be drastically reduced because
electrical stimulation consumes power on the order of only a few hundred
microwatts. Furthermore, insect–machine hybrid robots can be self-powered by
energy harvesters, such as implantable biofuel cells, implanted into the living
insect platform. In addition, these robots can be more robust in motion control
than robots that are entirely man-made. Complicated algorithms and controls,
which are necessary for entirely man-made robots to retain their postures or to
avoid or overcome obstacles, would not be necessary for insect–machine hybrid
robots because the insect's intrinsic control system can be utilized if needed.
For example, when an insect–machine hybrid robot encounters an obstacle, the
user shuts off the electrical stimulator and releases the insect from the user's
control system to allow it to avoid or overcome the obstacle by itself. Hence,
overall, insect–machine hybrid robots would exhibit high power efficiency and
excellent motion controllability.
Research related to insect–machine hybrid robots should be advanced from open-
loop control systems to closed-loop control systems to allow these robots' legs
to be regulated to follow the operator's predetermined motion path. Numerous
research groups have investigated locomotion control or appendage motion
response of various insects through electrical stimulation of insects' brain,
ganglia, and nerve cords or muscles. They have developed protocols and
methodologies of electrical stimulation of neurons and/or muscles to elicit
desired motion and overall locomotion (e.g., left–right turns in walking or in
flight) in an open-loop control (i.e., non-feedback control) manner. To date,
researchers have focused on the development of stimulation protocols, i.e.,
determining what stimulation applied to which neuromuscular sites can elicit the
desired motor actions and behaviors and evaluating the success rate and power
consumption. Consequently, various stimulation protocols have been developed,
but they have been demonstrated in an open-loop control manner. The questions
arise as to what the next step should be in the research into insect–machine
hybrid robots and how they can be practically used. Note that because of
animals' intrinsically complicated motion control systems and the unavoidable
physiological differences between individual animals, the elicited motions and
behaviors vary from trial to trial and from animal to animal, even under
identical stimulation protocols. As such, open-loop control techniques are
insufficient for achieving precisely controlled motions; thus, closed-loop
control must be introduced to reduce the deviations between the actually
elicited motion and the user's predetermined and desired motion. Therefore, the
next stage is the introduction of closed-loop control techniques to regulate
insect legs to follow predetermined angular positions.
When a closed-loop system is used with an insect–machine hybrid robot to
control, for example, an insect leg actuator, the electrical stimulation must
have two critical functions: (1) reliably control the direction of elicited leg
displacement and (2) induce a graded response in the magnitude of the leg
displacement to different electrical stimuli (e.g., different stimulation
frequencies and amplitudes). Reliability in the direction of elicited leg
displacement, as defined in this study, means that we can elicit leg motion in
the desired direction at a success rate of almost 100%, even though the elicited
magnitude of the leg displacement exhibits some variation. To achieve such
reliability in the displacement direction, we stimulated leg muscles instead of
neurons. Neurons are densely arrayed and stacked, which makes the separate
stimulation of individual neurons difficult. Even if an implanted electrode is
strongly fixed at the targeted neurons, small drifts of the electrode on the
order of the size of a single neuron would be impossible to avoid, and even a
tiny drift of the electrode could cause undesired motor action at unexpected
muscles. Compared with neurons, muscles are easier to visually identify and are
sufficiently large to be observed under a conventional optical microscope.
Variation in the position of an implanted electrode within the target muscle
and/or drift of the implanted electrode can cause small changes in the elicited
motion magnitude but might not affect the displacement direction. As such, we
expected to achieve a high success rate in eliciting leg motion in the desired
direction by stimulating leg muscles.
With respect to the other key operation needed for a closed-loop control, (2)
graded response, some variables must be identified to grade the magnitude of leg
displacement elicited by electrical stimulation. Such variables can be used as
manipulated variables in a closed-loop control system. Suppose a leg is
displaced by electrical stimulation of a leg muscle in the desired direction,
but the magnitude of the displacement is smaller than our predetermined value.
If we know a specific variable that governs the magnitude to be graded, such as
a higher value of the variable inducing a greater magnitude of leg displacement,
we update the electrical stimulation to generate a higher value of that variable
and output (apply) the updated stimulation to the muscle. For many insect
muscles, muscle contraction is enhanced by increasing the rate of neural input
(monitored as a spike in an electromyogram (EMG)) or increasing the electrical
stimulation frequency (summation or facilitation). Thus, the electrical
stimulation frequency can be used as a manipulated variable to grade the
magnitude of leg displacement and as an output from the final control element to
the leg muscle in a closed-loop control system. We measured the displacement of
leg motion elicited by various stimulation frequencies. We also measured the EMG
of a leg muscle group and associated it with the leg displacement. We then
confirmed the tendency for both a higher neural input rate and a higher
stimulation frequency to elicit a larger-magnitude leg displacement, and
consequently decided to use the stimulation frequency as the manipulated
variable in our closed-loop control system.
Overall, this paper reports the control of an insect's front leg motion by
electrically stimulating multiple leg muscle groups in a closed-loop control
manner. We successfully demonstrated a reliable biological microactuator with
multiple degrees of freedom (DoFs). The three pairs of antagonistic muscle
groups of the insect leg enable the leg to have three DoFs:
protraction/retraction, levation/depression, and extension/flexion. The
threshold stimulation voltage to elicit significant leg displacement in the
desired direction was determined. We then observed that the electrical
stimulation frequency is a variable that governs graded leg motion (i.e., the
magnitude of leg angular displacement) by measuring the elicited leg angular
displacement and velocity at various stimulation frequencies and the muscle EMG
associated with the leg motion. We then developed a closed-loop control system
in which the stimulation frequency was the manipulated variable from the final
control element to the leg muscle and the angular displacement of the leg was
the system response. This closed-loop control system can regulate the leg to set
at predetermined angular positions. Because the muscle configurations are
similar among all the six legs of an insect, the successful motion control of
the front leg will aid in developing motion controls for all other legs and
overall walking control in an insect–machine hybrid robot in the future.
# Materials and Methods
## Study animal
We used the beetle *Mecynorrhina torquata* (order Coleoptera; length: 62±8 mm;
mass: 7.7±1.9 g for all the beetles used in the experiments. Unless otherwise
stated, all data are represented as mean ± standard deviation) as our insect
platform for a biological actuator. The beetles were kept in separate plastic
terrariums (20 cm×15 cm×15 cm) with woodchips at the bottom. They were fed sugar
jelly every 2–3 days. The temperature and relative humidity in the terrariums
were maintained at 25°C and 60%, respectively. The use of this animal is
permitted by the Agri-Food and Veterinary Authority of Singapore (AVA, HS code:
01069000, Product code: ALV002). Invertebrates, including insects, are exempt
from ethics approval for animal experimentation according to the National
Advisory Committee for Laboratory Animal Research (NACLAR) guidelines.
## Electrode implantation
A beetle was anesthetized by placing it in a small plastic zip bag filled with
CO<sub>2</sub> gas for 1 min. For immobilization, the beetle was subsequently
placed onto a plastic or wooden plate and wrapped with dental wax (Cavex, Set Up
Modeling Wax), which had been softened in hot water (80°C) for 10 s. Four small
holes were made on its pronotum (positions indicated by red crosses) using an
insect pin (Indigo Instruments, enamel-coated \#5). Using the same technique,
eight more holes were made on the coxa and femur (positions indicated by red
crosses). A thin Teflon-insulated silver wire (A-M Systems, 127 µm uncoated
diameter, 178 µm Teflon-coated diameter) was used as the stimulating electrode.
The insertion depth of the electrode was 2 mm from the outer surface of the
cuticle. Both ends of the silver wire were heated in a flame to remove the
insulation and enable electrical contact at the ends.
## Electrical stimulation
To obtain a suitable threshold voltage, the non-implanted end of the wire was
connected to the output channel of a function generator (Agilent, 33220A). The
stimulation pulse width was fixed at 1 ms and the frequency was fixed at 30 Hz.
The stimulation voltage was varied in increments of 0.25 V, starting at an
initial stimulation voltage of 0.25 V.
To investigate the elicited leg motion due to different stimulation frequencies,
the three motion types of protraction/retraction, levation/depression, and
extension/flexion were analyzed individually. For example, when investigating
the protraction/retraction response to different stimulation frequencies, we
restricted the levation/depression and extension/flexion motions by inserting an
insect pin into the corresponding articulation. Two markers placed on the
beetle's leg were recognized by a 3D motion capture system as a solid line
segment, and the third marker placed on the beetle's body indicated the beetle's
position. The 3D motion capture system recognized and stored the *X*, *Y*, and
*Z* coordinates of all markers. Angular displacement was determined using the
following formula for calculating the angle between two vectors:where
*X*<sub>1</sub>, *Y*<sub>1</sub>, and *Z*<sub>1</sub> and *X*<sub>2</sub>,
*Y*<sub>2</sub>, and *Z*<sub>2</sub> are the initial coordinates of markers 1
and 2, respectively, and *X*<sub>1</sub>′, *Y*<sub>1</sub>′, and
*Z*<sub>1</sub>′ and *X*<sub>2</sub>′, *Y*<sub>2</sub>′, and *Z*<sub>2</sub>′
are the coordinates of the two markers as a consequence of the beetle's leg
motion. Therefore, all angular displacement values were calculated with respect
to the leg's initial (resting) position. The position of each leg segment at
rest (before electrical stimulation) was defined as the initial position (the
initial position varies from beetle to beetle; the variation is on the order of
a few degrees). Each time after the leg muscle was stimulated, we manually
positioned the leg to its initial position by checking the 3D coordinates of the
markers placed on the beetle's leg. For all experiments, the stimulation voltage
was fixed at 1.5 V and the pulse width was fixed at 1 ms.
The electrical stimulation power consumption was measured for all six muscle
groups present in the beetle's front leg. Current flow through a muscle was
measured using an oscilloscope (Yokogawa, DL 1640), and a function generator
(Agilent, 33220A) was used to supply a positive pulse train at 100 Hz, 1.5 V,
and a 1-ms pulse width.
## Measurement of leg-muscle EMGs synchronized with leg motion
A pair of thin silver wires (A-M Systems, 127 µm uncoated diameter, 178 µm
Teflon-coated diameter) were implanted into the muscle group of interest using
the technique described in the electrode implantation section. The electrodes
were glued to the outer surface of the cuticle with dental wax (Cavex, Set Up
Modeling Wax) to avoid potential artifacts due to the electrodes' drift. The EMG
signals were amplified 500-fold using an amplifier (LT1920, Burr-Brown
Products). A custom-programmed wireless microprocessor (Texas Instruments,
CC2431, 6×6 mm<sup>2</sup>, 130 mg, and 32 MHz clock) was used to collect the
EMG signals from the muscles at a sampling rate of 2000 Hz. The input/output
(I/O) pins of the microprocessor were set at inputs so that EMG signals from the
muscles were collected as input electrical potentials. The beetle's leg motion
was captured in the same manner as that described in the previous section
(“Electrical stimulation”) using the 3D motion capturing system. We developed a
customized software tool, BeetleCommanderEMG, which can simultaneously collect
and store EMG signals from the microprocessor and the beetle's leg motion
information from the 3D motion capturing system. The threshold voltage of the
EMG signal was determined individually for each experimental result to identify
the maximum number of EMG spikes captured. The EMG burst onset time was defined
as the time at which the voltage of an EMG spike exceeds a certain threshold
value. The EMG burst termination time was defined as the end time of the last
detectable EMG spike. The mean EMG frequency was calculated as the average of
the instantaneous frequencies within a single burst. The average angular
velocity was calculated as the linear regression slope of the angular
displacement during the time interval of motion. The onset time of leg motion
was defined as the time of first detectable leg retraction motion. The motion
offset time was defined as the beginning motion of the first detectable
protraction motion.
## Closed-loop control system
We developed a closed-loop motion control system to be introduced into
BeetleCommander. An electrical stimulation signal was generated using a custom-
programmed microprocessor (Texas Instruments, CC2431, 6×6 mm<sup>2</sup>, 130
mg, 32 MHz clock). Electrical stimulation signals generated from two separate
stimulation channels were used to control one pair of antagonistic muscle
groups. The BeetleCommander system could extract instantaneous marker position
information from the 3D motion capture system and calculate the immediate leg
angular position. Update time intervals (i.e., the time interval at which the
closed-loop system updated the instantaneous leg position and output stimulation
frequency) were user-adjustable. The concept of proportional control was also
used to adjust the magnitude of a step increment or decrement of the stimulation
frequency:where is the last output stimulation frequency from the final control
element, is the updated output stimulation frequency from the final control
element (its value is limited to the 10–200 Hz range), is the proportional gain
(user-adjustable), and is the instantaneous angular displacement error at time
*t*.
Protraction/retraction closed-loop motion control was used as an example of
operation, which was controlled by two separate channels from the final control
element. Electrical stimulation signals from the two channels were generated on
the basis of predetermined angular positions set by the user. Both stimulation
channels operated independently and concurrently. The electrical stimulation
frequency from one channel was initially increased to elicit the leg to move to
the desired position. If the actual leg angular position was greater than the
predetermined angle, the stimulation from the current working channel started to
decrease its stimulation frequency while that from the counter channel began to
decrease the angular position by increasing the stimulation frequency. Likewise,
if the actual angular position was less than the predetermined angle, the
counter channel stopped generating its stimulation signal and the other working
channel began to increase its stimulation frequency to increase the leg angular
position. Therefore, the two channels used to stimulate the pair of antagonistic
muscle groups operated simultaneously to ensure that the beetle's leg followed
the predetermined angular positions.
# Results and Discussion
## Threshold stimulation voltage to elicit leg displacement
We fixed the stimulation frequency at 30 Hz and used a 1 ms monophasic pulse
train with amplitudes ranging from 0.25 V to 2.50 V. shows the elicited
protraction/retraction angular displacements at various stimulation voltages
(number of beetles = 5 and 17≤ number of data points at each stimulation voltage
≤22). The threshold voltage required to elicit a beetle's leg movement was
approximately 1.0 V, and the maximum angular displacement was reached at
approximately 1.5 V (maximum protraction/retraction angle at 1.5
V = 18.09°±4.87°/12.73°±7.04°). When the stimulation voltage exceeded 1.5 V, the
maximum angular displacement for both protraction and retraction remained
relatively constant. For optimal results, we had to set the stimulation voltage
as low as possible to minimize any possible damage to the beetle's muscle while
simultaneously ensuring that the stimulation voltage was sufficiently high to
reliably elicit the desired leg motion. Thus, we fixed the stimulation voltage
at 1.5 V for all subsequent experiments. With a 1.5 V stimulation voltage, the
success rate for inducing leg movement in the desired direction was 100% (number
of beetles = 42). In addition, after repeatedly applying the electrical
stimulation to a single muscle group more than 200 times within one day during a
single experiment, we observed no obvious indications that the beetle's muscle
was damaged by the 1.5 V stimulation voltage.
## Stimulation frequency as a variable to grade the leg displacement magnitude
For all experiments, the stimulation voltage was fixed at 1.5 V and the pulse
width was fixed at 1 ms. The elicited leg motion at various stimulation
frequencies was studied. As shown in, the resulting angular displacements for
all six motion types monotonically increased with the stimulation frequency
(number of beetles = 10). The angular displacement of the beetle's leg reached
its limit when the stimulation frequency exceeded approximately 80 Hz for the
other five motion types, with the exception of levation motion (the maximum
angular displacement for levation was reached at stimulation frequencies of
approximately 40 Hz). This maximum angular displacement might be due to the
mechanical limitation of the beetle's leg structure. Similarly, the average
angular velocity also increased monotonically with the stimulation frequency.
When the stimulation frequency was greater than 250 Hz, the average angular
velocity of the leg motion reached the maximum value for the other five motion
types, with the exception of levation motion (, maximum average angular velocity
of levation was reached at a stimulation frequency of approximately 100 Hz).
## Comparison between muscle stimulation and sensory system stimulation
As discussed in the, the performance of a closed-loop control system in
stimulating a muscle is compared to that of a sensory system (e.g., a system
stimulated by an antenna or compound eye) on the basis of two key performance
requirements: (1) reliability in the direction of elicited leg displacement and
(2) a graded response in the leg displacement magnitude. As demonstrated in the
previous two sections, our leg-muscle stimulation satisfies both these key
requirements. However, numerous researchers have demonstrated various protocols
of stimulating sensory systems to control insect locomotion in walking and in
flight. For instance, Holzer and Shimoyama stimulated the antennae of a
cockroach (*Periplaneta americana*) to control its walking direction. Transient
(100–200 ms) turning behavior was observed when the ipsilateral antenna of a
walking cockroach was stimulated. However, the electrodes used to stimulate
sensory systems are not well secured, and the two aforementioned performance
requirements are not guaranteed. Unlike muscle stimulation, the stimulation of
sensory systems does not result in a 100% success rate in eliciting the desired
motor action or behavior. A graded response in a target muscle by stimulation of
sensory systems is possible and has actually been demonstrated, however, this
approach is relatively difficult and less reliable than muscle stimulation, as
discussed in the. Overall, the adaptation of muscle stimulation in closed-loop
motion control is preferred over the adaptation of sensory system stimulation.
## Comparison between the leg motions elicited by electrical stimulation and those by intrinsic neural input
To understand how effectively our electrical stimulation (synthetic input to
muscle) mimics an animal's intrinsic neuromuscular system (natural input to
muscle), we recorded the EMG signal of the retraction muscle group during
natural leg motion and synchronized the EMG with the leg displacement. A
decrease in the angular displacement corresponds to a retraction motion in the
figure. shows the average retraction velocity as a function of the mean muscle
EMG frequency (number of beetles = 4, total number of data points = 43). The
maximum EMG frequency recorded was approximately 70 Hz. A linear relationship
was observed between the angular velocity and the mean EMG frequency. shows a
plot of the average retraction velocity of the beetle's front leg as a function
of the electrical stimulation frequency (number of beetles = 5, number of data
points at each stimulation frequency = 25). Another linear relationship is
observed in this figure, which indicates that a higher stimulation frequency
resulted in higher angular velocity and hence elicited greater muscular force.
Despite the expected result that the angular velocity monotonically increases
with both average muscle EMG frequency and electrical stimulation frequency, the
slope of the linear regression line for the former case is approximately five
times steeper than that for the latter case; that is, provided that the EMG
frequency is the same as the electrical stimulation frequency, the resultant leg
motion is five times faster when the beetle moves voluntarily compared with when
it is electrically stimulated. This difference might be due to the basic
differences between the EMG signal (natural neural input) and the electrical
stimulation signal (synthetic input) in terms of amplitude and signal forms. In
addition, because the beetle might have resisted the electrically elicited
motion by activating the antagonist muscles and produced an opposing force, the
angular velocity of leg motion elicited by electrical stimulation could have
been smaller than that expected, on the basis of the tendency of the motion
associated with the EMG spike frequency.
## Demonstration of a closed-loop control system for insect leg motion
As each muscle group was stimulated by independent, isolated outputs from the
stimulator board, we could elicit the individual leg motion types either
separately or simultaneously, as demonstrated in and. Moreover, on the basis of
the findings that the leg angular displacement monotonically increased with
respect to the stimulation frequency, we developed a closed-loop control system
to make the leg move according to preset angular positions. shows typical
closed-loop control results (number of beetles = 5) for protraction/retraction
motion; these results were obtained by comparing the actual leg movement
achieved (blue path) with the predetermined leg angular position (red path) at
*K*<sub>p</sub> values of 0.1, 0.5, 1.0, and 1.5 and at update time intervals of
100, 200, and 300 ms (the update time interval is the time interval at which the
closed-loop system updates the instantaneous leg position and output stimulation
frequency). shows the overshoot and reaching time of the closed-loop control
experiment (number of beetles = 5, 35≤ number of data points at each experiment
setting ≤49). The statistical information for the experiments in which
*K*<sub>p</sub> = 0.1 is omitted from because the rate of increase of the muscle
stimulation frequency is not always sufficient to bring the leg to the
predetermined angular position due to the relatively small *K*<sub>p</sub> value
(see, *K*<sub>p</sub> = 0.1, update time interval = 100, 200, and 300 ms). Of
the 243 experimental data points for *K*<sub>p</sub> = 0.1, the beetle's leg
reached the predetermined angular position 104 times (42.8% success rate).
However, for *K*<sub>p</sub> = 0.5, 1.0, and 1.5, the beetle's leg was always
(100% success rate) brought to the predetermined angular position. As evident in
and and, when the *K*<sub>p</sub> values were increased, the leg response
overshoot generally increased, whereas the reaching time (i.e., the time
required to reach the predetermined angular position) decreased. For example,
when the *K*<sub>p</sub> value was increased from 0.5 to 1.5 using an update
time of 100 ms, the protraction overshoot angle increased from 10.47°±3.66° to
22.12°±7.75° and the retraction overshoot angle increased from 11.03°±5.06° to
17.48°±4.94°; in contrast, the protraction reaching time decreased from
0.518±0.133 s to 0.299±0.188 s and the retraction reaching time decreased from
1.249±0.917 s to 0.488±1.111 s. This tendency was consistent because larger
*K*<sub>p</sub> values would result in greater changes in the output stimulation
frequency (either increases or decreases) for the same instantaneous angular
displacement error. This larger output stimulation frequency change would then
elicit a greater angular displacement and a greater angular velocity of the
beetle's leg (i.e., a larger force elicited in the muscle). As a result, the
beetle's leg would move at a faster rate (decreased reaching time). However, at
the same time, the beetle's leg would be more likely to exceed the predetermined
angular position (increased overshoot). In general, as the update time interval
was incrementally changed, the leg response overshoot decreased, whereas the
reaching time increased (comparison across different *t* values, and). For
example, for *K*<sub>p</sub> = 1.0, as the update time was increased from 100 to
300 ms, the protraction overshoot angle decreased from 17.51°±5.79° to
16.01°±8.39° and the retraction overshoot angle decreased from 15.97°±5.38° to
6.52°±4.34°, whereas the protraction reaching time increased from 0.350±0.184 s
to 0.564±0.150 s and the retraction reaching time increased from 0.492±0.217 s
to 0.875±0.308 s. This tendency was consistent because a larger update time
interval would result in a longer required period for the beetle's leg to
respond to the electrical stimulation. This longer response period decreased the
likelihood that the 3D motion capture system would capture the leg position
before it reached its final position. Therefore, this longer response period
decreased the likelihood that the BeetleCommander closed-loop control system
would overly increase or decrease the stimulation frequency, which would result
in reduced overshoot. However, at the same time, with an increasing update time
interval, the reaching time would increase.
The statistically obtained reaching times and overshoots at the different system
settings (the proportional gain and update time interval) reveal certain
constraints and limitations in the design of insect–machine hybrid legged robots
in terms of the step cycle of the leg in the walking gait. We can refer to the
reaching-time data to determine the appropriate stepping frequency (e.g., the
duration of each step should be longer than the combined reaching time of all
motions involved in that step). Given the angular overshoot, we can estimate the
step-length error present in a given closed-loop control system. For example,
when *K*<sub>p</sub> and the update time interval *t* are set to 0.5 and 300 ms,
respectively, the overshoot for retraction motion is 5.05° on average. This
overshoot angle of 5.05° results in an estimated step-length error of
approximately 0.22 cm if the beetle's leg length is assumed to be 2.50 cm. As
such, even for future advanced close-loop control systems, we can refer to these
indices (e.g., the reaching time and overshoot) to reduce the constraint and
limitation of the step cycle and other relevant parameters in insect–machine
hybrid legged robots.
## Potential experimental errors
The use of living organisms in experiments can introduce several unavoidable
errors, which is why closed-loop motion control is necessary. For example, the
neutral or resting position of the leg differs within a few degrees from beetle
to beetle. Although some researchers have defined the neutral position of a
joint in terms of the angular position between the two leg segments (e.g.,
Guschlbauer et al. defined the neutral position of the femur-tibia joint of the
stick insect as 90°), ensuring that the leg appendage of a living insect rests
at the neutral position, as predefined by us, is difficult.
The CO<sub>2</sub> anesthetization of the beetle before implantation of the
stimulation wires (see the Electrode implantation section) can affect the leg
response. Several side effects of CO<sub>2</sub> anesthetization on insects have
been reported. CO<sub>2</sub> anesthetization can increase haemolymph acidity
and cause the heartbeat to stop. As a result, exposure to CO<sub>2</sub> can
impair oxygen delivery to tissues, thereby reducing oxidative phosphorylation
and adenosine triphosphate (ATP) production in cell mitochondria. These effects
can significantly influence the efficacy of a bio-actuator and are known to
affect insects' locomotion. Nonetheless, CO<sub>2</sub> exposure is one of the
most popular anesthetic methods in entomological research, even though its side
effects have not yet been fully elucidated. Improved anesthetic methods might be
helpful in future entomological research.
Despite these potential experimental errors, the magnitude of the leg
displacement is undoubtedly increased by increasing the stimulation frequency.
Inspired by this graded response to the stimulation frequency, we successfully
developed a closed-loop control system to regulate the leg to set at predefined
angular positions; this controlled response is independent of the potential
experimental errors in leg response because closed-loop control systems, in
general, reduce such errors.
## Power consumption
We confirmed that the power consumption of the insect leg actuator was
remarkably low (on the order of 100 µW to a few milliwatts). shows the positive
pulse train at 100 Hz, 1.5 V, and a 1 ms pulse width used as the muscle
stimulation signal. shows a typical current-flow profile through the depression
muscle group. The power consumption of electrical stimulation on all six muscle
groups of the front leg was measured across five different beetles. shows the
detailed numerical values of the mean and standard deviation in power
consumption (number of beetles = 5, number of data points collected from each
muscle group = 40). The average power consumption of the electrical stimulation
of a single muscle group was 148 µW. Assuming that the stimulations of the
middle and hind legs consume similar amounts of power, the power consumption of
an insect–machine hybrid legged robot using a beetle is approximately 5.3 mW in
the worst-case scenario, where all six muscle groups are stimulated
simultaneously for all six legs (stimulation of 36 leg muscle groups). Note that
the worst case scenario should be far from the actual case because just half of
the muscle groups would be simulated simultaneously in actual insect walking
control. Nonetheless, this power consumption is considerably low compared with
the 100–1000 mW order of power consumption in entirely man-made miniature legged
robots.
# Conclusions
The experimental results demonstrated that electrical stimulation with a
threshold voltage of 1.5 V elicited significant displacement of the leg in
desired directions (three DoFs, i.e., protraction/retraction,
levation/depression, and extension/flexion) at a 100% success rate. The
magnitude of the leg displacement was graded by the stimulation frequency: a
higher stimulation frequency elicited a larger-magnitude displacement. We used
the stimulation frequency as the manipulated variable in our closed-loop control
system, and the controlled leg was successfully set at predetermined angular
positions. In conclusion, coupled with the low power consumption compared with
that of entirely man-made legged robots, the ability to regulate a beetle's leg
motion under a closed-loop control system should contribute significantly to the
future design of biological actuators and hence biological legged machines
(i.e., insect–machine hybrid legged robots). Note that the leg-muscle
configurations are common or similar (i.e., a pair of antagonistic muscle groups
dominating the leg displacement in opposite directions) among many insect
orders. In addition, various stimulation protocols to elicit leg displacement in
the desired direction have been proposed and demonstrated for various insect
orders, including moth, stick insect, and locust. Therefore, the methodology and
experimental design demonstrated in this paper may be applicable to the
development of a closed-loop control of the leg motion of other insect orders.
# Supporting Information
The authors offer their appreciation to Mr. Cheo Hock Leong, Mr. Ow Yong See
Meng, Ms. Chia Hwee Lang, Mr. Poon Kee Chun, and Mr. Roger Tan Kay Chia at the
School of MAE, NTU. The authors thank Professor Ryohei Kanzaki (University of
Tokyo) and Professor Jiro Okada (Nagasaki University) for their helpful advice
and Professor Kris Pister (UC Berkeley) for providing the wireless communication
device.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: FC CZ TTVD YL DHS JSK NAH KI
PA MMM HS. Performed the experiments: FC CZ TTVD YL DHS JSK NAH MFBA HS.
Analyzed the data: FC CZ TTVD DHS JSK NAH MFBA HYC HS. Contributed
reagents/materials/analysis tools: FC CZ TTVD YL DHS JSK NAH MFBA HS. Wrote
the paper: FC CZ TTVD MMM HS. |
# Introduction
Half of UK workers are office based. Office workers spend approximately two-
thirds of the working day in seated tasks and are estimated to accumulate around
10.5h of sitting per waking day. Growing evidence has associated prolonged
sitting, characterised by very low energy expenditure (1.5 metabolic equivalents
or less), with increased disease and premature mortality risk. Sedentary office
work is an urgent public health issue. There is some evidence to suggest that
some of the deleterious health effects of sitting time can be offset by engaging
in moderate physical activity for at least one hour daily. However, based on
self-report data–which typically overestimate true activity levels–a third of
the UK population fails to meet the more modest target of 150 minutes of
moderate activity per week. It may be unrealistic to expect office workers who
are highly physically inactive and highly sedentary to adopt daily bouts of
activity lasting an hour or more. Displacing sitting with standing may offer a
more feasible sitting-reduction strategy.
Standing at work is often proposed as a way of reducing sitting. Standing has
been linked to lowered mortality rates, likely due at least in part to greater
energy expenditure, reduced glucose variability and oxidative stress. While
there are no government guidelines offering targets for standing and sitting
time in the UK, a recent expert-consensus document recommended that office
workers accumulate 2-4h of standing and light activity daily, and take regular
breaks from prolonged sitting. Achieving this target will require developing
strategies to displace sitting with standing. While the most commonly-evaluated
strategy has been to restructure the environment to facilitate desk work while
standing or moving, another commonly-proposed strategy is to promote standing in
normally-seated meetings.
There are multiple routes through which standing in meetings might be widely
adopted within the workplace. At the organisational and environmental levels,
managers could implement initiatives that explicitly support standing meetings
among employees, such as introducing standing-permissive meeting room furniture,
or enshrining standing meetings into workplace practices and procedures. Indeed,
standing meetings are commonplace in the software development sector, where they
are used for short daily team briefings. At the individual level, employees
might drive organisational change by voluntarily choosing to stand in meetings,
with the aim of normalising standing in typically-seated contexts. Such a
strategy, however, depends on the acceptability of standing in meetings to those
who attempt it.
Interview studies suggest that office workers believe standing in meetings to be
an acceptable sitting-reduction strategy *in principle*, but few studies have
documented *lived experiences* of attempting to stand in normally-seated
meetings. The scant available research into standing meetings to date has
focused on responses to the topic matter and meeting frequency, not the standing
experience itself. Initial experiences of novel behaviours are important
determinants of maintenance; the sustainability of standing in meetings where
all others are seated will depend not only on prior expectations, but also
whether experiences match expectations. Employees are unlikely to continue to
stand in seated meetings where they find the experience intolerable or
unsuitable.
This paper reports the first study to our knowledge to examine employees’
experiences of standing in seated meetings using qualitative data. Understanding
how employees experience standing in meetings, and the organisational, social
and psychological structures that surround such experiences, will help to inform
development and implementation of worksite standing interventions that go beyond
the provision of sit-stand workstations. Our study focused on university
workers. Universities are complex organisational structures offering diverse
office environments, with workers from across the socioeconomic spectrum, so
university workers’ experiences may potentially be relevant to many other office
workers and settings. Our research question was: “how do office workers
experience standing in normally-seated meetings?”
# Methods
## Participants
Participants were recruited from three UK-based universities, between January-
April 2016, using convenience sampling methods. Recruitment was conducted using
print and online advertisements through university communication channels
(internal email, poster, staff newsletter, Twitter). Participants were offered a
£50 (\~US\$65) voucher for taking part. Potential participants returned by email
an expression of interest form, and self-reported their demographics (work/job
role, gender, age, disability, ethnicity, income;) and eligibility, via items
assessing the following criteria. Eligible participants were: desk-based
employees; aged 18+ years with no intention to leave the organisation before
July 2016; able to identify three meetings, differing in size, with potential
for standing; and willing to be observed at meetings and interviewed. Those who
had engaged in standing up in meetings in the past 3 months, used a sit-stand
desk in the past 3 months, were full time students, or were unable to stand,
were excluded. Those meeting eligibility criteria took part in a face-to-face,
telephone or email inception meeting to clarify study requirements and agree
meeting dates.
Due to deadlines imposed by the broader project within which this study was
located, a six-month window was set for study planning, data collection and
analysis, which included three months for recruitment. Twenty-seven participants
were recruited during this time, of whom two (from Workplace A) dropped out,
citing insufficient time for participation, after the first meeting. The final
sample thus comprised 25 participants (7 from Workplace A, 10 from Workplace B,
8 from Workplace C). Of these, six (24%) were male, 18 (72%) female, and one
(4%) self-declared as ‘other’. The modal age category was 25-29y. Twenty-three
(92%) participants had degree level or higher qualifications, and two (8%) had
technical or professional qualifications.
## Procedure and interview schedule
As part of the pre-study information sheet, participants were given minimal
information on the potential health benefits of standing (“public health
researchers have suggested that office-based workers should stand up in
meetings, to promote health”), and were told that “the study aims to explore
what it is like for office-based employees to stand in meetings”. Participants
were asked to select three group workplace meetings that they had been due to
attend irrespective of study participation, and which differed in size (small:
3–10 attendees; medium: 11–19; large: 20+), such that each participant attended
one meeting of each size (i.e., three meetings in total). We determined three
meetings to be sufficiently conducive to variation in experiences for each
participant, and feasible within the study period.
We instructed participants to stand whenever they felt they wanted and for
durations decided by them. No further instruction was given. Except for five
meetings (covering three participants) to which access was denied to non-
invitees, a researcher attended each meeting to observe participants’ standing
behaviour, others’ responses, room layout and number of attendees, though these
field notes were not deemed sufficiently rich for analysis. A semi-structured
interview (face-to-face or telephone) was conducted as soon as possible (and no
longer than 48 hours) after each meeting (i.e. 3 interviews per participant), to
gather reflections on experiences of standing. One participant (Amelia) attended
her third meeting immediately following the second meeting, and so completed one
interview addressing both meetings. Interviews took place within the meeting
venue or another setting within the participant’s workplace, according to
participant preference. Nobody else was present during interviews.
An interview schedule was developed to cover the fundamental determinants of
standing (capability, opportunity, motivation), and piloted among three office-
based colleagues of the researchers. In all interviews, questions focused on
affective reactions to standing; others’ reactions; standing location, timing
and duration; occupational identity and status; workplace culture and norms; and
acceptability and feasibility of standing in meetings. In the third interview,
views towards workplace standing interventions were also sought. Each interview
was conducted by one of three female researchers, one of whom (JH) was a Social
Sciences doctoral student with previous experience of interviewing in the
workplace sedentary behaviour research domain, and two (MR, ER) were Health
Psychology Masters students trained in interviewing by the senior authors (LM,
BG). Each participant was interviewed by the same interviewer on three
occasions. The only interviewer-participant contact prior to the first interview
was via email or telephone, for the purposes of organising the first interview.
Participants were told, prior to the interview, that the interviewer was
conducting the study as part of a funded research project (JH), or as coursework
(MR, ER). No other interviewer characteristics were shared with participants
prior to the first interview. Interview duration ranged from 8-32mins (mean
20mins).
The study was approved by the Research Ethics Committee of each institution
(LRU-15/16-2533; 4385/002; 1875-LR-Jan/2016-1200). All participants provided
fully informed written consent to participate.
## Analysis
Interviews were digitally audio-recorded and transcribed verbatim, with
transcript quality and completeness verified by the researchers. We offered to
return transcripts, and our ongoing or completed analyses, to participants for
comment, correction, or any other purpose, but none expressed a wish to receive
them.
Interview transcripts and analysis were managed via NVivo 10 software. A
phenomenological methodological orientation was adopted, as this allows for
description (rather than quantification or explanation) of participants’
experiences, and exploration of the subjective meanings that they ascribe to
elements of these experiences. Data were analysed using Framework Analysis,
which allows inductive co-creation between multiple researchers of an initial
coding framework which guides subsequent analysis, and which is developed and
iteratively refined as coding progresses and new insights emerge. Repeated and
independent reading of a sample of interview data from one institution was
undertaken by two researchers (JH, LM), to develop a preliminary coding
framework. Next, selected interview transcripts (n = 9/25) were read and re-read
by four other researchers (MR, LS, ER, BG), who refined the coding framework to
ensure its accuracy and relevance. Discrepancies were resolved by discussion,
and resultant codes and themes verified by all researchers. The coding framework
was used (by JH, LM, and BG) to analyse the remaining data. Given that data
collection was constrained by the study timeline, data saturation was not
discussed prior to analysis.
Quotes are provided below as evidence of the validity of our analysis. All
participants were assigned pseudonyms. Punctuation was added to unambiguous
quotes and where necessary, words added in parentheses to clarify intended
meaning.
# Results
Four themes were derived from the data: physical challenges to standing;
implications of standing for meeting engagement; standing as norm violation; and
standing as appropriation of power. The first theme details experiences of
physical discomfort and of attempts to negotiate the physical environment to
permit standing. The second theme reports different ways in which standing
impacted on participants’ involvement in meetings. The third and fourth themes
describe discrete psychological challenges involved in negotiating the
organisational and inter-person context that frames workplace meetings.
## Theme 1: Physical challenges to standing
Some participants were aware of the detrimental impact of sitting and expected
that standing in meetings would confer health benefits (“*you just perceive
being standing up*, *it’s just better for your health and it just makes you feel
better overall”*; James, Workplace A). Yet, the physical experience of standing
in meetings rarely mapped onto participants’ expectations. Some reported
unexpected and unaccustomed discomfort from physical sensations in the muscles
of the feet, legs, back and shoulders, while others anticipated but did not
experience physical discomfort:
> *I just thought, oh your back will ache or legs ache or something, but >
actually it wasn’t borne out in reality*. > > (Tom, Workplace B).
The physical impact appeared partly due to the time spent standing, which was
self-determined by participants. Some expected to be able to stand for the
entirety of lengthy meetings, but on attempting to do so realised this was not
possible, due to physical discomfort:
> *After about twenty-five minutes … \[I was\] thinking oh my back is > killing
me! There’s the realisation that, oh I can’t stand for very > long*! > > (Joan,
Workplace B)
Aspects of the physical environment–furniture design, spatial configuration, and
the numbers and positions of attendees–also presented barriers and challenges to
standing. Participants’ descriptions of their meeting room environments
suggested that there were many chairs, but no standing areas, nor desks and
tables to support standing (a “*lack of furniture for standing”*; Angela,
Workplace C). The physical environment was felt to elicit sitting (*“the
\[physical\] environment … kind of shapes social expectations \[about standing\]
… we sit so much”*; Angela, Workplace C), which in turn reinforced perceptions
that standing in meetings was neither acceptable nor feasible:
> *Today, with the chairs all round, already set up, it makes it more of > a
barrier to actually saying I’m going to stand*… *it’s so easy just > to go, oh
I’m going to sit down* > > (Tom, Workplace B)
A lack of perceived environmental support for recording information when
standing posed practical difficulties for several participants, making engaging
in the meeting non-ergonomic and potentially physically uncomfortable:
> *Bending down and trying to take notes, it didn’t feel natural*. *It > was
just a question of the desk or table not being at a certain height > really* > >
(Charlie, Workplace B)
Suggestions were made for overcoming barriers presented by ill-suited physical
environments, including furniture adaptation, or using accessories to permit
usual meeting engagement while standing; for example, using a tablet instead of
pen and paper for note-taking.
## Theme 2: Implications of standing for meeting engagement
Many participants reported that standing affected their engagement in the
meeting. Some found that physical discomfort from standing motivated them to
increase engagement to minimise meeting length:
> *Because I was stood … \[I was\] kind of more, more ‘let’s get on with > it,
let’s get to the point’. \[…\] Because it’s not so relaxed as > being sat back
in a chair … the perception is the meeting is going on > for longer, or more
frustration if there’s no action being taken. > \[…\] It’s more efficient*. > >
(Tom, Workplace B)
Others felt that shifting from a relaxed, seated position to standing sustained
focus on the meeting, because standing prevented them from “*switching off*”
(James, Workplace A) as they would while sitting.
Many reported, at least in the first of the three meetings in which they stood,
unanticipated feelings of psychological discomfort from standing while all
others were sitting, together with a heightened awareness of the self, others,
and the interpersonal context. Participants variously felt *“disconcerted”*
(Joan, Workplace B), *“awkward”* (Brianna, Workplace C), or *“stupid”* (Charlie,
Workplace B), possibly due to being more visible than others. Some reported that
enhanced visibility made them feel more accountable to others:
> *I tend to drift out or find them a bit boring. \[…\] Because I was > both at
the front of the room and standing, I felt much more like I > had to, even if I
wasn’t engaged, look like I was more engaged, which > then made me more engaged.
So I actually listened to the whole thing! > \[…\] If I started drifting out …
I’d then get really self-conscious > and think, oh God, what if somebody saw me
with my glazed-over eyes or > something like that*! > > (Anne, Workplace C)
For others however, self-conscious thoughts were a distraction from the meeting:
> *When I did sit down, I was like (sighs). More relaxed. I could just > focus
on the meeting, not focus on my standing*. > > (Brianna, Workplace C)
Some participants sought to minimise psychological discomfort by standing in a
position within the room that they perceived to be less visible, so avoiding
obstructing others:
> *I positioned myself right towards the back in the corner… it was OK > to
stand because I wasn’t making a nuisance of myself to anybody, I > wasn’t in any
body’s way*. > > (Anusha, Workplace A)
However, physically removing themselves from others was experienced by some as
isolating, and potentially limited involvement in interactions within the
meeting:
> *Standing up made me … \[feel\] like I wasn’t part of the group. … > \[Once I
sat down\] I felt like I was then part of the meeting. And it > felt more like
we were a team group coming to some decisions and > stuff, because we were all
on the same eye level*. > > (Alisha, Workplace C) > > *I got missed out on the
signing register. I didn’t draw attention to > myself because I wanted someone
to notice me and give it to me, and > they didn’t*. > > (Selma, Workplace C)
Some felt that other attendees were preoccupied with their standing, which might
have compromised the engagement of others, and limited overall satisfaction with
the conduct of meetings:
> *They were kind of looking at me instead of looking at the director… I > think
I just felt like I was being a distraction for them \[others in > the meeting\],
I felt like I was taking away from the meeting*. > > (Brianna, Workplace C)
Some were concerned that standing could be interpreted by others as
unwillingness to fully engage in the meeting, and indeed, a minority of
participants reported being asked by others to sit for this reason:
> *\[In a meeting\] you expect someone to sit down and then \[if\] they > don’t
you think, are you not staying or do you not really want to have > the meeting*?
> > (Alisha, Workplace C) > > *She \[the chair of the meeting\] goes: “it’s
really distracting with > you standing up. It feels like you’re getting ready to
go, could you > sit please?” So then I sat*. > > (Brianna, Workplace C)
Many felt it particularly inappropriate not to sit in formal meetings, or those
addressing sensitive topics, as it risked diminishing the seriousness of the
meeting:
> *Knowing that \[the meeting topic\] is actually maybe quite > confidential or
sensitive, I don’t want to be standing up. I need to > be sitting down*. > >
(Else, Workplace C)
## Theme 3: Standing as norm violation
Many participants found that standing in all-seated meetings made salient the
prevailing implicit norm of sitting in meetings, and their deviation from this
behavioural standard.
> *As soon as it’s called a meeting, it formalises everything. There are > just
certain social expectations and standing is not one of them. > \[…\] I felt like
I was breaking the rule. \[…\] As with any social > norm, as soon as you’re in a
position where you might be going against > it, you suddenly feel the weight of
society’s expectations on you*. > > (Angela, Workplace C)
Participants worried that standing would be seen as a deliberate attempt to
challenge the sitting norm, and that they would be perceived by other attendees
as “*an attention seeker*” (Ben, Workplace B), willfully detracting from the
business of the meeting. Some participants were concerned that being seen to be
violating the norm could potentially detrimentally impact other attendees, and
so the progression and outcomes of the meeting:
> *I would worry that I was making them \[other attendees\] > uncomfortable and
worry that they wouldn’t be able to have the meeting > that they wanted, and
that they wouldn’t get out of it what they > wanted or not be able to talk as
freely as they would normally*. > > (Alisha, Workplace C)
Characteristics of the meeting context–the perceived formality, purpose, type,
length, and size of the meeting, and relationships between attendees–shaped the
standing experience for many, often affecting the extent to which participants
felt compelled to conform to the sitting norm. Meeting contexts that made
participants’ contravention of the sitting norm more prominent were most
aversively experienced. For example, meetings characterised by frequent
interaction between attendees, larger meetings, and those where other attendees
were unknown to the stander, were often cited as challenging. Many participants
sought to deflect unwanted attention and avoid misconceptions from others by
forewarning other attendees of their intention to stand, or seeking explicit
permission from the meeting leader, in advance or at the outset of the meeting.
Most felt that they had to explain their decision to stand to others, and while
many truthfully cited involvement in our study, some felt that this provided
insufficient justification, instead feigning ill-health to claim exemption from
the sitting norm:
> *I lied and told them I had a health reason for needing to stand*. > \[…\]
*It’s just one of those things that*, *unless you have a good > enough excuse to
stand*, *they’re going to assume that you’re just > being difficult*. > >
(Anusha, Workplace A)
Several participants recounted episodes in which their decision to stand was
misinterpreted by others as reflecting a lack of opportunity to sit, which in
turn was felt to obligate the participant to sit when such an opportunity was
provided:
> *I probably stood for about a minute … and then someone else looked at > me
and they were gesturing that they’d saved me a seat! \[…\] I felt > super
awkward and sat down*. > > (Angela, Workplace C)
## Theme 4: Standing as appropriation of power
Psychological discomfort appeared to arise not only from being seen to violate
sitting norms, but also because standing was felt to affect the power dynamics
of the meeting. Standing in an all-seated meeting was felt to symbolize status
and authority within the meeting:
> *You tend to think the more authoritative person, or the person that’s > going
to lead the conversation, might be the one that’s a bit higher*. > > (Brianna,
Workplace C)
Indeed, many reported that standing made them feel empowered:
> *I probably addressed everyone and raised my voice a little, projected > it a
bit more than I might do … if I was seated. \[…\] \[Standing is\] > a much more
confidence-boosting posture*. > > (Joan, Workplace B)
Where the participant was hosting the meeting, the additional power conferred by
standing was deemed useful for denoting and exercising leadership (“*I was the
lead … so it seemed natural that I had that authoritative position*”; Anusha,
Workplace A). Where participants were not leading the meeting, however, they
worried that standing would be misconstrued as a tacit attempt to appropriate
power by challenging the authority of the meeting leader, or other, more senior
attendees:
> *If everyone’s sat down there and you’re up there*, *there’s*, *you > know*,
*almost a visual representation of a hierarchy in a weird way*. > > (Joan,
Workplace B) > > *I stood up while \[my manager\] sat down. I felt uncomfortable
> because I felt like I was telling her what to do, like I was like a > teacher
and she was a student because I was standing over her. \[…\] I > think she
probably felt equally as uncomfortable*. > > (Angela, Workplace C)
Meetings featuring presentations by one attendee to the group were typically
less problematic, as were those in which senior attendees explicitly supported
standing, because participants felt that there was little risk of standing being
interpreted as an attempt to assert power in such situations:
> *As a junior member in a meeting, I wouldn’t really be willing to > stand up
and say, well I’m standing up because I want to stand up. > \[But\] I’d be more
willing to, if … someone more senior said I’m > standing up, I’d say, great, I’m
going to join you*. > > (Amelia, Workplace B)
Meetings held in familiar social or physical settings, especially locations over
which the participant felt they already had ownership and authority (e.g. the
participant’s office), were also less psychologically uncomfortable:
> *Just the familiarity of the people in the team now, my relationship > with
the people in the team makes it easy to stand, I’m comfortable, > you know if
anyone made, no one’s made really any negative comments, > but even if anyone
did, I’d be comfortable just being like, well this > is what I want to do*. > >
(James, Workplace A)
# Discussion
Standing in meetings is often proposed as a strategy by which to reduce
workplace sitting, but no study to our knowledge has yet documented how people
experience standing in meetings in which all other attendees sit. Interviews
with volunteers who stood in planned workplace meetings revealed barriers that
workers can expect to face if deciding to stand where others are sitting, and
potential facilitators. For many, the experience of standing in meetings was
uncomfortable in some way. Some participants experienced physical discomfort
from standing for self-imposed lengthy periods and spoke of practical challenges
posed by the lack of suitability of meeting room furniture to standing. Perhaps
moreover, participants felt psychologically uncomfortable about standing. This
apparently arose from concerns about being seen to violate a compelling social
norm favouring sitting, or being seen to be challenging the authority of other
attendees by standing while others sat. Many participants chose to reduce the
potential impact of their standing on others by removing themselves to the edges
of the meeting room, though this risked limiting their involvement in the
meeting. For meeting hosts, standing was often found to confer greater power,
and enhance confidence. Our findings provide a much-needed illustration of the
broader interpersonal and organizational contexts that frame workplace
behaviour, and the difficulties these pose for standing in meetings. Promoting
standing in normally-seated meetings requires that office-based organisations
and workers anticipate these challenges.
It is important to mitigate potentially aversive consequences of standing in
meetings; while office workers generally appear willing to try standing in
meetings, they are unlikely to continue to stand if initial attempts fail to
attain expected positive outcomes or yield predominantly negative outcomes. Our
findings revealed several such negative outcomes, many of which participants did
not foresee. Many reported physical discomfort, sometimes identified as pain,
which appears to have been due to prolonged standing; many felt that they had to
stand for the entirety of meetings, though we did not instruct them to do so.
This likely reflects a misplaced belief among the public that the health risks
of sitting can only be offset by prolonged standing. Yet, standing still for
long periods can also harm health. A recent expert consensus statement on
workplace sitting and standing advises that “prolonged, static standing postures
be avoided” (p1360), and that sitting be replaced by frequent sit-stand
transitions, standing, and light physical activity. While physical activity is
unlikely to be feasible in meetings, workers in lengthy meetings could
realistically be encouraged to build standing time gradually, stand only for as
long as is comfortable, and regularly alternate between sitting and standing.
Participants also described psychological discomfort resulting from ‘standing
out’ from others in the meeting. This echoes previous research showing that
people report feeling ‘weird’ or self-conscious from standing in normally-seated
workplace contexts. For many, such discomfort arose from knowingly violating a
strong perceived social norm exerting pressure to sit and not stand. These
findings support the centrality of social norms as a predictor of action, in
potential competition with one’s own attitudes, such that people sometimes act
in a counter-attitudinal way to conform to social pressures. Indeed, some
participants reported that, despite wanting to stand, they aborted their
standing attempts early, in response to implicit or explicit pressure from
others to sit. Experiences of standing appeared to vary in accordance with the
extent and visibility of perceived norm violation. In meetings in which standing
seemed to more strongly contravene the implicit sitting norm–such as larger
meetings characterized by discussion among attendees–standing produced stronger
feelings of awkwardness. Particularly where highly emotive topics–such as job
losses–were under consideration, many participants felt that their decision to
stand could be misinterpreted as a challenge to the seriousness of the meeting,
or the authority of attendees.
Standing also elicited psychological discomfort for some because they feared
that their standing would be interpreted as an assertion of power and authority
over other attendees. Indeed, for many animals, moving from sitting to standing
can be a sign of dominance and aggression, and previous research has documented
concerns among office workers about the potential for standing to be
misconstrued as aggressive or threatening. Concern about such misperceptions was
an especially powerful barrier to standing in meetings with more senior
colleagues. In meetings in which participants felt that standing could not
perceived to be an infraction–such as when standing to present information, or
when participants considered they had a legitimate reason to stand–standing did
not appear to elicit strong feelings. Interestingly, however, many felt that
current health problems precluding prolonged sitting offered the only legitimate
rationale for standing. This testifies to the perceived strength of the sitting
norm, and of being seen to respect established hierarchical relations within the
meeting; some believed that standing for the sake of health promotion (rather
than management of ill-health), or to honour commitments to participate in our
study, did not constitute sufficient reasons to disrupt norms or be seen to
challenge the authority of others. Thus, where meetings were held in settings
over which the participant perceived a sense of ownership and authority–such as
where the participant was leading the meeting–standing was seen as less of a
contravention of norms, and no threat to established power dynamic, so did not
evoke psychological discomfort.
Together, our findings suggest several potentially fruitful strategies for
overcoming norm barriers. First, as many of our participants found, notifying
others–especially meeting leaders–of intentions to stand, or relocating to a
less visible position in the room, can alleviate perceived social pressure to
sit. Relocation can, however, yield mixed consequences. In meetings
characterized by interaction among attendees, some participants felt that
standing physically and psychologically distanced them from collaborative
discussions. Others reported that standing could prompt more efficient meetings,
due to the desire to minimize anticipated physical discomfort from prolonged
standing. Indeed, previous research suggests that standing meetings tend to be
shorter in duration than seated meetings, with no impact on the quality of
meeting outcomes. Second, and more broadly, office-based organisations should
explicitly promote standing in meetings, to counter the perceived sitting norm,
and thus empower those who wish to stand without fear of infringing social
expectations. Meeting hosts should also encourage standing in meetings;
participants often felt more confident standing when they had secured prior
approval from those leading the meeting. Hosts might, for example, suggest that
attendees must stand when speaking in contribution to a group discussion, a
strategy shown to be acceptable in principle in a study of employees in Belgium.
Managers may be encouraged to support standing in meetings through emphasizing
potential benefits to productivity and staff time arising from shorter, more
efficient meetings. Third, there is an urgent need to promote standing for
health promotion purposes in the workplace, by developing messages that frame
standing as a legitimate strategy for sitting less. Given also the pervasive
culture of sitting cited by our participants, organisational buy-in, involving
creating and making salient and explicit a standing-permissive culture, will be
central to the effectiveness of promotional strategies for standing in meetings.
Organisations can also facilitate standing in meetings by providing standing-
appropriate infrastructure, such as meeting spaces with high tables and stools.
While many participants adapted to standing in sitting-conducive environments by
using accessories (e.g. using tablets to take notes), restructuring of the
physical environment has been shown to be an effective upstream method for
reducing sitting among workers.
Study limitations must be acknowledged. We did not assess participants’ prior
sitting time or the frequency with which they attended meetings, nor were
measures taken of the length of time for which participants stood, all of which
may have influenced responses to standing. Moreover, as we showed, the
experience of standing in meetings is influenced by the perceived responses of
other attendees, but we did not collect data from meeting attendees other than
the standing participant. Standing may create psychological discomfort among
non-standing attendees, in turn feeding negative perceptions of the stander.
While we have focused our practical recommendations on how to minimize feelings
of discomfort among standers, effectively promoting standing in meetings may
perhaps also require understanding and assuaging potentially negative
experiences of non-standing attendees.
We sought to explore real-world experiences of standing in meetings, but study
procedures may have influenced such experiences. Participants may at least in
part have been incentivized to stand by the gift voucher incentive, or by the
belief that participating in our study in this way would contribute to
scientific knowledge. It is thus possible that their reasons for and experiences
of standing among our sample may differ to those of employees who would stand in
meetings in more naturalistic settings, thus questioning the representativeness
of the experiences we documented. We sought to minimize our influence on the
standing experience by giving minimal instructions to participants, asking only
that they attempt to stand for a self-determined time. However, a lack of
further instruction ironically appears to have had an important influence on
experiences; the physical discomfort reported by many was apparently due to the
misconception that participants should stand for as long as possible.
Participants may have had more positive experiences had they, for example, been
advised on how best to integrate standing into meetings, including setting
realistic standing duration goals, and informing meeting hosts and other
attendees in advance. Nonetheless, our methods have documented the potential
importance of informing others of decisions to stand, and of the potential for
people to misunderstand advice to stand *more* as a recommendation to stand for
as long as possible. We are confident that our findings offer valid insights of
importance for informing future guidance for incorporating standing into
meetings.
Our sample was small and, while university employees span a socioeconomically
broad range, our participants were highly educated, which questions the
generalizability of findings. However, participants were recruited from three
office-based university organisations, and captured a diversity of meeting
types, job roles and seniority. Moreover, our aim was not to identify a
generalizable set of experiences, but rather to capture and explore a range of
reflections on the experience of standing in meetings. Indeed, while previous
research has suggested that office workers find the idea of standing in
normally-seated meetings acceptable in principle, ours is the first study to
document the rich complexity of the psychological, interpersonal and
organisational contexts that frame the standing experience.
# Conclusions
Displacing sitting with standing at work requires an in-depth understanding of
how to integrate standing into normally-seated work practices. While meetings
offer but one workplace context in which sitting time might be reduced, our
study demonstrates the complexity of this specific context, which should be
acknowledged by future workplace sitting reduction initiatives. Specifically, we
have highlighted some important physical, psychological and social barriers and
facilitators that may determine whether someone feels sufficiently capable to
break the mould and stand in normally-seated meetings. Office workers must
acknowledge that standing in meetings will involve a period of acclimatisation
to an unusual way of working. Many of our participants learned to adapt to
standing over the course of the three meetings, and so reduced initial physical
and psychological discomfort. Strategies that may enable office workers to
sustainably adopt standing in meetings as a sitting-reduction strategy include
building standing time gradually, and alternating between sitting and standing,
to alleviate physical discomfort, and notifying attendees of intentions to
stand, to avoid psychological discomfort from being seen to be challenging norms
and social hierarchy. Office managers should seek to provide visible
organizational support for standing, including the explicit promotion of the
acceptability of standing in the workplace as a health promotion strategy, and
provision of designated areas of standing-supportive furniture.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
The most widespread definition of underuse is *“the failure to provide a health
care service when it would have produced a favourable outcome for a patient”*,
while overuse occurs “*when a health care service is provided under
circumstances in which its potential for harm exceeds the possible benefit*”,
although recently it has been pointed out that overuse could include different
dimensions in relation to risk-benefit, cost-benefit and patient preference
approaches. While research has traditionally focused on identifying and reducing
the underuse of appropriate services in patients with a specific condition (e.g.
the use of antiplatelet agents in secondary prevention of ischemic heart
disease), the overuse of health care services has become an increasingly
recognized but understudied problem. Nonetheless, recent reviews have shown high
rates of overuse for a range of diagnostic tests, imaging tests and therapeutic
services in the US setting.
Overuse and underuse require operational definitions for each set of the
patient’s clinical condition and the service provided (**Fig A in**). Criteria
for these definitions may come from clinical trials, but more frequently come
from expert consensus or from the criteria established in clinical practice
guidelines (CPG). However, the latter may exhibit some variability in their
recommendations. In any case, and regardless of the method used, the assessment
of overuse and underuse requires having sufficient information to apply the
appropriateness criteria to each of the patients evaluated. This is particularly
important in the identification of underuse because it requires population
samples of non-treated patients with sufficient information to assess treatment
appropriateness, and in many cases these patients have no specific information
or may even have no contact at all with the healthcare system. Therefore,
availability of information allowing underuse assessment is extremely valuable.
Furthermore, studies have usually shown the overuse of specific services in a
particular condition or the underuse of other services in a different condition
(**Fig A in**). However, overuse and underuse may concur in the same healthcare
service and in the same clinical condition.
Regarding data on use of antiosteoporotic treatment, while Spain is one of the
European (and worldwide) countries with a lower incidence of osteoporotic
fracture, antiosteoporotic medications are widely prescribed. A recent report
analyzing the variability in the consumption of several therapeutic drugs in 15
developed countries (including the U.S., Canada, and several European countries)
identified Spain as the country with the highest utilization rates of
antiosteoporotic drugs. In addition, temporal trends show a very rapid and
disproportionate growth in osteoporosis drug consumption in recent years.
Concerning the appropriateness of antiosteoporotic drugs, previous studies
suggest that Spain and other countries are witnessing a massive use of these
treatments in young women with a very low risk of fracture, while there is a
significant underuse in women (and men) at a high risk of fracture, including
those who have already suffered a major osteoporotic fracture. These
estimations, however, could vary according to the criteria used to assess over
and/or underuse. Given the wide range of clinical practice guidelines on
osteoporosis existing globally, it would be desirable to determine the extent to
which estimations change according to such guidelines, and a setting with high
utilization rates of antiosteoporotic treatment could serve as a good example.
The FRAVO study is a population-based cross-sectional study designed to estimate
the prevalence of vertebral fracture and densitometric osteoporosis among post-
menopausal women over 50 years old living in Valencia (Spain). The
comprehensive information collected allows the estimation of the risk of
fracture and the operationalization of the criteria for antiosteoporotic
prescribing used in most CPGs, enabling the assessment of the impact on the
population of using different guidelines, as well as estimating the population
over or underuse of these treatments according to the criteria of each CPG. In
this study, we aimed to: 1) describe the population prevalence of
antiosteoporotic treatment among post-menopausal women of 50 and over and the
possible associations with socioeconomic factors, individual fracture risk
factors and the 10-year risk of hip fracture (assessed by FRAX), 2) estimate the
impact on the population of using different international and national
guidelines regarding antiosteoporotic treatments and, 3) estimate the over and
underuse of antiosteoporotic treatments among post-menopausal women of 50 and
over according to the criteria established by these guidelines.
# Methods
## Design
Population-based cross-sectional study conducted between February 2006 and March
2007, primarily designed to estimate the population prevalence of vertebral
fracture and densitometric osteoporosis among post-menopausal women of 50 and
over in the city of Valencia, Spain.
## Population and Sample
The study population was post-menopausal women of 50 years old and over living
in the city of Valencia, Spain, excluding women with cognitive impairment,
physical impediment preventing a woman from going to the radiology center by her
own means, race other than Caucasian and unwillingness to participate in the
study. The methods and main results of the FRAVO study have been fully described
elsewhere. Briefly, from an age-stratified random sample of 1758 women
resident in Valencia, a total of 824 fulfilling inclusion and exclusion were
included. Twenty cases for whom the X-Ray, the densitometry or the BMI was not
available were excluded in some analyses. As the final sample did not exactly
fit the population age distribution of the women of 50 and over in Valencia,
some estimates were weighted according to that population age distribution in
2006.
## Variables and definitions
Information about socio-demographic characteristics, lifestyle and risk factors
for vertebral fracture collected using the interviewer-administered
questionnaire included, among other variables, the subject's age, educational
level, body mass index, early menopause (defined as menopause before the age of
40), history of parental hip fracture, prior non-vertebral osteoporotic
fracture, treatment with glucocorticoids (use of oral glucocorticoid for at
least 3 months in the previous year) or other drugs that decrease bone mass (at
least one prescription of lithium, anticonvulsants, high dose thyroxin or
immunosuppressive treatment in the previous year), smoking, dietary calcium
intake, and secondary causes of osteoporosis (gastrectomy, bowel resection,
inflammatory bowel disease, thyroidectomy, diabetes mellitus, chronic liver
disease, chronic obstructive pulmonary disease, rheumatoid arthritis,
transplantation, chronic kidney failure). Spinal radiographs were performed
using standardized techniques and two radiologists, who were blind to all data
concerning the patients, performing the semi-quantitative evaluation of the
radiographs using the Genant method. Densitometric examinations were performed
with two calibrated densitometers and the World Health Organization (WHO)
osteoporosis classification criteria based on T-scores were used to classify
bone mineral density (BMD) results as normal, osteopenia or osteoporosis. Using
the FRAX tool calibrated for Spain
([www.shef.ac.uk/FRAX/index.htm](http://www.shef.ac.uk/FRAX/index.htm)) the
10-year risk of hip and major fracture was estimated for each patient. Regarding
antiosteoporotic medication, information was recorded on current treatments
(bisphosphonates, raloxifene, strontium ranelate, teriparatide, hormone
replacement therapy and calcitonins, which were the antiosteoporotic agents
available in Spain during 2006–7), duration of treatment and the specialty
(general practitioner, orthopaedic surgeon, gynaecologist, rheumatologist, and
other/unknown) of the prescriber of the first antiosteoporotic treatment.
## Selection of guidelines and operational criteria
We revised the guidelines chosen for inclusion in a previous review and selected
the closest to 2007 versions of four international guidelines (National
Institute for Health and Care Excellence (NICE, UK), National Osteoporosis
Foundation, (NOF, US); National Osteoporosis Guideline Group (NOGG, UK); and
Osteoporosis Canada), and six Spanish guidelines (Spanish Society for Family
and Community Medicine (semFYC), Spanish National Health System (SNS),
Spanish Society for Bone Research and Mineral Metabolism (SEIOMM), Spanish
General Medical Society (SEMERGEN), Spanish Orthopaedic Surgery and Traumatology
Society (SECOT), and the Spanish Rheumatology Society (SER)). These CPGs are,
from the authors’ point of view, the most well known and influential in the
Spanish setting, and many of them are also highly influential globally. This
selection was also based on a survey to around 75 professionals of different
specialities who rated the different guidelines according to their influence in
their clinical practice. This choice did not take into account the quality of
CPGs development and does not involve any judgment about the quality or validity
of these CPGs compared to other guidelines, nor any endorsement from the
authors. The guidelines criteria are described in **Table A in**. Some vague
criteria were unambiguously defined to allow their use in the study databases
(the corresponding specifications are also included in the **Table A in**).
## Ethical Aspects
The study was approved by the Institutional Review Board of the Primary Care
Departments of Valencia and Castellon. All of the participating women were
informed of the study’s characteristics and risks, and all gave signed informed
consent prior to enrolment.
## Analysis
First, we briefly described the clinical and demographic characteristics and
treatment rates of the participating women and conducted bivariate analyses to
determine which characteristics were related to osteoporosis treatment. We also
described the drugs used and the medical speciality of the physician who
prescribed the first antiosteoporotic treatment. Second, we used a multivariable
logistic regression (backward-forward stepwise method, with p\<0.05 for entrance
and p\<0.10 for removing variables) to retain the variables independently
associated with receiving osteoporosis treatment. Third, we used the information
from participants in the FRAVO study to estimate the percentage of women aged 50
years and over who would be recommended for treatment according to the
respective guidelines (impact on the population), with the corresponding 95%
confidence intervals (95% CI), calculated using the binomial approach.
Finally, we assessed the inappropriateness of treatments: the proportion of
treated women without a treatment recommendation according to the respective
CPGs (overuse) and the proportion of non-treated women with a positive
recommendation for treatment according to the above-mentioned guidelines
(underuse). All the analyses were performed using the STATA 11.0 (Stata Corp)
statistical software.
# Results
The study population included 824 post-menopausal women of 50 years old and over
living in the city of Valencia, Spain. Of these, 186 (22.0%) were on
antiosteoporotic treatment. After weighting the sample according to the
population age structure of Valencia, the estimated prevalence of
antiosteoporotic treatment in postmenopausal women of 50 years old and over in
Valencia was 20.9% (95%CI: 17.6–24.4). The most commonly prescribed drugs were
alendronate (36.6%), risedronate (24.7%) and raloxifene (22.5%), followed by HRT
(9.1%), calcitonins (3.7%), strontium ranelate (2.2%) and PTH (1.1%). Regarding
the origin of the initial prescription, orthopaedic surgeons were responsible
for most of them (37.1%), followed by gynaecologists (32%), general
practitioners (19.4%) and rheumatologists (8.1%). shows the antiosteoporotic
drugs prescribed according to the medical specialty responsible for the initial
prescription. Orthopaedic surgeons prescribed risedronate as their first choice
(40.6% of their treatments), followed by alendronate (36.3%), but prescribed
calcitonins and PTH more frequently than other specialties; gynaecologists
prescribed primarily raloxifene (40.0%), followed by hormone replacement therapy
(26.7%) and alendronate (21.7%); general practitioners preferentially prescribed
alendronate (58.3%), followed by risedronate (22.2%) and raloxifene (16.7%); and
for rheumatologists, their first choice was alendronate (40.0%), followed by
risedronate (26.7%), raloxifene (20.0%), and strontium ranelate (13.3%), the
latter being prescribed mainly by this speciality.
Regarding the sociodemographic, lifestyle, and clinical characteristics
considered, age at menopause, bone mineral density (BMD), morphometric vertebral
fracture, BMI and fracture risk score, were associated to antiosteoporotic
prescribing. Women with early menopause, densitometric osteoporosis and moderate
or severe morphometric vertebral fractures were more likely to be treated
(33.8%, 30.2% and 42.0%, respectively). Regarding the 10-year risk of hip
fracture (assessed by FRAX), the proportion of women treated was higher for
those with moderate (1–3) risk scores (28.8%). Obese women had lower treatment
rates (15.0%).
In the multivariable analysis, the factors independently associated with the
prescription of antiosteoporotic drugs were: early menopause (2.6 times greater
odds of having an antiosteoporotic drug prescription), morphometric moderate or
severe vertebral fractures (2.7 times greater odds), and densitometric
osteoporosis (1.5 greater odds). Women aged 65–69 years old were also more
likely to have a prescription than women in the lowest age group (reference
group). Obesity worked as a factor reducing the likelihood of being treated.
Regarding the potential impact on the population of applying the criteria for
treatment of the CPGs analyzed, between 8.7% (Osteoporosis Canada guideline) and
36.6% (National Osteoporosis Foundation guideline) of women would be recommended
for treatment according to the international CPGs, while the percentage of women
of 50 years old and over that would be treated according to the Spanish CPGs
would range between 17.7% (Spanish Society for Family and Community Medicine
guideline) and 44.3% (Spanish Rheumatology Society guideline).
Regarding inappropriateness of treatments, between 56.4% and 77.8% of women
under treatment did not meet any criteria to be treated according to the
international CPGs; these figures ranged from 41.5% to 66.0% when we applied the
Spanish CPGs criteria. According to all guidelines, the overuse of
antiosteoporotic treatments in postmenopausal women of 50 years old and over was
45.3% (61.3 and 46.4% according to the international and Spanish CPGs,
respectively). Concerning the underuse, between 6.6% and 34.6% of untreated
women should have received treatment according to the international CPGs,
whereas these figures were between 14.3% and 41.3% when the Spanish CPGs were
applied. According to all guidelines, the underuse of antiosteoporotic
treatments in postmenopausal women of 50 years old and over was 0.9% (3.4 and
2.0% according to the international and Spanish CPGs, respectively).
# Discussion
Our study shows that the prevalence of antiosteoporotic treatment in
postmenopausal women ≥ 50 in Valencia was 20.9% and the type of antiosteoporotic
drugs prescribed varied greatly depending on the medical specialty responsible
of the initial prescription. The impact on the population and the proportion of
treatments considered inappropriate when applying the most influential
osteoporosis guidelines, varied strikingly, with the percentage of women 50 and
over who should be treated ranging from less than 9% to over 44%. A large
proportion of inappropriate treatments was found when applying these guidelines
to the Spanish population, combining a high overuse (which ranged between 42 and
78%) and, to a lesser extent, underuse (ranging between 7 and 41%).
In the city of Valencia, one out of five women aged 50 and older were treated
with antiosteoporotic drugs. This treatment rate, lower than that reported for
the Valencia region in 2010, may be related to the healthier characteristics
of a real population sample (enrolled from a population registry, not from
medical practices), increasing treatment rates during the time period between
the two studies (the dispensing of antiosteoporotics in Spain rose by nearly 50%
between 2006 and 2008) or real differences in treatment rates between the
city of Valencia and the rest of the Valencia region. Nevertheless, our results
show high treatment rates in women with a low to moderate risk of fracture (19%
and 29% in women with≤1% and 1–3% 10-year risk of hip fracture assessed by FRAX,
respectively), and a huge proportion of unnecessary treatments (between 42% and
78%) according to the criteria of the most influential CPGs. These figures
suggest a striking amount of osteoporosis treatment overuse and an interesting
opportunity to reduce costs (including those related to adverse events from
unnecessary treatments) without compromising-and probably improving- patients’
health.
Regarding treatment underuse (7–41%, depending on the guideline used), although
lower than the treatment overuse observed in relative terms, it is based on a
larger population (the untreated women, 79%), also becoming a major issue in
osteoporosis management. Some of the figures described in the bivariate analysis
were highly suggestive of underuse. Although some acknowledged risk factors are
associated with an increased likelihood of being treated, other recognized risk
factors did not show such an association, or the proportion of women treated is
too sparse: only 33% of women with prior non-vertebral fractures and 42% of
women with moderate-severe vertebral fractures were receiving osteoporosis
treatment. Even some risk factors such as age or FRAX 10-year risk of hip
fracture showed paradoxical behaviour, with a greater likelihood of treatment at
intermediate values but no association with the highest-risk scores. These
figures confirm the existence of a relevant “osteoporosis care gap” in the
Spanish setting, something which has already been revealed in other
countries,and has remained unchanged over time.
Our study also shows the dramatic impact on the population treatment rates of
applying the diverse CPGs recommendations, varying the percentage of women who
should be treated according to different guidelines from less than 9% to over
44%. In real terms, from the approximately eight million women of 50 years old
and over in Spain, the number eligible for treatment would range from 0.7 to 3.8
million, depending on the guideline used. These results are consistent with
those reported by Bolland and Gray, showing treatment recommendations for 21%
and 48% of women after applying the NOGG and NOF guidelines criteria,
respectively, in a cohort of older women (mean age74 years) participating in a
clinical trial in New Zealand (which would be 11% and 37% for such CPGs in our
study population, which is 10 years younger).
Treatment decisions and the choice of a particular drug could be influenced by
patient characteristics, physician and organizational factors, pharmaceutical
promotion and healthcare system characteristics. One interesting result of
our study-limited by the small number of cases for analysis-is that the
selection of the specific antiosteoporotic agent seems to be more dependent on
the specialty of the physician starting treatment than on patient
characteristics. Several studies have found that the pharmacological management
of several conditions varies greatly by physician speciality; however, the
quality and relevance of those studies has been criticized. Moreover, little is
known regarding this variability in the pharmacological management of
osteoporosis. In our study, the prescribing patterns of gynaecologists were
particularly remarkable, treating only one third of patients with
bisphosphonates and almost two thirds with raloxifene (40%) or hormone
replacement therapy (25%)–five years after the publication of the Women's Health
Initiative trial results. These results could be explained, at least in part,
because these specialists often treat younger postmenopausal women. In this
sense, it is likely that such prescription patterns could be a significant
source of overuse, given the low risk of osteoporotic fracture in young women,
although it could also be a strictly local finding. Furthermore, we found that
orthopaedic surgeons were responsible for most of the initial antiosteoporotic
prescriptions. This might be explained by the relative lack of rheumatologists
within the Spanish National Health System, and the consequent tendency of
primary care physicians to refer these patients to the orthopaedic surgeons.
## Limitations
Our study has some potential limitations. First, we “applied” several U.S.,
U.K., and Canadian guidelines to a Spanish population that may have different
characteristics (e.g. prevalence of risk factors, incidence of fracture,
strength of the associations between risk factors and the incidence of fracture,
etc.) than the populations where the decision rules have been developed. Second,
although the dropout rate in the FRAVO study is similar to other population
studies, it was higher in the oldest group (with higher expected morbidity) but
also in younger working women, who were presumably healthier. Weighting for age
should have partially reduced this limitation, but it is difficult to assess the
effect and scope of any possible biases linked to missed cases in recruitment.
Third, the selection of particular guidelines from among the myriad of existing
documents (the International Osteoporosis Foundation website includes links to
more than 50 guidelines; see: <http://www.iofbonehealth.org/guideline-
references>) always has a subjective component, although we consider that those
selected are among the most representative, widely used, and influential in
Spain, Europe, the United States and Canada. Furthermore, these guidelines were
also rated by physicians of different specialities as the most influential in
their clinical practice. Fourth, some of the risk factors considered in the CPGs
evaluated were not available or had different definitions in the FRAVO data or
some CPGs include vague criteria that are impossible to operationalize
unambiguously. We operationalized these criteria, as indicated in Table A in,
using "reasonable" interpretations, but other possible interpretations may have
led to different estimates of population impact and inappropriateness.
# Conclusions and Implications
The pharmacological management of osteoporosis in women of 50 and over combines
an important overuse (mainly in young women with low risk of fracture) with an
important underuse (in women who are older, at high risk or with previous
osteoporotic fractures), although the level of inappropriateness varies
dramatically depending on the CPGs used. In a recent paper we described the high
variability among CPGs in recommending antiosteoporotic treatment, concluding
that such variability limits the effectiveness of those recommendations and,
given the heterogeneity of the criteria used, it should come as no surprise that
doctors and health care providers could become confused to the point of inaction
or misguided action. In the present study, we quantified the impact of this
variability on the number of women who should be treated and on the
inappropriateness of the prescribed treatments, and the overwhelming results
should not be overlooked by public health care policies in Spain (and probably
in other countries in similar situations) because osteoporosis is a frequent
condition and even small variations in treatment indication can account for
large differences in women treated and resources consumed.
Targeting high-risk populations is a strategic element for developing cost-
effective policies in the prevention of osteoporotic fractures. Predictive
modelling of fracture risk factors seems to be the main instrument for
stratifying the population into risk groups to which practical policies should
be applied, and CPGs should help integrate this information to identify people
who are more likely to benefit from treatment. The results of our study suggest
that the current CPGs, although based on the same evidence, seem to interpret it
differently and do not meet these clinical and policy needs sufficiently. The
development of more accurate predictive tools (especially for the intermediate
risks) could possibly contribute to the convergence of these interpretations, to
a consensus on more homogeneous guidelines and, eventually, to the reduction of
osteoporotic fractures. However, at present, it seems urgent to develop policies
to reduce treatment overuse (at least in those cases where there is wide
agreement) while reducing underuse should also not be neglected, especially in
women in secondary prevention for whom, beyond their scores of predictive risk
tools, a high risk of osteoporotic fracture has already been shown.
# Supporting Information
We are grateful to all doctors and nurses at the Valencia Health Agency primary
healthcare centres participating in the ESOSVAL study for their collaboration,
and to the Valencia Ministry of Health for its enthusiastic and continued
support of the ESOSVAL research projects.
[^1]: JSG has received speaking fees for symposia or other meetings from
Amgen and GlaxoSmithKline (GSK). SP has received speaking fees for symposia
or other meetings from Ferrer International and Health, Innovation and
Society Foundation of Novartis Spain. GS has received speaking fees for
symposia or other meetings from Ferrer and Boehringer Ingelheim
International. This does not alter the authors' adherence to PLOS ONE
policies on sharing data and materials.
[^2]: Conceived and designed the experiments: JSG GSG SP. Performed the
experiments: GSG IH CBP CLRB. Analyzed the data: GSG CBP IH CLRB JSG SP.
Wrote the paper: GSG SP CLRB. Interpretation of data, critical revision of
the manuscript for important intellectual content, and approved the final
version submitted for publication: GSG IH CBP CLRB JSG SP. |
# Introduction
Huntington’s disease (HD) is an autosomal dominant heritable disorder and is
characterized by progressive motor dysfunction, cognitive impairment and
disturbances in behavior. The worldwide prevalence of HD is 2–5 per 100,000
people. HD is caused by an expanded CAG trinucleotide repeat at the 5’ end of
the *Huntingtin* (*HTT*) gene and the length of the resulting polyglutamine
(polyQ) repeat in the HTT protein is inversely correlated with the age of onset
of the disease. CAG repeat lengths between 36 and 39 do not always cause signs
and symptoms of HD (due to reduced penetrance) but *HTT* containing 40 or more
CAG repeats leads invariably to HD. The expanded polyQ stretches result in
misfolded mutant huntingtin (mHTT) proteins by the formation of β-sheets, and
N-terminal mHTT fragments containing the polyQ expansion are aggregation prone.
Lowering the levels of soluble mHTT prior to aggregation through increased
degradation would be a therapeutic strategy to prevent or delay the onset of
disease. The two main systems responsible for protein degradation in the
eukaryotic cell are autophagy and the ubiquitin proteasome system (UPS)
(reviewed by Soares et al.). Whereas autophagy functions mainly in the
cytoplasm, the UPS is the main route for the degradation of misfolded proteins
in both the cytoplasm and the nucleus. The UPS is thus a promising candidate to
target both cytoplasmic and nuclear mHTT fragments. Initial studies suggested
that the proteasome cannot degrade polyQ stretches and that proteasomes are
irreversibly sequestered into mHTT aggregates. More recently, we showed that
proteasomes are dynamically recruited to mHTT inclusion bodies, that they remain
catalytically active and that they are accessible to substrates. Furthermore,
when polyQ-expanded mHTT fragments were targeted towards the proteasome with an
N-terminal degradation signal, they were efficiently and completely degraded.
Together this indicates that the proteasome remains active in cells with HTT
inclusion bodies and is able to degrade soluble mHTT when this is targeted for
degradation.
The proteasome consists of a latent 20S core particle, which is composed of four
heptameric α- and β-rings
(α<sub>7</sub>β<sub>7</sub>β<sub>7</sub>α<sub>7</sub>), of which the latter ones
contain catalytic subunits that face the interior of the cylinder and are
responsible for the proteolytic activity. Access to the central cavity is
regulated by a gate formed by the N-terminal protrusions of the α-subunits.
Modulation of the gate is required for substrate entry into the 20S core and is
mediated by the proteasome activators (PA), such as the 19S complex and PA28.
The 19S and PA28 can bind to both ends of the 20S proteasome core leading to
single (referred to as 26S when one 19S complex binds 20S), double (referred to
as 30S when two 19S complexes bind 20S) or even hybrid-capped proteasomes (with
a 19S and PA28 complex on opposing ends of the 20S). The 19S is a complex of
distinct subunits and is involved in the recognition, unfolding and de-
ubiquitination of poly-ubiquitinated substrates in an ATP-dependent manner. PA28
is involved in ATP-independent degradation and has three homologues: PA28α and
PA28β, forming heterodimers, and PA28γ, forming a homo-heptameric ring, which is
only expressed in the nucleus. Although PA28αβ exists as a heterodimer,
homomeric PA28α is sufficient to activate proteasome activity. By binding to the
proteasomal α-ring, PA28 allows entrance of peptides into the 20S core and
increases proteasome activity. PA28 overexpression also increases degradation of
oxidized proteins in cells, with increased PA28αβ binding to the 20S proteasome
immediately upon H<sub>2</sub>O<sub>2</sub> treatment, followed by increased
PA28αβ expression during oxidative stress adaptation. Interestingly, PA28αβ has
been shown to prevent aggregation in the mouse hippocampus during aging.
Furthermore, PA28γ overexpression relieved HD pathology and lowered the number
of inclusion bodies in cells and *in vivo*. The role of PA28αβ, which is present
in both the nucleus and cytoplasm, on mHTT turnover remains elusive.
In this study, we examined whether changes in proteasome complex formation occur
during disease progression in HD mouse models. To determine which consequences
these alterations have on mHTT degradation, we modulated PA28αβ activated
proteasomes in various *in vitro* models and determined the effects on both
polyQ fragments and mHTT proteins.
# Materials and methods
## HD mouse models
All procedures were in accordance with the Animals (Scientific Procedures) Act
1986 and were approved by the King’s College London (KCL) Ethical Review Process
Committee. In this study *Hdh*Q150 mice expressing endogenous full-length mouse
*Htt* with an expanded CAG repeat and R6/2 mice expressing a human exon 1 *HTT*
transgene were used. Animals were genotyped by PCR and CAG length was determined
as previously described. The *Hdh*Q150 homozygous mice were on a CBA/Ca and
C57BL/6J F1 background and had a CAG repeat size of 166±9. The R6/2 mice were
bred by backcrossing R6/2 males (CBA/Ca x C57BL/6J) to F1 females
(B6CBAF1/OlaHsd, Envigo, Netherlands) and had a CAG repeat length of 209±2.
*Hdh*Q150 mice were sacrificed by cervical dislocation at 2, 16 and 22 months of
age, and R6/2 mice at 4 and 14 weeks of age. The dissected brain regions were
immediately snap frozen.
## DNA constructs
PA28α and PA28β (kindly provided by Prof. PM Kloetzel, Charité
Universitätsmedizin Berlin, Germany) were cloned into a pcDNA3 vector using
EcoRI. GFP-Ub-Q54, DNAJB6 and m*HTT*(Q25/Q97)exon1-H4 were generated as
described before, respectively.
To create a stable cell line with inducible expression of mHTT, a pINDUCER
m*HTT*(Q25/Q46/Q97)exon1-IRES-*GFP*-Q16 construct was generated. First,
m*HTT*(Q97)exon1 was amplified with Xho on the 3’ end (fw (T7)
5’-`TAATACGACTCACTATAGGG`-’3, rv 5’-`GTTCTAGATTAAGGTCGGTGCAGAGGCTC`-’3) and
cloned into pIRES2-*GFP*, using XhoI and SmaI, creating
m*HTT*(Q97)exon1-IRES-*GFP*. Next, *GFP*-Q16 was amplified with BstX1 and Not1
on the 3’ and 5’ ends, respectively (fw
5’-`CGATGATAATATGGCCACAACCATGGCCACCATGGTGAGCAAGGGCGAGG`-3’, rv
5’-`TGATCTAGAGTCGCGGCCCCGCTTACCTGGGGCTAGTCTC`-3’) and cloned into
m*HTT*(Q97)exon1-IRES-*GFP* using BstX1 and Not1, to replace *GFP*.
Subsequently, m*HTT*(Q97)exon1-IRES-*GFP*-Q16 was amplified with EcoRI on the 3’
and 5’ ends (fw 5’-`GTCCAGTGTGGTGGAATTCTCGAGGTCGACCGCCATGG`-’3, rv 5’-
`CTGGATATCTGCAGaattCCGCTTACCTGGGGCTAGTCTC`-‘3) and introduced into pENTR/D-TOPO
by EcoRI. Finally, the construct was transferred from the pENTER Gateway vector
into the pINDUCER20-Blast lenti-viral dox-regulated expression vector. To create
cells expressing m*HTT*(Q25/Q46)exon1-IRES-*GFP*-Q16 m*HTT*(Q25/Q46)exon1 was
amplified with BamHI on the 3’ and 5’ ends (fw
5’-`TGGTACCGAGCTCGGATCGCCACCATGGCGACCCTGGAAAAGCTG`-’3, rv
5’-`GAGGGAGAGGGGCGGATCTTAAGGTCGGTGCAGAGGCTC`-’3). Next m*HTT*(Q25/Q46)exon1 was
cloned into pENTR m*HTT*(Q25/Q46)exon1-IRES-*GFP*-Q16 using BamHI, replacing
m*HTT*(Q97)exon1. All plasmids were verified by sequencing before use.
## Cell culture
HEK293 and ST*Hdh*<sup>Q7/Q7</sup> cells were cultured in DMEM (Gibco)
supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco) and 0.2mM
L-glutamine (Gibco) and grown in a humidified chamber with 5% CO<sub>2</sub> at
37°C or 32°C, respectively. ST*Hdh* cells expressing doxycycline inducible
m*HTT* were generated by retroviral transfection of ST*Hdh*<sup>Q7/Q7</sup>
cells with pINDUCER m*HTT*(Q25/Q46/Q97)exon1-IRES-*GFP*-Q16.
## Treatment and transfection
HEK293 cells were stimulated with 100U/ml IFNγ (ProSpec) for 72 hours and
proteasomes were inhibited with 250nM epoxomicin (Sigma) for 16 hours. HEK293
cells were transfected with jetPEI one day after plating according to the
manufacturer’s instructions (Polyplus transfection). Neon Transfection System
(Invitrogen) was used to overexpress constructs in ST*Hdh* cells. For silencing
experiments with shRNA, the MISSION® TRC-Mm 1.0 (Mouse) library was used. The
PA28α targeting sequence (5’-`CCCGATCCAGTCAAAGAGAAA`-3’, MISSION® TRC shRNA
TRCN0000066420) was delivered through retroviral transduction. The pLKO.1-puro
Non-Mammalian shRNA Control Plasmid (SHC002) was used as a control. SiRNA
targeting PA28α (ON-TARGETplus Mouse Psme1 siRNA SmartPool, Horizon Discovery)
or non-targeting siRNA (siGENOME Non-Targeting siRNA Control Pool, Horizon
Discovery) was delivered to the cells by using Lipofectamine RNAiMAX
Transfection Reagent (Invitrogen) directly with plating the cells.
## Native PAGE
Brain tissue and cell pellets were suspended in TSDG buffer (10mM Tris/HCl
pH7.4, 25mM KCl, 10mM NaCl, 1.1mM MgCl<sub>2</sub>, 0.1mM EDTA, 10% glycerol,
1mM ATP fresh) and brain tissue was further homogenized by the use of Dounce
tissue homogenizer. Lysis was performed by 3–5 freeze/thaw cycles in liquid
nitrogen. After centrifugation (15 minutes, 14.000 rpm at 4°C), the protein
concentration of the clarified lysate was determined by Bradford protein assay
(Serva). After the addition of 4x native sample buffer (20mM Tris pH8.0, 50%
glycerol, bromophenol blue) samples were separated on 3–12% NativePAGE Novex
Bis-Tris gels (Invitrogen). For western blotting, native gels were transferred
to PVDF membranes (Millipore, Bedford, MA, USA) in transfer buffer (25mM Tris
pH7.5, 192mM Glycine, 20% MeOH) using the Criterion blotter (Biorad). After
blocking in 5% milk, membranes were incubated with the antibody of interest and
Odyssey detection system (LICOR Bioscienses) was used for scanning and analysis.
## Visualizing proteasome activity and peptide degradation in gel
Activity based probe (ABP) labeling was performed either in the lysate or after
running native PAGE. For in-lysate labeling, the samples were incubated with
0.5μM ABP for 30 minutes at 37°C before adding sample buffer and loading on
native PAGE. In-gel ABP labeling or in-gel peptide degradation was performed
after protein separation by native PAGE. For these overlay assays the wet gel
slab was incubated in 10ml overlay buffer (20mM Tris pH7.5, 5mM
MgCl<sub>2</sub>, 1mM ATP fresh) with 25nM ABP or 400μM of the quenched peptide
for 20 minutes at 37°C. Fluorescent intensities were measured directly on a
Typhoon imager (Ge Healthsciences) using the 580 BP 30 filter. In order to
inhibit proteasome activity in these assays, the lysate was incubated with 0.5μM
epoxomicin (Sigma), or similar amounts of DMSO in control samples, for 1 hour at
37°C, prior to in lysate labeling or native PAGE separation.
## Western blot
Cells pellets were lysed in Triton-x buffer (50mM Tris/HCl pH7.4, 150mM NaCl,
1mM EDTA, 1% Triton-X100, supplemented with complete mini protease inhibitor
cocktail (Roche)). After centrifugation (15 minutes, 14.000 rpm at 4°C), the
protein concentration of the supernatant was determined by Bradford protein
assay (Serva). Samples were boiled in 6x sample loading buffer (350mM Tris/HCl
pH6.8, 10% SDS, 30% glycerol, 6% β-mercaptoethanol, bromophenol blue) and
separated on 12.5% SDS-PAGE gels. Proteins were transferred to nitrocellulose
membranes (Biorad) with the use of the Trans-Blot Turbo Transfer System
(Biorad). After blocking in 5% milk, membranes were incubated with the antibody
of interest and Odyssey detection system (LICOR Bioscienses) was used for
scanning and analysis.
## Filtertrap assay
For filtertrap assay, the pellet obtained after centrifugation of the cell
lysate was resuspended and treated with endonucleases for 1 hour at 37°C (1mM
MgCl<sub>2</sub>, 50mM Tris/HCl pH8.0, with 0.02U/μl DENARASE® (c-LEcta) added
fresh). This reaction was stopped by adding 2x termination buffer (40mM EDTA, 4%
SDS, 100mM DTT fresh) and samples were diluted in 2% SDS buffer (2% SDS, 150mM
NaCl, 10mM Tris/HCl pH8.0). Cellulose acetate membranes (Schleicher & Schuell)
with a pore size of 0.2 μm were pre-equilibrated in 2% SDS buffer. After sample
loading through the Bio-Dot microfiltration apparatus (Biorad, Hercules, CA,
USA), the membrane was washed twice with 0.1% SDS buffer (0.1% SDS, 150mM NaCl,
10mM Tris pH8.0) and further treated like western blot membranes.
## Antibodies
The following primary antibodies were used: anti-PA28α, directed against
RVQPEAQAKVDVFRED, (1:3000, kindly provided by Prof. M Groettrup, University of
Konstanz, Germany) \[100\], anti-PA28α (1:1000, Enzo Life Sciences, BML-PW8185),
anti-polyQ 1C2 (1:1000, Millipore, MAB1574), anti-polyQ (1:1000, Sigma-Aldrich
3B5H10, P1874), anti-β-actin (1:1000, Santa Cruz, SC-130656 and SC-47778),
anti-α2 (1:1000, MCP236, kindly provided by Prof. Rasmus Hartmann-Petersen,
Biologisk Institut, University of Copenhagen, Copenhagen), anti-α7 (1:1000,
MCP72, Enzo Life Sciences, PW8110), anti-HTT (1:5000, Abcam, ab109115), anti-
tubulin (1:1000, Cell Signaling Technology, CST2148) and anti-RPT (1:1000, Enzo
Life Sciences, PW8825). IRDye 680 and IRDye 800 (1:10.000; LI-COR Biosciences)
were used as secondary antibodies.
## In vitro degradation assays
mHTT(Q25/Q97)exon1-H4 was purified as described before. 100ng purified
HTT(Q97)exon1-H4 protein was incubated with 0.3μg mammalian open-gated 20S
proteasomes in 1x 20S buffer (10mM Tris/HCl pH7.4, 30mM NaCl, 1mM MgCl2, 400μM
DTT fresh) for 8 hours at 37°C. For proteasome inhibition, 1μM epoxomicin
(Sigma), or similar amounts of DMSO in control samples, was added. For
proteasome activation, 3μg isolated PA28αβ caps, 50μM RPT peptides (both kindly
provided by Prof. M. Rechsteiner, University of Utah School of Medicine, USA) or
0.01% SDS were added to the reaction. ATP Regeneration solution (Enzo Life
Sciences) was added to reactions involving the 26S proteasome. After the
incubation period, 0.5μM ABP was added for an additional 30 minutes at 37°C. The
reaction was stopped by boiling the samples in 6x sample loading buffer (350mM
Tris/HCl pH6.8, 10% SDS, 30% glycerol, 6% β-mercaptoethanol, bromophenol blue).
The complete reaction was used for 12.5% SDS PAGE to visualize ABP signal and
for immunoblotting.
## Fluorescent microscopy and quantification of aggregate formation
STHdh mHTT(Q97)exon1-IRES-GFP-Q16 cells were rinsed with PBS and fixed with 4%
formaldehyde in 1x PBS for 1 hour at RT. Nuclei were stained with 0.01 mg/ml
Hoechst 33324. Images were obtained using automated microscopy (ImageXpress
Pico). To measure the aggregates, GFP-Q16 was used as a fluorescent reporter.
The amount of aggregation was defined by using the MATLAB algorithm. In short,
the algorithm defined the single nuclei on the pictures and it calculated the
average intensity of the nuclei, which was later used to calculate the amount of
cells on the images. Nuclei were defined based on size, intensity and shape.
Aggregates were defined based on their size and intensity that needs to be above
certain threshold based on the GFP overexpression in the cells. More information
on the MATLAB script can be found as supplementary information.
## Data quantification
Experimental results were normalized to the control condition and presented as
mean ± SEM. Outliers were identified using Grubbs’ test statistic, and removed.
Statistical differences between groups were determined by the one sample t-test
or one-way ANOVA with Dunnett’s Multiple Comparison. Analysis was performed
using GraphPad™ Prism v.9 (GraphPad Software, Inc.). An alpha level of 0.05 was
used to define statistical significance. All data is available through the
online platform FiglinQ
(<https://create.figlinq.com/~k.geijtenbeek/92/collection>/).
# Results
## Loss of PA28 activated proteasomes during HD progression in HD mice
To explore whether proteasome complex formation changes during HD development,
we investigated proteasome complex composition in two different HD mouse models:
the *Hdh*Q150 mice expressing endogenous full-length mouse *Htt* with an
expanded CAG repeat and the R6/2 mice expressing a human exon 1 *HTT* transgene.
For the visualization of proteasome complexes, we used ABP labeling. These
probes bind to the catalytic sites of 20S proteasomes and can be visualized via
their fluorescence tag. Only proteasome complexes associated with proteasome
activating complexes such as PA28 and the 19S are accessible for substrates.
Therefore, ABPs will specifically label incorporated, active proteasome
subunits. When the lysates are subsequently separated on native PAGE gels to
keep protein complexes intact, and are analyzed for fluorescence, various bands
can be distinguished representing various proteasome complexes. To represent
different disease stages, proteasome complexes were analyzed at 2, 16 and 22
months of age for homozygous *Hdh*Q150 mice, and at 4 and 14 weeks of age for
R6/2 mice , which show a faster disease progression due to the expression of the
N-terminal exon 1 HTT fragment. Of these mice cortex, striatum, hippocampus,
cerebellum and brain stem were dissected and analyzed, with HD pathology being
more prominent in the first regions. The frozen sections were lysed, labeled
with ABP and subjected to native PAGE analysis for fluorescence gel analysis. A
decrease in ABP labeling at the height of PA28 capped proteasomes was observed
in late disease stages in various brain regions, which suggests an alteration in
these complexes (upper panels showing ABP labeling). Interestingly,
immunostaining of PA28α subunits confirmed a decrease in PA28αβ activated
proteasomes (middle panels, black arrowheads). In addition, an increase in the
free pool of PA28αβ subunits (not bound to 20S proteasomes) was observed in late
stage models (open arrowheads at the bottom of the gel), suggesting disassembly
of the PA28αβ activator from the 20S core. Quantification of the fraction capped
PA28αβ (black arrowheads) versus the total pool of PA28α (black and open
arrowheads) showed a significant decrease in PA28αβ proteasome activation at
late disease stages in cortex, striatum and hippocampus of *Hdh*Q150 mice, and
in hippocampus of R6/2 mice (graphs in lower panels).
## PA28αβ overexpression improves degradation of polyQ peptides
To examine whether the observed reduction in PA28αβ proteasome activation in HD
mice would affect mHTT degradation directly, we first studied the effect of
PA28αβ-activated proteasomes towards degradation of polyQ repeats using quenched
Q8 (qQ8) peptides, which become fluorescent after cleavage by endopeptidases.
When lysates are separated on native PAGE gels, proteasome complexes do not only
remain intact, but also remain active. If these gels are subsequently incubated
with the qQ8 peptides in an overlay assay, local fluorescence will appear at the
height of the responsible enzyme when the peptide is cleaved. When HEK293 cell
lysate were subjected to an overlay assay with the qQ8 peptide, a pattern of
fluorescent bands was observed, demonstrating polyQ peptide degradation (left
panel). Treatment with epoxomicin prevented cleavage of the polyQ peptides,
indicating that the fluorescence was specifically generated by proteasomal
cleavage. In addition, ABP labeled proteasomes showed a similar fluorescence
pattern (middle panel), and merging both fluorescent channels demonstrated the
ability of proteasomes to cleave polyQ substrates (right panel). To examine the
effect of PA28αβ activation on the degradation of these polyQ-peptides in cells,
HEK293 cells were transfected with PA28αβ. A native PAGE overlay with qQ8
peptides showed that PA28αβ overexpression led to an increase in qQ8
degradation. Together these data demonstrate that proteasomes are able to cleave
within polyQ sequences and that activation by PA28αβ accelerates this
degradation.
Next, we examined whether overexpression of PA28αβ would enhance the degradation
of polyQ peptides that exceed the pathological threshold. We transfected HEK293
cells with GFP-Ub-Q54, which generates pure Q54-peptides since GFP-Ub is
separated by C-terminal hydrolases directly after synthesis. Cells were co-
transfected with PA28α, PA28β or PA28αβ. The chaperone DNAJB6 was transfected as
a positive control to detect soluble Q54, as it prevents aggregation of polyQ
peptides. Although no significant decrease in soluble mHTT levels (arrowhead)
was observed, overexpression of PA28α and PA28αβ led to a decrease in insoluble
Q54 levels as shown by filtertrap analysis. To study whether the PA28αβ-induced
effects on polyQ peptide levels were due to improved degradation through the
proteasome, we treated the cells with the proteasome inhibitor epoxomicin.
Epoxomicin slightly reduced the effects of PA28 overexpression. The fact that
epoxomicin could not completely prevent the increased degradation of Q54 by
PA28αβ can be explained by the relatively short incubation with the inhibitor
compared to the long expression time of PA28 and Q54. Together this shows that
PA28αβ improves degradation of polyQ peptides by the proteasome.
## PA28αβ hampers mHTT degradation by the 20S in vitro
Since PA28αβ improved degradation of polyQ-expanded peptides, we next examined
whether PA28αβ would also improve the degradation of polyQ-expanded mHTT protein
fragments. First, we used isolated mHTT fragments to study the degradation *in
vitro*. For this we isolated N-terminal mHTT(Q25/Q97)exon1 fragments from N2a
cells. These proteins resemble the fragments that are also expressed in the R6/2
mice. The mHTT fragments were incubated with purified 20S proteasomes in the
absence or presence of isolated PA28αβ. Both HTT(Q25) and mHTT(Q97) (arrowheads)
were degraded by 20S proteasomes. However, when 20S proteasomes were activated
by PA28αβ, as demonstrated by ABP labeling, the degradation of HTT(Q25) was
reduced and mHTT(Q97) degradation was completely prevented. Interestingly,
artificial opening of the 20S did result in increased mHTT(Q97) (arrowheads)
degradation. We stimulated 20S opening by using RPT peptides that represent the
C-termini of the 19S subunits RPT2 and RPT5, which are responsible for opening
of the α-ring. Additionally, we used SDS to open 20S proteasomes. It should be
noted however that SDS can also affect protein denaturation and thereby its
accessibility into the proteasome. Toghether these results indicate that
(artificial) opening the entrance of the 20S core can improve degradation of
mHTT, but that PA28αβ only improves the accessibility of the 20S for small
peptides, while entrance of larger protein fragments including mHTT exon1 is
completely blocked.
## PA28αβ overexpression does not affect mHTT levels in cells
Following the experiments with purified proteasomes, we examined the effects of
PA28αβ activation on mHTT exon1 degradation in cells, which contain all other
components of the proteostasis network that may affect the role of PA28αβ on
mHTT degradation. HEK293 cells transfected with N-terminal mHTT(Q97) and PA28α,
PA28β or PA28αβ for 72 hours showed an increase in activity of PA28αβ activated
proteasomes, as shown by ABP labeling and immunoblotting, yet no statistically
significant changes were observed in either soluble (arrowhead) or insoluble
mHTT levels upon PA28αβ overexpression. Since cell-type specific differences
were observed in mHTT aggregation and sensitivity and proteasome composition
differs between different human cell lines, we next examined whether proteasomal
complexes differ between HEK293 cells and striatal ST*Hdh*<sup>Q7/Q7</sup>
cells, which are more relevant in HD. When proteasome complexes of both cell
lines were analyzed by native PAGE ST*Hdh* cells showed higher levels of hybrid
and 30S proteasome complexes, while the 26S proteasome is more abundant in
HEK293 cells. Interestingly, PA28αβ seems to be more abundant in ST*Hdh* cells,
both as a free pool and in complex with 20S proteasomes. Modulating proteasomal
complexes could therefore have distinct consequences in these cell lines. To
examine the effect of PA28αβ levels on mHTT degradation in ST*Hdh* cells, we
generated ST*Hdh*<sup>Q7/Q7</sup> cells that express N-terminal mHTT(Q97)-exon1
under a doxycycline inducible promotor. These ST*Hdh*(Q97) cells were
electroporated with PA28αβ and mHTT expression was subsequently induced for 48
hours. Resembling the data observed in HEK293 cells, no changes in soluble or
insoluble mHTT(Q97) levels were detected after PA28αβ overexpression in ST*Hdh*
cells.
## PA28αβ silencing in STHdh cells increases mHTT aggregation
To examine the effects of PA28αβ silencing on mHTT(Q97) clearance in ST*Hdh*
cells that express high PA28αβ levels, and thereby mimic the decrease in PA28αβ
capped proteasomes observed in HD mice, we reduced PA28α levels by 80% using
retroviral transduction of shRNA targeting PA28α. Native PAGE showed efficient
reduction of PA28αβ activated proteasomes, also leading to less proteasome
activity as shown by ABP labeling. Subsequently, mHTT expression was induced for
eight hours (pre-aggregation state) or 48 hours and the effects of PA28α
silencing on mHTT protein levels were determined. While reduced PA28αβ activated
proteasome levels did not affect soluble mHTT(Q97) protein levels, levels of
insoluble mHTT(Q97) increased significantly. These results were confirmed using
fluorescence microscopy experiments, using ST*Hdh* cells expressing doxocycline
inducible mHTT(Q97)-IRES-GFPQ16. Here the separately expressed GFPQ16 acts as a
fluorescent reporter for mHTT aggregation, with the short polyQ sequence being
sequestered into aggregates formed by untagged mHTT exon1, while in the absence
of mHTT aggregation the GFP-Q16 reporter will be diffusedly distributed
throughout the cell. This approach enables the visualization of aggregation of
untagged mHTT in cells, which is preferred since a tag can influence a protein’s
stability. Following siRNA transfection targeting PA28α and induction of mHTT
expression, the cells were analyzed by automated microscopy and the percentage
of cells with aggregates was determined by a generated Matlab script. This data
showed that, similar to the filtertrap assay, PA28α silencing increased
aggregation. When HTT(Q25) or mHTT(Q46) were used as wildtype or short polyQ-
expanded HTT fragments, respectively, which do not form aggregates after 48
hours of expression, no effects of PA28α silencing on soluble wildtype HTT or
mHTT levels were observed. To investigate whether PA28α silencing leads to
accelerated aggregation HTT(Q25) and mHTT(Q46) samples were analyzed in a
filtertrap assay. Although five times more protein was loaded for HTT(Q25) and
mHTT(Q46) compared to mHTT(Q97), there was no signal above background visible.
This indicates that there were no aggregates present, despite silenced PA28α
levels. Altogether this data indicates that downregulation of PA28αβ accelerates
aggregate formation, but does not affect soluble HTT turnover directly.
# Discussion
By studying changes in proteasome complexes during disease progression in HD
mouse models, we observed that PA28αβ disassembles from the 20S core most
obviously in the cortex, striatum and hippocampus during HD disease progression.
This suggests that the changes in PA28αβ were most abundant in the HD-affected
regions. When examining the consequences of alterations in PA28αβ activated
proteasomes on mHTT turnover in cell models and using purified proteasomes, we
observed that while the degradation of polyQ peptides is improved by PA28αβ
activation of (purified) proteasomes, degradation of the mHTT protein fragment
is hampered by the addition of PA28αβ to purified proteasomes. This shows that
PA28 obstructs the entrance of the folded mHTT protein into the 20S core. Indeed
it has been suggested before that PA28αβ selectively blocks the passage of
larger protein fragments. However, PA28αβ overexpression did not affect mHTT
levels when overexpressed in cells. This can suggest several things: PA28αβ
activation is already sufficient; another limiting factor necessary for
degradation is required; other proteasome complexes including the 26S proteasome
mainly target mHTT; or mHTT is not efficiently targeted towards the proteasome.
However, as reducing PA28αβ levels increased mHTT aggregation but did not affect
the degradation of soluble (m)HTT, this suggests that PA28αβ is critical for
overall proteostasis and only indirectly affects mHTT aggregation.
The indirect role of PA28αβ affecting mHTT aggregation may be the result of its
reported chaperone-like functions. The 90-kDa heat shock protein, HSP90, binds
unfolded proteins to prevent aggregation and is, together with HSC70 (cytosolic
HSP70), and HSP40 (cytosolic DnaJ homologue), involved in protein refolding.
PA28 serves as a linker between HSP90 and HSC70 and is a necessary cofactor for
protein remodeling. Since PA28 can act as a chaperone cofactor, the observed
effects on mHTT(Q97) could be mediated by the ability of PA28 to interfere with
aggregate formation. Indeed, hippocampal extracts from PA28α overexpressing
mice, prevented aggregation of heat sensitive luciferase. In these samples
activity of PA28α activated proteasomes was not increased and the total amount
of damaged proteins was not altered, suggesting that chaperone-like activity is
responsible for the observed effects. Overexpression of PA28γ in the striatum of
HD mice reduced HD pathology as demonstrated by behavioral tasks. Furthermore,
in these mice PA28γ overexpression led to reduced levels of mHTT aggregation,
but no changes in mHTT levels were observed. This could again point towards a
potential chaperone function of PA28 in obstructing mHTT aggregate formation.
Another known function of PA28αβ is its role in the cell’s protection mechanism
against oxidative stress. Rapidly after the induction of oxidative stress by
hydrogen peroxide, PA28αβ and PA28γ bind to free 20S subunits. *In vitro*
experiments show that PA28αβ activated proteasomes improve the selective
degradation of oxidized proteins, but not their native form. In cells,
overexpression of PA28α also improves the capability of the proteasome to
degrade oxidized proteins. Reducing PA28αβ levels on the other hand, leads to an
increase in carbonylated proteins in differentiating embryonic stem cells,
without altering proteasome content. HD progression is associated with increased
oxidative damage to DNA, proteins and lipids, which contributes to
neurodegeneration (reviewed by Kumar and Ratan and Gkekas et al.). mHTT disrupts
nuclear integrity and both mHTT protein and expanded CAG RNA impair DNA repair
mechanisms. In combination with the increased oxidative damage to DNA, this
contributes to neuronal pathology. Furthermore, HTT itself can be oxidized,
which leads to stabilization of mHTT oligomers and accelerates aggregation, by
facilitating aggregate interactions. By the dissociation of PA28αβ from the 20S
core, the proteasome is less adapted to degrade oxidized proteins, which could
accelerate oxidative stress and subsequent pathology in HD.
The question remains whether dissociation of PA28αβ activators from the 20S core
in the cortex, striatum and hippocampus of HD mice is a cause or consequence of
increased mHTT aggregation. During normal homeostasis, proteasome complex
formation is a dynamic process, which is altered upon several stimuli, including
inhibition of catalytic activity, pro-inflammatory stimuli and competition
between the different PAs. The pool of free PA28αβ heptamers, which are not
bound to 20S core subunits, suggests that the cell has free PA28 activators that
can be used to quickly increase PA28αβ-mediated proteasome activation. This
underlines the ability of cells to dynamically regulate the number of PA28αβ
activated proteasomes. Upon hydrogen peroxide treatment, levels of PA28αβ
activated proteasomes increase within the first hour prior to synthesis of new
PA28αβ complexes. Moreover, PA28γ activated proteasomes increase within hours
after proteasome inhibition, without an increase in PA28γ transcription.
Although the exact signaling pathways remain elusive, phosphorylation of PA28αβ
is found to be involved in its binding to the 20S core subunit. Several studies,
however, show that kinases are dysregulated in HD (reviewed by Bowles and
Jones). This may imply that during HD, general dysregulation of the signaling
pathways responsible for proteasome conformational changes may lead to the
disassembly of PA28αβ activated proteasomes, after which normal functioning is
impaired. However, PA28αβ-20S disassembly may also be a specific response to the
increase in mHTT aggregates in affected cells. Since PA28αβ is involved in the
clearance of aggregates though its chaperone like function, the increased need
for free-PA28αβ may cause the disassembly from the 20S core. Based on this
hypothesis, the PA28αβ-20S disassembly observed in mouse brain would not lead to
increased aggregation but would rather be a coping mechanism to deal with the
aggregates already present in the cell. Indeed, aggregates are already present
at the disease stages at which we observe the change in PA28αβ activated
proteasomes.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
In the perspective image of a slanted textured surface, oriented components of
the texture that are aligned with the 3-D slant converge to form orientation
flows, while components orthogonal to the slant increase in frequency. On casual
observation, the horizontal component appears perceptually more salient than
other components when a surface is slanted (top left and right) than it does
when the surface is parallel to the frontal plane (top center). The increase in
saliency is more pronounced in complex texture patterns, e.g. the octotropic
plaid, which consists of eight gratings of the same frequency, equally spaced in
orientation ( bottom). Since these converging orientation flows play a critical
role in conveying the perceived 3-D slant and shape of the surface, an increase
in their saliency should enhance the 3-D perceived slant. The goal of this work
is to examine the neural mechanisms that enhance the visibility of orientation
flows.
Many surface textures contain components of roughly the same frequencies at many
different orientations, with most of the frequencies in the higher frequency
declining segment of the human CSF. Slanting the surface increases the
frequencies of components not aligned with the slant, thus leading to a
reduction in visibility. If different oriented components were processed
independently by the visual system, the increase in saliency of the components
parallel to the slant could be due just to the reduced visibility of the other
components. However independent processing of different orientations is not a
feasible premise.
The response of oriented neurons in cat and primate striate cortex to a stimulus
at a preferred orientation is suppressed by the superposition of a second
oriented stimulus, even at the null orientation. Parallel to these results,
psychophysical studies have reported that the contrast threshold of an oriented
stimulus is increased in the presence of a superimposed orthogonal stimulus.
Physiologically measured cross-orientation suppression (COS) is broadband for
orientation and occurs over a wide range of spatial frequencies.
Psychophysically measured COS appears to be broadband for orientation, but with
mixed evidence for frequency-selectivity. Thus it is possible that
psychophysically measured COS has components that are distinct from the COS
measured in V1 neurons.
In this study we identify the mechanism underlying the change in salience of
orientation flows. In the first experiment, we show that the visibility of
orientation flows increases as a function of surface slant. In the second
experiment, we show that the increased salience results from the frequency-
selectivity of COS and not the frequency dependent visibility of the masking
components.
# Methods
All research followed the tenets of the World Medical Association Declaration of
Helsinki and informed consent was obtained from the subjects after explanation
of the nature and possible consequences of the study. The research was approved
by the Queens College Institutional Review Board.
## 1. Apparatus and Presentation
Stimuli were presented on a 22″ Mitsubishi Diamond Pro 2070 flat screen CRT
monitor with an 1024×768 pixel screen running at a refresh rate of 100 Hz via a
Cambridge Research Systems ViSaGe Visual Stimulus Generator controlled through a
3.2 GHz Pentium 4 PC. Observers' head positions were fixed with a chinrest
situated 1 m from the stimulus monitor. All stimuli were presented so that the
center of each image was level with the observer's eye. Viewing was monocular in
a dimly lit room, and there was no feedback.
## 2. Stimuli and Procedure
Planar surfaces were patterned with horizontal-vertical (h–v) and octotropic
plaid patterns and projected in perspective. All stimuli were presented such
that the horizontal grating component was interleaved with non-horizontal
components in alternating frames at 100 Hz. This technique enabled us to alter
the contrast of the horizontal component independently from the other
components. For the h–v plaid, the contrast of the vertical grating was fixed at
50%, the highest possible for interleaved frames. Similarly, the contrast of
each of the non-horizontal gratings in the octotropic plaid was fixed at the
highest possible level, 7.1%. The phases of the plaid pattern components were
randomized on each trial. Stimuli were presented in circular apertures spanning
6.5 deg against a grey background at the mean luminance of 58 cd/m<sup>2</sup>.
Contrast thresholds of the horizontal component were determined using a 2IFC
paradigm. Each session was preceded by a grey screen with a central black
fixation cross that remained onscreen for 1 minute. The fixation cross remained
onscreen for the duration of the session. After the initial adaptation, a tone
signaled the start of the trials. Each of the two stimulus intervals in each
trial lasted 500 msec, separated by a 400 msec inter-stimulus interval. Audible
beeps of different frequencies signaled the presentation of each of the two
stimulus intervals. Test contrast was varied in interleaved 3-down/1-up double-
random staircases to ascertain the 79% correct point. Each staircase completed
two reversals at 1.8% contrast steps, then eight reversals at 0.4% contrast
steps. Threshold was estimated as the average of the last six reversals.
## 3. Experiment 1: Orientation Flow Visibility as a Function of Surface Slant
Surfaces were patterned with 3 cpd h-v and octotropic plaids. For each of the
two plaid types, observers completed eight sessions which were grouped as
follows. One baseline session measured contrast thresholds for the horizontal
grating alone in the fronto-parallel orientation. Each of three other baseline
sessions measured contrast thresholds of the horizontal grating alone at left
and right slants of 25, 50, and 65 deg. In the other four sessions, contrast
thresholds were measured in the presence of the non-horizontal components. Thus
there were a total of 16 sessions per observer. For each pattern type, the four
baseline sessions were run first in random order, then the remaining sessions
were run in random order. Each session took approximately 10–15 minutes.
## 4. Experiment 2: Frequency-Selectivity of Cross-Orientation Suppression Mechanism
Fronto-parallel surfaces were patterned with an iso-frequency h–v plaid or an
h–v plaid consisting of a vertical grating of half the frequency of the
horizontal grating. The same surfaces were also presented slanted at left or
right at 60 deg which acts to approximately double the vertical frequency in the
image. Consequently, the frequencies in the image of the slanted 6 cpd iso-
frequency plaid become 6 cpd horizontal and 12 cpd vertical and the frequencies
in the image of the 6 cpd horizontal and 3 cpd vertical plaid become
approximately equal at 6 cpd. To test whether suppression is a function of the
similarity of frequencies between the test and mask, or of the salience of the
mask, we needed to select frequencies from the small set that are highly
visible, effectively convey surface slant, and are significantly less salient
when doubled. We used frequencies of 4 and 6 cpd which satisfy these
requirements by being just past the peak of the human CSF.
Contrast thresholds for the horizontal grating were measured using the
interleaved staircase procedures. Observers ran four different sessions, three
times each: one baseline session for the horizontal grating alone at fronto-
parallel, rightward slanted by 60 deg and leftward slanted by 60 deg
orientations, one session for fronto-parallel plaids and two sessions for
slanted plaids. The slanted sessions were blocked in order to contain both types
of plaids and both types of slants within each session, while keeping the length
of sessions similar to the sessions testing the fronto-parallel stimuli.
## 5. Observers
One of the authors and two experienced but uninformed observers participated in
this study. All had normal or corrected-to-normal visual acuity.
# Results
## 1. Experiment 1: Orientation Flow Visibility as a Function of Surface Slant
Contrast thresholds of the horizontal components in the different conditions are
shown for the three observers in in separate columns. The panels in the top row
plot contrast thresholds of the horizontal grating alone (filled circles), in
the presence of the vertical grating in the h–v plaid (open triangles), and in
the presence of the seven non-horizontal components in the octotropic plaid
(filled squares) as a function of surface slant. The axis along the top of each
panel represents the frequency of the vertical grating component as it changes
with surface slant relative to the frequency of the test. Thresholds of the
grating alone (filled circles) are relatively unchanged by surface slant,
reflecting the fact that the spatial frequency of this component is relatively
unchanged. The presence of the vertical grating (open triangles) increases
thresholds for all surface slants (except for the steepest slants for observer
DT), reflecting an overall decrease in visibility of the horizontal component.
Thresholds increased even more in the presence of the seven non-horizontal
components of the octotropic plaid (filled squares).
We quantified the suppression induced by non-horizontal components by dividing
thresholds of the horizontal grating in the presence of other components by
thresholds in the absence of other components. The suppression factors for the
simple and octotropic plaids are plotted as functions of surface slant, with
solid and dashed lines respectively, in the bottom panels of. Suppression for
both patterns decreases as surface slant increases, with substantially greater
and steeper changes in suppression for the octotropic plaid.
We have previously shown that perceived orientation flows determine the
perception of 3-D shape from texture. 3-D shape is not perceived when the flows
are physically present if they are masked by other components (see, Figure 10).
The results in indicate that orientation flows are more visible for the h–v than
the octotropic plaid at shallow slants, but equally visible at steep slants.
Hence, slants should be easier to see for the h–v plaid than the octotropic
plaid at shallow angles, but the two should be equally perceptible at steep
angles. This prediction is borne out in where the orientation flows and thus
slants are easier to see in the h–v plaid than the octotropic plaid at ±40 deg,
but are equally visible for the two plaids at ±70 deg.
## 2. Experiment 2: Frequency-Selectivity of Cross-Orientation Suppression
It is clear from the results in that contrast thresholds are raised by
orthogonal masks, which is a signature of COS. Since the frequencies in the
fronto-parallel plane were 3 cpd which is near the peak of the human CSF, the
question remains whether the peak suppression is a function of the similarity of
frequencies between the test and mask, or of the salience of the mask.
The four conditions of Experiment 2 provided two independent comparisons of
these hypotheses. In (bottom), mean suppression factors averaged across the
three observers are plotted for all conditions for the 4 cpd horizontal grating
(left) and the 6 cpd horizontal grating (right). Error bars represent one
standard error of the mean. Data in each panel are plotted in the same order
from left to right as the four stimulus conditions shown above. First, the
similarity hypothesis predicts that thresholds should be higher in the iso-
frequency fronto-parallel plaid than for the unequal frequency fronto-parallel
plaid, whereas the salience hypothesis predicts the opposite. Thresholds for
both the 4 cpd and 6 cpd test gratings were raised more by the iso-frequency
mask than the more salient unequal frequency mask. Second, the increase in
suppression for the 4 cpd condition when the unequal frequency plaid is slanted
(leading to an iso-frequency image pattern) also supports the similarity
hypothesis over the salience hypothesis. In addition, in comparing the two
slanted plaids, suppression was greater when the image pattern was iso-frequency
than when the surface pattern was iso-frequency. Since we expected suppression
to decrease with increasing slant for the iso-frequency condition and increase
with slant for the unequal frequency condition, we tested for interaction
between the frequency conditions and the slant conditions in a 2×2 ANOVA. The
interaction was in the correct direction for both spatial frequencies, and
statistically significant at the.05 level for the 4 cpd test (F(1,12) = 23.12,
p = .0406) but not for the 6 cpd test (F(1,12) = 12.00, p = .0742).
These results indicate that the COS from the vertical grating is greatest when
the frequency in the projected image is equal to that of the horizontal grating,
even when the frequency is one to which we are less sensitive. Previous
measurements of the spatial frequency tuning of COS showed a decrease in masking
for a 4 cpd test when the mask frequency increased from 4 to 8 cpd, but did not
determine whether spatial-frequency mismatch or a decrease in mask saliency was
the cause.
## 3. Feed-Forward Models of Cross-Orientation Suppression
COS is well-documented in cortical area V1, the first site in the visual pathway
containing orientation tuned cells. COS has been attributed to compressive
contrast nonlinearities in LGN, but a cortical component has also been revealed.
Although several electrophysiological studies examining the frequency
selectivity of COS suggest that suppression mechanisms are broadly tuned, it is
unclear whether this kind of tuning plays out psychophysically. It would be
remarkable if the facilitation of 3-D shape perception occurs automatically
through the neural processes that lead to COS, so to ascertain its locus, we
have explored the possibility of frequency selectivity in an LGN based model.
Although intra-cortical inhibition was the original suggestion for COS, the fact
that suppression is not reduced by prior monocular or binocular adaptation to
the masking stimulus, that suppression is robust for masks at temporal
frequencies beyond the limits of cortical neurons, and that COS has an early
onset led to the suggestion that the suppression results from the depression of
thalamo-cortical synapses. More recent papers quantifying the fast recovery
times of COS and the suppression of both synaptic inhibition and excitation by
orthogonal masks challenge the notion of synaptic depression. Instead these
models suggest that COS results from contrast saturation and rectifying
nonlinearities in the LGN, and expansive spike threshold nonlinearities in the
cortex.
To test the frequency-selectivity of COS in the models of Li et al. and Priebe
and Ferster, we computed cortical responses to a vertical test grating in the
presence of superimposed horizontal masks of the same or different frequency.
The model simulates responses of a simple cell as determined by excitation of
LGN cells tuned to the spatial frequency of the test grating. The receptive
field of each ON-center cell is modeled as the difference of two Gaussians:where
σ<sub>c</sub> is the variance of the central Gaussian, and σ<sub>s</sub> is the
variance of the surround Gaussian. OFF-center receptive fields were modeled as
negatives of ON-center receptive fields. Linear outputs of LGN cells at each
location of the stimulus were approximated by convolving ON- and OFF-center
receptive fields with the stimulus (either a single vertical grating, or a
vertical grating added to a horizontal mask). The outputs were then subjected to
a compressive contrast nonlinearity in the LGN of the form:where *R* is the
compressed response, L is the linear response, and the value of k dictates the
strength of the compression (greater compression for greater values).
Excitation and inhibition in the cortical simple cell receptive field has been
modeled by summed responses of LGN cells in “push-pull” form. Excitation from
ON-center LGN cells and inhibition from OFF-center LGN cells form an ON sub-
region of the simple cell receptive field, while excitation from OFF-center LGN
cells and inhibition from ON-center LGN cells form an OFF sub-region of the
simple cell. Summed excitatory and inhibitory responses are then squared,
representing an accelerating cortical spike-voltage non-linearity. (A range of
different expansive nonlinearities yielded the same qualitative patterns in our
simulation.) This model simple cell gives null responses to horizontal (mask)
gratings in isolation.
Responses of the model to the test grating plus the mask were computed for masks
that were the same frequency as the test, or half, twice, and three times the
frequency of the test. We defined response suppression as the response to the
grating alone divided by the response to the grating plus the mask.
The graph in the bottom of plots response suppression as a function of the
frequency of the orthogonal mask relative to the frequency of the test. The
points at zero mask frequency represent model responses to the vertical test
alone. To test the generality of the simulations, we implemented two different
center-surround variance ratios and two different compressive nonlinearities.
Each of the four curves represents one combination of these variables: solid
lines represent conditions in which the variance of the surround of the LGN
cells is twice the variance of the center, dashed lines represent conditions in
which the variance of the surround is three times the variance of the center.
Square symbols represent NL1 conditions in which k = e in the compressive
nonlinearity (Equation 2), and the triangles represent the more compressive NL2
conditions in which k = max(abs(L)). All combinations of receptive fields and
nonlinearities lead to frequency selectivity, with suppression greatest when the
frequency of the mask matches that of the test. Increasing the surround variance
acts to slightly broaden the frequency tuning, and increasing the strength of
the compression acts to increase the overall suppression and sharpen the
frequency tuning. The magnitudes of suppression reported in Experiment 1 fell
between the suppression values for the two model nonlinearities.
# Discussion
The suggested roles of COS in visual encoding have included orientation tuning,
contrast gain control, and redundancy reduction in the coding of natural images.
Here we postulate a potential role for COS in the decoding of 3-D slant. We have
shown that when textured surfaces are slanted, the release of COS makes the
critical orientation flows more visible, which correlates with better perception
of 3-D slant. We have shown that COS is frequency specific, and that this
specificity can arise in simple feed-forward models of COS. To our knowledge,
feed-forward explanations of frequency selectivity of COS have not been
suggested previously. The LGN models of COS were formulated on the basis of cat
physiology, where almost all cell response functions are compressive as a
function of contrast. In primate LGN, M-cells are compressive, but P-cells are
fairly linear. Our model thus provides the M-cell component of COS. Since V1
cells get input from P and M-cells, some component of COS involves cortical
interactions.
Purpura et al. examined whether neurons in V1 and V2 facilitate the extraction
of 2-D orientation patterns for the perception of 3-D shape. Of the 29 neurons
in macaque V1 and V2 that were isolated from tetrode recordings, flat plaids
induced significant suppression in 78% of the neurons compared to optimal single
gratings. Suppression was significantly reduced in 45% of the neurons for plaids
slanted along or orthogonal to the optimal orientation. In addition, 28% of V1
and 56% of V2 neurons showed enhanced responses to orientation flows per se,
indicating that asymmetries may be more prominent in the 2-D structure of V2
receptive fields. Since COS and surround suppression significantly reduce
responses to patterns in natural scenes, stimuli that undermine these sources of
suppression may allow V1/V2 to mark areas that have a higher probability of
containing 3-D shape. In particular, release from cross-orientation suppression
serves to enhance the visibility of orientation flows that are the keys to
decoding 3-D shapes as signaled by texture, shading, and specular reflection.
This work was presented in part at the Vision Sciences Society meeting in
Naples, FL, May 2008. The authors wish to thank John Zhu for programming.
[^1]: Conceived and designed the experiments: AL QZ. Performed the
experiments: AL. Analyzed the data: AL. Contributed
reagents/materials/analysis tools: AL QZ. Wrote the paper: AL QZ.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
Atrial fibrillation (AF) is the most common cardiac arrhythmia, causing
substantial cardiovascular morbidity and mortality. Although risk factors have
been described, there are no available blood tests to predict AF risk.
The activation of renin-angiotensin system (RAS) definitively plays an important
role in the pathogenesis of atrial fibrillation. The angiotensin II is a primary
mediator of RAS which is mainly produced from angiotensin I by angiotensin-
converting enzyme (ACE, EC 3.4.15.1, CD143). Patients with AF are known to have
increased level of ACE expression in the heart tissue, in particular, in atria.
Study on the transgenic mice also demonstrated that the increased ACE expression
in the heart might be a causative factor for AF and sudden cardiac death. Both
ACE inhibitors and angiotensin receptor blockers reduce AF incidence and may
prevent AF-related complications in patients and in experimental models.
ACE is a Zn<sup>2+</sup> peptidyldipeptidase which plays key roles in the
regulation of blood pressure by producing angiotensin II and degrading
bradykinin and in the development of vascular, including cardiac, pathology and
remodeling. ACE is constitutively expressed on the surface of endothelial and
some epithelial cells, as well as cells of the immune system (macrophages,
dendritic cells) reviewed in. ACE expression in the normal and pathological
human hearts has been studied previously. To date, several new substrates for
ACE have been identified (AcSDKP, angiotensin 1–7) and new functions for ACE
have been proposed, such as outside-in cell signaling, antigen presentation. The
anti-fibrotic and anti-inflammatory actions of a substrate for ACE, AcSDKP, seem
to be especially important for AF pathogenesis in patients with the enhanced ACE
level in the heart.
Despite the strong relationship of RAS activation to arrhythmias, plasma levels
of ACE do not correlate to AF and ventricular arrhythmias. Generally, plasma ACE
accurately reflects the level of tissue ACE. Blood ACE originates from
endothelial cells, mostly lung capillaries, which exhibit nearly 100% ACE
expression compared to only 5–15% ACE-positive capillaries in the systemic
circulation. Based on that, we estimated that ACE shed from heart capillaries
could represent not more than 1% of total ACE in the blood. This is why a total
plasma ACE level in patients does not seem to be a predictive parameter for
patients with AF. However, as atrial ACE increases 3-fold in patients with AF,
the quantification of the definite heart-derived ACE (mainly, atria-derived ACE)
in plasma theoretically could be used as a predictive test for the risk of AF.
We proved the concept that the conformation of ACE is cell- and tissue-specific
and stems likely from different glycosylation of the enzyme on the example of
ACE from lung endothelial cells versus ACE from macrophages and dendritic cell
of sarcoid granuloma and ACE from epithelial cells of prostate. The ACE
conformational fingerprint based on the pattern of the binding of a set of mAbs
to different epitopes on the surface of ACE has, therefore, a potential for the
disclosure of the cells/organs from which ACE originates.
We applied this approach here to demonstrate conformational differences of ACEs
originated from human heart and lung and showed differences for purified ACEs,
for ACEs in tissue homogenates, and ACEs in the blood after *in vivo* perfusion.
We believe that such differences will allow the generation of monoclonal
antibodies (mAbs) able to distinguish ACEs shed to the blood circulation from
these two organs and, therefore, form the base for the blood test for predicting
the risk of AF.
# Experimental section
## ACEs from different sources
The work was carried out in accordance with The Code of Ethics of World Medical
Association (Declaration of Helsinki) and was approved by the Institutional
Review Boards of the Bakulev Center of Cardiovascular Surgery, Moscow State
University, and the University of Illinois at Chicago. None of the donors were
from the vulnerable populations and all donors or next of kin provided written
informed consent that was freely given. Human citrated plasma and tissue
homogenates (1:9 and, in some cases, 1:3 w/v ratio) from 10 donors (five male
donors, age 54–66, and five female donors, age 28–68), including three donors
with atrial fibrillation, were used as sources of somatic ACEs. Lung and heart
ACEs were purified from tissue homogenates using anion-exchange chromatography
on DEAE-Toyopearl 650M and then lisinopril affinity chromatography—as in “”.
Purified ACE preparations were proved to be homogeneous by electrophoresis in
7.5% SDS-PAGE “”.
## ACE activity assay
ACE activity in blood plasma, homogenates of human organs or homogenates of
heart chambers was measured using a fluorimetric assay with two ACE substrates,
2 mM Z-Phe-His-Leu (ZPHL) and 5 mM Hip-His-Leu (HHL). Inhibition of ACE activity
with anti-catalytic mAb 5F1 to the N domain of ACE and mAb 1E10 to the C domain
was performed at mAbs concentrations 100 μg/μl and 10 μg/μl, correspondingly,
with 1mM ZPHL or 2.5 mM HHL as substrates.
## Substrate specificity of ACE
The kinetic parameters of the hydrolysis of several synthetic tripeptide
substrates and natural substrate decapeptide angiotensin I by purified human
heart and lung ACEs were determined in 0.05 M phosphate buffer, pH 7.5,
containing 0.15 M NaCl and 1 μM ZnCl<sub>2</sub>, at 25°C. The rates of
enzymatic hydrolysis of Z-Phe-His-Leu, Hip-His-Leu and angiotensin I were
determined fluorimetrically, whereas the kinetics of the hydrolysis of FA-
containing substrates, FA-Phe-Gly-Gly and FA-Phe-Phe-Arg, was studied
spectrophotometrically.
## Immunological characterization of ACE (Plate immunoprecipitation assay)
Ninety six-well plates (Corning, Corning, NY) were coated with anti-ACE mAbs via
goat anti-mouse IgG (Pierce, Rockford, IL) bridge and incubated with different
sources of ACE, which were equilibrated for ACE activity. After washing off
unbound ACE, plate-bound ACE activity was measured by adding a substrate for
ACE, Z-Phe-His-Leu, directly into the wells. Sixteen mAbs to human ACE were
generated in our lab, while mAb BB9 was kindly provided by Paul J. Simmons (then
Brown Foundation of Molecular Medicine, University of Texas Health Science
Center, Houston, TX, USA).
## Dialysis and filtration of human plasma and heart and lung homogenates
Dialysis of the heart and lung homogenates was performed in 10 kDa dialysis
cassettes (Pierce, Rockford, IL) and in 100 kDa Biotech dialysis tubes (Spectrum
Inc., Houston, TX) against 0.05 phosphate buffer, pH 7.5, 0.15 M NaCl and 1 μM
ZnCl<sub>2</sub>, at 4°C. Filtration of the homogenates was performed on
Vivaspin filtration membranes (GE Healthcare, Sartorius Corp., Bohemia, NY) with
30 kDa and 100 kDa limits at 12 000g.
## Aminopeptidase activity
Aminopeptidase activity in tissue homogenates at different dilutions was
estimated by the rates of the hydrolysis of 0.01–0.1 mM His-Leu in PBS-BSA
buffer, pH 8.3, at 37°C.
## ACE perfusion in rats
All *in vivo* rat methods/experiments were approved by and performed in
accordance with Moscow State University Committee on the Ethics of Animal
Experiments guidelines and regulations that conform to the NIH guidelines (Guide
for the care and use of laboratory animals). Adult male Wistar rats weighing
\~300 g were put under pathogen-free condition in individual plastic cages with
sawdust bedding in an air-conditioned room under constant temperature (23±1°C)
and 70% humidity. Rats were provided with water and standard diet (LabDiet, 5053
(LabDiet; St. Louis, MO, USA)) *ad libitum*. All animals were monitored for body
health on a daily basis for a week before an experiment. All efforts were made
to minimize rats suffering. Purified human heart and lung ACEs, 1000 mU in 100
μl, pH 7.4, were injected into the tail vein of rats (two rats per group) with
anesthesia with ketoprofen. After 30 min, euthanasia was performed by
decapitation, the blood was collected and citrate plasma prepared. At that
moment, about 30% of human ACE was still in the rat blood. Precipitation of
human ACE from rat plasma by a set of mAbs to ACE was performed as described
above but corrected for trace precipitation of the rat ACE by these mAbs.
## Statistical analysis
All data are means ± SEM. Significance was analyzed using the Mann-Whitney test
with STATISTICA 6 (StatSoft, Inc., OK).
# Results and discussion
## ACE activity in the human heart
We estimated ACE expression in different human tissues, namely, heart, lung,
kidney and spleen, by the comparison of ACE activity in homogenates of these
tissues as well as in human citrated plasma. ACE activity in the human heart
homogenate (expressed in mU per gram of tissue) was about 3-fold more than in
human plasma and 8-12-fold less than in human lung. This estimation correlates
with the density of radioligand ACE inhibitor binding sites in human heart and
lung, as well as with high ACE RNA transcription in the lung, while negligible
in the heart, as in Human Protein Atlas.
ACE activity measured simultaneously by two substrates in the homogenates of
different whole heart chambers showed statistically significant difference in
atrial and ventricular ACE activity, this activity being the highest in the
right ventricle and the lowest in the right atrium (insert).
Mammalian tissues and blood contain endogenous ACE inhibitors and ACE effectors,
including putative ACE binding proteins. In order to demonstrate the presence of
endogenous ACE inhibitors in human tissues we compared an apparent ACE activity
in the heart and lung homogenates at serial dilutions. An apparent ACE activity
significantly increased 3-4-fold during dilution of both homogenates. We
excluded the presence of aminopeptidases in human tissues as a reason for that
effect “”. Thus, an effect of dilution reflects the presence of endogenous ACE
inhibitors in heart and lung tissues. Dialysis (10 or 100 kDa) and filtration
(100 kDa) of the heart and lung homogenates also resulted in a similar
(200–400%, p\<0.05) increase of an apparent ACE activity (data not shown).
Therefore, in order to estimate correctly ACE activity in the heart (or lung)
homogenate it is not necessary to perform time-consuming dialysis, as simple
dilution of homogenates 10-fold successfully induces dissociation of low
molecular weight (LMW) ACE endogenous inhibitors from their complex with ACE.
We discovered that the effect of the dilution of tissues homogenates on relative
ACE activity with two substrates, Z-Phe-His-Leu and Hip-His-Leu (ZPHL/HHL
ratio), was substrate specific. We have shown previously that the selective
inactivation/inhibition of the C domain increases ZPHL/HHL ratio for somatic,
two-domain ACE, whereas selective inactivation/inhibition of the N domain
decreases this ratio, as these substrates are cleaved by the two domains with
different rates. Therefore, we can interpret the decrease of ZPHL/HHL ratio and
increase of this ratio upon dilution of heart and lung homogenates,
respectively, as an evidence of the presence of different sets of endogenous ACE
inhibitors in the heart and lung tissues.
## Conformational fingerprinting of heart and lung ACEs
We characterized the conformation of the heart and lung ACEs using a panel of
mAbs directed against 17 different epitopes and mapped on the surface of the N
and C domains of catalytically active human ACE–a method of “conformational
fingerprint of ACE”. As apparent in, the immunoprecipitation profile of heart
ACE significantly differed from that for lung ACE, the difference being observed
both for the purified enzymes and ACEs in homogenates. This allowed us to
conclude that heart ACE, originated from heart endothelial cells and perhaps
myofibroblasts and lung ACE, originated from lung endothelial cells, exhibit
different local conformations of their surface, probably, due to tissue- and
cell-specific post-translational modifications (PTM). PTM, which are common for
various proteins, can precisely regulate the functions of proteins by inducing
conformational changes, which subtly or dramatically alter the protein surface
or its overall tertiary structure. The most common PTM is glycosylation.
Somatic ACE represents a type-I N-glycosylated membrane glycoprotein, exact
glycan structures of which, as well as locations of actually present
oligosaccharide chains on the surface of the protein globule, can vary with a
protein source. The amino acid sequence of human somatic ACE contains 17
potential sites for N-glycosylation. The structure and exact positions of glycan
moiety in human somatic ACE from different tissues were not investigated
completely.
Heart and lung ACEs possess rather equal masses about 170 kDa as was revealed by
SDS-PAGE “”which indicates on the absence of major differences in the degree of
glycosylation of these ACEs. It was shown earlier that common sialylated
biantennary complex oligosaccharide can contact the surface of the enzyme within
an area about 200–300 Å. As epitopes for mAbs are usually 600–900 Å, it is
obvious that the presence of the oligosaccharide within an epitope (as well as
its definite structure, i.e. the number of antennae, sialylation, fucosylation,
etc.) can hugely influence mAbs binding. Previously, we showed that different
glycosylation of ACE in endothelial and epithelial cells could be the main
reason for the differences in mAbs binding to ACEs from lung and seminal fluid.
Moreover, the difference in binding efficiency for some particular mAb can be
attributed to the different glycosylation of the corresponding glycosylation
site within epitope for this very mAb. So, the main reason for the difference
observed for immunoprecipitation profiles of the heart and lung ACEs could be
due to different glycosylation of ACE, even in endothelial cells but from
different organs, induced by different environment of these cells in different
organs. Conformational fingerprints of heart and lung ACEs allowed us to suggest
that different glycosylation of these ACEs occurs in the following glycosylation
sites on ACE: Asn25 in the epitopes for mAbs BB9, 3A5 and i1A8, and Asn117 in
the epitope for mAb 5F1 on the N domain, as well as Asn666 in the epitope for
mAbs 4E3 and 1E10, and Asn731, which is within the epitopes for mAbs 1B8 and
3F10 and close to the epitope for mAb 1B3 on the C domain of ACE.
The purified heart and lung ACEs were isolated from different donors and
strictly speaking the differences in mAbs binding to these purified ACEs could
be attributed to the inter-individual differences in the protein glycosylation
in these donors, in our case, ACE glycosylation. In order to exclude this
possibility, we performed the conformational fingerprints of heart and lung ACEs
in homogenates obtained from the same donor. For statistical reasons and
reproducibility, we used 10 pairs of homogenates obtained from the tissues from
10 donors. As the general pattern of mAbs binding to ACE in pairs of homogenates
was similar to the pattern of mAbs binding to purified heart and lung ACEs from
different donors we can conclude that different conformational fingerprints of
heart and lung ACEs demonstrate a real conformational tissue specificity of the
enzyme. It is worth noting that ACE from donors with AF in anamnesis
demonstrated similar differences in mAbs binding to heart and lung ACEs (not
shown), that is this difference is not caused by the disease but by the nature
of ACE-producing cells.
In order to confirm that the differences in local ACE conformation of heart and
lung ACEs retain in the blood we perfused purified ACEs from heart and lung (and
seminal fluid ACE as a representative of ACE produced by epithelial cells) via
alive rat circulation. Circulation of glycoproteins (including ACE) in blood
after shedding from the cell membranes leads to the enrichment of serum
glycoproteins with molecules with a higher content of sialic acid residues as a
result of the selective removal of asialo-molecules (with terminal galactosyl
residues) by hepatic lectin, while sialylated glycoproteins remain in the
circulation. The results clearly demonstrate that the effect of such perfusion
on the pattern of mAbs binding to heart and lung ACEs is significantly
different, once again demonstrating that the patterns of glycosylation
(sialylation, in particular) of the heart and lung ACEs are different. In
addition, the binding of mAbs 5F1 and 1E10, which better bind to ACE molecules
when glycan in the epitopes for these mAbs is less sialylated (Danilov and
Trakht, unpublished observation), decreased after perfusion of the lung ACE into
the rat in accordance with expected relative enrichment of ACE moiety with
sialic acids. As a result of the perfusion of the heart ACE, however, the
binding of these very mAbs unexpectedly increased, which indirectly confirms
that the local conformation of heart ACE and its sensitivity to the structure of
glycan differs from that for lung ACE.
It is important, that even after circulation in the blood, the
immunoprecipitation profiles of soluble ACEs from heart and lung remained
significantly different ( and ““) proving the possibility to distinguish ACEs
produced by different organs in a real blood.
Therefore, an analysis of ACE conformation provides a structural base for the
generation of mAbs able to distinguish ACE produced by a definite cell type or
definite organ, in particular, by heart, and to generate a blood test for the
detection and quantification of this particular ACE in the blood for the
prediction of AF risk.
## ACE effectors in human heart tissue
As we discussed above, tissue homogenates contain high amounts of ACE
inhibitors/effectors. When we performed purification of heart and lung ACEs by a
combination of anion-exchange and affinity chromatography, we noticed
significant influence of this purification procedure on the ACE conformational
fingerprint (and “”), that is a microenvironment significantly influenced on ACE
conformation in the tested tissues. Purification of both ACEs from the
corresponding homogenates led to a decrease of the binding of mAbs 1G12, 6A12,
and i2H5 having overlapping epitopes on the N domain, which could be attributed
to the dissociation of endogenous ACE inhibitors from the complex with ACE.
In addition, several antibodies to the C domain, mAbs 1B3, 1B8, 3F10, 1E10, and
4E3, dramatically increased their binding to ACE as a result of the enzyme
purification. It is important that we did not observe such remarkable increase
in the binding of these mAbs neither after dialysis nor after filtration of
homogenates through filters with 30 kDa limit (not shown) or even 100 kDa limit.
These data allowed us to suggest that, in addition to the presence of endogenous
easily-dissociating (due to dilution, dialysis or filtration) LMW ACE inhibitors
(Figs) in both heart and lung tissues, these tissues contain some high molecular
weight (HMW) ACE effectors/binding proteins, which could not be eliminated via
filtration or dialysis but are eliminated during ACE purification procedures (“”
and “”).
## Kinetic characteristics of heart and lung ACE
In order to determine whether these differences in conformation of heart and
lung ACEs are reflected in different functions of these ACEs, we compared
kinetic characteristics of these enzyme.
The pH-dependencies of enzymatic activity of heart and lung ACEs in the
hydrolysis of Z-Phe-His-Leu at 0.15 M NaCl were quite similar with pH-optimum
about 7.0 in accordance with previous results obtained for bovine ACE. However,
the pH-optima for the hydrolysis of Hip-His-Leu were equal to 7.5 for heart ACE
and 7.8–7.9 for lung ACE, thus confirming different conformations of the two
enzymes.
The inhibitory effect of anti-catalytic mAb 5F1 to the N domain and mAbs 1E10
and 4E3 to the C domain, while these mAbs differently bound to the heart and
lung ACEs, did not show, however, significant difference in the extent of
inhibition of these two ACEs (not shown).
The kinetic parameters, *k*<sub>cat</sub> and K<sub>m</sub>, of the hydrolysis
of several synthetic substrates, as well as a natural substrate angiotensin I,
by purified heart and lung ACEs are presented in. Two of the substrates, Z-Phe-
His-Leu and Hip-His-Leu, are C-terminal analogs of angiotensin I, while a
substrate with Phe-Arg on its C-terminus could be considered as a C-terminal
analog of another natural ACE substrate, bradykinin, as well as C-terminal
analog of atriopeptin 2.
The absolute k<sub>cat</sub> values calculated for the hydrolysis of angiotensin
I by the human ACE markedly differ in the literature, from 3.5 s<sup>-1</sup> to
40 s<sup>-1</sup> for recombinant human ACE expressed in CHO cells, to 250
s<sup>-1</sup> for human heart ACE, and to 66 s<sup>-1</sup> for human kidney
ACE (Kost OA, unpublished data), likely due to different conditions of
angiotensin synthesis, different conditions of its hydrolysis, and different
conditions and extent of ACEs purification. So, to minimize the putative errors,
we determined the kinetic parameters of the hydrolysis of definite substrates by
the two ACEs, from heart and lung, simultaneously.
The K<sub>m</sub> values of the hydrolysis of any substrate by the two ACEs
appeared to be similar, as well as the k<sub>cat</sub> values for most
substrates. However, for a short substrate Hip-His-Leu, the k<sub>cat</sub>
values of the hydrolysis differed two-fold for the heart and lung ACEs.
Some differences in the catalytic properties of heart and lung ACEs were shown
earlier for human and bovine ACE, as well as different ability of these enzymes
to be inhibited by a set of inhibitors. There are some examples demonstrating
the influence of glycosylation on the kinetic properties of the enzymes, and
references herein, including ACE which activity could vary 2-fold due to
different glycosylation. Thus, it is likely that different ACE conformations
induced by different protein glycosylation in different tissues could be the
reason for different kinetic characteristics of these ACEs. We could not even
exclude the possibility that unique ability to hydrolyze atriopeptin II which
was reported only for heart, but not lung, ACE could be partially explained by
the differences in the conformations of the two ACEs due to different
glycosylation.
## ACE phenotyping in different heart chambers
In order to estimate putative heterogeneity of ACE expression in heart chambers
we performed ACE phenotyping in homogenates of the whole heart chambers from 10
donors. The presence of endogenous ACE inhibitors in heart chambers was
demonstrated by the comparison of the apparent ACE activity in atria and
ventricles at different dilutions. The apparent ACE activity in heart chambers
markedly depended on homogenate dilution, as was observed for the whole heart
and lung homogenates. Effect of homogenates dilution confirmed the lower
apparent ACE activity in atria in comparison with ventricles. The amount of
inhibitors in heart chambers, however, varied in different donors. Bearing in
mind that ventricles comprise the main part of the whole heart it is obvious
that ventricle tissues are the main contributor to the amount of ACE inhibitors
in the heart tissues.
When we compared the amount of immunoreactive ACE protein determined by
precipitation of ACEs by mAb 9B9 with an apparent ACE activity in different
heart chambers, we have found that their ratio in the right atrium is
significantly bigger than in other chambers, especially for undiluted
homogenates, which is likely explained by the higher content of ACE inhibitors
in the atrium.
Conformational fingerprinting of ACE in heart chambers revealed statistically
significant increased binding of mAbs 1B8 and 3F10 to ACE from the right atrium,
while the difference in the binding of mAbs to ACE from the right ventricle or
the left atrium was negligible.
This difference was observed both in undiluted and diluted homogenates, which
means that the reason is the conformational difference between ACEs expressed in
atrium and ventricle, not the presence of different effectors. Moreover,
conformational fingerprinting of ACE was separately performed on 10 sets of
homogenates of heart chambers obtained from 10 donors for reproducibility and
statistics. These two mAbs, 1B8 and 3F10, have highly overlapping epitopes on
the C domain which contain potential glycosylation site Asn731. Highly probable
that different microenvironment of ACE in the heart chambers (Figs) can lead to
different glycosylation of Asn731 in ACE in the right atrium, compared to the
glycosylation of this particular glycosylation site in ACE in other chambers,
and, as a results of that, lead to different effectiveness of the binding of
mAbs 1B8 and 3F10.
It is worth noting that atrial tissues are likely to perform specific
biochemical functions. As an example, only atrial bovine tissue was reported to
contain a metallodipeptidyl carboxyhydrolase (EC 3. 4.15.4) which resembles ACE,
as it is able to hydrolyze ACE common substrate Hip-His-Leu and be inhibited by
ACE inhibitors. This enzyme, however, differed from ACE in a number of molecular
and kinetic properties, the major difference being the ability to hydrolyze
atriopeptin II, which could be considered as heart-specific substrate, and its
analogue Hip-Ser-Phe-Arg, and references herein.
Thus, there could be major differences in the biochemical processes within the
heart chambers due to different enzymatic content. However, there could be also
more subtle differences due to the changed properties of the same enzyme
expressed in different heart chambers.
## Conclusions
The significant differences in the local conformations of heart ACE (originated
from heart endothelial cells and likely from myofibroblasts) and lung ACE
(originated from lung endothelial cells) allow us to suggest that the properties
and functions of ACE could be sensitive to the microenvironment and be regulated
by accompanying constituents of tissues and blood, this regulation could depend
on a set of possible ACE effectors in heart and lung tissues.
Therefore, significant structural differences in ACE from heart and ACE from
lung demonstrated in this study may be the base for the generation of mAbs,
which will distinguish these two ACEs. Possible limitation of this study is that
we did not generated yet these mAbs. We have to say that this is a pioneer and
extremely difficult work because the discriminative power of such mAbs should be
very high (about 100-fold) in order to detect only about 1% of heart-derived ACE
in the blood where the majority of ACE molecules comes from lung. Therefore, the
occurrence of hybridomas with required specificity would be low demanding to
test thousands of hybridomas producing anti-ACE mAbs.
However, such mAbs (if we will be able to select them) may have a potential for
the development of blood-based assay for quantification of heart-derived ACE in
the blood for identification of the patients with the increased level of heart
ACE, i.e. with the increased risk of atrial fibrillation, which cannot be
achieved by estimation of ACE activity in the blood.
# Supporting information
The authors are grateful to Prof. V. Pozdnev (Institute of Biomedical Chemistry,
Moscow) for the substrate Fa-Phe-Phe-Arg and to Dr. V. Popov (M.V. Lomonosov
Moscow State University) for the help with the experiments with rats.
This work was supported by the Ministry of Science and Education of Russian
Federation (grant number 14.Z50.31.0026).
ACE angiotensin-converting enzyme
AF atrial fibrillation
FA- furanacryloyl-
HHL (Hip-His-Leu) hippuryl-L-histidyl-L-leucine
HMW high molecular weight
LMW low molecular weight
PTM post translational modifications
RAS renin-angiotensin system
ZPHL (Z-Phe-His-Leu) carbobenzoxy-L-phenylalanyl-L-histidyl-L-leucine
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Diabetes Mellitus (DM) refers to a group of common metabolic disorders that
share the phenotype of hyperglycemia. It is one of the four common non-
communicable diseases causing major morbidities and mortalities. World Health
Organization (WHO) estimated that 422 million adults had diabetes in 2014.
International Diabetes Federation (IDF) estimated this number to be 629 million
by 2045. In Africa, the prevalence of diabetes adults between 20–79 years was 16
million in 2017 and projected to be 41 million in 2045. About 69.2% were
undiagnosed. Africa attributes 77% of the deaths under 60 years to diabetes
mellitus. The figure is the highest proportion in the world. International
Diabetes Federation in 2015 estimated that 5.2% of Ethiopian adults had
diabetes.
Self-management is the ability of the patient to deal with all that a chronic
illness entails, including symptoms, treatment, physical and social
consequences, and lifestyle changes. Since health care cost for the treatment of
acute and chronic complications of diabetes is high, self-management is
compulsory. Ethiopia is one of the low-income countries. Thus, supporting
patient self-management practice plays a key role in effective chronic illness
care and improve patient outcomes. Effective management of diabetes requires
predominantly self-directed practices, where the individuals become responsible
for the day-to-day decisions related to controlling their disease. The emerging
evidence supports the implementation of practice strategies that are conducive
to patient self-management and improved patient outcomes among chronically ill
patients. Implementation of self-management needs to be individualized, and
people with diabetes should be assisted to understand the impact of medication,
food and physical activity on blood glucose control. Adequate self-management
can minimize the disease and disease-related complications, while the frequency
of self-monitoring can be determined based on the individual’s self-management
goals. The key goal of self-management is controlling blood glucose, improving
quality of life and reduction of diabetes-complications. A systematic review
conducted in 2018 in Sub-Saharan Africa on self-management of Type 2 diabetes
shows that patients rarely self-monitored their glucose levels; had low
duration/frequency of physical activity; moderately adhered to recommended
dietary and medication behavior and had a poor level of knowledge about diabetes
complications.
Numerous studies conducted in different parts of Ethiopia shows slightly more
than half of patients living with diabetes had good self-management practice.
Different cross sectional survey indicated that dietary and medication adherence
of diabetic patients was low. Internationally, only two-thirds of diabetic
patients perform daily self-monitoring of blood glucose. However, self-
monitoring of blood glucose was good in Ethiopia. Inadequate attention to
diabetes center and lack of knowledge about self-management was reported as main
factor in Ethiopia.
Self-management practice in clients with chronic diseases is essential to
maintain good health while taking the medications. However, the practice may
vary from client to client due to several factors. Different researches have
investigated that age, current occupation, lack of awareness, absence of self-
practice health education, years of suffering from DM, having family members
suffering from the illness and lack of knowledge about the illness were the
factors that affect the level of self-management behaviors. Besides, belief in
treatment effectiveness, family support, self-efficacy, awareness about the
disease and social support were also the factors that affect self-management
practice.
Despite the knowledge about the factors that affect self-management practice,
there was no comprehensive study conducted on self-management of chronic illness
especially DM covering the different hospitals located in western Oromia,
Ethiopia, as far as the researchers’ knowledge is concerned. This study thus
focused on the assessment of self-management practice and its predictors in the
study facilities. The findings of this study believed to give useful input for
policymakers mostly in enforcing and establishing self-management practice
interventions which, in turn, may encourage immediate health care providers to
consider it in their routine care practices.
# Methods and materials
## Study design and setting
A hospital-based cross-sectional study was conducted among diabetic patients on
follow-up at diabetic centers from November 2017 to February 2018 in public
hospitals found in western Oromia, Ethiopia, namely Nekemte specialized
hospital, Gimbi general hospital, and Nedjo general hospital.
## Source population
All diabetic patients on follow-up attending hospitals were considered as source
populations.
## Study population
All diabetic patients attending diabetic centers and wards of the study
hospitals during the study period were subjects of the study.
## Eligibility criteria
Known diabetic patients who visited the diabetic centers for follow-up and wards
to receive care were included in the study while patients with diabetic
emergencies like diabetic ketoacidosis and diabetic coma were excluded.
## Sample size determination
A single population proportion formula was used to determine the sample size.
The proportion of patients who performed self-management practice (54.7%
according to a study done at Nekemte referral hospital, Ethiopia in 2013) was
considered in sample size calculation. Marginal error between sample size and
population parameter of 5%, and 95% confidence level, and 5% non-response rate
was considered. A total of 400 patients with known diabetes mellitus
participated in the study.
## Sampling techniques
All known diabetic patients visited the study hospitals for follow-up, and those
admitted to the wards were taken into consideration. The average monthly client
load was taken from the daily average DM client flow of the hospital and the
registry book. Systematic random sampling was used. The interval was calculated
at each hospital. The sample was allocated proportionally to the client flow of
the respective facilities. The first client who arrived at the waiting area on
the first day of data collection, and who met the eligibility criteria was taken
as the first candidate for the study. This process continued until the desired
sample size was attained.
## Data collection tools and methods
The data was collected directly by interviewing diabetic clients after getting
informed consent. The questionnaire was prepared in English by modifying from
different literature sources with similar areas of interest. It was translated
from English to Afaan Oromo, a local language, and re-translated back to English
to ensure consistency. The questionnaire was pre-tested in Dambidolo hospital.
Three trained diploma nurses were used as data collectors under the close
supervision of one B.Sc degree nurse, and the data were collected in a face-to-
face interview. For the presence of co-morbid, we had observed the patent’s
folder. The only physician confirmed and recorded disease(s) was taken as a co-
morbid disease.
## Data processing and analysis
Data were entered into Epi Info 3.5.4 software package and cleaned first. Then,
the analysis was made using a statistical package for the social sciences (SPSS)
software package version 20. Analysis of overall self-management practice was
done by transforming the scores on closed-ended questions related to self-
management practices. Using the odds ratio (OR) with a 95% limit of the
confidence interval, the association of dependent and independent variables was
analyzed, and their degree of associations was computed. Potential confounding
variables were controlled using binary and multivariate logistic regression.
Statistical significance was considered at P\<0.05. Finally, the analyzed data
were presented using frequency, percentage, and texts.
## Data quality control
A pre-test was conducted at Dambidolo hospital on 5% of the total sample size to
check clarity, understandability, and consistency of the data collection tool.
Then, the necessary amendments were made to the questionnaire before the full-
scale data collection was implemented. Data collection was conducted under the
close supervision of supervisors and the collected data were checked for
completeness.
## Study variables
### Dependent variable
Diabetes self-management practice.
### Independent variables
Sociodemographic characteristics like sex, age, occupation, marital status,
religion, level of education, lack of self-management education, patient
education, getting family support, presence of DM-related complications and
other health problems.
### Operational definitions
**Self-management**: The practice of diabetic patient’s self-initiated and
performed activities to control disease and maintain life, health, and
wellbeing.
**Good self-management practice**:—Diabetic patients with average and above
scores on closed-ended questions related to self-management practices.
**Poor self-management Practice**:—Diabetic patients with less than average
score on closed-ended questions related to self-management practices.
**Hyperglycemia**—an abnormally increased concentration of glucose in the blood
(≥ 126 mg/dl at FPG).
## Ethics statement
Ethical approval was obtained from the Research and Ethics Committee of Wollega
University. An official letter was written to each hospital to get official
permission. Participants were informed that privacy and confidentiality were
maintained. Written consent was taken from the study participants.
# Results
## Sociodemographic characteristics of the respondents
depicts the overall characteristics of the study participants. A total of three
hundred ninety-eight diabetic patients participated in this study, raising the
response rate to 99.5%. More than half of the respondents were male (225,
56.5%). The average age of all respondents was 41.33 ± 18.93 (SD) years.
Majority of the participants were married (255,64.1%), Oromo people (377,
94.7%), living in urban area (204, 51.3%) and Protestant Christians (219,
55.0%). The remaining (106, 26.8%) were single, 20 (5%) Amhara people, (194.
48.7%) living in rural areas and (136, 34.12%) followers of Orthodox Christian
religion. One hundred eighteen (29.6%) respondents had attended
college/university, whereas about 92 (23.1%) can not read and write, and only
20.4% had attended secondary school education.
Student patients were relatively high (21.6%) followed by housewives (20.4%) and
government employees (20.4%). Based-on participants’ believe, more than half,
54%, of the participants had a middle-level income compared to their neighbors.
About 30.7% of the participants were poor and a very small proportion of the
participants were very rich (0.3%). The average family size was 5 ± 2 (SD).
About three fourths of the participants (74.6%) were husbands and wives in the
family, followed by sons and daughters (22.9%). Most of the study participants
(91.0%) reported that they need family support for disease treatment. About two-
thirds of the participants (65.8%) received support from their families.
More than half of the participants, 220 (55.3%) did not know the type of
diabetes they have. One hundred twenty-three (31.0%) of the participants had
Type 1 diabetes, while the other had Type 2 diabetes. One hundred eleven (28.0%)
had the disease for more than a decade, whereas about 72% live with DM for less
than a decade. One-third of the study participants (66.8%) believe that they can
cured of the disease, and the majority (71.6%) thought that the medication they
were taking could cure them. Near half of the participants, 195 (49.0%) believe
that health care providers had a good approach towards them. And two-thirds of
the participants, 266 (66.8%) alleged that they would recover from the disease
while others did not.
The majority of the participants (93.8%) had either of the following chronic
illnesses: hypertension, cancer, edema, Human Immunodeficiency Virus/Acquired
Immunodeficiency Syndrome (HIV/AIDS), dyspepsia and bronchial asthma. About
three-fourth (74.1%) of them had diabetes-related manifestations and
complications. Again, more than half of the participants had shock (55.9%)
followed by diabetes ketoacidosis (52.9%). Some of the respondents also reported
nerve (32.9%) and eye problems (23.4%).
Study participants had practiced different self-management interventions. They
had also been taking medications and most of them had a monthly follow-up. About
half (49.7%) of them had been taking insulin regimen followed by oral
hypoglycemic agents (39.7%) for treatment and glycemic control. Only 42 (10.6%)
were taking combined medications. The adherence to medication was varying. About
the participants’ diet intake, the participants had been consuming food three
times a day with the different food menu. However, only one-third of them used
the food menu. About half of the respondents used to consume vegetables followed
by starch at lunchtime. At dinner, 39.2% of them used starch, while the others
(43.0%) used vegetables. Close to three-fourths of the participants (72.4%) did
not use to consume snacks, and starch was the most consumable food item. About
half (52.0%) of them performed regular physical activity while the remaining did
not (48.0%). Again, more than half of the respondents (57.8%) performed regular
foot care, whereas 168 (42.2%) did not. Participants did not perform annual foot
care check-up.
## Predictors of self-management practice
Overall, about 63.6% of the study participants self-management practice was
good, while 36.4% self-management practice was poor. One hundred eighty-four
(46.2%) of the participants knew about diabetes self-management management, of
which 129 (32.4%) were practicing it. Cross-tabulation of the variables showed
that those who are female, living in urban, married, rich and merchant tended to
practice diabetes self-management. Females practice self-management more than
males, 74.2% and 68% respectively. The magnitude of self-management practice was
relatively higher among married clients (73.5%) than never married (66.7%).
About 73.7% of participants from urban and 64.3% of rural had practiced self-
management. No difference in self-management practice was observed between
participants who had developed DM-related complications and who had not. Self-
management practice was high in participants who believed DM is curable, than
those who did not believe so. Participants who were merchants practiced more
diabetes self-management, followed by daily laborers (79.2%). Those economically
rich tended to practice more self-management than their poor counterparts.
The logistic regression analysis results indicated that only two variables,
namely occupation and having family support had shown statistically significant
association with the practice of self-management management in both binary and
multiple logistic regressions. Accordingly, merchants were observed practicing
self-management about six times higher than clients with other occupations with
AOR of 5.945 (1.177–30.027 at 95% CI). Clients who had family support in DM-
related care were again observed practicing self-management 2.87 times more than
those who had no family support with AOR of 2.835 (1.386–5.801 at 95% CI). No
difference was observed among other variables entered into the regression model.
# Discussion
Among 398 study participants, 63.6% of them had good self-management practice.
Self-blood glucose monitoring, physical activity, diet adherence, medication
adherence, and foot care are the components of self-management practice. This
finding is comparable with the finding from Northern Ethiopia. The result of
this study demonstrates that self-management practice of people living with
diabetes mellitus attending hospitals in western Oromia is higher than the
result of previous studies conducted in Nekemte referral hospital, Jimma
University teaching hospital, and Harar, Ethiopia, and Lesotho. This maybe due
to day to day improvement of awareness of the disease management and cultural
variation.
Physical activity believed as essential in controlling blood glucose. The result
of this study revealed that more than half of participants practiced regular
physical activity. The participants level of physical activity is greater than
the finding of the study conducted in Harar, Ethiopia. This discrepancy may be
due to increased awareness regarding diabetic self-management and differences in
the measurement tools. American Diabetes Association (ADA) recommends that
physical activity performance five days/week that lasts for 30 minutes. In this
study, only half of the respondents self-reported as they perform regular
physical activity which is inconsistent with the recommendation of ADA. Under
performance of physical activity perhaps a lack of awareness about
recommendations on physical activity and lack of infrastructures. ADA recommends
diabetic patients should perform a comprehensive foot evaluation at least
annually to identify risk factors for ulcers and amputations. More than half of
diabetic patients performed foot care. Foot care practice is less than the
practice of community-dwelling Philippine diabetic patients, and adult patients
attended Black lion hospital, Ethiopia. On the other hand, foot examination and
foot self-management were not consistent with recommendations of ADA. This may
be due to a variation in awareness creation, poor income, and increased age of
the participants.
World Health Organization recommends the consumption of a healthy diet. A
healthy diet includes consumption of fruit, vegetables, legumes, nuts, and whole
grains; less than 10% of total energy intake from free sugars; 30% of total
energy intake from fats and less than 5g of iodized salt. American diabetes food
pyramid recommends bread, rice, pasta, and rice as the first food menu, followed
by vegetables and fruits. The majority of the participants were not using the
food menu which is inconsistent with the recommendations of WHO and ADA. The
result of this study is unlike the results of the studies from Northwest
Ethiopia, and Harar, Eastern Ethiopia. Inconsistency on food menu may related
with a level of awareness, the purpose of using food menu, absence of diabetes
food education, and difference in data collection tools. This implies the
participants had no awareness about food menu.
The vast majority of diabetic patients (74.1%) had diabetes mellitus-related
manifestations and complications. More than half of the participants had
diabetes-related shock and diabetic ketoacidosis. The prevalence of DM-related
complications was higher than the results of the study conducted at Jimma
University teaching hospital. Nerve diseases were reported by one-third of the
study participants which was slightly higher than the study done in Jimma,
Ethiopia. However, the reported DM-related eye problems were more prevalent than
the result of the study conducted in Jimma university referral hospital. High
prevalence of eye problems may be be due to the public awareness about diabetes-
related complication and lack of consistent health education. This implies that
diabetes self-monitoring of blood glucose was poor in the participants of this
study.
Being female, living in urban, married, rich, merchant and having family support
were associated with self-management practice. Occupation and family support
were tended to be the predictors of self-management. The finding of this study
shares a similarity with that of studies done in Iran and Malaysia Family
support was reported to increase adherence to self-management. Increase in self-
management practice is comparable with the finding of the systematic review and
meta-analysis which reported the social support significantly improved self-
management. However, these factors were inconsistent with factors reported by
Amente, Belachew, Kaehaban, Hongsranagon and Formosa and Muscat from Ethiopia,
Thailand, and Malta, respectively. This variation may be demonstrated due to day
to day changes in diabetes education that increases awareness and culture
variation.
The present study has some strengths. First, the response rate was high. Second,
it tried to find out the predictors of self-management practice using
appropriate data analysis methods. Limitations of the study were using
unstandardized and validated tools.
# Conclusions
Compared to the findings of previous studies, the diabetes self-management
practice of the participants of this study was good. The study participants’
regular physical activity, food intake, medication adherence, and foot self-
examination was moderate. Being a merchant and having family support were found
to be the predictors of self-management practice. Randomized controlled trials
involving the participants is needed to proof. Predictors of self-management
should be considered to boost self-management practice.
# Supporting information
Our gratitude goes to the study facility administrators, service providers and
participants for their collaboration and information. The authors would also
like to thank colleagues who contributed their valuable suggestions throughout
this research work.
ADA American Diabetes Association
AOR Adjusted Odds Ratio
CDC Centre for Disease Control
CI Confidence Interval
DKA Diabetes Ketoacidosis
DM Diabetes Mellitus
IDF International Diabetes Federation
OR Odds Ratio
SBGM Self-monitoring of Blood Glucose
SPSS Statistical Package for the Social Sciences
WHO World Health Organization
10.1371/journal.pone.0232524.r001
Decision Letter 0
Assefa Woreta
Solomon
Academic Editor
2020
Solomon Assefa Woreta
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
31 Oct 2019
PONE-D-19-26947
Predictors of self-management practice among diabetic patients attending western
Oromia hospitals, Ethiopia
PLOS ONE
Dear Dereje Chala Diriba
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
a revised version of the manuscript that addresses the points raised during the
review process.
==============================
The topic appears to be the most studied in most part of Ethiopia including
Oromia region.
Abstract:
The methods are very short sighted to provide proper information. The result
looks fragmented. The conclusion drawn not based on the finding?
Introduction:
This section included literature review from different part of the world
including Ethiopia. I found this necessary to make a strong argument in the
discussion section. However, the write up looks very inconsistent and juggling
from the beginning to the end. You should follow introduction writing guide
line. Since, the topic is highly researched in various part of Ethiopia I
recommend authors to incorporate as many literature from Ethiopia as possible.
Methods:
I have noticed similar study had been conducted one year prior to this study in
Nekemet, Western Oromia region. What is your rationale to Nekemete Hospital in
this study?
How you operationally defined the study variables in this research? What is your
dependent and independent variables?
How you developed the questionnaire? How you validate your questionnaire? Where
you pretested the questionnaire?
Result:
The socio-demographic section seems to have included junk of information and
redundancy. You must take account of socio-demographic variables than unrelated
information. In general, the result section is full of in consist, in
comprehensive and fragments of data. Most of the paragraph appears to have a
copy and paste. I would recommend the authors to give appropriate time to
evaluate the manuscript from the beginning to the end before submission to make
sure the information built-in is clear and vivid information to understand.
Discussion:
As it has been said this topic is one of the most studied, hence the chance to
get appropriate literature to make a strong argument on the finding is likely
very high even in Ethiopia. However, the arguments are shallow, not based on
evidence and weak. Why you cited literature which doesn’t have any similarity to
the study setting, for instance America and others. This makes the discussion
very shallow and inadequate.
What is the limitation of this study?
Conclusion:
How do you measure whether self-care is good or bad? There is no single
statement in the methods section that clearly defined self-care and how it was
measured. Overall, the conclusion not inferred from the analyzed result.
==============================
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**Additional Editor Comments (if provided):**
The topic appears to be the most studied in most part of Ethiopia including
Oromia region.
Abstract:
The methods are very short sighted to provide proper information. The result
looks fragmented. The conclusion drawn not based on the finding?
Introduction:
This section included literature review from different part of the world
including Ethiopia. I found this necessary to make a strong argument in the
discussion section. However, the write up looks very inconsistent and juggling
from the beginning to the end. You should follow introduction writing guide
line. Since, the topic is highly researched in various part of Ethiopia I
recommend authors to incorporate as many literature from Ethiopia as possible.
Methods:
I have noticed similar study had been conducted one year prior to this study in
Nekemet, Western Oromia region. What is your rationale to Nekemete Hospital in
this study?
How you operationally defined the study variables in this research? What is your
dependent and independent variables?
How you developed the questionnaire? How you validate your questionnaire? Where
you pretested the questionnaire?
Result:
The socio-demographic section seems to have included junk of information and
redundancy. You must take account of socio-demographic variables than unrelated
information. In general, the result section is full of in consist,
incomprehensive and fragments of data. Most of the paragraph appears to have a
copy and paste. I would recommend the authors to give appropriate time to
evaluate the manuscript from the beginning to the end before submission to make
sure the information built-in is clear and vivid information to understand.
Discussion:
As it has been said this topic is one of the most studied, hence the chance to
get appropriate literature to make a strong argument on the finding is likely
very high even in Ethiopia. However, the arguments are shallow, not based on
evidence and weak. Why you cited literature which doesn’t have any similarity to
the study setting, for instance America and others. This makes the discussion
very shallow and inadequate.
What is the limitation of this study?
Conclusion:
How do you measure whether self-care is good or bad? There is no single
statement in the methods section that clearly defined self-care and how it was
measured. Overall, the conclusion not inferred from the analyzed result.
\[Note: HTML markup is below. Please do not edit.\]
Reviewers' comments:
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**Comments to the Author**
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Reviewer \#1: Partly
Reviewer \#2: Yes
Reviewer \#3: Partly
\*\*\*\*\*\*\*\*\*\*
2\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#1: Yes
Reviewer \#2: No
Reviewer \#3: Yes
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: No
Reviewer \#2: No
Reviewer \#3: No
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: No
Reviewer \#2: No
Reviewer \#3: No
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Reviewer \#1: This is a study on self-management practice among diabetes
patients in Ethiopian hospital setting. The authors do a great job in providing
the big picture of diabetes in Ethiopian context and identifying those
predictors of self-management practice. They followed a rigor procedure in their
study design, analysis and reporting. This study adds a valuable information in
the management of diabetes in western Oromia hospitals by providing some
predictors for self-management practice.
This study has several issues that need to be addressed before being considered
for publication.
• The introduction is well written but does not inform the reader about the
importance of measuring the self-management practice among diabetes patient. Why
we should seek to measure the self-management practice of diabetes among other
chronic illness should be explained.
• What self-management is not described to a reader in the introductory section,
rather the definition happened in a latter section of the manuscript (at the
beginning of the discussion). This is not good without introducing the concept
at the beginning but presenting the result and defining the term in the
discussion.
• As the authors reviewed there are several studies conducted in Ethiopia
(Harar, Nekemte, Addis Ababa). Therefore, why they are interested to do same
topic in Ethiopia at western Oromia is not clear. The justification made as to
why this study is conducted is not very convincing. For instance, 45% of
participants at Nekemte referral hospitals had poor diabetes self-management
though 54.3% of them had diabetic related knowledge, it worth doing a study why
such level of practice with higher level of knowledge and what are the factors
for such low self-care practice using other study design such as case-control
than repeating same study in same place.
• The last section of introduction mentioned the absence of diabetic self-
management education program in the country. However, the relevance of
mentioning this concept in this section is not explained. The authors know this
idea in advance and makes them even to forward a recommendation on establishing
the education center. The claims are not placed properly in the context of the
previous literature. Generally, the introduction should be re-written signifying
the importance of doing this study in the light of “effective patient self-
management is necessary to prevent adverse health outcomes of diabetes” among
diabetes patients in lower-middle income countries including Ethiopia.
Reviewer \#2: Abstract:
• Under method section number of study participants needs to be included
• Under result number of those who have either good or poor self care management
needs to be indicated.
• The conclusion should be in line with the pertinent finding described under
abstract
• Recommendation is not in line with conclusion.
Introduction
• Definition of self care management needs to be included.
• References needs to be in logical order.
Methods
The study setting is not well described.
Tools used to assess self care management was not clearly indicated.
Analysis method for self care management is not indicated.
Under ethical clearance future tense was used.
Result
• Standard deviation of mean age should be reported in plus or minus, not only
plus
• Under table 1 you put asterisk on the other category of religion, but not
define it below the table as footnote.
• Eye problems which is considered as one complication of DM is indicated by two
different numbers.
• Rather than including other information better narration follows similar
table.
• There is redundancy of narration about patients believe of diabetic treatment
before and after table 2
• Under statements which is written above the title “predictors of self
management” under result section, there is a statement which says figure 2, but
there is no figure in the document.
• Better you include all variable you consider for logistic regression to table
3 with their respective confidence interval and odds ratio rather than
duplicating it under table 4
• How you found at the end two predictor variable using logistic regression is
not clearly explained.
• Revise your interpretation of logistic regression especially your comparison
of occupation.
• How self care management is measured?????????? And classified as good and poor
under the title “level of self care practice” are not clearly indicated.
Discussion
Your discussion needs revision. Implications given by the researcher is not
satisfactory.
Conclusion
The conclusion is not in line with that of the title of the study and there are
repeated copying and pasting of what is already described under result section.
Reference
Some of the references listed are not utilized for preparation of the manuscript
Finally competing interest and list of abbreviations/acronyms are not included
to the manuscript
Reviewer \#3: The authors tried to make allignment between title, objective,
results and conclussion. However the manuscript need further revision and
modification particularly the reporting format and language used during
reporting.
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: No
Reviewer \#2: No
Reviewer \#3: Yes: Markos Desalegn Beyene
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10.1371/journal.pone.0232524.r002
Author response to Decision Letter 0
24 Dec 2019
We really appreciate the genuine comments of the editors and reviewers. For the
comments given by reviewers and editor, we reacted and corrected promptly. We
are ready to correct if any further comments provided. The point by point
response to reviewers commented was uploaded.
10.1371/journal.pone.0232524.r003
Decision Letter 1
Assefa Woreta
Solomon
Academic Editor
2020
Solomon Assefa Woreta
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
5 Feb 2020
PONE-D-19-26947R1
Predictors of self-management practices among diabetic patients attending
hospitals in western Oromia, Ethiopia.
PLOS ONE
Dear Dr. Dereje Chala,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
a revised version of the manuscript that addresses the points raised during the
review process.
==============================
I gain would like to applaud for putting together the effort to make the
necessary improvement on the manuscript. Most of the comments and feedback that
have been given by reviewers appears to be corrected and incorporated
accordingly, but there are few issues need to be addressed in order to proceed
to the next step. For instance, the definition used to describe knowledge has a
preconceived notion. Did you evaluate knowledge based on individual question
mean or the summation mean of the knowledge questions? Secondly, the argument
used to discuss the finding seems to be so frail and not enough to provide vivid
clarification. In general, this section needs to have literary supported
evidence with clear reference or citation for your argument. For example, the
first paragraph in the discussion section has compared and contrasted other
studies with your current, but the statement used to ornate the argument read as
“This could be related to change to diabetic education in different ways. Thus,
sustainable scheduled diabetic education is very crucial to increase self-
management practice.” Where did you get this information? Is this information
research driven or based on the predetermined knowledge? Hence, all the argument
you might reach need to have study based explanation or argument. Make sure you
make the necessary changes in this section to eliminate the inconsistency.
==============================
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sustainable scheduled diabetic education is very crucial to increase self-
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10.1371/journal.pone.0232524.r004
Author response to Decision Letter 1
11 Mar 2020
Question 1: Did you evaluate knowledge based on individual question mean or the
summation mean of the knowledge questions?
Response: Thank you for thorough evaluation and observation. We didn’t evaluate
the diabetes knowledge score; however, we reported the general knowledge about
diabetes management. Thus, we have removed from operational definition part.
Question 2. Secondly, the argument used to discuss the finding seems to be so
frail and not enough to provide vivid clarification. In general, this section
needs to have literary supported evidence with clear reference or citation for
your argument.
Response: Thank you for your comment. We noted that our discussion is weak, thus
we made modifications with implications of the results. We also cited the
literature we have used.
10.1371/journal.pone.0232524.r005
Decision Letter 2
Tu
Wen-Jun
Academic Editor
2020
Wen-Jun Tu
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
17 Apr 2020
Predictors of self-management practices among diabetic patients attending
hospitals in western Oromia, Ethiopia.
PONE-D-19-26947R2
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10.1371/journal.pone.0232524.r006
Acceptance letter
Tu
Wen-Jun
Academic Editor
2020
Wen-Jun Tu
This is an open access article distributed under the terms of the
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provided the original author and source are credited.
22 Apr 2020
PONE-D-19-26947R2
Predictors of self-management practices among diabetic patients attending
hospitals in western Oromia, Ethiopia.
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[^1]: The authors have declared that no competing interests exist. |
# Introduction
Despite the availability of highly effective COVID-19 vaccines to prevent
hospitalization and reduce mortality, variants continue to fuel the surge of
COVID-19 across the U.S.. High-quality diagnostic and serology tests are
essential tools to better understand the epidemiology of COVID-19 and immunity
after infection. Viruses and antibodies are primarily detectable within certain
temporal windows. However, many individuals infected with SARS-CoV-2 are
asymptomatic or may not seek medical care because of mild symptoms. In contrast
to molecular diagnostic tests, serologic tests are informative even once the
SARS-CoV-2 infection is no longer present.
Currently, there are 90 authorized SARS-CoV-2 serology/antibody tests approved
for Emergency Use Authorization (EUA). However, they have not undergone the same
evidentiary review standards required for Food and Drug Administration (FDA)
clearance due to the COVID-19 national emergency. There is a need to assess the
real-world performance of these tests. Further, while large studies have shown
that greater than 91% of people with active SARS-CoV-2 infection seroconvert,
the factors associated with seroconversion (e.g., pre-existing conditions, the
severity of COVID-19 presentation) remain elusive.
From a public health perspective, confidence in the ability of serological tests
to identify those with recent infections is critical for effective pandemic
planning. Estimates of disease prevalence directly inform dynamic population
estimates of susceptible, infected, and recovered, which are needed to
understand the infectiousness of SARS-CoV-2. From a clinical perspective, an
accurate understanding of SARS-CoV-2 exposure is necessary to understand disease
presentation and a clinical course of action, especially when patients do not
present with symptoms or present late in their disease course (e.g., post-acute
sequelae of SARS-CoV-2). Additionally, identifying factors associated with
seropositivity may elucidate potential mechanisms of action that may be
foundational in the development of therapy and treatment plans.
To address these gaps, we characterize the performance of serology tests by
estimating the positive percent agreement (PPA) of serological samples obtained
from people known to be positive for SARS-CoV-2 infection by molecular assay
(e.g., PCR). We also sought to identify factors associated with seropositivity.
Findings from this study may facilitate understanding of the real-world
performance of serology tests, many of which were issued under EUA, and may help
inform our understanding of the immune response to SARS-CoV-2.
# Materials and methods
## Study population and setting
Six health systems (i.e., datasets) collaborated on the Diagnostics Evidence
Accelerator (EA): Health Catalyst, Mayo Clinic, Optum Labs, Regenstrief
Institute, the University of California Health System, and Aetion and
HealthVerity. The EA is a consortium of leading experts in health systems
research, regulatory science, data science, and epidemiology, specifically
assembled to analyze health system data to address key questions related to
COVID-19. The EA provides a platform for rapid learning and research using a
common analytic plan. Health Catalyst, Mayo Clinic, and the University of
California Health System all utilized electronic health records (EHR) data from
their respective healthcare delivery systems. The Regenstrief Institute accessed
EHR and public health data from the Indiana Health Information Exchange, while
Aetion sourced healthcare data from HealthVerity Marketplace encompassing
medical claims, pharmacy claims, hospital chargemaster, and data collected
directly from laboratories. Optum Labs data included de-identified medical, and
pharmacy claims as well as laboratory results data utilized medical, and
pharmacy claims from a single, large U.S. insurer as well as data directly from
laboratories. We refer to these health systems as datasets A-F for the purposes
of anonymity. Data sources included in the analysis are generally categorized as
either payer (claims) or healthcare delivery systems. As illustrated in, data
were drawn from across the U.S. with heavy representation in California,
Illinois, Ohio, and Michigan. Characteristics of participating data sources and
representative populations are described in the.
## Study design
In this retrospective cohort study, we identified patients across different
settings (e.g., inpatient, outpatient, emergency department (ED), or long-term
care facility) who tested positive for SARS-CoV-2 ribonucleic acid (RNA) by
molecular test between March–September 2020 and who received at least one
subsequent serological test for SARS-CoV-2 immunoglobulin (Ig) G or Total
antibody (Ab) from 14–90 days after the positive RNA test. We analyzed the first
serology test in the 14–90-day follow-up period, which ended on December 31,
2020. “Date of RNA positive” served as the index (cohort entry) date and was
defined hierarchically as either the date of 1) sample collection; 2) accession;
or 3) result. Because the optimal time to observe a positive serology is at
least two weeks after the index date, we only include patients who had at least
one serology test 14–90 days after the index date.
To minimize the effect of differential missingness between datasets, we applied
the following rules: 1) included all persons with an office or telephone visit
in the +/- 14 days around the index date to enable as complete an assessment of
presenting symptoms as possible; 2) in claim systems, included only persons with
at least six months of enrollment in the year before index; 3) estimated the
proportion of patients at each site who had zero encounters in the prior year to
contextualize our capture of pre-existing conditions, and 4) excluded variables
from analysis if ≥30% of values were missing.
The Western—Copernicus Group (WCG) Institutional Review Board (IRB), the IRB of
record for the Reagan-Udall Foundation for the FDA, reviewed the study and
determined it to be non-human subjects research. Additionally, all legal and
ethical approvals for use of the data included in this study were submitted,
reviewed, and/or obtained locally at each contributing dataset by an IRB and/or
governing board.
## Measures
### Outcomes
The primary outcome of interest for the validation analysis was the PPA of
positive antibody (IgG or total) from serology tests with positive RNA from
molecular tests (e.g., PCR), which served as the reference standard. Serology
tests reported in this analysis included: Abbott Architect IgG, Euroimmun IgG,
Diazyme DZ-Lite SARS-CoV-2 IgG CLIA kit, Beckman SARS-CoV-2 IgG, Ortho Vitros
IgG, Diasorin Liaison SARS-CoV-2 S1/S2 IgG, and Roche Elecsys Total Ab. The
Ortho Vitros was the only test used across multiple (3) datasets. We refer to
these manufacturers—serological tests as Δ, Θ, Π, Λ, Ξ, Γ, and Ψ for anonymity.
Molecular tests most reported in this analysis included: Hologic Panther Fusion,
Hologic Aptima, Roche Cobas, Quest rRT-PCR, and Thermo Fisher Scientific Combo
Kit. We refer to these manufacturers—molecular tests as Σ, Φ, Ω, X, Y, and j for
anonymity.
### Covariates
We collected demographic, behavioral, and environmental characteristics,
baseline clinical presentation, key comorbidities, and test characteristics,
including manufacturer, according to a diagram illustrating potential factors
associated with serology testing. We identified comorbidities and clinical
presentation using phenotypes defined by the International Classification of
Diseases 10 (ICD-10), and/or National Drug Codes. We identified comorbidities
(pre-existing conditions) in the 365 days before the index date through 15 days
before the index date. We provided coding algorithms for groups to use, while
some groups used existing algorithms generated by their site. The ICD-10 codes
used to identify comorbidities are listed in the. We also stratified analyses by
RNA tests conducted before June 15, 2020, which marked the beginning of the
summer wave of infections in the first year of the pandemic, compared to on or
after that date.
## Statistical analysis
Each contributing dataset ran its analysis according to a common protocol.
Results were reviewed as a group to ensure alignment with the protocol and to
review any protocol deviations. We calculated PPA as: *(Number of positive
antibody results ÷ Number of positive RNA results) x 100*. We calculated PPA
based on the first eligible serology test in the follow-up period overall and by
age, sex, race, ethnicity, U.S. region, pregnancy status, pre-existing
conditions, including but not limited to cardiovascular disease, obesity,
hypertension, kidney disease, asthma, dementia, chronic liver disease, and
smoking status. We also report the PPA by presenting symptoms, and serology
tests at the time of the first serology test. We examined variations in PPA by
serology tests and time, and serology tests and symptom presentation. We also
examined variations in PPA by geography and care setting over time. We
calculated exact (Clopper-Pearson) 95% confidence intervals (CI). We report
significant differences where 95% CI have complete separation—although we did
not conduct formal statistical comparisons of PPA between groups.
To study the odds of seropositivity, we estimated a model for the association to
identify independent risk factors for seropositivity, assuming a binomial
distribution for seropositivity status. Results are presented as the odds ratio
(OR) and 95% CI that was calculated using score confidence intervals or exact
CI. All variables were treated as categorical. Symptoms were reported as a
binary variable: “1” if any of the following symptoms were present: fever
\>100.4, abnormal chest imaging finding, high respiratory rate, low blood
pressure, diarrhea, hypoglycemic, chest pain, delirium/confusion, headache, sore
throat, cough, shortness of breath, pneumonia, acute respiratory infection,
acute respiratory distress, cardiovascular presentation, renal presentation; and
“0” otherwise. For datasets with data covering \>1 geographic catchment area,
geography was included as either one of four U.S. Census regions, or nine U.S.
Census divisions based on patient home zip code. Variables with \>30%
missing/unknown values were excluded from models (except for pregnancy, pre-
existing condition, or presenting symptoms, all of which were included). Each
dataset used automated backward selection to remove non-significant pre-existing
conditions while forcing all other covariates into the model. All analyses were
performed using SAS software, version 9.2 or higher (SAS Institute, North
Carolina, U.S.); or the Aetion Evidence Platform v4.13 (including R v3.4.2),
which includes audit trails of all transformations of raw data and a quality
check of the data ingestion process.
# Results
Samples sizes across datasets ranged from 660–7,115; a total of 15,615 people
with at least one serology test 14–90 days after the index date were included in
the analyses. Between 35–65% of patients identified from health care delivery
systems had no documented encounter in the system between 365 and 15 days before
the index date. In contrast, only 11% of patients from national insurers
reported having zero claims in the baseline period. As shown in, the serotested
population was primarily 45–64 years of age (\>40%), with a history of
cardiovascular disease, including hypertension (8–70%). Race and ethnicity data
were robust (\<30% missing) in four datasets. The serotested population in those
datasets was primarily White (\>53%) and non-Hispanic (\>65%), From datasets
with national representation, persons from the Northeast (New England and Mid-
Atlantic) were most represented in this serotested population. In datasets that
represent regionally-based healthcare delivery systems, their population
reflected their locations: Pacific and Midwest. Information on manufacturer test
names was provided in four datasets. Generally, 2–3 primary tests were utilized
in each dataset; 4 of 7 tests reported were used in \>1 dataset. We did not
observe any difference by age or sex in those for whom the test name was known
versus unknown. In a single dataset with \<30% of missing data on
race/ethnicity, we observe over-representation of White and Hispanic people in
those for whom the test name was known.
## Positive percent agreement (PPA) of serology among molecularly confirmed SARS-CoV-2
The overall PPA ranged from 65–90% across analytic datasets. The real-world PPA
met the EUA requirement of ≥87% in three datasets (A, B, D). Two of these
datasets represented national administrative claims and associated results with
the date the sample was collected or received by the laboratory; the third
represented data from EHRs and associated results with the date the test was
conducted, which is lagged further from the clinical interaction than the
former. Overall PPA was likely influenced by the mix of serology tests
represented in each dataset. Seven serological tests were reported in this
analysis, of which two (Δ and Γ) met the EUA PPA requirements. Two tests were
used across multiple datasets and performed similarly above the EUA requirement.
PPA by serology test type varied across datasets; with three of five reporting
significantly lower PPA from total antibody (PPA range: 69–90%) compared to IgG
(PPA range: 87–92%); and two showing no difference. We observed no difference in
PPA with antibody tests that target spike compared to nucleocapsid proteins.
PPA was significantly higher in Black (PPA range: 86–92%), as compared to White
(PPA range: 78–86%), persons in at least two of the four datasets reporting
robust race/ethnicity data. PPA was significantly higher in Hispanic (PPA range:
79–96%), compared to non-Hispanic (PPA range: 60–86%), patients. PPA appeared
highest in those with diabetes (PPA range: 75–94%) and kidney disease (PPA
range: 75–95%), and lowest in those with conditions that leave them
immunocompromised (PPA range: 56–93%). We observed higher PPA in the inpatient
(PPA range: 70–97%) or ED (PPA range: 93–99%) setting compared to outpatient
(PPA range: 63–92%). There was some evidence of higher PPA among patients with
at least one COVID-19 related symptoms as compared to those with none (PPA
range: 63–91%) among two datasets (B and D); and was particularly high for
select conditions like pneumonia (PPA range: 82–97%).
However, differences in the PPA by the presence of symptoms do not appear to be
explained by the test. A stratified analysis by test comparing those with and
without symptoms showed no significant difference in PPA. PPA trends by calendar
time were not consistent across datasets.
## Factors associated with seropositivity
In adjusted models (Figs –), the OR for seropositivity was significantly
elevated in Hispanic compared to non-Hispanic ethnicity (OR range: 2.59–3.86);
among those with pre-existing diabetes (OR range: 1.49–1.56) and obesity
(1.63–2.23) as compared to those without pre-existing conditions; and among
those observed in the ED compared to outpatient (OR range: 2.49–10.97). The OR
for seropositivity was significantly lower in those with pre-existing
immunocompromised or autoimmune conditions compared to those without such
conditions (OR range: 0.25–0.70). In two of three datasets that included pre-
existing cardiovascular disease in the OR model, the OR for seropositivity was
significantly lower in persons with, compared to those without, such conditions
(OR range: 0.49–0.57). The OR for seropositivity tended to be lower on or after
June 15 compared to prior in half the datasets, but differences were not
significant in the other half.
# Discussion
Serology tests are an important instrument in the toolkit to understand the
epidemiology of COVID-19 because of their ability to identify persons with prior
infection who may present too late in the infectious period due to mild
symptoms, or no symptoms at all. Serology results may inform diagnoses of post-
acute SARS-CoV-2 (PASC) and the appropriate treatment course, which may depend
on whether patients are at increased risk for severe illness due to insufficient
antibody response. The reported sensitivity of the serology tests included in
this analysis that were submitted for EUA approval were all \>95%. Our analysis
of multiple large datasets of patients with confirmed SARS-CoV-2 infection
suggests that serology tests performed lower than = expected–with PPA ranges (a
measure analogous to sensitivity) from 65–90%.—Our results align with results
from smaller, detailed laboratory evaluations that suggest a lack of
harmonization, including optimization of cut-off values, may contribute to
decreased overall performance. Additionally, our results align with studies that
include more representative samples of milder or asymptomatic persons. Two of
seven tests reported across datasets achieved the EUA requirement of PPA ≥ 87%.
As we did not have data on specific serology-molecular pairs or meta-information
on the tests (including fidelity to protocols for serology and molecular test
analysis), these results reflect more on the real-world implementation of the
tests rather than the true quality of the tests. Specifically, where the same
test was used across multiple datasets, they all performed similarly. For
example, the serology test Γ performed similarly high (PPA \>90%) across three
datasets. However, the overall PPA for tests performed in datasets A and B were
higher than in dataset E. A major factor that may have contributed to this
difference is that the other serological tests reported to datasets A and B
performed above the EUA requirement. In contrast, the other tests reported in
dataset E performed below the EUA requirement. Additionally, datasets A and B
leveraged administrative claims data and associated RNA and serology results
with sample collection or sample receipt date, while dataset E associated
results with the date the test was run.
Dataset E also represents those from a healthcare delivery system where serology
tests were initially only used for symptomatic patients with at least 12 days of
symptoms. This practice shifted after approximately two months (June 1, 2020) to
a protocol that required both molecular and serological testing for SARS-CoV-2
as part of pre-procedure screening. This protocol was in effect for another
three months (August 31, 2020), after which the healthcare system shifted to
unrestricted testing for both molecular and serology tests and saw a substantial
drop in the use of serological testing. We expected that procedural “lags” to
serotesting, combined with additional lags due to associating results with a
date downstream from the clinical interaction, may have further extended the
time between infection/symptom onset and the actual time of serology sampling.
The impact of this misclassification may be most important for serology samples
at the upper bounds of 90 days; where samples were likely \>90 days from the
point of infection and humoral antibodies more likely to have declined. Despite
changes in the protocol over time, we observed no overall or test-specific
difference in PPA before or since June 15, 2020 in dataset E. Nevertheless,
administrative protocols create lags in serotesting that challenge our
assumptions of whether the observed molecular “test date” is a good proxy for
symptom onset. Absent any knowledge of such policy, it’s difficult to make broad
assumptions regarding patterns in molecular or serology testing unless
established clinical protocols are known.
We observed that patients of Hispanic ethnicity compared to non-Hispanic
patients, with pre-existing obesity and those who presented in the ED had a
higher OR for seropositivity; and similarly higher PPA. These results further
support what others have observed that persons with unmanaged diabetes, who are
disproportionately people of color, are vulnerable to hyper-inflammation related
to COVID-19. Furthermore, hyper-inflammation, including pro-inflammatory
cytokine storm, has been associated with severe disease, reduced viral
clearance, and sustained antibody production. Although a recent small study
showed that while a low viral load is associated with lower antibody response,
clinical illness does not guarantee seroconversion. Other studies have
demonstrated people with cancer have a lower probability of mounting an immune
response from the vaccine, as demonstrated by seroconversion, viral
neutralization, and T-cell response. Our results demonstrating lower odds of
seropositivity among those with cancer and other immunodeficiencies suggest that
the same may be true regarding their antibody response to infection.
## Strengths
Our study has many strengths. This was a large assessment of serotesting across
the U.S. in diverse datasets leveraging either EHR or claims data. We developed
a protocol that incorporated the unique characteristics of each data source and
provided a forum to transparently communicate and collaborate on study design
and interpretation. We also established a platform to rapidly collect and
analyze data from various systems to evaluate process improvement and identify
important trends over time. Such a platform may be used to evaluate process
improvement and comparisons within data systems. We did extensive
characterization of missing data to guide model development and help with
interpretation. Additionally, this study was conducted before public
availability of COVID-19 vaccines across the U.S., which minimizes the potential
for confounding related to vaccine-induced antibodies.
## Limitations
A major limitation in this real-world analysis is a large number of missing test
names and relevant meta-data, including quality control measures adopted, for
both molecular and serological tests. As such, we were unable to account for
molecular-serology pairs when assessing PPA or the fidelity with which these
tests were performed. A large amount of missing test name information limited
our ability to describe trends by the manufacturer. Although, a thorough
examination of missing data does not suggest differential missingness by age or
sex. Importantly, the intent of this analysis was not to evaluate individual
tests, but the performance of serology in the context of real-world
implementation of test protocols and varying reference standards. As discussed
in our prior manuscript, the sample included in this study included those who
were more likely to be serotested for SARS-CoV-2: White, 45–64 years of age,
with prior history of cardiovascular disease. Nevertheless, there was still
sufficiently large number of people to assess PPA trends among younger ages and
in those with and without other pre-existing conditions. Finally, this study was
conducted before the surge of the Omicron variant, which has been shown to have
a number of mutations on the N-gene and S-gene that reduce the sensitivity of
some diagnostic tests. As such, our inference is limited to the SARS-CoV-2
variants prior to Omicron, primarily alpha.
# Conclusion
Across large samples of patients with molecularly confirmed SARS-CoV-2, serology
tests did not consistently meet the EUA requirement of PPA ≥ 87% in the post-
market setting. However, given the limited availability of test names, this
analysis serves as a signal that further investigation into how serology and
molecular tests are used, including protocol fidelity, is needed to understand
ways to improve the real-world performance of serology tests.
Despite differences in testing protocols and data availability, the similarity
in performance of serology tests across datasets suggests that serology tests
were robust to differences in care settings. However, the real-world PPA for
several serology tests did not meet EUA requirements; and the exclusive
representation and low use of such tests in certain datasets look to have
impacted the overall performance of serology tests in those datasets. Where data
were sufficiently robust, we observed that people of Hispanic ethnicity had a
higher odd of seropositivity than non-Hispanics. Higher odds of seropositivity
in those with pre-existing diabetes or obesity further support the hypothesis
that these conditions are associated with more severe disease, reduced viral
clearance, and the sustained presence of antibodies. Conversely, lower odds of
seropositivity among those with cancer and other immunodeficiencies suggest that
immunopathology in these groups associated with the vaccine may extend to
infection.
Interpreting results from real-world data collected from clinical and
administrative databases is challenging. A clear understanding of testing
protocols at the point of care is needed to validate assumptions regarding proxy
variables and to interpret results. Incomplete information on race/ethnicity and
test name limited our ability to address racial disparities in testing and real-
world performance of serological tests. Nevertheless, implementing best
practices for analyzing and reporting results from observational data across
multiple datasets yields confidence in trends that are repeated. And where
results are divergent, we were able to explore how differences in data sources
may explain findings and target areas for future investigation. Improved data
interoperability to link test names and clinical/demographic data is critical to
enable rapid assessment of the real-world performance of in vitro diagnostic
tests, particularly in the face of fast-mutating pathogens.
# Supporting information
Special thanks to our advisors on this project from the U.S. Food and Drug
Administration: Aloka Chakravarty, Tamar Lasky, Gina Valo, Mary Jung, Stephen
Lovell, Jacqueline M Major, Daniel Caños, Sara Brenner, and Wendy Rubinstein;
and Duke-Margolis: Christina Silcox. We thank all members of the Evidence
Accelerator Workgroup for their support and feedback: Roland Romero, James
Okusa, Elijah Mari Quinicot, Amar Bhat, Susan Winckler, Alecia Clary, Sadiqa
Mahmood, Philip Ballentine, Perry L. Mar, Cynthia Lim Louis, Connor McAndrews,
Elitza S. Theel, Cora Han, Pagan Morris, and Charles Wilson. A special thanks
and recognition for the contributions and sacrifice of Dr. Michael Waters, our
dear colleague, and friend who will be forever in our thoughts. We thank Amir
Alishahi Tabriz MD, PhD for his assistance with manuscript preparation.
10.1371/journal.pone.0279956.r001
Decision Letter 0
Banada
Padmapriya P
Academic Editor
2023
Padmapriya P Banada
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
15 Jul 2022
PONE-D-22-11773Real-World Performance of SARS-Cov-2 Serology Tests in The United
States, 2020.PLOS ONE
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Reviewer \#1: The Rodriguez-Watson et al. manuscript describes a synthesis of
real-world serology testing for SARS-CoV-2. It analyses agreements between SARS-
CoV-2 PCR testing and antibody detection assays in 6 US health systems across
different setting (inpatient, outpatient, ED, long-term care). The aims of the
manuscript were to address gaps in understanding exposure to SARS-CoV-2 and
identifying factors associated with seroconversion.
The manuscript is well written and understandable even though presents a large
amount of data.
Although the odd of seropositivity according to demographics gives a good
understanding of factors that might affect seroconversion, the authors failed to
address the gaps in understanding exposure.
Comments:
1- The manuscript describes agreement (PPA) between PCR testing and serology
results at 14-90 days post PCR. The approach to analyzing agreement is not
complete with no mention of kappa (Cohen, McNemar test, etc.).
2- Is there a loss of agreement depending on day serology was done?
3- When comparing PPA observed according to ethnicity or other factors, are the
differences significant?
4- Line 284, the authors mention test quality. Many studies looking at the
quality of several serology tests used in this manuscript have been published,
can the authors discuss further and refer to these manuscripts? Does lower
agreement observe in this study match the published data?
4- In the section results, no odd ratio is given, only 95%CI. Please add.
5- Factors associated with seropositivity, line 257-266. Are the differences
described significant?
6- Line 135 change e.g. by i.e. or remove altogether
7-Line 135, remove comma after IgG, “IgG, \[21\],”
8- Table 1, “Na” in lowercase, all other table “NA” is capitalised. Please
homogenise throughout.
9- Line 313, should read sustained antibody production.
10- Line 359, should read the sustained presence of antibodies.
11- Line 371, should be “in vitro”.
Reviewer \#2: Summary:
In this important study, Dr. Rodriguez-Watson and colleagues studied 6 large-
scale datasets to understand the 2020 performance of real-world use of EUA-
approved SARS-CoV-2 serology testing after a positive molecular test. The group
demonstrates substantial real-world variance in the PPA of these tests across
health contexts.
Major comments
1\. What was done statistically regarding individuals with a 2nd serology test?
Line 161 implies only the first test was used. What was the concordance/timing
between 1st and 2nd tests? Did 2nd tests, where done, have a different PPA?
2\. Dataset C seems to have a broadly lower PPA vs the other datasets, has the
smallest N, and is relatively geographically restricted. This dataset does not
appear to have any manufacturer molecular test names available, but there is no
PPA reported in the Unknown/missing category in table 2 for that variable. Is
this dataset usable? It would seem that as both the serological and molecular
test characteristics would contribute to the PPA, not knowing the molecular test
name at all makes using this dataset problematic.
3\. What is known about the contribution of “other” molecular tests to this
dataset, such as the adoption of “rapid” PCR testing and “in-house” testing that
some institutions produced during this time period? Is it possible to address
those tests where both the serological and molecular tests are known? As above,
the confounding factor of molecular test characteristics could influence the PPA
of the serological tests in question.
4\. Time between the molecular and serological test seems like a key point as
well. Do you have that data? Does time between tests affect the results?
Minor comments
1\. The figures are not showing well (scattered pixels) in my Adobe Acrobat Pro
DC view of the PDF. Please ensure that high quality images are used during
publication. I can access the tif files, which look right.
2\. I do not understand why figures 2-6 list the study period as starting in
2019. Is this a typo or is this correct? The methods list 2020 as start date,
which would make sense given the dates of the pandemic.
3\. Please check line 380, there may be a comma instead of a period.
4\. Perhaps figures 2-7 should be combined into a single summary figure (with
total N for the entire study) and the individual flowsheets by cohort might be
moved to supplemental. The reader might better grasp the overall study with a
simpler summary figure.
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Reviewer \#1: No
Reviewer \#2: No
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10.1371/journal.pone.0279956.r002
Author response to Decision Letter 0
3 Nov 2022
Dear Dr. Padmapriya Banada:
We thank you and the reviewers for your thoughtful comments on our manuscript.
We appreciate the opportunity to respond and believe the revisions have improved
the manuscript. Below, please find a table with the summary of reviewer comments
and our responses. Please be advised that all line numbers referenced in the
responses below correspond to line numbers in the tracked changes version of the
manuscript. Please reach out with any additional questions, or if more
clarification is required. We look forward to hearing from you.
Regards,
Carla Rodriguez-Watson, PhD, MPH
Director of Research
Reagan-Udall Foundation for the FDA
Comments/Questions Response
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Financial support for this work was provided in part by a grant from The
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from the FDA Foundation.
AJB funded by award number A128219 and Grant Number U01FD005978 from the FDA,
which supports the UCSF-Stanford Center of Excellence in Regulatory Sciences and
Innovation. Its contents are solely the responsibility of the authors and do not
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The funders had no role in study design, data collection and analysis, decision
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consultant to Samsung, Mango Tree Corporation, and in the recent past, 10x
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Illumina, Nuna Health, Assay Depot (Scientist.com), Vet24seven, Regeneron,
Sanofi, Royalty Pharma, Pfizer, BioNTech, AstraZeneca, Moderna, Biogen, Twist
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and several other non-health related companies and mutual funds; and has
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licensed to NuMedii and Personalis. AJB’s research has been funded by NIH,
Northrup Grumman (as the prime on an NIH contract), Genentech, Johnson and
Johnson, FDA, Robert Wood Johnson Foundation, Leon Lowenstein Foundation,
Intervalien Foundation, Priscilla Chan and Mark Zuckerberg, the Barbara and
Gerson Bakar Foundation, and in the recent past, the March of Dimes, Juvenile
Diabetes Research Foundation, California Governor’s Office of Planning and
Research, California Institute for Regenerative Medicine, L’Oreal, and
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serves as a scientific advisor to IDbyDNA (San Francisco, CA and Salt Lake City,
UT); and is on the Board of the Commonwealth Fund.
CK is a paid employee of Aetion and hold Aetion stock options.
NES is an employee of Optum Labs and owns stock in the parent company
UnitedHealth group.
NDL was an employee of Health Catalyst at the time the work was performed.
JLG is a full-time employee of Regenstrief Institute, which provides independent
research services to entities including those within the pharmaceutical and
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SJG serves as Chief Medical Information Officer for the Indiana Health
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Yale University, Mayo Clinic, and the U.S. Food and Drug Administration (FDA)
(3U01FD005938).
CK, CMF, SJG, PJE, EHE, NDL, and JLG work was funded by a designated sub-grant
from the FDA Foundation.
AJB funded by award number A128219 and Grant Number U01FD005978 from the FDA,
which supports the UCSF-Stanford Center of Excellence in Regulatory Sciences and
Innovation. Its contents are solely the responsibility of the authors and do not
necessarily represent the official views of the HHS or FDA.
The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.”
Thank you for bringing this to our attention. We have removed the funding
information from the Acknowledgement section and include all funding information
as updated above under “Funding Information.”
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7\. One of the noted authors is a group or consortium “Evidence Accelerator
Workgroup”. In addition to naming the author group, please list the individual
authors and affiliations within this group in the acknowledgments section of
your manuscript. Please also indicate clearly a lead author for this group along
with a contact email address.
Thank you for bringing this to our attention. The Evidence Accelerator Workgroup
refers to our consortium. We only had included those who met the ICMJE
authorship as co-authors, but wanted to acknowledge all those who worked behind
the scenes to support the work. Given this advice, we will include their names
as acknowledgements. We have added the following phrase “We thank all members of
the Evidence Accelerator Workgroup for their support and feedback: \[list
names\]” to lines 395-399.
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Thank you for bringing this to our attention. We had previously attached the
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reprint information to the figure 1’s caption.
Reviewer 1 comments:
1\. The manuscript describes agreement (PPA) between PCR testing and serology
results at 14-90 days post PCR. The approach to analyzing agreement is not
complete with no mention of kappa (Cohen, McNemar test, etc.).
Thank you for the comment. This analysis focused on serology agreement with
positive RNA tests. As such, we did not collect any RNA negative results, which
are required to assess kappa.
2\. Is there a loss of agreement depending on day serology was done?
Yes, there was loss of agreement depending on the day serology test was done,
and one of the results we intend to present in a subsequent publication. The
current manuscript is intended to discuss the overall agreement between serology
and PCR during the window for which agreement is most expected (days 14-90 after
PCR) and by key demographic and clinical factors. A subsequent publication will
look at how that agreement changes over different time periods.
3\. When comparing PPA observed according to ethnicity or other factors, are the
differences significant?
We report differences in PPA by race and ethnicity only where there are
sufficient data (i.e. missing \<30% and sample size ≥ n=40). We consistently
observed higher PPA in Hispanic ethnicity compared to non-Hispanic ethnicity, as
demonstrated by complete separation of confidence intervals (Clopper-Pearson).
We did not conduct direct comparison of PPA across groups. We clarified the
statistical analysis section (lines 171-173) to describe the meaning of
'significant differences’ outside of direct comparisons: “We calculated exact
(Clopper-Pearson) 95% confidence intervals (CI). We report significant
differences where 95% CI have complete separation - although we did not conduct
formal statistical comparisons of PPA between groups.”
4\. Line 284, the authors mention test quality. Many studies looking at the
quality of several serology tests used in this manuscript have been published,
can the authors discuss further and refer to these manuscripts? Does lower
agreement observe in this study match the published data?
Thank you for the great suggestion. We have now included in the Discussion a
comparison of our PPA results to those from sensitivities reported in original
EUA submissions; as well as in context of other external evaluations (see lines
290-298):
“The reported sensitivity of the serology tests included in this analysis that
were submitted for EUA approval were all \>95% \[36\]. Our analysis of multiple
large datasets of patients with confirmed SARS-CoV-2 infection suggests that
serology tests performed lower than =expected – with PPA ranges (a measure
analogous to sensitivity) from 65-90%. - Our results align with results from
smaller, detailed laboratory evaluations that suggest a lack of harmonization,
including optimization of cut-off values, may contribute to decreased overall
performance. Additionally, our results align with studies that include more
representative samples of milder or asymptomatic persons \[37–39\].” Although
many of these evaluations are still limited by much smaller sample sizes than we
report (though they are more detailed laboratory studies) and appear limited in
the replicability of the same assay result across different laboratories, they
do contain more diverse populations and note lower performance compared to
initial certification related evaluations.
5\. In the section results, no odd ratio is given, only 95%CI. Please add.
Apologies for the confusion, the results given in the parenthesis in this
section are actually ranges of OR, not the 95% CIs. We have clarified this in
reporting results in this version.
6\. Factors associated with seropositivity, line 257-266. Are the differences
described significant?
Thank you for the comment. The differences described in the text were
significant. Significant differences can be observed in the figures as those
whose 95% CI does not cross the ‘1’on the X axis. Across the board, we found age
20-44 yrs to have a lower odds of seropositivity than those 45-54 yrs; Hispanic
Ethnicity to have higher odds than non-Hispanic; immunocompromised to have a
lower odds of seropositivity than those with no pre-existing conditions. ORs for
obesity and presenting with \>1 COVID symptom also were also significantly
elevated in \>1 data source. We have clarified this in the text by adding the
word “significantly” to lines 269 and 273.
7\. Line 135 change e.g. by i.e. or remove altogether
Thank you for calling this out. To clarify, PCR was not the only molecular
conducted. The list included NAAT, RT-PCR so we respectfully leave it as “e.g”
8\. Line 135, remove comma after IgG, “IgG, \[21\],”
Thank you for bring this to our attention. We have removed the comma after IgG.
Please see line 141.
9\. Table 1, “Na” in lowercase, all other table “NA” is capitalised. Please
homogenise throughout.
Thank you for bring this to our attention.
We have updated Table 1 to read NA instead of Na to match the other table.
10\. Line 313, should read sustained antibody production.
Thank you for bring this to our attention. We have updated the language in line
331.
11\. Line 359, should read the sustained presence of antibodies.
Thank you for bring this to our attention. We have updated the language in line
377-378.
12\. Line 371, should be “in vitro”.
Thank you for bring this to our attention. We have updated the language in line
389.
Reviewer \#2 comments:
1\. What was done statistically regarding individuals with a 2nd serology test?
Line 161 implies only the first test was used. What was the concordance/timing
between 1st and 2nd tests? Did 2nd tests, where done, have a different PPA?
The majority of person in our cohort had just one serology test done. In order
to compare consistently, we picked the first test done on an individual
occurring 14 or more days after their positive molecular test. This choice has
the added benefit of avoiding bias that would occur if we counted the same
individual more than once knowing that individuals are more likely to retest if
they get a result which is unexpected.
2\. Dataset C seems to have a broadly lower PPA vs the other datasets, has the
smallest N, and is relatively geographically restricted. This dataset does not
appear to have any manufacturer molecular test names available, but there is no
PPA reported in the Unknown/missing category in table 2 for that variable. Is
this dataset usable? It would seem that as both the serological and molecular
test characteristics would contribute to the PPA, not knowing the molecular test
name at all makes using this dataset problematic.
Keen observations! Not all partners reported the name of the molecular test and
thus, did not estimate PPA by molecular test (reference). All partners did
estimate PPA by serology test. We acknowledge in the limitations that we did not
analyze molecular-serology pairs. As you note, characteristics of each test may
influence PPA results, though we account for many other factors that may affect
results. Because of this limitation, we chose not to report results by specific
test name as it may suggest a deficiency that we could not accurately explain.
3\. What is known about the contribution of “other” molecular tests to this
dataset, such as the adoption of “rapid” PCR testing and “in-house” testing that
some institutions produced during this time period? Is it possible to address
those tests where both the serological and molecular tests are known? As above,
the confounding factor of molecular test characteristics could influence the PPA
of the serological tests in question.
Rapid tests were not included in this analysis. One site included an ‘in-house’
test that was not FDA approved or for whom an EUA was not issued; the majority
used only FDA approved or EUA tests. As such, we did not conduct the suggested
analysis.
4\. Time between the molecular and serological test seems like a key point as
well. Do you have that data? Does time between tests affect the results?
Yes, we agree with your comment. Analysis of PPA since the time of the molecular
test is the focus of a subsequent manuscript. In the current analysis, we focus
on tests 14-90 days from positive molecular test to maximize sensitivity of the
test.
5\. The figures are not showing well (scattered pixels) in my Adobe Acrobat Pro
DC view of the PDF. Please ensure that high quality images are used during
publication. I can access the tif files, which look right.
Thank you for letting us know. We will resubmit the figures as.tiff files.
6\. I do not understand why figures 2-6 list the study period as starting in
2019. Is this a typo or is this correct? The methods list 2020 as start date,
which would make sense given the dates of the pandemic.
Thank you for the question. This is correct. March 2019 represents the
collection of baseline data such as comorbidities and socioeconomic data. The
study follow-up period begins in March 1, 2020 and continues until September 30,
2020.
7\. Please check line 380, there may be a comma instead of a period.
Thank you for bring this to our attention. We have removed the comma and changed
it to a period. Please see line 399
8\. Perhaps figures 2-7 should be combined into a single summary figure (with
total N for the entire study) and the individual flowsheets by cohort might be
moved to supplemental. The reader might better grasp the overall study with a
simpler summary figure.
Thank you for the suggestion. We created a summary figure (Fig 2) that depicts
the general study design and the sample size of each partners study cohort; thus
renumbering the remaining figures and moving the individual study diagrams to
the supplemental figures. Each of the final cohorts (A-F) include patients that
have both molecular test and follow up serology test as indicated in the
methods. The parallel analysis approach entails that each cohort was analyzed
separately according to a common protocol. Since this was not an aggregated
analysis, we did not aggregate the numbers across partners.
10.1371/journal.pone.0279956.r003
Decision Letter 1
Banada
Padmapriya P
Academic Editor
2023
Padmapriya P Banada
This is an open access article distributed under the terms of the
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, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
19 Dec 2022
Real-world performance of SARS-Cov-2 serology tests in the United States, 2020.
PONE-D-22-11773R1
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Two very minor typos if this can be changed before publication
line 111 : Figs 2 - remove s
line 161 add space between CI and reference \[33\]
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10.1371/journal.pone.0279956.r004
Acceptance letter
Banada
Padmapriya P
Academic Editor
2023
Padmapriya P Banada
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
24 Jan 2023
PONE-D-22-11773R1
Real-world performance of SARS-Cov-2 serology tests in the United States, 2020.
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[^1]: AJB is a co-founder and consultant to Personalis and NuMedii;
consultant to Samsung, Mango Tree Corporation, and in the recent past, 10x
Genomics, Helix, Pathway Genomics, and Verinata (Illumina); has served on
paid advisory panels or boards for Geisinger Health, Regenstrief Institute,
Gerson Lehman Group, AlphaSights, Covance, Novartis, Genentech, Merck, and
Roche; is a shareholder in Personalis and NuMedii; is a minor shareholder in
Apple, Facebook, Alphabet (Google), Microsoft, Amazon, Snap, Snowflake, 10x
Genomics, Illumina, Nuna Health, Assay Depot (Scientist.com), Vet24seven,
Regeneron, Sanofi, Royalty Pharma, Pfizer, BioNTech, AstraZeneca, Moderna,
Biogen, Twist Bioscience, Pacific Biosciences, Editas Medicine, Invitae,
Doximity, and Sutro, and several other non-health related companies and
mutual funds; and has received honoraria and travel reimbursement for
invited talks from Johnson and Johnson, Roche, Genentech, Pfizer, Merck,
Lilly, Takeda, Varian, Mars, Siemens, Optum, Abbott, Celgene, AstraZeneca,
AbbVie, Westat, several investment and venture capital firms, and many
academic institutions, medical or disease specific foundations and
associations, and health systems. AJB receives royalty payments through
Stanford University, for several patents and other disclosures licensed to
NuMedii and Personalis. AJB’s research has been funded by NIH, Northrup
Grumman (as the prime on an NIH contract), Genentech, Johnson and Johnson,
FDA, Robert Wood Johnson Foundation, Leon Lowenstein Foundation, Intervalien
Foundation, Priscilla Chan and Mark Zuckerberg, the Barbara and Gerson Bakar
Foundation, and in the recent past, the March of Dimes, Juvenile Diabetes
Research Foundation, California Governor’s Office of Planning and Research,
California Institute for Regenerative Medicine, L’Oreal, and Progenity. CLB
has intellectual property in and receives royalties from BioFire, Inc. She
serves as a scientific advisor to IDbyDNA (San Francisco, CA and Salt Lake
City, UT); and is on the Board of the Commonwealth Fund. CK is a paid
employee of Aetion and hold Aetion stock options. NES is an employee of
Optum Labs and owns stock in the parent company UnitedHealth group. NDL
was an employee of Health Catalyst at the time the work was performed. JLG
is a full-time employee of Regenstrief Institute, which provides independent
research services to entities including those within the pharmaceutical and
medical device industries. SJG serves as Chief Medical Information Officer
for the Indiana Health Information Exchange, and is a founding partner of
Uppstroms, LLC. This does not alter our adherence to PLOS ONE policies on
sharing data and materials. |
# Introduction
*Dichelobacter nodosus* is a gram-negative fastidious anaerobic bacterium and
the causative agent of ovine footrot. The disease has a global presence and is
endemic in many countries. In Switzerland, the true prevalence of virulent *D*.
*nodosus* in sheep on animal level is estimated at 16.9% and on farm level at
16.2%. Clinical symptoms range from mild interdigital dermatitis in benign
footrot to severe underrunning and separation of the hoof horn from the
underlying tissue in virulent footrot. Clinical symptoms start as early as 2
weeks after first contact and the disease leads to pain, lameness, decreased
meat and wool production as well as animal welfare issues. The economic and
intangible costs of the disease are considerable. In Switzerland, costs for
management and growth reduction without control measures were estimated at CHF
172.3 million for 2014–2030. To address these problems, various countries
started to develop and implement footrot control or elimination programs.
In Switzerland, a mandatory footrot control program started first in the canton
of Grisons in 1994 whereas in other cantons sheep owners could voluntarily join
the control program offered by the Swiss Consulting and Health Service for Small
Ruminants. The successful control in Grisons, progress in laboratory diagnostics
allowing PCR-detection and discrimination of virulent and benign *D*. *nodosus*,
and the ongoing unsatisfactory situation in other cantons led to the political
decision for a nationwide footrot control program. A cost-benefit analysis
confirmed positive epidemiological and economic effects of this approach. The
Federal Food Safety and Veterinary Office is currently preparing the nationwide
footrot control program, which is planned to start in 2022. The goal is to
reduce flock prevalence of virulent *D*. *nodosus* to less than one percent
within five years.
The control program consists of three phases: i) swab sampling for detection of
virulent (*aprV2*-positive) *D*. *nodosus* by PCR, ii) treatment of
*aprV2*-positive herds by claw-trimming and weekly footbaths in disinfectant
solution, iii) surveillance of treated herds.
Most frequently used disinfectants in footbaths in Switzerland are 4%
formaldehyde, 10–20% zinc sulfate and 5–10% copper sulfate. In a recent proof-
of-concept study, weekly footbaths in 10% zinc sulfate was shown to eliminate
*aprV2*-positive *D*. *nodosus* from feet of sheep within 6–19 weeks. Despite
their effectiveness, these substances have undesirable characteristics for use
in a nationwide control program. Formaldehyde smells pungent and irritates
airways. It is a known cause of allergic contact dermatitis and a carcinogen in
both humans and animals. Repeated use of formaldehyde footbaths in sheep caused
keratinization of the interdigital skin, which can lead to secondary infection
and lameness. Zinc and copper are both essential trace elements, acting as
catalytic or structural components of larger molecules and are therefore
indispensable for live. However, similar to more toxic heavy metals they are a
major contaminant of soil and groundwater, accumulating in water, sediment,
aquatic plants and fishes, posing a potential health threat to aquatic, human
and animal life.
Hence, the aim of this study was to identify an alternative disinfectant
solution, which is highly effective against *D*. *nodosus*, non-carcinogenic,
environmentally acceptable, inexpensive, available as concentrate and suitable
for licensing as biocide for treating footrot in Switzerland. The effects of
different disinfectant solutions on virulent *D*. *nodosus* were investigated by
culture based *in vitro* testing and *ex vivo* evaluation applying a newly
established PMA (propidium monoazid) real-time PCR (PMA-qPCR) using the improved
dye PMAxx<sup>™</sup> allowing discrimination of viable and dead *D*.*nodosus*.
# Materials and methods
## *In vitro* disinfectant testing
Disinfectants were selected from literature considering their toxicity,
degradability and availability as concentrate. Three disinfectants already in
use for treatment of footrot in Switzerland and 19 substances or products
selected by expert opinion based on the results of a literature search were
evaluated *in vitro* for their antimicrobial activity against virulent *D*.
*nodosus*. Disinfectants, which resulted in a ≥ 5 log reduction of the number of
colony-forming units (CFU), were further tested with organic soiling.
Formaldehyde, copper sulfate, zinc sulfate, DESINTEC® Hoof Care Special D
(Desintec) and its main compounds acetic acid, glycolic acid and glutaraldehyde
were tested three times, all other substances once, with and without simulating
soiling. The composition of Desintec is given in.
Virulent (*aprV2*-positive) *D*. *nodosus* ATCC 25549<sup>T</sup> was cultured
on Brucella Blood Agar with Hemin and Vitamin K1 (Becton Dickinson) at 37°C in
an anaerobic chamber (80% N<sub>2</sub>, 10% CO<sub>2</sub> and 10%
H<sub>2</sub>; Scholzen Microbiology Systems AG). After 4–5 days, cultures were
transferred with a cotton swab into Difco<sup>™</sup> LB Broth (Becton
Dickinson) and suspended until McFarland 4 was reached in Densichek
(bioMérieux). To simulate high-level soiling, a solution of 10% Bovine Serum
Albumin (Sigma-Aldrich) and 10% yeast extract (Becton Dickinson) was filtered
through a 0.2 μm Acrodisc syringe filter (Pall Corporation) and complemented
with 10% defibrinated sheep blood (Thermo Fisher Scientific) resulting in a 10x
soiling-solution.
To determine the CFU/ml in the test mixture at the beginning of contact time
(*N*<sub>*0*</sub>*)*, the number of CFU/ml in the test mixture after
disinfectant treatment at the end of contact time (*N*<sub>*D*</sub>), and in
the positive control, 1 ml of LB or 1 ml soiling-solution were prepared in a 15
ml Falcon tube (Sarstedt). Then 8 ml of LB, 8 ml 1.25x disinfectant solution or
8 ml 5% formaldehyde (Sigma-Aldrich) were added, respectively. Finally, 1 ml of
*D*. *nodosus* suspension was added before mixing the tubes. After 5 min contact
time at room temperature, the tubes were centrifuged for 5 min at 4255xg, the
supernatant discarded and the pellets resuspended in 10 ml LB. This was repeated
twice, the last time with resuspension in 1 ml LB. Subsequently, tenfold
dilution series down to 10<sup>−7</sup> were prepared with 100 μl test
suspension and 900 μl LB out of which 500 μl were plated on Brucella Blood Agar.
The plates were incubated for 4–5 days as described before.
After counting, plates with CFU in the range of 14 to 330 were included for
calculation of the weighted mean count of *N*<sub>*0*</sub> and
*N*<sub>*D*</sub>. If possible, two dilutions were evaluated, otherwise only
one.
*N*<sub>*0*</sub> and *N*<sub>*D*</sub> were calculated as follows: $$N =
\frac{c}{\left( {n_{1} + 0.1n_{2}} \right)vd}$$ where
*c* is the sum of CFU taken into account;
*n*<sub>*1*</sub> is the number of plates taken into account in the lower
dilution;
*n*<sub>*2*</sub> is the number of plates taken into account in the higher
dilution;
*v* is the volume plated in ml;
*d* is the dilution factor corresponding to the lower dilution.
Example of two dilutions: $$N = \frac{244 + 33}{\left( {1 + 0.1 \cdot 1} \right)
\cdot 0.5 \cdot 10^{- 5}} = \frac{277}{1.1 \cdot 0.5 \cdot 10^{- 5}} = 5.0 \cdot
10^{7}\ (in\ CFU/ml)$$
The reduction (R) was expressed as decimal logarithm: log<sub>10</sub>R =
log<sub>10</sub> *N*<sub>*0*</sub> –log<sub>10</sub> *N*<sub>*D*</sub>. Whenever
*N*<sub>*D*</sub> was zero, the value "1" was applied. Disinfectants
demonstrating a ≥ 5 log<sub>10</sub> reduction at 5% disinfectant concentration
with and without soiling were considered useful.
## *Ex vivo* experiments
### PMAxx<sup>™</sup> treatment conditions and linear range of PMA-qPCR
A 1 ml McFarland 4 suspension of virulent *D*. *nodosus* was prepared as
described before. Half of the suspension was heated at 99°C for 10 min. With
both, the living and heat-treated *D*. *nodosus* suspension, tenfold serial
dilutions were prepared down to 10<sup>−5</sup>.
For determining CFU in the suspension, 10 μl of each untreated 10<sup>−3</sup>,
10<sup>−4</sup> and 10<sup>−5</sup> dilution were mixed with 500 μl 0.85% NaCl,
plated on Brucella Blood Agar and incubated for 4–5 days. Killing of heat-
treated *D*. *nodosus* was confirmed by plating 10 μl of the heated-treated
suspension in the same way.
For PMA-qPCR enumeration of viable and dead *D*. *nodosus* in the dilution
series, 10 μl of dilution were mixed with 40 μl of 0.85% NaCl in transparent 1.5
ml tubes (Sarstedt). In a darkened room 100 μM (0.25 μl) of PMAxx<sup>™</sup>
(Biotium) was added and the tube was vortexed and incubated for 3 min at room
temperature in a metal box impervious to light. After incubation, tubes were
placed on ice and exposed to LED light (Optonica LED floodlight Item No. FL5836,
white light, 100 W, 6000 K, 8500 lm) at a distance of 20 cm for 5 min. Following
light treatment, the tubes were centrifuged at 15'000xg for 5 min in a
microcentrifuge. To remove the supernatant, the tubes were held in a horizontal
position and twisted tissues were used to absorb the liquid. The pellets were
resuspended in 500 μl SV-lysis buffer (4 M guanidine thiocyanate, 0.01M
Tris–HCl, 1% β-mercaptoethanol).
DNA extraction was done following established protocols including the
VetMax<sup>™</sup> Xeno<sup>™</sup> IPC (Thermo Fisher Scientific) on an
automated purification system (KingFisher<sup>™</sup> Duo-Prime, Thermo Fisher
Scientific). Extracted DNA was used directly or stored at -20°C until further
qPCR analysis was undertaken.
Genomic DNA of *D*. *nodosus* ATCC 25549<sup>T</sup> was used as an external
standard in the qPCR with seven points corresponding from 10<sup>7</sup> to
10<sup>1</sup> genome equivalents per well. Dilution stages of live and dead
*D*. *nodosus* were analyzed in duplicate, the external standard in triplicate.
Assay conditions consisted of a 25 μl reaction mixture containing 1 x
TaqMan<sup>™</sup> Fast Advanced Master Mix (Thermo Fisher Scientific), 300 nM
primers DnAprTM-L and DnAprTM-R, 100 nM Probe DnAprTm-v, 250 nM Probe DnAprTM-b,
pyrogen-free water, 1 μl Xeno<sup>™</sup> LIZ Primer Probe Mix (Thermo Fisher
Scientific) and 2.5 μl of DNA template. Amplification was done in a 7500 Real-
Time PCR-System instrument (Applied Biosystems), using cycles of 2 min at 50°C
and 10 min at 95°C followed by 40 cycles with 15 s at 95°C and 1 min at 60°C.
Results were analyzed using the 7500 Software (v 2.3.) with the threshold set at
0.015. Mean cycle threshold (Ct) values of duplicates and triplicates were
calculated. The Ct values of heat-treated and live *D*. *nodosus* dilution
series were plotted versus the log quantity of standard DNA. Determination of
linear range was done three times and average values calculated.
The protocol for detection of live *D*. *nodosus* by PMA-qPCR is publicly
available at <http://dx.doi.org/10.17504/protocols.io.bbh9ij96>
### *Ex vivo* disinfectant testing
Clinically affected feet from sheep with footrot score ≥ 2 were collected at the
abattoir in Thun and transported at room temperature to the laboratory within 30
min. Gross manure was cautiously removed from claws and the interdigital space,
the open articulation of the carpal or tarsal joint was covered with gloves and
an identification number was assigned to each foot. A cotton swab was soaked in
0.85% NaCl and a first sample (prevalue) was taken from the interdigital space.
The swab was once rotated 360 degrees and subsequently soaked in 500 μl of 0.85%
NaCl in a 1.5 ml tube. The feet were then attached to the cords of an in-house
foot-dipping machine, which allowed foot-dipping of eight feet in parallel and
had a frequency of five down movements per minute and a total contact time with
the disinfectant of 50 s per minute.
The machine was turned on, and feet were dipped and moved in plastic beakers
containing 800 ml of disinfectant solution. After 10 min, the machine was
stopped and the feet were left hanging for another 60 min outside the
disinfectant solution at room temperature. The dipping machine is supposed to
imitate the situation of the sheep treated alive as described by Greber et al..
In short, sheep are standing and moving their feet in the bath for 10 min. Foot
movement is supposed to increase the contact with the disinfectant. After
footbathing sheep are contained for 60 min on a clean and dry concrete floor.
A second interdigital swab (postvalue) was afterwards collected from the same
area in the same way as described above. Prevalue and postvalue tubes were
centrifuged for 5 min at 15'000xg and the supernatants were discarded. The
remaining pellets were resuspended in 50 μl of 0.85% NaCl. The PMA-qPCR was
performed as described before. Each swab sample was analyzed in duplicate, and
the external standard was used for quantification.
For each foot, the reduction of live *D*. *nodosus* was calculated as follows:
$${\mathit{\log}_{10}R} = {\mathit{\log}_{10}(N\ prevalue)} -
{\mathit{\log}_{10}(N\ postvalue)}$$
With the exception of Desintec, all other substances from the *in vitro*
experiment were not approved as biocide for use in footbaths with sheep.
Therefore, only 3%, 6% and 9% Desintec was tested *ex vivo* and compared to 4%
formaldehyde and 10% zinc sulfate. A 0.85% NaCl solution was chosen as negative
control.
The software NCSS12 (NCSS Statistical Software) was used for the statistical
analysis of the logarithmized reduction values. Assumption of normal
distribution was checked using histograms and Shapiro-Wilk test. The values were
not normally distributed and equal variance was rejected. Therefore, Kruskal-
Wallis Test with Dunn's Test for multiple comparisons was used for determination
of significant differences among disinfectants. The significance level was
calculated at z-value \> 1.9600. Post hoc power testing was performed using Two-
Sample T-Tests allowing unequal variance.
# Results
## *In vitro* disinfectant testing
Results for each disinfectant with the corresponding reduction of virulent *D*.
*nodosus* are given in for triplicate testing and for single testing. The
disinfectants formaldehyde (4%; 7.2/6.7) and copper sulfate (5%: 5.7/6.1; 10%:
7.2/6.7) achieved a ≥ 5 log reduction without (x/) and with (/x) organic soiling
whereas 10% zinc sulfate (4.9/4.7) failed to meet the envisaged log reduction.
The 20% zinc sulfate solution could not be evaluated, because centrifugation in
the solution failed due to its high density.
The individually tested active ingredients of Desintec at 5% (acetic acid;
glycolic acid; glutaraldehyde) showed log reductions ≥ 5 (7.2/7.2; 7.7/6.0;
6.2/6.6) without and with organic soiling, respectively.
Desintec itself showed log reductions ≥ 5 in 1:10 and 1:100 dilutions without
and with soiling (6.8/6.8 and 6.8/6.4, respectively). In a 1:1000 dilution, the
product evoked a 5.8 log reduction without soiling and 0.3 log reduction with
soiling whereas in 1:10 000 dilution both test conditions failed to meet the
envisaged ≥ 5 log reduction (0.4/0.2).
In single experiments also lactic acid, propionic acid, hydrogen peroxide,
sodium hypochlorite, octenidine dihydrochloride, chlorocresol and Ampholyt 20
achieved the required ≥ 5 log reduction in the number of *D*. *nodosus* even
under soiling conditions. While calcium magnesium tetrahydroxide, calcium
hydroxide and calcium oxide showed the requested reduction without soiling but
not with soiling, sodium benzoate, tartaric acid, calcium magnesium oxide and
electrochemically activated water failed to meet the minimum ≥ 5 log reduction
without and with soiling.
## *Ex vivo* experiments
### Linear range of PMA-qPCR
Enumeration of viable *D*. *nodosus* by culture and PMA-qPCR revealed good
correlation. Samples containing 10<sup>7</sup> to 10<sup>3</sup> of *D*.
*nodosus* showed a decrease of DNA in the dead cell sample by PMAxx<sup>™</sup>
treatment between 4 log and 2 log. At 10<sup>2</sup> CFU/ml, the decrease
dropped to 1 log unit. Therefore, part of the DNA corresponding to approximately
10<sup>1</sup> to 10<sup>3</sup> CFU/ml remained intact.
### *Ex vivo* disinfectant testing
Efficiency of disinfectants is shown in. Medians of 10% zinc sulfate versus
0.85% NaCl were not different (z = 1.8705), whereas 4% formaldehyde evoked a
significant reduction (z = 3.8794). For this reason, the more effective
disinfectant formaldehyde was chosen for comparison with Desintec. Both 6% and
9% Desintec showed no significant differences compared to 4% formaldehyde (z =
0.8710 and 0.2646). Post hoc power testing between 4% formaldehyde and 6%
Desintec with group sample sizes of 13 and 14 achieved 80.078% power to reject
the null hypothesis of equal means when the population mean difference (delta)
is 1 with standard deviations of 1.2 for group 1 and 0.8 for group 2, and with a
significance level (alpha) of 0.050. A 4% formaldehyde solution is significantly
more effective than 10% zinc sulfate (z = 1.9719). Unequal variances of
disinfectants and a dose-responsive reduction of Desintec are visible in the box
plot.
# Discussion
The aim of this study was to identify and test alternative disinfectants for
future use as footbath solutions in a footrot control program. Based on the
requirements for an effective, non-carcinogenic, environmentally acceptable,
inexpensive and licensable concentrate, around 22 substances or products were
selected by expert opinion. It became clear in discussions with representatives
of veterinary drug companies that only an already registered product for
footbaths would be considered for marketing since a new registration of a
substance for the limited market would be too expensive. The product DESINTEC®
Hoof Care Special D (Desintec) had already been registered as a biocide in
Germany under the name PediSept G20 and became therefore the focus of the study.
Thus, Desintec and its main components glutaraldehyde, acetic acid and glycolic
acid, as well as the "gold standards" formaldehyde, copper sulfate, zinc sulfate
and other chemical substances were tested.
The *in vitro* study confirmed the effectiveness of formaldehyde, copper sulfate
and zinc sulfate as disinfectants in footbaths. These have been used for a long
time and are an effective treatment option for footrot in sheep. However, the
10% zinc sulfate solution was slightly below the requested ≥5 log reduction and
the 20% solution could unfortunately not effectively be tested due to its high
density, which prevented *D*. *nodosus* and erythrocytes from sedimentation by
centrifugation.
Desintec fulfilled the targeted reduction of ≥5 log at 1:10 and 1:100 dilution
in accordance with the manufacturer recommendation to use it at a concentration
of 3–5%. The product was even effective in 1:1000 dilution without organic
soiling, however, organic soiling substantially reduced its effect. When testing
the components of Desintec (glutaraldehyde, acetic acid, glycolic acid)
individually at 5% concentration, each of them was able to reduce the number of
*D*. *nodosus* at the requested scale even under soiling conditions.
Glutaraldehyde has a broad spectrum of activity and a rapid microbial killing
rate. It is supposed to destroy all forms of microbial life, including bacterial
and fungal spores, tubercle bacilli and viruses. Glutaraldehyde is part of many
disinfectant solutions for livestock, listed by the committee for disinfection
of the German Veterinary Medical Society. Organic acids are known to be used as
food preservatives due to their antimicrobial potential. Like that, acetic acid
reduced the microbial load of foodborne pathogens on several fresh fruits and
vegetables. In medicine, acetic acid has been used for wound disinfection. Even
though microorganisms vary in their susceptibility, acetic acid solution proofed
to be bactericidal for *D*. *nodosus* in the current study as well. Glycolic
acid is well known for its keratolytic properties. The small molecular weight
allows for easy penetration of the skin, targeting the corneosomes and resulting
in desquamation of the stratum corneum. *D*. *nodosus* can be found in a depth
of 2200 μm in footrot affected tissue. The keratolytic effect of glycolic acid
may contribute to a deeper penetration of disinfecting ingredients of biocides
into skin and hoof thus improving their effectiveness. Apart from the
keratolytic effect, glycolic acid shows antimicrobial properties like other
carboxylic acids although it is less commonly used for this purpose. It proofed
to be an effective postmilking teat disinfectant, and the strong reduction of
*D*. *nodosus* in our *in vitro* experiments is another example of its
antimicrobial activity.
Lactic acid, propionic acid, hydrogen peroxide, sodium hypochlorite, octenidine
dihydrochloride, chlorocresol and Ampholyte 20 were effective in reducing the
number of viable *D*. *nodosus in vitro* even under soiling conditions. On the
other hand, calcium magnesium tetrahydroxide, calcium hydroxide and calcium
oxide showed the requested reduction without soiling but not with soiling, while
sodium benzoate, tartaric acid, calcium magnesium oxide and electrochemically
activated water failed as disinfectant without and with soiling. Since all these
components lack the chance of becoming registered biocides, they were only
tested once and not considered in the *ex vivo* assays.
Based on the promising use of Desintec this product was further investigated in
*ex vivo* tests using sheep feet in a dipping machine. In contrast to the *in
vitro* assay, culturing of *D*. *nodosus* from a swab sample of heavily
contaminated feet is not a sensitive method and requires subculturing, which
makes a quantification of viable *D*. *nodosus* as needed for assessing
effectiveness of disinfectants in the *ex vivo* part of our study impossible.
Therefore, an alternative approach able of detecting viable cells by PCR as e.g.
presented by Nogva et al. was established. By this approach, the distinction
between viable and non-viable cells is possible, based on membrane integrity.
For that purpose, the samples containing *D*. *nodosus* were treated with the
improved nucleic acid intercalating propidium monoazid (PMA) dye
PMAxx<sup>™</sup> that selectively enters cells with compromised cell membranes,
whereas the intact cell membrane presents a natural barrier for this molecule.
After exposure to strong light, it covalently binds to the DNA, preventing DNA
from being amplified by PCR, thereby enabling differentiation of viable from
non-viable cells. At the same time when the cross-linking with DNA occurs, any
unbound excess PMAxx<sup>™</sup> reacts with water. The resulting molecule is no
longer reactive, preventing reaction of PMAxx<sup>™</sup> with DNA extracted
from intact cells.
The PMA-qPCR proved to be a valid method for comparison of antimicrobial
efficiency in the *ex vivo* experiments. Thereby, 10% zinc sulfate did not show
a significant difference to the NaCl control. Therefore, 4% formaldehyde that
showed a significant reduction of live *D*. *nodosus* was chose to compare to
Desintec. The Desintec solution diluted down to 6% was still effective not
showing any significant difference to the "gold-standard" of 4% formaldehyde.
Variability of reduction within disinfectant groups was observed and is
explainable by various factors influencing the *ex vivo* experiment. *D*.
*nodosus* loads vary naturally among feet due to individual differences and the
different clinical status of footrot affected feet, leading to different
prevalues. Furthermore, the total number of microorganisms can also affect the
activity of a disinfectant. Higher inoculum levels can attenuate the efficacy of
disinfection by adding to the level of soiling and by providing protection to
other organisms at the site. In addition, disinfection inactivation follows
first-order kinetics. Starting from a high prevalue, the absolute reduction is
high whereas the relative reduction is small. On the other hand, starting from a
small prevalue, the effect is reversed with small absolute and high relative
reduction. Reduction values obtained from smaller prevalues possibly lead to
higher relative reductions, widening the variability. Negative reduction can be
explained with the low precision of the swab sample. Another reason for the
variability could be the extent of soiling. Soiling can affect both the
disinfection process and PMA-qPCR. Even though claws and interdigital space were
cleaned and open articulations were covered, disinfectant solutions were soiled
with manure and blood to varying degrees after the footbath. Soiling can affect
microbial activity by direct interference with the disinfectant, by interaction
and protection of the target organism and formation of microbial aggregates.
Moreover, complex matrices found in environmental samples can negatively
influence the PMAxx<sup>™</sup> treatment by chemical adsorption of the dye and
interference with photoactivation.
Glutaraldehyde contained in Desintec is not listed by the International Agency
for Research on Cancer (IARC) and there is no evidence for carcinogenic
activity, genetic or reproductive toxicity. However, glutaraldehyde is
irritating and corrosive to the skin, eyes and respiratory tract and is a known
cause of allergic contact dermatitis and occupational asthma. It is toxic to
aquatic life and should not be discharged into water bodies. On the other hand,
a smaller amount of glutaraldehyde in combination with acetic and glycolic acid
is needed to achieve the same antimicrobial effect as 4% formaldehyde. In a 6%
Desintec solution (recommended final concentration) there is 0.36%
glutaraldehyde compared to 4% formaldehyde currently used in footbaths.
Furthermore, glutaraldehyde is rapidly degradable in air, water, and soil, does
not bioaccumulate and is less toxic than formaldehyde. Desintec applied as 6%
solution in footbaths is therefore an environmentally acceptable biocide that
after use is recommended to be disposed on the manure pile or in the slurry
basin.
# Conclusion
The study showed that Desintec is an effective alternative to formaldehyde (4%),
zinc sulfate (10%) and copper sulfate (5%) for the use in sheep footbaths to
eliminate virulent *D*. *nodosus*. The product is not only effective but also
non-carcinogenic, is biodegradable and available as concentrate, making it an
improvement over the currently used disinfectants. The results of this study
represent a step forward on the way to a footrot control program that will
mainly be based upon herd-level footbathing.
# Supporting information
We would like to thank the various members of the expert opinion group (Yuval
Tempelman, Giochen Bearth, Maria Christina Härdi-Landerer, Rita Lüchinger, Mina
Gómez Góngora, Flurina Stucki, Alwin Meichtry, Richard Eicher, Heinz Burkhardt)
for their advice concerning requirements, registration and practical
implementation of an alternative disinfectant. We would also like to thank
Beatriz Vidondo and Brian Friker (Veterinary Public Health Institut, Liebefeld,
Switzerland) for assistance with the statistics used in this work. Furthermore,
we are grateful to Isabelle Brodard and Simon Feyer (Institute for Veterinary
Bacteriology, Bern, Switzerland) for excellent technical support and the
abattoir Thun for providing the footrot diseased feet of sheep.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
A variety of animals, from nematode to human, show social behavior. The social
behaviors allow for individuals to create an entity greater than the sum of the
individuals and provide the key to successful adaptation to the environment. One
of the most striking features of the highly-ordered animal society is the
ability to share information among individuals. In turn, higher communicative
ability is a fundamental basis that enables animals to maintain a more evolved
society.
Honeybees (*Apis mellifera* L.) organize a highly-ordered society and have a
sophisticated communicative ability known as the ‘dance communication’. Worker
honeybees that find a rich food source return to the hive and might transmit the
information on the location of the food source to their nestmates using a
symbolic dance. The dance information is decoded into the spatial information of
the food source by the other worker bees (followers) that follow the dancers.
During foraging flights, worker honeybees integrate the incoming sensory
information: they estimate the distance of food sources based on the amount of
optic flow they perceive, and direction based on the position of the sun, which
are the essential bases for the expression of dance communication. Although
there is a considerable amount of research concerning the sensory basis of these
remarkable abilities, almost nothing is known about the underlying neural
mechanisms.
As a first step in elucidating the neural mechanisms of these remarkable
abilities, it is important to identify active brain regions in dancing and
foraging honeybees that might be involved in dance communication and/or
information integration during foraging flight. Although methods to detect the
expression of immediate early genes (IEGs) as markers of neural activity are
widely used in vertebrates, neural IEGs have not yet been identified in insects.
In the present study, we identified a novel IEG that can be used as a neural
activity marker and found that the neural activity of a mushroom body (MB)
neuron subtype is preferentially increased in foraging honeybees, suggesting its
involvement in information integration during foraging flight.
# Results
## A novel non-coding IEG, *kakusei*, can be used as a marker to visualize neural activity in the honeybee brain
To identify IEGs, we used the differential display method to search for honeybee
genes that are immediately induced in the brain by neural activity. To evoke
strong neural activity in the brain, seizures were induced by awakening workers
from ice-cold induced anesthesia, because some of the IEGs were identified by
inducing seizures in the animals. When the workers are awoken from anesthesia,
they show seizure-like movement with their legs and body shaking. Using
differential display screening of approximately 6500 bands, which were derived
from mRNAs extracted from the brains of seizure-induced and non-treated bees, 49
candidate bands were identified. Among them, we selected nine candidates that
showed a pronounced difference in band intensity between the seizure-induced and
non-treated bees. After preliminary Reverse transcription-polymerase chain
reaction (RT-PCR) analysis of these candidates, we finally focused on a single
candidate that showed the most prominent and reproducible seizure-induced
transcript increase. As a result, we identified a novel IEG that we named
*kakusei* after the word ‘awakening’ in Japanese (the whole sequence of
*kakusei* was deposited as DDBJ accession number AB252862). To examine the size
of the *kakusei* transcript, we performed Northern blot analysis using total RNA
isolated from whole brains of bees anesthetized with CO<sub>2</sub> and bees
awakened from CO<sub>2</sub>-induced anesthesia. The results indicated that the
induced *kakusei* transcript was approximately 7 kb long. There was no
significant open reading frame in any of the three possible reading frames of
the *kakusei* cDNA sequence, suggesting that the *kakusei* transcript functions
as a non-coding RNA. RT-PCR experiments and sequence analysis also confirmed
that the contig *kakusei* sequence is expressed as continuous transcripts.
*Kakusei* expression was transiently induced in the whole brain after awakening
the workers from anesthesia induced by either CO<sub>2</sub> \[; Sz
(seizure)-induced\] or ice-cold (data not shown). Real-time RT-PCR revealed that
*kakusei* is expressed predominantly in the brain, suggesting a brain-specific
function. *In situ* hybridization revealed that *kakusei* expression can be
detected in every brain region, including the MBs, optic lobes (OLs), and
antennal lobes (ALs) in a seizure induction-dependent manner, suggesting that
*kakusei* can be used as a marker in broad brain regions. In addition, *kakusei*
signals (purple) were detected exclusively in the nuclei (green) of brain
neurons, reflecting the characteristics of the *kakusei* transcript as a non-
coding RNA. This notion was clearly demonstrated when the *kakusei* transcript
localization was compared to that of *actin*, which is transported to the
cytoplasm to be translated into protein and is detected as a broadly-distributed
signal in the cytoplasm. This characteristic *kakusei* signal staining enabled
us to count and quantify the number of *kakusei*-positive neurons.
We next examined whether *kakusei* expression reflects physiological neural
activity by testing whether *kakusei* expression was induced in the brain upon
light exposure. First, workers were dark-adapted by keeping them in the dark
overnight. The next day, experimental bees (light-exposed) were exposed to white
light for 30 min, and then used for *in situ* hybridization. Dark-adapted bees
were used as a control. *Kakusei* expression was detected in the OL and MB
neurons of the light-exposed bees. The expression of *kakusei* was particularly
prominent in the lamina neurons, which receive direct input from retinal
neurons. In contrast, there was no strong *kakusei* expression in the AL
neurons. These results indicate that *kakusei* can be used as a marker to
visualize physiological neural activity in the honeybee brain.
To further examine whether *kakusei* expression can be detected in the brains of
bees that exhibit physiologic behaviors, we studied *kakusei* expression in the
brains of bees exhibiting phototactic behavior. According to a previous report,
we collected foragers that moved to the lighted side and nurse bees that did not
move to the lighted side 30 min after the start of the phototactic behavior, and
investigated *kakusei* expression in the brains (see for the experimental
procedures). Almost the same *kakusei* expression pattern was observed in the
brains of both the foragers and nurse bees: a large number of neurons in the
optic lobes were *kakusei*-positive, which was similar to the findings in the
light-exposed bees. These results suggest that *kakusei* expression is not so
sensitive as to be induced by the neural activity specific to phototactic
behavior, and that the activity in the optic lobes, which could be induced by
visual inputs upon light illumination, is predominant even in the brains of bees
that exhibited phototaxis. The fact that *kakusei* expression was observed in
the optic lobes of bees that were not dark-adapted and had natural phototactic
behavior strongly suggests that *kakusei* expression reflects neural activity
under normal physiologic conditions, although we still cannot exclude the
possibility that light-exposure is a stressor to the honeybee.
## Neural activity is increased in the small-type Kenyon cells of the dancer brain
The workers shift their tasks from nursing their brood (nurse bees) to foraging
for nectar and pollen (foragers) according to the number of days after eclosion.
When foragers successfully find food sources, some of them perform a dance to
enroll followers to forage. Therefore, we next examined *kakusei* expression in
the brains of the dancers, followers, and nurse bees to identify the brain
regions involved in dance communication. The individual workers were caught
immediately after confirming their behaviors in the observation hives, and used
for *in situ* hybridization. The bees caught from the observation hives were
immediately anesthetized by ice-cold water and kept on ice until use for *in
situ* hybridization to maintain the current state of the *kakusei* transcripts
in the brain. There was a characteristic *kakusei* expression pattern in the
dancer brains, especially in the MBs. The honeybee MBs consist of three types of
intrinsic neurons termed large-type Kenyon cells (lKCs), small-type KCs (sKCs),
and class II KCs. The most prominent *kakusei* expression was observed in the
sKCs , whose somata are located in the center of the MBs. In contrast, only a
small number of positive cells were detected in the brains of followers and
nurse bees. *Kakusei* expression in the central complex neurons was not clear,
as we could not identify the central complex neurons in our *in situ*
hybridization experiments.
Quantitative analysis revealed that the number of *kakusei*-positive cells in
the sKCs was approximately 20 times higher in the dancers than in the followers
or nurse bees. In addition, *kakusei* expression was also weakly induced in the
lKCs and class II KCs, as well as in the dorsal and ventral OL neurons (dOL and
vOL, respectively). A three-factor ANOVA \[F1: bee type; F2: brain region
(repeated measure); F3: brain hemisphere (repeated measure)\] revealed that
there was a significant difference between the bee type and brain region (F1 and
F2: *P*\<0.0001, respectively; significant interaction between F1 and F2:
*P*\<0.0001). In contrast, there was no significant difference between the right
and left hemispheres (F3: *P* = 0.9455). Significant differences between bee
types were observed in the dOL, vOL, lKCs, sKCs, and class II KCs (\*,
*P*\<0.05; \*\*, *P*\<0.01; Tukey-Kramer's test). These results indicate that
the neural activity in these brain regions, especially the sKCs, is increased in
the dancer brain. The differences in *kakusei* expression between dancers and
followers could not be due to their different developmental stages but only to
their behavioral differences, because both the dancers and followers are thought
to be of the same behavioral stage. Thus, the prominent neural activity in the
sKCs observed in the dancer brains is likely due to their characteristic
behaviors.
## Neural activity of the small-type Kenyon cells is also increased in the forager brain
According to the expression profile, *kakusei* expression reflects neural
activity that occurred 15 to 60 min prior to sampling of the bees. In our
observation, dancers repeated the dance every 5 to 7 min and the foraging every
10 to 15 min. Thus, the neural activity detected in the dancer brains might be
due not only to the dancing behavior, but also to the preceding foraging
behavior. To address this question, we examined *kakusei* expression in the
brains of foragers. Only some of the foragers that succeed in finding food
display dance behavior. Thus, when we analyze foragers, only some of them are
expected to be dancers.
Therefore, we collected foragers with pollen loads (an indication that they were
successful in finding food) in front of the hive entrance before we checked
whether or not they danced in the hive. The results indicated that every forager
(N = 12) had a *kakusei* expression pattern similar to that of the dancers
(N = 6). In addition, there was no significant difference in the density of
*kakusei*-positive cells in the MB neurons, including in the sKCs, between these
bees \[; *P*\>0.05, two-factor ANOVA (F1: bee type; F2: brain hemisphere)\].
Thus, these results suggest that the increased sKC neural activity in the dancer
brain is associated with foraging behavior rather than dancing behavior,
although we cannot exclude the possibility that the foragers we examined also
exhibited the dance behavior shortly before the observation period.
## Re-orienting bees showed different *kakusei* expression patterns from foragers and dancers
Foraging behavior might involve multiple behavioral processes, such as visual,
olfactory, tactile, and gustatory experiences, flying, landmark learning and
memory, information processing for the dance communication, etc. We next
examined whether the sKC-preferential *kakusei* expression is associated with
forager-specific behavioral components or components that are common to the
other behaviors, such as flying, visual experience, or landmark learning and
memory. We investigated *kakusei* expression in the re-orienting bees, which
were collected as the workers that fly around the hive to re-orient themselves
to the hive when the location of the hive has changed. For this, we moved the
hives at night with the entrance closed. The next morning, we opened the
entrance for 5 min and then caught the bees flying around the hive 0, 15, and 30
min later. *Kakusei* expression was increased in the MBs in a time-dependent
manner in the re-orienting bees, suggesting that this neural activity is induced
by re-orienting behavior. One-way ANOVA among re-orienting bees revealed that
the time effect was significant for every MB neuron type (:
*P'*s\<0.0001–0.003). In contrast to sKC-preferential *kakusei* expression in
the foragers, however, *kakusei* was induced in all KC types in the re-orienting
bees. The proportion of *kakusei*-positive cells in the sKCs compared to that in
the lKCs was significantly higher in the foragers than in the re-orienting bees.
To exclude the possibility that the neural activity in the re-orienting bees is
due to the increased light exposure when they leave the hive, a similar
experiment was performed using re-orienting bees from transparent observation
hives. In this experiment, a significant time-dependent increase in *kakusei*
expression was observed in every MB neuron subtype in the re-orienting bees
(*P*\<0.0001, one-way ANOVA), like in the re-orienting bees from the normal
hives, indicating that the increased *kakusei* expression in the re-orienting
bees is due to re-orienting behavior, and not merely to light-exposure. Here,
the workers that performed re-orientation flights were probably foragers, also
suggesting that the differences in the *kakusei* expression pattern between the
brains of re-orienting bees and foragers/dancers are due to differences in
behavior, but not age. These results demonstrate that the active brain regions
are different between the re-orienting bees and foragers/dancers, suggesting
that the sKC-preferential *kakusei* expression in forager/dancer brains is not
due to behavioral components in common with those of re-orienting bees, like
visual experience, flying, or landmark learning and memory. Rather, the forager-
specific behavioral components are likely to be responsible for the sKC-
preferential *kakusei* expression.
# Discussion
In the present study, using novel methods to visualize neural activity in the
honeybee brain, we demonstrated that neural activity in the MBs, especially the
sKCs, is prominently increased in the brains of dancer and forager honeybees. In
contrast, the neural activity of both the sKCs and lKCs was increased in the
brains of re-orienting workers. These findings strongly suggest that sKC-
preferential activity, but not mere MB-preferential activity, is associated with
behavioral components that are specific to foraging flight.
The MBs are believed to be important for higher sensory integration in the
insect brain. Partial ablation of the MBs impairs only complex olfactory
learning without affecting simple olfactory learning. In addition, in the
honeybee brain, all sensory modalities investigated (visual, olfactory,
gustatory, and mechanosensory) project to the MBs, ,. Furthermore, foraging
experience greatly influence the MB neuropile volume increase and the MB neuron
outgrowth. Although these findings imply that the MBs are the appropriate brain
regions to process complex information during foraging behavior, such as the
calculation of distance and direction, there was no direct evidence whether the
MB neurons are actually active in forager brains. The methods that we
established in this study provide the first opportunity to investigate neural
activity in the brains of naturally behaving honeybees and revealed that the MB
neurons are active in the dancers and foragers, although these methods are
limited to labeling only cell bodies and not the neuropile because *kakusei*
encodes a non-coding RNA whose expression is confined to the nuclei.
Our study demonstrated that neural activity of the sKCs, one of the MB neuron
subtypes, was prominently increased in the dancer and forager brains. Although
the inputs and outputs of the sKCs have been well investigated, the role of the
neural circuitry composed by the sKCs is unknown. The calyx (input region) of
the honeybee MB is divided into three zones: lip, collar, and basal ring. The
sKCs project dendrites to the basal ring zone, whereas the lKCs project to the
lip or collar zone. On the other hand, all sensory modalities investigated
(visual, olfactory, gustatory, and mechanosensory) project to both the basal
ring and lip/collar zones, which are then relayed to both sKCs and lKCs,.
Studies of the anatomy of the bee brain suggest that the basal ring zone
receives multi-modal (visual, olfactory, gustatory, and mechanosensory) inputs
and extensive recurrent inputs from extrinsic MB neurons, whereas the lip and
collar zone receive mono-modal (olfactory and visual, respectively) inputs. In
addition, the sKCs are composed of neurons with different morphologies. Thus, we
speculate that the sKC-preferential neural activity observed in the
forager/dancer brains reflects the complex activity within the MB neural
networks required for sensory integration during the foraging flight. Recent
studies suggest functional differences among the KC types based on their gene
expression patterns,. For example, activation of *Amfor*, one of the genes
preferentially expressed in the sKCs, affects the onset of foraging behavior.
These findings are also consistent with our notion that the sKCs play roles in
higher integration of the complex information that accompanies foraging
experience.
Prominent *kakusei* expression was detected only in the forager brains, not in
the follower brains, possibly due to the differences in their foraging
experience during the hour before sampling. The followers often show reduced
foraging activity before they start following the dance. Thus, considering the
short half-life of *kakusei* expression, it is possible that this behavioral
difference is reflected in the *kakusei* expression in the follower brains. In
addition, there were strikingly different *kakusei* expression patterns in the
MBs between the re-orienting and foraging workers. Because the re-orienting bees
fly around to form spatial memory regarding their hive location, they have
several behavioral components in common with the foragers. How then are the
differences in *kakusei*-expression between re-orienting and forager/dancer bees
explained? Foraging behavior is different from orienting behavior in some ways:
for example, foragers need to calculate the distance and direction of food
sources, memorize them in association with food information, and recall them
repetitively to repeat foraging, which involves much broader and multi-modal
sensory integration than just orienting. Thus, it is possible that the increased
activity in the sKCs in the forager brains is associated with these behavioral
components and reflects such sensory integration. Alternatively, it is also
possible that the sKC activity is important for both the foraging and re-
orienting behaviors and the relative repression of the lKC activity is important
for the foraging behaviors. If this is the case, the integration of sensory
information during foraging flight might result from interactions between active
sKCs and inactive lKCs.
Here we identified the transcript of novel IEG, *kakusei*, as a non-coding
nuclear RNA. Although microRNA, which is expressed in response to neural
activity, has been reported in vertebrates, *kakusei* is the first example of a
long non-coding nuclear RNA that shows an immediate early response to neural
activity. Long nuclear RNAs regulate gene expression, whereas microRNA post-
transcriptionally regulates gene expression. In general, vertebrate IEGs encode
transcription factors and have roles in modulating neural functions in an
activity-dependent manner. Thus, *kakusei* might regulate gene expression as a
long non-coding RNA to modulate neural function.
Although methods using IEGs as markers of neural activity are widely applied in
vertebrates, no IEG has yet been reported in the insect brain. Thus, this is the
first use of an IEG to identify an active brain region in the insect. Future
studies examining *kakusei* expression after a well-defined experience of
various sensory modalities such as gustatory, tactile, olfactory, and visual
(colors, patterns, etc.) should dramatically enhance our ability to interpret
the present data. Moreover, in future studies, the link between *kakusei*
expression and neural activity will need to be clarified to reveal the kind of
neural activity that is reflected by *kakusei* expression.
Due to limitations of the experimental methods, detailed behavioral components
that induce *kakusei* expression in the sKCs remain to be examined. Nonetheless,
the present study provides important insight into the neural basis of sensory
integration during foraging flight, which might be related to the dance
communication. It also describes a useful method for mapping active brain
regions involved in behaviors of interest in the honeybee.
# Materials and Methods
## Bees
European honeybees (*A. mellifera* L.) were purchased from a local dealer and
maintained at the University of Tokyo. Observation hives were made as previously
described with some modification.
## Differential display
Worker honeybee brains were dissected out from each of 10 bees anesthetized on
ice and bees awakened from ice-induced anesthesia, which showed a seizure-like
phenotype. Total RNA was isolated with TRIzol (Invitrogen), treated with DNase
I, and reverse transcribed with SuperScript II (Invitrogen). The differential
display method was performed as described previously using a Fluorescent
Differential Display Kit and LA *Taq* polymerase with a combined total of 216
primer sets (Takara). Bands of interest were excised, reamplified, and subcloned
into a pGEM-T vector (Promega).
## Northern blotting
Whole brain total RNA was isolated from each of 10 bees anesthetized by
CO<sub>2</sub> and bees awakened from CO<sub>2</sub>-induced anesthesia. RNA was
subjected to 1% formaldehyde-agarose gel electrophoresis, transferred to a nylon
membrane, and hybridized with <sup>32</sup>P-labeled riboprobes.
<sup>32</sup>P-labeled riboprobes were synthesized by T7 polymerase with Strip-
EZ<sup>TM</sup> RNA Kit (Ambion) from a template containing the fragment
isolated by differential display (DD fragment; from+4511 to+5159).
## cDNA cloning
To identify the whole length of *kakusei* cDNA, 5′-and 3′-rapid amplification of
cDNA ends (RACE) methods were performed repeatedly using the SMART RACE cDNA
Amplification Kit (Clontech).
## RT-PCR
RT-PCR experiments were performed using LA Taq (Takara) according to the
manufacturer's protocol and the SMART RACE cDNA as templates. Primers were
designed to amplify the regions shown in ; (a)
5′-CACGCTCGTCGTCGTGCCTTGCTCAGATAA-3′ and 5′-TTCAGAGCACGTTGGAACTAATCTCGCG-3′, (b)
5′-ACCTTGGAACGTGAAAGCGCATTTTCGA-3′ and 5′-AACCGTGTCCTTCTGCAGACACCTGACA-3′
## Quantification of the *kakusei* transcript
The expression of *kakusei* was induced by awakening bees from
CO<sub>2</sub>-induced anesthesia. Control bees were kept in CO<sub>2</sub>.
Total RNA was extracted from three to five bees for each sample. Real-time RT-
PCR was performed with Light Cycler-DNA master hybridization probes (Roche)
according to the manufacturer's protocol, using gene-specific primers
(*kakusei*; 5′-GGAAACAGGTGGTTTGATGACCATTG and 5′-CACGTTCCAAGGTTTAACGATGCG,
*actin*; 5′-GAAATGGCAACTGCTGCATC and 5′-TCCACATCTGTTGGAAGGTG) and fluorescent
probes (*kakusei*; fluorescein isothiocyanate (FITC) probe,
5′-CGCTGTAGTGCGTTTTCACTCGGATCGA, and LC-Red640 probe,
5′-TCCGAGGAAATCCGAGCAAAGTTCGTTC, *actin*; FITC probe,
5′-CCATGAAAATTAAGATCATCGCGCCAC, and LC-Red640 probe,
5′-CGAGAAGAAATATTCCGTATGGATTGGTG). The amount of *kakusei* transcript was
normalized with that of *actin* and is shown as relative to the value of control
bees at 0 min or to the whole brain. There was no significant difference in the
levels of *actin* expression between control and seizure-induced bees.
## *In situ* hybridization and image analysis
*In situ* hybridization was performed as described previously with some
modification. Frozen coronal brain sections (10 µm thick) were fixed in 4%
paraformaldehyde in phosphate buffered saline, pretreated, and hybridized with
digoxigen (DIG)-labeled riboprobes. The DIG-labeled riboprobes were synthesized
by T7 or SP6 polymerase with a DIG labeling mix (Roche) from a template
containing the fragment isolated by differential display (from+4511 to+5159).
After stringent washes, DIG-labeled riboprobes were detected
immunocytochemically with peroxidase-conjugated anti-DIG antibody (1:500; Roche)
and TSA Biotin System (Perkin Elmer). Sense probes were used as negative
controls and the signals were confirmed to be antisense probe-specific in every
experiment. Micrographs of fluorescent *in situ* hybridization were taken using
an IX71 confocal microscope (Olympus). SYTOX Green (Molecular Probes) was used
to stain the nuclear DNA. Intensity and brightness of the micrographs were
processed with Photoshop software (Adobe).
For quantification, the brain regions were defined as shown in. The rostral
section was defined as the section containing the AL, and the caudal section was
defined as the section containing the DL. The numbers of *kakusei*-positive
cells were manually counted from rostral and caudal sections. For each animal,
one rostral and one caudal section were analyzed. The area of each brain region
was measured using ImageJ analysis software (NIH, <http://rsb.info.nih.gov/ij>).
The amount of signal was divided by the area and the values from two sections
were averaged, when there were two data points for one individual (e.g., MB and
OL neurons). Sections to be counted were randomly selected from many sections.
The density of *kakusei*-positive cells was shown as a value relative to 100000
µm<sup>2</sup>. Micrographs were numbered and signals were counted by an
investigator blind to the bee type. The number of examined bees is shown in the
figure. Statistical analyses were conducted by *F*-test and Student's *t*-test
using Microsoft Excel (Microsoft) or JMP (SAS) software. Multiple comparisons
were performed using one-way analysis of variance (ANOVA) and Tukey-Kramer's
test. Statistical comparisons were made within the same brain hemisphere. Data
are shown means±standard error (SEM) throughout this paper.
# Supporting Information
[^1]: Conceived and designed the experiments: T. Kiya. Performed the
experiments: T. Kiya. Analyzed the data: T. Kiya. Wrote the paper: T. Kiya,
T. Kunieda, T. Kubo.
[^2]: This work was supported by the Program for Promotion of Basic Research
Activities for Innovative Bioscience (PROBRAIN). T. Kiya is the recipient of
a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS)
Fellows. |
# Introduction
Defensins are important small, basic, cysteine-rich, antimicrobial, and cationic
peptides that are abundant and widely distributed. Defensins are widely
distributed in multiple tissues in the body, most notably leukocytes and
epithelial surfaces. They are often present at high concentrations and
play an essential role in the innate immunity of their hosts from insects and
plants to amphibians and mammals. Membrane permeabilization is the crucial
step in defensin-mediated antimicrobial activity and cytotoxicity.
Defensins from different origins exhibit structural and functional similarities
with phylogenetic relationships between different types of defensins. Mature
defensins amino acids sequences are highly variable in each defensin family and
subfamily. Accurately identifying the types of defensins will be helpful in
analyzing their specificitis for various microbial targets, provide novel
insights for understanding their function, and facilitate antimicrobial drugs
targets discovery.
Biochemical experimental methods are highly reliable for elucidating types of
defensins, such as nuclear magnetic resonance (NMR) spectroscopy. However, such
the experimental techniques are time-consuming and expensive. Bioinformatics
methods can timely provide useful information and insights for both basic
research and antibiotics design. Thus, better understanding the distinct
functions of defensin proteins requires an automated method for timely and
reliably annotating the families of many defensin proteins. In our previous
study, four defensin families (vertebrate, plant, insect and other defensins)
were successfully classified using the increment of diversity (ID) method. In
another work the authors developed the DEFENSINPRED classifier to predict human
defensin proteins and their types based on pseudo amino acid compositions.
However, further work is necessary because the datasets constructed in those
methods were too small to reflect a statistical profile and did not impose a
rigorous cutoff threshold to exclude the redundant samples in the existing
defensin datasets. Moreover, a better web-server for defensins is also needed.
In the present work, we constructed a more rigorous benchmark dataset to train
the program, and a support vector machine (SVM) classifier was further proposed
to classify these five defensin families. An 4% improvement was obtained
compared with the previous method. For the convenience of experimental
scientists, a free online server **iDPF-PseRAAAC** was first established. A
friendly guide was further provided to describe how to use the web server.
# Materials and Methods
## Dataset
With rapidly increasing interest in defensins, the Defensins Knowledgebase is
available, which is a manually curated database and information source devoted
to the defensin family of antimicrobial peptides. The benchmark data set
$\mathbb{S}$ for the defensin proteins in this study was taken from the
Defensins Knowledgebase, which currently contains more than 500 defensin
sequences ranging from prokaryotes to eukaryotes. To prepare a high-quality
dataset, the program CD-HIT was used to remove the defensin proteins with ≥
80% pairwise sequence identity to any other protein. Highly similar data will
surely lead to overestimation of the performance of the proposed methods. If the
sequence identity cutoff is set to a lower percentage (such as 25%), the results
will be more objective and reliable. However, in this study we did not use such
a stringent criterion because the currently available data do not allow us to do
so. The proposed method is a sequence-dependent predictor, the input feature
vectors are only derived from the primary amino acids sequence. So there is
needed enough amino acids and dipeptide compositions to train the multi-
classifier module. For the defensin peptides are polypeptides of fewer than 100
amino acids (See). Besides, for ensure the data reliable, this dataset is a
manually curated database. All of the families annotation are gathered from
bibliographic databases and sequence databases literature sources. If the
sequence identity cutoff is set to a lower percentage (such as 25%), the numbers
of proteins for family subsets would have been too few to have statistical
significance. And the imbalanced data cause classifiers to tend to overfit and
to perform poorly in particular on the minority class. Finally, we
obtained a dataset $\mathbb{S}$ composed of 333 defensin proteins classified
into five families, as formulated by the following equation: $$\mathbb{S} =
\mathbb{S}_{1} \cup \mathbb{S}_{2} \cup \mathbb{S}_{3} \cup \mathbb{S}_{4} \cup
\mathbb{S}_{5}$$ where the subset $\mathbb{S}_{1}$ contains 60 insect defensins,
$\mathbb{S}_{2}$ contains 34 invertebrate defensins, $\mathbb{S}_{3}$ contains
42 plant defensins, $\mathbb{S}_{4}$ contains 40 unclassified defensins and
$\mathbb{S}_{5}$ contains 157 vertebrate defensins, and ∪ represents the symbol
for “union” in set theory. The length distribution of the five families is
depicted in. For the readers’ convenience, the 333 defensin proteins sequences
and codes are in.
## Reduced Amino Acid Alphabet
In this study, the reduced amino acid alphabet composition(RAAAC) clustered by
Protein Blocks(PBs) are used to predict defensins family and subfamily,
which is composed of 16 average protein fragments of 5 residues in length. The
Protein Blocks have proven their efficiency both in description and prediction
of longer fragments\[–\]. Once the databank was encoded in terms of PBs,
sequence specificity was computed. Each PB was so associated with a set of
enlarged sequence windows \[–w; +w\] of length l. An amino acid occurrence
matrix of dimension 20 × l was computed for each PB. Then, each matrix was
transformed into propensities matrix. Finally, all the matrices were compiled to
create a matrix F of size 20 ×m with m, a vector of length 16l. The distance
between two kinds of amino acids *i* and *j* was computed by using the D(aa*i*,
aa*j*). Then a hierarchical clustering using all the amino acid occurrence
matrices of the 16 PBs was performed, each resulting amino acid cluster
represents amino acids that showed the same over- and under-representations upon
all the PBs.
Different defensin peptides usually have specific functional regions, such as
β-sheet-rich fold and framework of six disulphide-linked cysteines. Based on the
similarity of their functional and physicochemical features in proteins, the 20
amino acids can be clustered into some smaller groups. The reduced amino acids
not only can simplify the complexity of the protein system, but also improve the
ability in finding structurally conserved regions and the structural similarity
of entire proteins. The reduced amino acid alphabet derived from Protein
Blocks method has the ability for abstracting useful functional and conservative
feature. And it also is helpful for simplifying the amino acids composition of
defensin peptide and improving the ability in finding structurally conserved
regions and the structural similarity of entire proteins.
Up to now, the Protein Blocks method has successful been used to analyze long
protein fragments and to predict functional regions, and the results have
proven their efficiency both in description and prediction of longer fragments,
such as protein structure mining, outer membrane proteins analysis and
backbone structure prediction of proteins. Our previous researches have also
demonstrated that this feature selection method will be useful for analyzing the
conservative domain and understanding function evolution of defensin protein.
## Support Vector Machine (SVM)
SVM is a powerful and popular method for pattern recognition that has been
widely used in biology classification based on statistical learning theory\[–\].
In training an SVM classification system, proteins are represented by sequence-
derived properties and are projected onto a hyperspace where the proteins in a
family are separated from proteins outside the family by a hyperplane. By
projecting a new sequence onto this hyperspace, the SVM system can determine
whether or not the corresponding protein belongs to the family based on its
location with respect to the hyperplane.
In the current study, the LIBSVM 3.0 package was used to implement of SVM;
it can be downloaded for free from the website
(<http://www.csie.ntu.edu.tw/_cjlin/libsvm>). Four types of kernel functions, a
linear function, polynomial function, sigmoid function and radial basis function
(RBF), can be used for predictions in this software. Empirical studies show
demonstrated that the RBF outperforms the remaining three types of kernel
functions in nonlinear classification. Thus, the RBF kernel function was
used in the current work. All the computations were performed using LIBSVM-3.0
standard package (Chang and Lin, 2001). The various user-defined parameters,
e.g., kernel parameter γ and regularization parameter C were optimized on the
training dataset. The predictor obtained via the aforementioned procedure is
called **iDPF-PseRAAAC**, where “i” stands for “identify”, “DPF” for “defensin
peptide family”, “Pse” for “pseudo”, “R” for “”reduced”, “AAA” for “amino acid
alphabet”, and “C” for “composition”.
## Multi-class SVM
Prediction of defensin family classes is a multi-classification problem. SVM is
regarded as a typical binary classifier. The methods of applying SVM to
solve multi-class classification problems have one-against-one(OAO), one-
against-all(OAA) and directed acyclic graph SVM (DAGSVM). In the present
study, we adopt the “One-against-one” approach to transfer it into a two-class
problem. This method involves construction of individual binary SVM classifier
corresponding to each pair of the classes. Hence, if there are K classes, OAO
will construct a total of K(K-1)/2 classifiers. Each classifier plays a role in
classifying of one class and another class. Classifier *i j*, named
*f*<sub>*ij*</sub>, is trained using all the patterns from class *i* as positive
instances, all the patterns from class *j* as negative instances, and
disregarding the rest. The classifiers then are combined using majority voting
scheme. Predictions are made with each binary classifiers and label is assigned
to a class with maximum number of votes. In case when tie arise, i.e. two
classes have identical votes, label assignment to the class is made on the basis
of smallest index. More details for one-against-one(OAO) of SVM classification
can be found in can be found in.
## Performance Evaluation
This method's performance was measured based on sensitivity (Sn), specificity
(Sp), Matthew’s correlation coefficient (MCC) and overall accuracy (OA), which
were defined as follows: $$\left\{ \begin{array}{l} {\mspace{13mu}\text{Sn}(i) =
\frac{\text{TP}(i)}{\text{TP}(i) + \text{FN}(i)}} \\ {\mspace{13mu}\text{Sp}(i)
= \frac{\text{TN}(i)}{\text{TN}(i) + \text{FP}(i)}} \\ {\quad\text{MCC}(i) =
\frac{\text{TP}(i) \times \text{TN}(i) - \text{FP}(i) \times
\text{FN}(i)}{\sqrt{\lbrack\text{TP}(i) + \text{FP}(i)\rbrack\lbrack\text{TP}(i)
+ \text{FN}(i)\rbrack\lbrack\text{TN}(i) +
\text{FP}(i)\rbrack\lbrack\text{TN}(i) + \text{FN}(i)\rbrack}}} \\
{\quad\text{OA} = \frac{1}{N}{\sum\limits_{i = 1}^{M}{\text{TP}(i)}}} \\
\end{array} \right.$$ where TP(*i*), TN(*i*), FP(*i*), and FN(*i*) represent
true positive, true negative, false positive and false negative of family *i*;
*M* = 5 is the number of subsets while *N* the number of the total samples in
$\mathbb{S}$.
# Results and Discussion
## Cross Validation
Three cross-validation methods, namely the sub-sampling (or K-fold cross-
validation) test, independent dataset test and jackknife test, are often used to
evaluate the quality of a predictor. Among the three methods, the jackknife
test is the least arbitrary and most objective as demonstrated in and can
always yield a unique result for a given benchmark dataset hence. The jackknife
test has been widely recognized and increasingly adopted by investigators to
examine the quality of various predictors\[–\]. Accordingly, the jackknife test
was used to examine the performance of the model proposed in the current study.
## Defensin Family Prediction
The jackknife results obtained using the **iDPF-PseRAAAC** and the benchmark
dataset $\mathbb{S}$ based on different sizes(S) and N-peptide compositions(N)
are depicted in. shows the prediction results for the overall accuracy of the
defensin families based on N-peptide composition with S size alphabet (N, S). As
the dimensions increases, N-peptides provide progressively more detailed
sequential information. However, the predictive ability did not increased
linearly with dimension increase; for example, when the tripeptides composition
(3, 20), 8000 dimensions, was selected as the input parameter, the overall
accuracy for predicting five defensins families was only 79.28%. The results
reflect the notion that a larger dimension does not necessarily result in better
performance, and the prediction ability is not always better when the feature
dimensions increase. Excessively large dimensions typically lead to information
redundancy or noise, which results in bad prediction accuracy.
The heatmap shows the adjacent correlation of 13 reduced amino acids for five
different defensin families. From the prediction performance based on different
vector dimensions depicted in, we observed that the overall accuracy reached a
maximum 85.59% based on 2-peptide composition of 13 reduced amino acids (N = 2,
S = 13). shows the Jackknife results obtained using **iDPF-PseRAAAC** to
identify defensin family with dipeptide (N = 2) composition based on different
reduced amino acid alphabet approaches. As shown in, 2-peptide compositions with
alphabet of 13 (N, S) outperformed the other reduced amino acid alphabet sizes.
The largest defensin family, vertebrate defensins, yielded the best success rate
at 99.36%. The 10-fold cross-validation has been performed to examine the
comparability of our method. The prediction results are similar to the jackknife
test (Total accuracy: 83.78% Vs 85.59%).
For further comparison, the amino acid (i.e., N = 1) and tripeptide (i.e., N =
3) results were also calculated and were in, which shows that none of the
results exhibit a higher success rate than N = 2. The data indicat that the
reduced amino acid composition provides a greater weight of compositional bias
to proteins with a signal at different sequence regions. Subsequently, the
adjacent correlation for 13 reduced amino acids was analyzed and depicted using
a heatmap plot.
## Vertebrate Defensin Subfamily Prediction
Vertebrates include three distinct defensin subfamilies, Alpha-, Beta-, and
Theta-defensins, which exhibit a broad spectrum of antimicrobial activities
against bacteria, fungi, and viruses. Subsequently, the proposed method was used
to predict the vertebrate defensins subfamily. The prediction results in show
that the best overall accuracy was 98.39%, and the Mathew's correlation
coefficients (MCC) for the Alpha-type, Beta-type and Theta-type are 0.97, 0.96
and 0.89, respectively. Such high accuracies demonstrate that the proposed
method is an effective and powerful approach for predicting defensin
subfamilies.
## Comparison with Previous Methods
To further demonstrate the performance of the proposed method, it is necessary
to be compared with other existing methods. However, directly comparing the
results is not objective and strict examination due to the different benchmark
datasets used. Therefore, we repeated the feature selection and prediction
process on the previous dataset. The jackknife cross-validated accuracies are
depicted in. Obviously, our proposed method yields the highest predictive
success rate.
According to, when the 169 reduced dipeptides are used, our method can achieve a
maximum overall accuracy (OA) of 95.10% for the defensin family, and 98.39% for
the vertebrate subfamily, which is higher than the maximum accuracy obtained
using other methods. Although the success rate for others family and subfamily
obtained using our method is not better, the accuracy of the other families and
subfamilies are dramatically better than using other methods, and clearly
indicate that the proposed method is more powerful than our previous method.
Subsequently, it is instructive to compare the overall success rate from **iDPF-
PseRAAAC** with the success rate for weighted random guess (WRG). The
overall success rate based on the OA to identify the defensin proteins among
their four subfamilies WRG is given by. $$\text{OA}(\text{WRG}) =
\frac{{(N_{1})}^{2} + {(N_{2})}^{2} + {(N_{3})}^{2} + {(N_{4})}^{2} +
{(N_{5})}^{2}}{N^{2}}$$ where *N* is the number of defensin proteins in the
benchmark dataset $\mathbb{S}$, *N*<sub>1</sub> the number of defensin proteins
in the subset $\mathbb{S}_{1}$, *N*<sub>2</sub> the number of defensin proteins
in the subset $\mathbb{S}_{2}$, and so forth. Substituting these data into, we
obtain the following.
<img src="info:doi/10.1371/journal.pone.0145541.e021" id="pone.0145541.e021g" />
OA
(
WRG
)
=
(
60
)
2
\+
(
34
)
2
\+
(
42
)
2
\+
(
40
)
2
\+
(
157
)
2
333
2
=
29.55
%
In contrast, the best overall success rate for **iDPF-PseRAAAC** was 85.59%.
Compared with the results in, the overall success rate for **iDPF-PseRAAAC** is
approximately 56% higher than using a weighted random guess, which indicate that
**iDPF-PseRAAAC** may be an easy and useful tool for timely identifying defensin
proteins families.
## Web-Server Guide
For the convenience of most experimental scientists, below, we provide a step-
by-step guide on how to use the **iDPF-PseRAAAC** web-server to achieve the
desired results.
**Step 1.** Open the web server at
<http://wlxy.imu.edu.cn/college/biostation/fuwu/iDPF-PseRAAAC/index.asp> and you
will see the top page for **iDPF-PseRAAAC** on your computer screen, as shown
in. Click on the 'Read Me' button to see a brief introduction about the
predictor and the caveat for using it.
**Step 2**. Either type or copy/paste the query defensin peptide sequence into
the input box at the center of. The input sequence should be in the FASTA
format. A sequence in the FASTA format consists of a single initial line
beginning with a greater than symbol (“\>”) in the first column, followed by
lines of sequence data. The words immediately following the “\>” symbol in the
single initial line are optional and only used for the identification and
description. The sequence ends if another line starting with a “\>” appears;
this indicates the start of another sequence. Example sequences in FASTA format
can be viewed by clicking on the 'Example' button right above the input box.
**Step 3**. Click on the 'Submit' button to see the predicted result. For
example, if you use the query defensin protein sequences in the 'Example' window
as the input, after clicking the 'Submit' button, you will see the "Result page"
shown on the screen of your computer. All these results are fully consistent
with the experimental observations. It takes approximately a few seconds for the
above computation before the predicted result appears on your computer screen;
for query sequences and longer each sequences, more time is typically required.
**Step 4**. Click on the 'Citation' button to find the relevant papers that
document the detailed development and algorithm of **iDPF-PseRAAAC**.
**Step 5**. Click on the 'Data' button to download the benchmark datasets used
to train and test the **iDPF-PseRAAAC** predictor.
# Conclusions
Defensins also play important regulatory roles in the immune systems of animals
and plants, acting as a bridge between innate and adaptive immunity in
vertebrates. In this study, a promising method, **iDPF-PseRAAAC,** was developed
to improve prediction performance for defensin proteins. The use of reduced
amino acid alphabets not only provides an efficient and accurate means of
protein vectorization for sequence-based protein classification systems but also
remarkably improves computational efficiency. High predictive accuracies
demonstrate that our proposed method is a potentially useful tool for
classifying defensin family.
# Supporting Information
We wish to express our gratitude to the editor and three anonymous reviewers
whose constructive comments were very helpful in strengthening the presentation
of this paper.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: YCZ GLF. Performed the
experiments: YCZ YL. Analyzed the data: LY GLF. Contributed
reagents/materials/analysis tools: ZYW GPL. Wrote the paper: YCZ YL GLF. |
# Introduction
The immunoglobulin G (IgG) class of antibodies plays an important role in the
adaptive immune defense of the human host against pathogens. IgG consists of two
identical heavy chains and two identical light chains, which in turn are
composed of variable and constant domains. Papain treatment of the IgG molecule
generates two separate monovalent Fab fragments recognizing antigens and an
intact Fc fragment, a recognition site for host receptors and a site of
interaction with a number of effector molecules, including the classical
complement pathway starting with factor C1q. IgG is a glycoprotein containing a
conserved complex carbohydrate structure attached to the asparagine 297 residue
in the CH2 domain of each IgG Fc part. It consists of a biantennary core of
*N*-acetylglucosamine and mannose with added terminal and branching carbohydrate
residues such as *N*-acetylglucosamine, fucose, sialic acid, and galactose. The
presence of this carbohydrate is crucial for proper antibody structure and for
interactions with cellular immunoglobulin G Fcγ receptors (FcγRs) and the
complement system. Altered glycosylation of IgG have been associated with
autoimmune disorders like rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE) and Crohńs disease. Several forms of autoimmune vasculitis
show a differentiating glycosylation with decreased Fc galactosylation and
sialylation. Additionally, it has been suggested that sialylated IgG *in vivo*
switch from its anti-inflammatory activity with subsequent reduced antibody
effector activity, to a pro-inflammatory/toxic activity upon decreased Fc
sialylation. IgGs, classified into four subclasses, IgG1, IgG2, IgG3 and IgG4,
are described to interact with different types of FcγRs giving different
activation profiles.
FcγRs provide a linkage between the humoral and cellular immune responses.
Phagocytic cells express members of three classes of IgG-Fc receptors, FcγRI,
FcγRII and FcγRIII, characterized by structural and functional homology and by
the specific recognition site on the CH2 region of IgG. Binding of pathogen-IgG
complexes to FcγRs mediates an essential response from the host against
pathogens by initiating a cascade of signals causing antibody-dependent-
cellular-cytotoxicity (ADCC), complement-dependent-cellular-cytotoxicity (CDCC),
endocytosis, phagocytosis, oxidative burst, the release of inflammatory
mediators, etc.. Complexed IgG-FcγR can besides activation of the C1q component
of complement also activate other ligands e.g. mannan binding lectin (MBL), the
neonatal receptor FcRn, the mannose receptor (MR), etc.. FcγRs may be expressed
constitutively on haematopoietic cells and may also be induced or up-regulated
by cytokines and other agents. FcγRs are responsible for balancing activation
(FcγRI, FcγRIIa and FcγRIIIa) and inhibitory signals (FcγRIIb) of the immune
system with the ability of both activating and inhibiting the IgG mediated
effector stimulation.
*Streptococcus pyogenes* is one of the most common human pathogens causing
pharyngitis, scarlatina and more severe infections like necrotizing fasciitis
and sepsis. Like other bacteria it expresses several virulence factors and uses
several immune evasion strategies to successfully invade its host. The recently
discovered enzyme Endoglycosidase S (EndoS) is secreted by *Streptococcus
pyogenes* and has a specific endoglycosidase activity on native IgG by
hydrolyzing the conserved asparagine-linked glycans on the heavy chains of IgG.
This 108 kDa-enzyme is encoded by the gene *ndoS* that is highly conserved and
is present in virtually all examinated isolates. EndoS is the first known
bacterial enzyme with a unique specificity for native IgG. This is in contrast
to other related endoglycosidases as EndoF1-3 from *Elisabethkingia
meningoseptica* (previously *Flavobacterium meningosepticum*), which show
enhanced hydrolytic activities on the denaturated forms of basically any
glycoproteins with the appropriate N-linked glycan, or EndoE from *Enterococcus
faecalis* that in addition to activity on the glycan of native IgG also
hydrolyzes high-mannose glycans on other denatureted glycoproteins. EndoS is
N-terminally processed by the cysteine proteinase SpeB that could be of
importance in regulating EndoS activity. Furthermore, the molecular requirements
for EndoS glycosidase activity have recently been elucidated revealing the
importance of glutamic acid 235 (Glu-235) and tryptophans. EndoS activity
affects the functionality of opsonizing IgG by decreased binding to Fc-receptors
on a monocyte-like cell line and impaired classical complement activation *in
vitro*.
In the present study we elucidated the effect(s) of EndoS on IgG subclasses and
IgG-FcγR interactions. The results revealed that EndoS hydrolyses the heavy
chain of all four human IgG subclasses (IgG1–4), both soluble and in a plasma
environment. Additionally, we found that EndoS hydrolysis of the IgG glycan
dramatically influences the binding of IgG to soluble, immobilized FcγRIIa and
FcγRIIb as well as to FcγR-expressing cells. Moreover, IgG pre-bound to these
cells dissociates due to treatment of cells with EndoS. Furthermore, an inactive
form of EndoS generated by site-directed mutagenesis binds with high affinity to
IgG1–4, while the active form only transiently interacts with its substrates.
These results provide novel information about the mechanisms behind enzymatic
modulation of the host immune defense by bacteria, provide novel information
about the molecular interactions between an IgG glycan-hydrolyzing enzyme and
IgG, and emphasize the importance of IgG glycosylation for correct antibody
effector functions.
# Results
## EndoS has glycosidase activity on all four IgG subclasses
It has previously been shown that EndoS hydrolyzes the chitobiose core of the
conserved N-linked glycan on the γ-chain of human polyclonal IgG. It was
therefore of interest to elucidate whether EndoS has activity on all four
subclasses of human IgG (IgG1–4). Purified recombinant EndoS was incubated with
purified human IgG1–4. SDS-PAGE analysis revealed that EndoS-treated IgG of all
subclasses migrated at an apparent molecular weight of approximately 3 kDa lower
than untreated IgG, which is consistent with hydrolysis of the chitobiose core
of the IgG glycan. To confirm glycan hydrolysis, samples were also analyzed by
lectin blot using a *Lens culinaris* agglutinin (LCA) lectin recognizing
α-linked mannose residues. Lectin blot analysis of the samples revealed that all
IgG subclasses lose the reactivity with LCA after incubation with EndoS
consistent with complete or nearly complete hydrolysis of the glycan.
Additionally, the glycosidase activity of EndoS on IgG1–4 in a plasma
environment was investigated. In this experiment human plasma was incubated with
purified EndoS or buffer, followed by affinity purification of the IgG fraction.
These fractions were subsequently subjected to a LCA ELISA using immobilized
monoclonal antibodies against IgG1–4 to capture IgG. This revealed that all four
IgG subclasses reacted with lectin when plasma was incubated with buffer,
indicating presence of the glycan (data not shown). In contrast, when plasma was
treated with EndoS, a dramatically reduced IgG1–4 reactivity with LCA lectin was
observed. IgG1 was hydrolyzed to 87±11%, IgG2 was hydrolyzed to 81±13%, IgG3 was
hydrolyzed to 74±23%, and IgG4 was hydrolyzed to 72±3%. Taken together, these
results clearly show that EndoS has the ability to hydrolyze human IgG of all
subclasses, in purified form as well as in whole plasma.
## The inactive form of EndoS binds IgG
We have previously partly elucidated the molecular requirements for EndoS
hydrolysis of IgG. Site directed mutagenesis of glutamic acid 235 to glutamine
(EndoS(E235Q)) at the proposed orifice of the catalytic tunnel abolishes
enzymatic activity. In addition, chemical blocking of tryptophanes revealed that
these amino acid residues are important for activity. To further investigate the
physical interaction between enzyme and substrate, the binding of EndoS and
EndoS(E235Q) to immobilized polyclonal IgG and IgG1–4 subclasses was studied
using slot-binding experiments with immobilized IgG probed with EndoS and
EndoS(E235Q). Purified, soluble IgG subclasses 1–4, each immobilized onto a
nitrocellulose membrane, were probed with EndoS and EndoS(E235Q) followed by
incubation with antibodies against EndoS. This experiment revealed a strong
binding of EndoS(E235Q) to polyclonal IgG, IgG1 and IgG2, and a weaker
association to IgG3 and IgG4, while only very weak interactions between active
EndoS and all subclasses could be seen. To calculate the affinity constants
between EndoS and immobilized IgG1–4, surface plasmon resonance technology was
used. Similarly to slot-binding results, this showed that EndoS(E235Q) binds all
IgG subclasses with high affinity, while there is no detectable binding of EndoS
to IgG.. The kinetic parameters of EndoS (E235Q) binding to immobilized IgG
subclasses were of similar character and the strongest interaction was
demonstrated between IgG1 and EndoS(E235Q) with a binding affinity constant
(*K<sub>D</sub>*) of 0.42 µM. No binding of either EndoS or EndoS(E235Q) to
IgG1–4 subclasses, which were hydrolysed by EndoS before immobilization, was
detected. These findings indicate that the intact IgG glycan is necessary for
the interaction between EndoS and IgG. Furthermore, the experiments comparing
the interactions between EndoS, EndoS(E235Q) and IgG indicates that EndoS binds
IgG with a high affinity, but the active enzyme is instantly released after
glycan hydrolysis in a “touch and go” manner.
## EndoS influences the binding of IgG1–4 to FcγRs
Since the nature of the interactions between FcγRs and the Fc domain of IgG is
highly dependent on the IgG glycosylation state, we explored the effects of
EndoS activity on IgG interactions with FcγRs. Thus, in an ELISA experiment the
soluble FcγRIIa, FcγRIIb and FcγRIIIa were immobilized and probed with purified
IgG1–4 subclasses. In line with other observations it was here seen that FcγRIIa
and FcγRIIb binds IgG1. This binding was nearly abolished after treatment of
IgG1 with EndoS. The binding of the other IgG subclasses to these receptors was
weak and was even more reduced after treatment with EndoS. In general, ELISA
studies revealed the IgG subclass binding affinity pattern
IgG1\>IgG3\>IgG4\>IgG2 for FcγRIIa and IgG1\>IgG4\>IgG3\>IgG2 for FcγRIIb.
Furthermore, we observed that the EndoS hydrolysed IgG2 had a different outcome
regarding the binding to FcγRIIa/FcγRIIb with more extensive binding ability,
compared to the untreated IgG2. FcγRIIIa was negative in binding of all IgG
subclasses (data not shown). The interaction between IgG1–4, with or without
EndoS treatment, with FcγRs was further analyzed by surface plasmon resonance.
Each IgG subclass was tested for binding to a surface with an immobilized
FcγRIIa, FcγRIIb or FcγRIIIa. Consistent with the ELISA data, the results showed
that IgG1 had the strongest affinity for both FcγRIIa and FcγRIIb with similar
binding affinity constants, 97 nM and 170 nM respectively. In agreement with our
previous findings, no binding of IgG1 to these receptors was detectable when
EndoS treated IgG1 was used. There was no detectable interaction between
FcγRIIa/FcγRIIb and IgG2 or IgG3, or between IgG4 and FcγRIIa. No binding of
soluble IgG1–4 subclasses to immobilized FcRIIIa could be detected. These
results indicate that EndoS hydrolysis dramatically decreases IgG's affinity for
FcγRs.
## EndoS decreases IgG binding to blood cells
Based on results from ELISA and surface plasmon resonance, we continued to
analyze the effect(s) of EndoS glycosidase activity on the interaction between
FcγRs and IgG. For this purpose we used an erythroleukemic cell line (K562)
exclusively expressing FcγRIIa. Since soluble FcγRI was not available to us, we
also investigated human monocytes that predominantly bind IgG through this
receptor. Thus, IgG was purified from plasma treated with EndoS or PBS, labeled
with <sup>125</sup>I and incubated with the K562 cells. The radioactivity of the
cell pellets was measured. This revealed significantly decreased binding of
radioactive IgG, originally purified from plasma treated with EndoS, to K562
cells. In a control experiment, the specific IgG binding to these cells was
calculated by addition of cold human IgG, which inhibited the binding of
radioactive IgG to 93% (data not shown). A strong binding of IgG to K562 cells
after incubation of cells with human plasma was confirmed by Western blot and
the reactivity of cell lysates with antibodies against human IgG. In contrast,
there was a clear decrease in binding of IgG to K562 cells incubated with plasma
pre-treated with EndoS. Likewise, the binding of <sup>125</sup>I-IgG to
monocytes as analyzed by SDS-PAGE was totally inhibited when IgG was treated
with EndoS. To further analyze EndoS' influence on the interaction between FcγRs
on monocytes and IgG, flow cytometry analysis of whole blood was performed.
Human blood was pre-incubated with EndoS before addition of the leukocyte
activator fMLP. Monocytes were gated based on forward and side scatter and the
reactivity of monocytes with monoclonal anti-human IgG was evaluated. The result
revealed that 87% of monocytes were positive for IgG binding, while only 43% of
monocytes in blood incubated with EndoS were positive. These results indicate
that EndoS-hydrolyzed IgG is significantly reduced in its binding capacity to
human cells expressing different sets of FcγRs.
## IgG dissociates from FcγRIIa upon treatment with EndoS
Our experiments this far have revealed that EndoS hydrolysis of IgG inhibits
binding to FcγRs on cells and surfaces, but it remained unclear if EndoS has
activity on IgG already bound to FcγRs and if such activity could release the
IgG bound to FcγRs. Therefore, we investigated the effects of EndoS on IgG bound
to K562 cells that had been exposed to human plasma and subsequently treated
with EndoS. The cell lysates were analyzed by SDS-PAGE and Western blot using an
antibody against human IgG. There was a significant binding of IgG to K562 cells
as judged by the results presented in. Interestingly, no IgG-band was visible on
a blot when cells were treated with EndoS, suggesting a total IgG dissociation
from the cells. A control experiment, using EndoS(E235Q), revealed an IgG signal
on the surface of K562 cells comparable to the untreated cells. These results
strongly suggest that IgG dissociates from the cell surface due to N-glycan
hydrolysis of IgG by EndoS. Similarly, the effect of EndoS on IgG bound to
monocytes was analyzed. This showed that most of the monocyte-bound IgG
dissociated from cells due to the treatment with EndoS as compared to untreated
cells. As expected, monocytes treated with EndoS, in contrast to control cells,
showed no reaction with the LCA lectin, indicating that the minute amounts of
IgG remaining on the cells as detected in the IgG blot had most likely been
hydrolyzed by EndoS. The results demonstrated above were further confirmed by
surface plasmon resonance experiments. Soluble IgG1 and FcγRII receptor were
chosen because of our earlier observation that IgG1 is the strongest binder of
FcγRII. After binding of IgG1 to pre-immobilized FcγRIIa and reaching a steady-
state dissociation phase, the IgG1 injection was aborted and replaced by EndoS
injection or running buffer. This revealed that EndoS injection causes the
dissociation of IgG1 from immobilized FcγRIIa receptor while the IgG1
dissociation from FcγRIIa was unaffected when adding running buffer. Taken
together, these results clearly demonstrate that EndoS by IgG glycan hydrolysis
can release IgG bound to FcγRs on cells and surfaces.
# Discussion
In the present study we attempted to elucidate the physical interaction between
EndoS and IgG and the physiological relevance of EndoS IgG *N*-glycan
hydrolyzing activity for IgG-FcγR interactions. We present for the first time
that EndoS specifically acts as an endoglycosidase on all human IgG subclasses,
both in purified form and in a plasma environment. As expected, there is a
physical interaction between the enzyme and all IgG subclasses, that we
successfully demonstrated using an enzymatically inactive, mutated form of
EndoS. In this study we could not separately investigate the EndoS effects on
the isolated binding of IgG to FcγRI. However, we observed that EndoS-hydrolyzed
IgG did not bind to monocytes, and that there was a nearly complete dissociation
of IgG from monocytes upon hydrolysis by EndoS. Since monocytes express FcγRI
and this receptor has the highest affinity for IgG, we conclude that EndoS
influences IgG binding even to FcγRI because the effects of EndoS observed must
be predominantly due to involvement of FcγRI. EndoS seems to have an effect on
both isotypes of FcγRII receptors, thus influencing both activating and
inhibiting IgG mediated effector stimulation. Interestingly, IgG2 treated with
EndoS, in opposite to what was observed for the other subclasses of IgG, showed
increased binding to FcγRIIb, and slightly also to FcγRIIa, immobilized to
microtiter plate. One possible explanation for this could be aggregation of IgG2
upon hydrolysis by EndoS leading to increased binding to FcγRs. However, no
binding of IgG2 to FcγRs was detected using surface plasmon resonance which is
in agreement with earlier publications. This could be explained by the constant
flow of IgG2 over the immobilized receptors in the case of surface plasmon
resonance, in contrast to ELISA where IgG2 is allowed to aggregate and interact
with the receptor.
The activity of EndoS on IgG has obvious benefits for *S. pyogenes* expressing
this enzyme with potential modulation and/or evasion of an IgG-mediated response
against the bacteria. We have previously demonstrated that EndoS treatment of
human opsonizing IgG antibodies directed towards the cell-wall anchored M
protein significantly enhances the bacterial survival in blood. Therefore, in
the context of an intact infecting *S. pyogenes,* EndoS is a potentially harmful
molecule to the human host that contributes to the bacterial virulence. In
contrast to this, the purified form of EndoS has substantial potential as a
biotechnological tool and/or a therapeutical agent that could be beneficial for
future experimental science and possibly also health care.
Our results reveal that EndoS possesses a capacity to inhibit the IgG binding to
FcγRs and detach IgG bound to FcγRs on cell surfaces. We have recently been able
to show that pre-treatment of arthritogenic antibodies abrogates development of
arthritis in a mouse model of collagen-induced arthritis. This suggests that
EndoS may have potential for being further developed as a therapeutical agent in
other antibody-mediated autoimmune disorders.
We suggest two principally different biotechnological uses of EndoS, one based
on the IgG-glycan hydrolyzing activity of the wild-type enzyme, and the other
based on the high affinity IgG-binding of EndoS(E235Q). The active enzyme could
be used for *in vitro* treatment of whole blood or purified blood cells in order
to remove IgG already bound to various FcγRs on these cells. This could
facilitate the analysis of effects of specific IgG preparations added to the
cells, regarding receptor binding and cellular activation, without the
interference of pre-bound IgG. The inactive form of EndoS (EndoS(E235Q)) has a
great potential as a specific IgG purification and detection tool. In this study
we have demonstrated that EndoS(E235Q) interacts equally well with all
subclasses of IgG. This is comparable to what can be seen for protein G, one of
the major molecules currently used for IgG preparation and detection, but
advantageous compared to protein A that does not bind IgG3. Protein A also binds
IgM and IgA to a certain extent. We have previously shown that there is no
interaction between EndoS and IgM or IgA. Furthermore, we could show here that
EndoS(E235Q) does not interact with IgG lacking its heavy chain glycans. This is
in contrast to both protein G and protein A that bind IgG irrespective of its
glycosylation state. This could be especially important when only intact IgG
with a certain functional effector region is required. When using currently
available reagents like protein G, a second purification step using for instance
a lectin column is required to obtain only the glycosylated fraction of IgG.
This property of EndoS(E235Q) could be used in combination with for instance
protein G to assess the glycosylation state and the functional quality of an IgG
preparation.
In conclusion, EndoS is a bacterial immunomodulatory protein with a great
potential. Our results provide novel information about bacterial pathogenesis,
i.e. how the pathogens evade the immune system of the host organism by affecting
the functions of IgG/FcγRs. Moreover, EndoS could be used as an important
biochemical tool for specific IgG *N*-glycan hydrolysis and IgG
purification/detection, or perhaps as a potential immunosuppressing agent that
could be used to interfere with antibody-mediated pathological processes.
# Materials and Methods
## Proteins and reagents
Blood was drawn from healthy individuals and collected in heparin-containing
tubes. Full-length EndoS with glutathione-S-transferase (GST) as a fusion was
recombinantly expressed and purified from *Escherichia coli* harboring the
plasmid pGEXndoS. When appropriate, the GST-tag was removed using Factor Xa as
previously described. The mutation of glutamic acid 235 of EndoS into glutamine
was performed using QuickChange II Site-Directed Mutagenesis Kit according to
manufacturer's instructions (Stratagene, La Jolla, CA) with following
verification of the mutation by sequencing. Soluble purified Fc-receptors were
generated by co-transfection of CHO-K1(CHO) cells with pNT-neo-FcγRII or pNT-
neo-FcγRIII plasmids with subsequent selection in 1 mg/ml genetecin. IgG-
subclasses were produced by transient transfection in 293T cells. RPMI 1640
medium and Hank´s balanced salt solution (HBSS) were from GIBCO, Paisley, U.K.
All other reagents were purchased from Sigma-Aldrich unless indicated otherwise.
## EndoS-treatment of antibodies
Purified human IgG1–4 was hydrolyzed with GST-EndoS purified as previously
described. Enzyme/substrate molar ratio was 1∶20 in PBS and samples were
incubated for 2 h at 37°C. GST-EndoS was removed from the samples by passing
three times over a glutathione-Sepharose column (Amersham Biosciences, Uppsala,
Sweden). 1 µg of treated and untreated IgG1–4 was separated on 10% SDS-PAGE
followed by staining with Coomassie Blue or *Lens culinaris* agglutinin
(LCA)-lectin (Vector Laboratories, Burlingame, CA, USA) blot analysis (se
below).
## Treatment of human plasma with EndoS and purification of IgG
A volume of 2 ml human plasma was incubated with 20 µg EndoS or a PBS equivalent
for 1.5 hours at 37°C. The IgG fraction was purified using Protein G Sepharose
(GE Healthcare Bio-sciences AB, Uppsala, Sweden). Briefly, 200 µl Protein G
Sepharose suspended 1∶1 in PBS (phosphate-buffered saline; 10 mM phosphate
buffer, pH 7.4, 120 mM NaCl, 3 mM KCl) was added to plasma samples and incubated
at 4°C for 2 hours or over night. After centrifugation for five minutes at
8000×*g*, the supernatant was discarded and the pellet washed three times with
PBS. IgG was eluated with 0.1 M glycine pH 2.0 and neutralized with 1 M Tris-HCl
pH 8.0. The IgG concentration was determined to 8 mg/ml using the Advanced
Protein Assay (Cytoskeleton, Denver, CO, USA).
## Cell preparations
The K562 cell line was cultured in RPMI 1640 medium supplemented with
Glutamax-I, 100 µg/ml antibiotics (penicillin and streptomycin) and 10% fetal
calf serum at 37°C in an atmosphere containing 5% CO<sub>2</sub> and 95%
humidity. Nunclon flasks for cell culture were used (Nunc A/S, Roskilde,
Denmark). Cells were cultured in a serum free medium for 20 hours before being
used in experiments. Monocytes were isolated from human whole blood using the
Polymorphprep kit (AXIS-SHIELD, Oslo, Norway) or Ficoll-Paque Plus (Amersham
Biosciences, Uppsala, Sweden) according to instructions provided by the
manufacturers. After isolation, the cells were counted and resuspended in PBS or
RPMI-medium.
## Enzyme linked immunosorbent assay (ELISA)
For glycan detection, microtiter plates (NUNC, Roskilde, Denmark) were coated
with 100 µL monoclonal mouse anti-human IgG1, IgG2, IgG3 or IgG4 (SIGMA®, Saint
Louis, MO, USA) diluted to final concentrations of 1.5–0.5 µg/ml in a coating
buffer containing 16 mM Na<sub>2</sub>CO<sub>3</sub> and 35 mM
NaHCO<sub>3,</sub> pH 9.6 and kept at 4°C overnight. The plates were washed
three times with lectin buffer containing 10 mM HEPES, pH 7.5, 0.15 M NaCl, 0.01
mM MnCl<sub>2</sub>, 0.1 mM CaCl<sub>2</sub> and 0.1% v/v Tween 20 and blocked
in the same buffer for one hour at room temperature. In the next step purified
IgG fraction (dilution 1∶100) was added and the incubation proceeded for another
2 hours at 37°C. After three washes with lectin buffer, 1 µg/ml biotinylated
LCA-lectin was added and incubation continued for 1 hour at 37°C. Following
three more washes, 0.1 µg/ml peroxidase-labeled streptavidin (Vector
Laboratories) was added and the plate was incubated for 1 hour at 37°C. The
color reaction was developed with 0.1 M citric acid monohydrate, 0.1 M
Na<sub>2</sub>HPO<sub>4</sub>×2H<sub>2</sub>O buffer pH 4.5 containing 0.012%
v/v H<sub>2</sub>O<sub>2</sub> and 1.7 mM 2,2′-azino-
bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The absorbance was read on a
model 550 micro plate reader (BIO-RAD, Hercules, CA, USA) at 415 nm. For
detection of binding of human IgG subclasses to FcγRs, the plate was coated with
soluble FcγRIIa or FcγRIIb or FcγRIIIa at a concentration 5 µg/ml for 20 hours
at 4°C. Next day, the plate was blocked with PBS supplemented with 0.05% v/v
Tween 20 (PBST) and 2% w/v bovine serum albumin for 2 hours at room temperature.
After this step, the purified IgG subclasses, 0.1 µg of each, were added. The
plate was washed three times with PBST after the coating step and between each
of the following incubation steps. A peroxidase-conjugated protein G (dilution
1∶5000) (BIO-RAD, Hercules, CA, USA) was used for detection. The color reaction
was performed as above. All experiments were made in triplicates.
## Radioactive labeling
Proteins were labelled with 0.2 mCi Na <sup>125</sup>I (PerkinElmer, Upplands-
Väsby, Sweden) using the IODE-BEADS Iodination reagent kit (PIERCE, Rockford,
IL, USA) according to the manufacturer's instructions. The unbound radioactivity
was removed by desalting the proteins on PD-10 Sepharose (Pharmacia, Sweden).
The activity of the labeled proteins was estimated to 4 µCi/µg protein.
## Detection of IgG binding to cells
IgG was purified from human plasma treated with EndoS or PBS as described above
and thereafter labeled with <sup>125</sup>iodine (<sup>125</sup>I). For
detection of <sup>125</sup>I-IgG binding to K562 cells, 2×10<sup>6</sup> cells
were incubated with 0.5×10<sup>6</sup> cpm of <sup>125</sup>I-IgG or
<sup>125</sup>I-deglycosylated IgG for 30 minutes at room temperature. After
five washes with PBS and centrifugations at 1000×g for three minutes, the
radioactivity of the cell pellets was detected using Wallac Wizard™ 1470
Automatic Gamma Counter (PerkinElmer, Waltham, MA, USA). To evaluate the
specificity of the binding of radioactive IgG to K562 cells a control experiment
was performed. The cells were incubated with 20 µg human IgG in addition to
radioactive IgG during the similar incubation conditions as mentioned above. In
another experiment, 1×10<sup>6</sup> monocytes were incubated with
0.5×10<sup>6</sup> cpm of <sup>125</sup>I-IgG or <sup>125</sup>I-EndoS treated
IgG for 30 minutes at room temperature. After five repeated washes of cells with
PBS and final pelleting of cells by centrifugation at 1000×g for five minutes,
the cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.4,
0.150 M NaCl, 1% v/v Triton-100 and 0.25% v/v NP40 for ten minutes at 4°C. Next,
the samples were centrifuged for ten minutes at 14000×g and supernatants applied
on a polyacrylamide gel. After separation, the gel was dried and samples
analyzed by phosphoimaging in a Fujix BAS 2000 Bioimaging analyzer (Fujifilm
Sverige AB, Stockholm, Sweden). In an experiment where the binding of IgG to
cells was analyzed by Western blot, 0.5–1×10<sup>6</sup> cells were incubated
with plasma treated with either EndoS or buffer (as described above), at 37°C
for 1 hour. Afterwards, the cells were washed three times with PBS or RPMI
medium, resuspended in 100 µL lysis buffer and the bound IgG in cell lysates
analyzed by Western blot.
## Incubation of cells with EndoS
K562 cells or monocytes, 2×10<sup>6</sup> and 8×10<sup>6</sup> respectively,
were incubated with two ml of human plasma for 30 minutes at 37°C. The cells
were washed five times with PBS and centrifuged at 1000×g for ten minutes after
every wash. EndoS, 40 µg in PBS or PBS alone was added to cells and incubation
followed for one hour at 37°C. Cells were washed three times with PBS and
resuspended in 100 µL lysis buffer. Samples were centrifuged for five minutes at
14000×g, pellets discarded and supernatants analyzed for IgG and glycan contents
using SDS-PAGE and Western blot.
## Slot-blotting analysis
IgG1–4, 0.3, 015 and 0.075 µg of each in PBS were applied to PVDF membranes
using a slot-blot apparatus from Schleicher and Schuell, Inc., Kene, NH 03431,
USA. The membranes were incubated with PBST and 5% skim milk for 1 hour, washed
with PBST and incubated with EndoS or EndoS (E235Q), 0.05 mg/ml in PBST and 5%
skim milk for 1 hour. After washing, the membranes were incubated with rabbit
EndoS-antiserum and subsequently with peroxidase conjugated goat anti-rabbit
antibodies. The color development was made using ABTS as peroxidase substrate.
All incubation steps were performed at room temperature.
## Surface plasmon resonance interaction analysis
Receptors, IgGs and deglycosylated IgGs were diluted with 10 mM sodium acetate
pH 4 and immobilized via amine coupling to different flow cells of CM5
sensorchips (BIAcore, Uppsala, Sweden). Immobilization levels were optimized to
around 8000–10000 response units. After determining EndoS(E235Q) as a non-binder
to all deglycosylated IgG variants, these flow cells were considered as controls
for bulk refraction index changes for EndoS(E235Q) binding to IgG1 throughout
IgG4, respectively. In experiments determining IgG1-IgG4 affinity for the
receptors FcγRIIa, FcγRIIb and FcγRIIIa, a flow cell subjected to the
immobilization protocol but without addition of protein was used as control. For
affinity measurements, the binding and dissociation phases were monitored in a
BIAcore 2000 instrument. In control experiments for possible mass transfer
limitations, the IgGs were injected over the receptors and the EndoS variants
over the IgG sub-classes at different flow rates. No differences in initial
binding were observed at 5 µl/min or above indicating no limitations to any
combinations. Interactants were injected in different concentrations (typically
10–1.25 µg/ml) at 35 µl/min and 25°C over the different coated surfaces (flow
cells) (in running buffer: 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.005% surfactant
P20, and 3.4 mM EDTA). Between experiments, the surfaces were strictly
regenerated with pulses of running buffer containing 2 M NaCl followed by an
extensive wash procedure after reaching baseline. For EndoS digestion of IgG
bound to pre-immobilized FcγRIIa, an IgG1 concentration (10 µg/ml) was chosen to
give a suitable steady-state dissociation phase at a time point were the IgG1
injection was aborted and replaced by running buffer. This experiment was
considered as a control and as such compared to an EndoS injection at the same
time point after IgG1 binding to FcγRIIa. After X and Y normalization of data,
the blank curves from control flow cells of each injected concentration were
subtracted. Where applicable, the association (*k<sub>a</sub>*) and dissociation
(*k<sub>d</sub>*) rate constants were determined simultaneously using the
equation for 1∶1 Langmuir binding in the BIA Evaluation 4.1 software (BIAcore).
The binding curves were fitted locally and the equilibrium dissociation
constants (*K<sub>D</sub>*) were calculated from mean values of the obtained
rate constants.
## Flow cytometry analysis of whole blood
A volume of 15 ml blood was incubated with 0.4 mg EndoS or PBS for 35 minutes at
37°C. An activator of leukocytes, formyl-methionyl-leucyl-phenylalanine (fMLP),
(dilution 1∶10000) (SIGMA, Saint Louise, MO, USA) was then added and the
incubation continued for 10 minutes at 37°C. Next, blood samples were
centrifuged 1000×*g*, five minutes. Plasma and buffy coat were transferred to
another tube and centrifuged for five minutes at 1000×*g*. The cells were then
washed three times with HBSS containing 30% v/v RPMI and finally resuspended in
100 µl of the same medium. Monoclonal mouse anti-human IgG was prepared by
mixing equal amounts of mouse anti-human IgG1 (51 mg/ml), IgG2 (22 mg/ml), IgG3
(16 mg/ml) and IgG4 (24 mg/ml). Five µl of this mixture was added and samples
incubated for ten minutes at room temperature. In the next step 5 µl of FITC-
conjugated goat anti-mouse IgG (DakoCytomation, Glostrup, Denmark) was added
before erythrocytes were lysed using the DakoCytomation Uti-Lyse erythrocyte kit
(Carpinteria, CA). Signals were analyzed on a FACSCalibur flow cytometer (Becton
Dickinson, Franklin Lakes, NJ, USA). Monocytes were identified by forward
scatter and side scatter characteristics (FSC/SSC).
## SDS-PAGE and Western blot analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed using Mini Protean II cell equipment from BIO-RAD (Hercules, CA, USA)
or equipment from LKB (Bromma, Sweden) using the buffer system described by
Laemmli. Samples were mixed 1∶1 (v/v) with sample buffer supplemented with 5%
mercaptoethanol, and boiled for five minutes before loading onto the 10%
polyacrylamide gel. PageRuler™ Protein Ladder Plus (Fermentas, Burlington,
Canada) was used as high-molecular-mass standards. The polyacrylamide gels were
stained with Coomassie Brilliant Blue R-250 and in some cases dried. For
immunoblotting, the gels were transferred to polyvinylidenefluoride (PVDF)
membranes (Immobilon P, Millipore, Bedford, MA) as described by Matsudaira (18).
After blotting, membranes were blocked in PBS supplemented with 0.05% v/v Tween
20 (PBST) and 5% w/v skim milk (DIFCO, Detroit, MI) for 20 minutes at room
temperature. For detection of IgG, the blots were subsequently washed in PBST
and then incubated with rabbit anti-human IgG (diluted 1∶3000) (BIO-RAD,
Hercules, CA) for one hour at 37°C. After a washing step, membranes were
incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (BIO-RAD)
(dilution 1∶1000). For lectin blot analysis, membranes were blocked for 20
minutes in lectin buffer (10 mM HEPES, pH 7.5, 0.15 M NaCl, 0.01 mM
MnCl<sub>2</sub>, 0.1 mM CaCl<sub>2</sub> and 0.1% v/v Tween 20) at room
temperature and incubated with biotinylated LCA lectin (diluted 1∶5000). After
repeated washes in lectin buffer the membranes were incubated with peroxidase-
labeled streptavidin (Vector Laboratories) (diluted 1∶10000). All membranes were
developed using SuperSignal West Pico (PIERCE, Rockford, IL) according to the
manufacturer's instructions before analyzing by the Chemidoc XRS imaging system
and Quantity One image analysis software (BIO-RAD).
The authors are indebted to Dr. Jeffrey V. Ravetch's laboratory for preparation
of Fc-receptors and IgG-subclasses.
[^1]: Conceived and designed the experiments: MA MC AO FN. Performed the
experiments: MA MC AO. Analyzed the data: MA MC AO. Contributed
reagents/materials/analysis tools: FN. Wrote the paper: MA.
[^2]: A patent application on the in vitro use of EndoS has been filed with
MA, AO, FN, and MC listed as inventors. The application is pending. |
# Introduction
Periodontal disease is an infectious disease of the supporting structures of
teeth that affects over 47% of American adults. In the elderly, 65 and older,
the prevalence rate increases to over 70%. The cost that is associated with the
treatment of PD represents a significant fraction of all of the expenses related
to dental care, which totals on average, \$113 billion a year in the US.
Periodontitis is characterized by an acute inflammatory process, osteoclast
activity, as well as connective tissue destruction, which is then characterized
by a pro-inflammatory profile of cytokine release, known as Th1. Among the
cytokines with a Th1 profile, we can cite IL-1β, IL-6 and TNF-α. Periodontal
disease is often associated with other chronic systemic conditions, such as
cardiovascular diseases, diabetes, and asthma..
Asthma is defined as a chronic inflammation of the airways, with recurrent and
reversible episodes of dyspnea, chest stiffness, coughing and wheezing. Its
prevalence can vary from 1% to 18%, depending upon the studied population.
According to the World Health Organization (WHO), it estimates that 235 million
people worldwide suffer from asthma. The socio-economic implications are
considerable, when one considers work absenteeism, hospitalization costs,
medicines, a decrement in the quality of life, and premature death. The
respiratory system becomes hyperresponsive and the major consequence is a
reversible mechanical obstruction of the airways. Although the primary cause may
vary, it can be triggered by several factors, classified as predisposing, causal
and contributory. After the sensitization phase, the asthmatic patient presents
with eosinophil infiltrate, activated mast cells on the airways surface,
together with activated T lymphocytes, with a profile of cytokine release, known
as Th2. Among the cytokines with a Th2 profile, we can highlight IL-4, IL-5 and
IFN-γ, due their effects during the allergic responses. In addition, the Th2
profile has also been described as being involved in the progression of
periodontitis.
Some studies linking periodontitis and asthma have been proposed. The lack of
standardization for a periodontitis diagnosis and the inclusion of patients with
different ages make it difficult to compare studies. As for asthma, a diagnosis
is often made by a self-reporting of the disease. Further studies are needed in
order to elucidate the link between these two pathologies. In a recent study
with 5.976 patients, a positive association was found between periodontitis and
asthma, while there is an inverse association when the patients have been taking
antiasthmatic medication. The causal relationship between them is still unclear.
Allergies have been negatively associated with clinical attachment loss
hypothesizing that a periodontopathogenic colonization of an oral cavity could
have a protective effect on an allergic disease. Other authors have found an
inverse association between a clinical attachment loss and asthma. Corroborating
with human studies, some authors evaluated the immunoregulatory mechanism of
asthma during a periodontitis. This study determined if a subcutaneous infection
with *Porphyromonas gingivalis* exerted a regulatory effect on the allergic
airway inflammation. The authors showed a reduction of lung inflammatory cells,
as well as cytokines, after a *P*. *gingivalis* infection, prior to an allergen
sensitization with ovalbumin (OVA).
The standard PD treatment aims to reduce the biofilm microorganisms through
scaling and root planning, followed by an oral hygiene control by the patient.
However, it is not able to eliminate the subgingival pathogens and the calculus.
There may be a bacterial re-colonization at inaccessible periodontal sites.
*Aggregatibacter actinomycetemcomitans* and *Porphyromonas gingivalis* have been
found in infected pulmonary fluids. Nowadays, antibiotics are indicated for a
restricted group of patients, as an adjuvant to a periodontal treatment, due to
the risk of a bacterial resistance development. Antimicrobial photodynamic
therapy (PDT) is an adjuvant to a periodontal treatment, in order to reduce the
amount of microorganisms in the localized infections of the subgingival sites.
The principal advantage of this adjuvant is that the light and the
photosensitizer reach places where the conventional treatment with curettes only
has partial access. The mechanism is based upon the activation of a
photosensitizer drug, by light at a suitable wavelength, generating oxidative
species, such as hydroxyl radicals, superoxide and singlet oxygen. These species
act in the bacteria organelles, damaging structures and loosing essential
functions for survival. However, there is an absence of literature reports
regarding a bacterial resistance to PDT. Cationic photosensitizers bind to
bacteria due to electrostatic interactions, since there is a negative potential
in the cell’s surface. Regarding this concept, phenothiazinium dyes have been
widely studied as photosensitizers, with methylene blue and toluidine blue being
the main ones. Methylene blue presents a broad spectrum of activity against
bacteria, being effective in the inactivation of *P*. *gingivalis*, *P*.
*intermedia*, and *A*. *actinomycetemcomitans* bacteria, with a key role in a
periodontitis. Despite the advantages of PDT, there is a lack of well-designed
clinical studies for a proper evaluation of this therapy. There are no reports
of a bacterial resistance or side effects, with a preservation of the oral
microbiota and a low toxicity, unlike a treatment with antibiotics or a
mouthwash with chlorhexidine. Notwithstanding the advantages of PDT, well-
designed studies on this subject are needed.
There is an important gap in the literature, evaluating whether a periodontitis
is capable of influencing the development of a pulmonary disease. In addition,
if the suppression of the infectious agent of the periodontium is able to
influence the inflammatory parameters of asthma. Therefore, the hypothesis of
this study has been to evaluate whether periodontitis modulates lung
inflammation in an experimental model of asthma as well as the photodynamic
therapy (PDT) is associated with a reduction of lung inflammation
# Material and methods
This study was approved by the Animal Ethics Committee of the University Nove de
Julho (UNINOVE), São Paulo, Brazil, under \#020/2015. The mice were maintained
in a constant temperature of 22°C to 25°C, with a 12-h light/dark photoperiod,
under artificially controlled ventilation, with a relative humidity ranging from
50% to 60%. Rations (NUTRILAB CR-1<sup>®</sup>) and water were provided *ad
libitum*. Seventy-two BALB/c male mice (\~2 months) were randomly divided into 8
groups (n = 9): 1) Basal—without any induction of a disease, 2) P—Induction of
Periodontitis, 3) P+TP—Induction of Periodontitis + Standard Periodontal
Treatment, 4) P+PT+PDT—Induction of Periodontitis + Standard Periodontal
Treatment + Photodynamic Therapy, 5) Asthma—Induction of Asthma, 6) Asthma
+P—Induction of Asthma + Induction of Periodontitis, 7) Asthma+P+TP—Induction of
Asthma + Induction of Periodontitis + Standard Periodontal Treatment, 8)
Asthma+P+TP+PDT—Asthma Induction + Induction of Periodontitis + Standard
Periodontal Treatment + Photodynamic Therapy. All of the manipulations of the
animals (periodontitis induction, periodontal treatment, PDT) were performed by
only one operator (Candeo LC). Seventy-two animals were identified. The
randomization of the animals was performed (Microsoft Excel, Version 2013) by
separating them into 8 blocks (groups) of 9 animals. For the induction of
asthma, the animals were injected subcutaneously with 4 μg ovalbumin (OVA)
(SIGMA<sup>™</sup>), together with aluminum hydroxide solution, on the first day
of the experiment (sensitization), and then 14 days thereafter (booster). From
the 14<sup>th</sup> day, the animals were submitted to a nebulization
(challenge) with 10 μg OVA, 3 times a week, for 2 weeks.
Periodontitis was induced by using a modification of a previously described
method of ligature-induced periodontitis in mice. The ligature method is
detailed at. The method of the periodontitis induction was realized by one
operator when using an ophthalmological nylon silk ligature of 6–0
(SHALON<sup>®</sup>, São Paulo, Brazil). It was gently introduced into the
interproximal area between the first mandibular molar and the second mandibular
molar with two curved needle-holders (DLMICOF<sup>®</sup>, Sao Paulo, Brazil)
that were developed for this study. The silk ligature was gently tied in order
to avoid damaging the periodontal area around the first molar. In order to
perform these procedures, the mice were anesthetized with an intraperitoneal
injection of ketamine (100 mg/kg) (DOPALEN<sup>®</sup>, VETBRANDS, São Paulo,
Brazil) and the muscle relaxant xylazine (10 g/kg) (ANASEDAN<sup>®</sup>,
VETBRANDS, Brazil). They remained sedated for approximately 90 minutes. After 15
days, the ligatures were removed and the periodontal treatments were performed
in a standardized manner in the groups P+PT, P+PT+PDT, Asthma+P+PT, and
Asthma+P+PT+PDT. No antibiotics or anti-inflammatories were administered. A Mini
Five 5/6 curette was used and crown-root scrapings/ planning were performed on
the vestibular lingual, mesial and distal faces. PDT was then employed as an
adjuvant therapy to the periodontal treatments in the P+PT+PDT and
Asthma+P+TP+PDT groups. For such, the photosensitizer methylene blue
(0.005%—CHIMIOLUX, DMC, São Paulo, Brazil) was administered with a syringe,
cartridge and needle (with a stop and without a bevel) at the two sites
(vestibular and lingual). After three minutes, the periodontal pockets were
irradiated with a red laser (λ = 660 nm ±10nm) (THERAPY XT, DMC, São Carlos, São
Paulo, Brazil—ANVISA 80030810157). The radiant power of the appliance is 100 mW.
The spot (area) was 0.02827 cm<sup>2</sup>. The radiant energy delivered per
point was 9J in 90 seconds. The radiant exposure was 318 J/cm<sup>2</sup> and
irradiance was 3.5W/cm<sup>2</sup>. In the vestibular face 9J were applied and
another point in the lingual face (9J) of the right first molar also applied.
followed by abundant rinsing with water for complete removal of the methylene
blue. We irradiated with a small spot (0,02827mm<sup>2</sup>) because the tooth
size of the 1<sup>st</sup> mandibular molar of mice is about 1.44 ± 0.011 mm at
vestibular face and 0.80 ± 0.019 mm in the buccolingual length.. All periodontal
treatment (with or without PDT) was performed in a single session Euthanasia and
posterior analyzes of the material were performed, 43 days after the beginning
of the study The complete timeline of the induced ligature (15 days), the
periodontal treatment (7 days), the PDT (7 days) and the euthanasia (43 days),
for the 8 groups of this study, is detailed in. After the experimental period,
the mice were euthanized with an overdose of ketamine (1.6 g/10 ml of solution)
and xylazine (3g/100ml of solution).
The blood samples were collected from the aortic artery of the mice by
exsanguination, for a cell count by using Sysmex<sup>™</sup> C9.0 Software for
the hemogram test. For an evaluation of lung inflammation in the BAL
(bronchoalveolar lavage), the animals were tracheostomized and cannulated. Their
lungs were washed with 3x0.5ml phosphate buffered saline (PBS). The volume of
recovered BAL was centrifuged (1600rpm, 5 min at 4°C). The supernatant was
collected and stored at -70°C for the cytokine analyzes by enzyme-linked
immunosorbent assays (ELISA). The cell button was resuspended in 1 ml of PBS and
was used for the total cell count. Ten microliters of the samples were added to
Trypan Blue for a total cell counting with a Neubauer chamber. One hundred
microliters were used to prepare the laminae for a differential counting of the
cells (5 min, 1900rpm, 4°C) (Cytospin II—Shandon Instruments, Sewickley, PA,
USA). The staining of the slides was performed with Instant-Prov. Three hundred
cells were counted per laminae. The lung fragments were fixed in a 4% solution
of paraformaldehyde, with 0.1 M of Sorensen’s phosphate buffer, at pH 7.4, at
4°C, for 24 h in anhydrous alcohol, followed by dehydration in alcohol and by
diaphanization in xylol. The fragments were embedded in paraffin, sectioned to 5
μm with the aid of a microtome (HYRAX M60, Zeiss, GR), de-paraffinized, cut to a
thickness of 5 μm, and then stained with periodic acid-Schiff for analyzes of
the mucus. The protocol was detailed in. The internal and external limits of the
respiratory epithelium was delimited (Image Pro-Plus 7.0) The mucus area was
determined by area of glycoprotein component relation to total area of the
respiratory epithelium. The results are expressed as the percentage (%). The
measurements were performed in the five airways of each animal at ×400
magnification.
The hemi-jaws were dissected and fixed in 10% buffered formalin solution (Merck
& Co. Inc, New Jersey, USA) at pH 7 for a period of 24–48 hours. They were
decalcified in 4% EDTA for 2 months. The mandibles were then dehydrated in
alcohol solutions and diaphanized in xylol. The fragments were embedded in
paraffin, sectioned to 5 μm with a microtome (HYRAX M60, Zeiss, GR), de-
paraffinized, cut to a thickness of 5 μm and stained with hematoxylin and eosin.
Serial cuts were used involving the best specimens.
The morphometric analyzes were performed by a single examiner. Kappa values were
used in order to measure the intra-observer agreement of measures. The observer
was an experienced pathologist, França CM, and he had an intra-observer
agreement of 0.85. This pathologist followed some references in order to proceed
with the morphometric analyzes. The histological sections were photographed by
using an Olympus Bx43 microscope with the help of Olympus cellSens<sup>™</sup>
software. The measurements of bone loss were performed with Image J Software
Version 1.45. The distance between the cementum-enamel junction (CEJ) to the
alveolar bone (the distal region of the first molar) was measured in
millimeters. The linear measurements of each slide were summed in order to
obtain an average value for each animal. The concentrations of cytokines were
determined in the supernatant samples of the lavage fluid and the serum. The
results were expressed as picograms of cytokine produced per mL. The Interleukin
Th2 (IL-10, IL-4, IL-5, IFN-γ) and the Th1 (IL-1, IL-6, INF-γ) profiles were
quantified by using ELISA (BioLegend, San Diego, USA). The determinations were
performed in duplicate for each sample by using standard curves and following
the manufacturer’s specifications. The quantification of serum alkaline
phosphatase was also quantified by using ELISA (BioLegend, San Diego, USA).
Statistical analyzes were performed by a blind statistician using the GraphPad
Prism program (GraphPad Software, Inc). The Kolmogorov-Smirnov test was used in
order to determine the data distribution. Since the data was parametric, a one-
way analysis of variance (ANOVA) was employed, followed by the Student-Newman-
Keuls test. A p-value \< 0.001 was considered indicative of statistical
significance.
# Results
The quantification of the cells that were recovered in the bronchoalveolar
lavage (BAL) of the asthmatic mice shows that the total count of the
inflammatory cells (x10<sup>4</sup>/ml) in the bronchoalveolar lavage (BAL) was
increased in the periodontitis (P) group (p \<0.05) and in the asthma group
(Asthma) (p \<0.001) when compared with the basal group (B). The periodontal
treatment (P + PT) (p \<0.05) and the periodontal treatment associated with PDT
(P + TP + PDT) (p \<0.05) were able to decrease the total amount of inflammatory
cells in the bronchoalveolar lavage when compared to the periodontitis (P)
group. The association of the periodontitis with asthma (P + Asthma) reduced the
number of the total cells that were recovered in the BAL when compared to the
group with asthma (Asthma) (p \<0.001). There were no differences in the total
number of BAL cells after the standard periodontal treatments in the asthmatic
mice (Asthma + P + TP) when compared with the asthmatic mice in the
periodontitis group (Asthma + P). On the other hand, when photodynamic therapy
was associated (Asthma + P + TP + PDT), there was a significant increase in the
total number of cells (p \<0.001) in relation to the periodontitis with asthma
group (Asthma + P).
In the quantification of the differential number of cells that were recovered
from the BAL there was an increase (p\<0.001) in eosinophils for the (P) and
(Asthma) groups in relation to the Basal group. The treatment with PDT decreased
(p \<0.001) the amount of eosinophils in the (P + TP + PDT) group when compared
to the (P) group. When associated with asthma, the two modalities of TP (Asthma
+ P + TP) and (Asthma + P + TP + PDT) increased (p\<0.001) the number of
eosinophils when compared with the (Asthma + P) group. There was a reduction
(p\<0.05) in the macrophage differential count (x10<sup>4</sup>/ml) of the BAL
for the (P+PT) group in relation to the (P) group. When we associated asthma
with P (Asthma + P), there was a reduction of cells (p\<0.001) in relation to
the Asthma group. The periodontal treatment (Asthma + P + TP) and PDT (Asthma +
P + TP + PDT) in the asthmatic mice increased (p\<0.001) the amount of
macrophages when compared to the (Asthma + P) group. The amount of lymphocytes
increased (p\<0.001) for the (P) group and for the (Asthma) group (p \<0.01)
when compared to the (Basal) group. For the (P+PDT) group, there was a decrease
(p \<0.001) in relation to the (P) group. For the (Asthma + P) group, there was
a decrease (p \<0.05) in relation to the (Asthma) group. When the treatments
were associated, the (Asthma + P + TP) group and the (Asthma + P + TP + PDT)
group had increased (p \<0.001) lymphocytes in relation to the (Asthma + P)
group. There was an increase in neutrophils (p\<0.001) for the (Asthma + P + TP)
and (Asthma + P + TP + PDT) groups when compared to the (Asthma + P) group.
For the quantification of mucus in the airway (Figs) there was an increase
(p\<0.001) in the mucus production in the asthma (A) and periodontitis (P)
groups (p \<0.01) when compared to the basal (B) group. The association of P
with asthma (Asthma + P) decreased the mucus production when it was compared to
the (Asthma) group.
For the analyzes of the IL-1β, IL-6 and TNF-α cytokines in the BAL supernatant,
the inflammatory cytokines related to the Th1 mechanism were analyzed. An
increase (p \<0.01) of IL-1β production in the periodontitis (P) group was
observed when compared to the basal group (B). There were no differences in the
other groups that were analyzed (p\> 0.01). There was an increase (p\<0.001) in
the IL-6 levels in the BAL of the (P) group when compared to the basal (B)
group. On the other hand, there was a reduction (p\<0.001) in the IL-6
production in the treatment groups (P + TP) and (P + TP + PDT) when compared to
the (P) group. We also observed an increase (p\<0.001) in the production of
TNF-α for the (P) and (A) groups when compared with the basal (B) group. It was
observed that the standard periodontal treatment (P+TP) alone or in a
combination with the photodynamic therapy (P + TP + PDT) decreased the
production of TNF-α in the BAL, respectively (p\<0.001 and p\<0.01), when
compared with the (P) group. The association of P with asthma (Asthma + P)
decreased (p \<0.05) the TNF-α production when compared to its control group,
the (Asthma) group.
For the analyzes of the IL-4, IL-5 IL-10 and IFN-γ cytokines in the BAL
supernatant–the cytokines related to the mechanism (Th2) of asthma were
evaluated in the BAL supernatant. There was an increase (p \<0.001) in the
production of IL-4 in the (A) group when compared to the basal (B) group. The
association of P and asthma (P + A) decreased (p\<0.001) the production of IL-4
in relation to the (Asthma) group. In addition, the periodontal treatment
(Asthma+P+TP) and the periodontal treatment with PDT in the asthmatic mice with
P (Asthma+P+TP+PDT) decreased (p\<0.01) the level of IL-4 in the BAL when
compared to the (Asthma+P) group. There was an increase (p\<0.01) in the IL-5
levels of the BAL in the (P) and (Asthma) groups when compared with the basal
(B) group. On the other hand, there was a decrease (p\<0.01) in IL-5 when the
periodontal treatments of (P+TP) and PDT were performed, (P+TP+PDT) when
compared to the (P) group. It was observed that the PDT treatment (A+P+TP+PDT)
in asthmatic mice increases the IL-10 production in the BAL when compared to the
Asthma + P group (A+P). A decrease in the IFN-y levels (p\<0.05) in the
periodontitis (P) and asthma (Asthma) groups when compared to basal (B) group
was observed in. The standard periodontal treatment (P+TP) increases IFN-γ.
Quantification of serum alkaline phosphatase—there was an increase in the
production of alkaline phosphatase (U/L) in the (P) group when compared to the
basal (B) group (p\<0.001). PT that was associated with PDT (P+ PT+PDT)
decreased the alkaline phosphatase production (p\<0.01) in relation to the (P)
group.
An increase (p\<0.001) in bone resorption was observed in the (P) group when
compared to the basal (B) group. PT that was associated with PDT (P+PT+PDT) was
able to decrease the bone resorption (p\<0.01) in the alveolar bone region when
compared to the (P) group. The asthmatic mice in which P was induced (Asthma+P)
presented a greater resorption (p\<0.001) than did the group in which only
asthma (A) was induced. The PT (Asthma+P+PT) and PDT (Asthma+P+PT+PDT) groups
had lower values of bone resorption (p\<0.01) than did the asthmatic mice with
periodontitis (Asthma+P). These values are shown in and illustrated in.
# Discussion
Periodontitis is defined as a chronic inflammation that destroys the supporting
tissues of the teeth. It was characterized based on high levels of bone
resorption, and alkaline phosphatase which are well-established methods in the
literature. As we described before, periodontitis is characterized by an acute
inflammatory process, which is then characterized by a pro-inflammatory profile
of cytokine release, known as Th1. Among the cytokines with a Th1 profile, we
can cite IL-1β, IL-6 and TNF-α. Periodontitis group (P) increased the amount of
alkaline phosphatase and bone resorption, thus validating the experimental model
of periodontitis and increase all the Th1 cytokines profile.
The asthma model involved the induction of an allergic reaction using ovalbumin,
which is recognized as mimicking signs and symptoms found in asthmatic patients.
The experimental model for the induction of asthma has increased cellularity in
the BAL represented by eosinophils, lymphocytes and neutrophils, as well as an
increase in cytokines with a Th2 profile (IL-4 and IL-5), together with the
production of mucus in the airways. This was an expected result for the
asthmatic animals. To better understand the mechanism linking these two
diseases, we studied the Th1/Th2 immunoregulatory control through its different
cytokine profiles.
Interestingly, the combination of asthma and periodontitis led to a significant
reduction in the inflammatory cells (eosinophils, lymphocytes, macrophages) as
well as the IL-4 cytokines release in the lungs. This decrease was accompanied
by a reduction in the amount of mucus. It can therefore be inferred that
periodontitis exerted some influence on pulmonary inflammation. In this group
(A+P), bone resorption remained high, showing that periodontitis was still
active. These findings are in agreement with data described previously by
researchers studying the association between periodontitis and allergy. This
study revealed an inverse relationship between periodontitis and allergy in a
sample of 2.837 individuals. The authors discussed whether exposure to
periodopathogens may influence the asthma progression. In other words, the
regulation of the Th1-Th2 balance was deviated to the Th1 response in the
asthmatic mice when periodontitis was associated with asthma. For these reasons,
we have inferred that our results have an inverse relationship.
Conversely, in a recent study with 5.976 patients, a positive association was
found between periodontitis and asthma, while there is an inverse association
only when the patients have been taking antiasthmatic medication. The causal
relationship between them is still unclear. Further studies are needed to
elucidate the link between these two pathologies.
Considering the controversial data regarding the correlation between
periodontitis and asthma, we have evaluated the effects of its treatment with
conventional periodontal treatment and with PDT.
When comparing the effectiveness of the two periodontal treatments in the mice
without asthma, we noticed that the conventional treatment decreased the
macrophages, as well as IL-6 and TNF-α, corroborating with the literature.
However, it was interesting to note that the treatments appeared to act slightly
differently, since PDT decreased the number of eosinophils and IL-5 in the mice
without asthma and caused less bone resorption as well. Further studies are
necessary to better understand the influence od PDT in Th2 mechanisms.
In the asthmatic mice, after conventional periodontitis treatment, an increase
in macrophages, lymphocytes, neutrophils and eosinophils were found. This
variation in cellularity was followed by decreased IL-4 and TNF-α production.
Moreover, standard treatment was able to interrupt the bone resorption process,
confirming what occurs in clinical practice. Interestingly, PDT in asthmatic
mice also increased the total cell count (macrophages, lymphocytes, neutrophils
and eosinophils) in the BAL. Although the increase in defense cells is a
disadvantage of this form of treatment, the cytokine release pattern was
favorable, as the anti-inflammatory cytokine IL-10 was released in a greater
quantity only for this group. These results suggest that PDT could stimulate the
cellular immune response as demonstrated by some authors. Further studies could
investigate if both M2 (macrophages) and Treg (regulatory T cells) are
implicated in these processes producing anti-inflammatory cytokines.
Although the mechanisms of the action are different, when photobiomodulation was
directly applied in the lungs, has reduced the TNF-α expression after an acute
immunocomplex lung injury in rats. They tested different doses of laser and they
found a dose-dependent reduction of TNF-α levels in acute inflammation. In our
study we also found a decrease in TNF-α levels in the BAL after a PDT. One
limitation of this study was not to study the systemic alterations (serum) of
cytokines release. More studies are needed in order to understand these systemic
interactions.
Photodynamic Therapy is known as the first order of antimicrobial action in a
periodontal treatment and has been largely studied. Photosensitizers affect
bacterial cells, damaging structures and impeding essential functions for
bacterial survival. We selected methylene blue because it is a widely studied
photosensitizer for antimicrobial PDT, due to its absorption around 650nm and
its photophysical and photochemical properties. Besides that, it has a positive
charge compound which binds to the bacteria due to an electrostatic interaction.
Some studies have also shown an effective antibacterial action. Methylene blue
is commercialized by different companies and it may be used in different
formulations/concentrations. Some well-known brands are HELBO<sup>®</sup> and
Chimiolux. In Brazil, we have selected methylene blue because is approved by the
Brazilian Health Regulatory Agency (ANVISA), and is widely used for
periodontitis treatment. Besides, different chemical names for this molecule are
also used, such as 3,7-Bis(dimethylamino)phenazathioniumchloride, Basic Blue 9,
Tetramethyl Thionine Chloride and Phenothiazine Chloride. The nomenclature
“phenothiazine” is not the most suitable of products since phenothiazines are
tricyclic yellow compounds and not photosensitizers.
Finally, it was interesting to note that we had an unexpected result in one of
our control groups (P+PT). Our data has shown that periodontitis group (P) *per
se* caused an increase in the total number of cells, mainly in the eosinophils
and lymphocytes, as well as in the IL-5 cytokine release and mucus production.
Thus, this work has contributed in elucidating, in part, the relationship
between P and asthma. Additional studies involving functional measurements of
airway hyperreactivity and the investigation of structural changes in the
airways are needed to gain a better understanding of the effects of periodontal
treatment in patients with asthma.
In conclusion, periodontitis in the asthmatic mice reduced the inflammatory
migrated cells in the BAL (eosinophils, lymphocytes, macrophages), as well as in
reduce the levels of the IL-4 and TNF-α cytokines, which was additionally
accompanied by a decreased mucus production. After the removal of the causative
agent of the periodontal inflammation/infection (PDT treatment), the total cell
counts increased, but this increase was not accompanied by a pro-inflammatory
cytokines release. Only in the PDT group an anti-inflammatory cytokine (IL-10)
was increased. More studies are needed in order to understand these mechanisms
of action.
# Supporting information
We would like to thank Alana Dias dos Santos for her laboratorial assistance
[^1]: The authors have declared that no competing interests exist.
[^2]: ‡ These authors also contributed equally to this work. |
# Introduction
The neurotrophins (NT) are a family of proteins comprising nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), and
NT-4/5, initially identified through their crucial function in nervous system
development, growth control, and neuron and astrocyte apoptosis. However, the
effects of these growth factors are much more broad, and extend to a wide range
of cell types, including immune cells. Indeed, growing evidence suggests that
NGF, BDNF, and NT-3 participate in inflammatory responses, including the
modulating and regulating immune function in inflammatory and autoimmune
diseases.
Neurotrophin function in immune regulation has been assessed in several reports
that demonstrate that immune cells both secrete and are targets of the three
major NTs (NGF, BDNF and NT3). Indeed, after activation, B cells, plasmocytes
and T cells express NT receptors (TrkA, TrkB, TrkC and p75<sup>NTR</sup>) and
produce functional NGF, BDNF, and NT-3, which is involved in lymphocyte
maturation, proliferation, and activation. Concerning B lymphocytes, NGF is
secreted during B cell activation, which triggers their proliferation and
differentiation into plasma cells. Immunoglobulin secretion (IgG and IgM or IgE)
is enhanced by NGF or BDNF. In addition, BDNF also plays an important role in B
cell development. This was demonstrated in BDNF<sup>-/-</sup> mice, which show a
developmental arrest in B cell maturation at the pre-BII stage. Lastly, both NGF
and BDNF are autocrine factors for mature B and plasma cell survival, whereas
this function for NT-3 has only been reported during inflammatory conditions.
NTs also exert important functions in T lymphocytes: they may promote T cell
activation of a Th2 or Th1 profile. Indeed, NGF enhances Th2 cell proliferation
and modulates T-cell-dependent antibody synthesis and T cell production of
gamma-interferon (INF-γ). Moreover, CD40L, interleukin-1β (IL-1β), IL-4 and
*tumor necrosis factor-alpha* (TNF-α) upregulate production of NGF in
lymphocytes. In contrast, INF-γ decreases NT synthesis. Th2 cytokines can also
upregulate BDNF production in immune cells. NT3 synthesis is enhanced in
Th1-activated human lymphocytes. Thus, these data support a potential crosstalk
between NTs and Th1 and Th2 cytokine profiles during the inflammatory response.
Data on immune cell expression of NT-4/5 are sparse. NT-4/5 is expressed by 25%
of human circulating peripheral blood mononuclear cells (PBMC), activated human
T cells, and murine alveolar macrophages. However, the function of this
neuropeptide, known to interact with the TrkB receptor in neural cells, remains
unknown in immune cells.
The relationship between NT-secreting immune cells and the resulting tissue
damage has been evaluated in some chronic inflammatory-autoimmune diseases.
During rheumatoid or psoriasis arthritis, synovial CD3+ T lymphocytes and
monocytes/macrophages produce high levels of NGF, which enhance both fibroblast-
like cell proliferation and synovial T cell activation via TrkA ligation and Akt
phosphorylation. In sarcoidosis, epithelioid and multinucleated giant cells of
the granuloma, alveolar macrophages and T cells produce NGF, BDNF and NT-3. CD4
and CD8 NT expression correlates with the sarcoidosis radiological damage index.
In contrast, in Crohn’s disease, local secretion of NT, especially NGF and BDNF
by mast cells, reduces enteric glia cell apoptosis induced by pro-inflammatory
cytokines.
Together, these findings suggest that NT, excessively produced by immune cells
in autoimmune diseases, may participate in disease progression by modulating
both immune cell function and tissue lesions.
Based on this foundational data, other studies have evaluated serum NT levels in
various autoimmune and pro-inflammatory diseases. However, these reports have
mainly dealt with NGF. Indeed, serum NGF concentrations are increased in
juvenile arthritis, Kawasaki disease, Behçet’s disease, systemic sclerosis and
primary Sjögren’s syndrome. Increased BDNF levels in sera have also been
reported in primary Sjögren’s syndrome, which correlates with systemic activity
and B and T cell activation. In contrast, serum BDNF levels are decreased in
systemic sclerosis, reflecting the vascular aspect of the disease. It has also
been reported that NT-3 is upregulated only in autoimmune diseases strongly
affecting the joints. Serum NT-4/5 levels are upregulated in mood disorders but
have not been yet evaluated in autoimmune disease.
There is little data on lymphocytic NT expression in human inflammatory disease.
BDNF-secreting T cells are reduced in untreated multiple sclerosis patients and
increased after interferon beta treatment, while NGF, NT-3 and NT-4 production
by PBMCs in multiple sclerosis patients is enhanced in the post-relapse phase.
In contrast, BDNF production is unchanged in B and T cells in systemic sclerosis
patients compared to healthy controls.
In SLE, few studies have focused on NT expression and its relationship to
disease activity. In NZB/W mice, serum NGF concentrations are significantly
increased, correlating with an accumulation of NGF-containing cells in the
kidney and spleen. NGF levels are higher in the sera of SLE patients than
healthy controls and reflect systemic activity of the disease as assessed by the
SLEDAI (SLE Disease Activity Index) score. However, reports on serum BDNF
concentration in SLE are contradictory and limited to neuropsychiatric forms of
the disease. Though serum BDNF levels are decreased in neuro-SLE according to
one case report, they are increased in two other studies.
The aim of the present study was to evaluate serum and lymphocytic levels of
NGF, BDNF and NT-3 in SLE patients and identify their relation to clinical
features (systemic activity assessed by SLEDAI score, joint, skin, neurological
and kidney involvement, vasculitis), SLE-related immunological activity (anti-
native DNA antibodies, complement activation via CH 50, C3 and C4 levels), and
anti-phospholipid antibodies. Furthermore, we evaluated B cell activation
parameters that could be modulated by SLE (serum BAFF levels and autoantibody
production) and their association with enhanced levels of NT in sera.
Additionally, we analyzed the cytokine profiles and T-regulatory cell population
that could be modified by SLE activity. IL-10 and IFN-γ, two cytokines belonging
to the TH2 and TH1 profile, respectively, were measured in sera from controls
and patients. Several studies have previously demonstrated that IL-10 levels in
sera are significantly higher in untreated SLE patients than in healthy controls
and strongly reflect SLE activity. Their dynamics are closely linked to those of
autoantibody synthesis. Strikingly, circulating IL-10 levels decrease after
treatment and correlate with a change in the SLEDAI score, indicating that sera
IL-10 level is a biological marker of SLE activity. Moreover, the evaluation of
IL-10 levels in SLE is also supported by the direct effect of IL-10 on NGF
secretion in a murine astrocyte model. Likewise, a relationship between IFN-γ
and BDNF cell activation has been established in microglial cells, in that BDNF
inhibits IFN-γ-induced activation.
*Considering that no reported experimental data support the involvement of NT4/5
in B and T cell activation or autoimmune disease, this study mainly focused on
the relationship between serum levels, B-lymphocyte expression of NGF, BDNF and
NT-3, and SLE activity.*
# Materials and Methods
## Patients and control population
Twenty-six successive SLE patients, including 24 women (median age 44±12 years),
were included in a one-year cross-sectional study (2011) in Limoges University
Hospital. All participants fulfilled the revised American College of
Rheumatology (ACR) criteria for SLE. Disease activity was evaluated using the
SLEDAI score. Neurological complications were ruled out by clinical examination
combined with normal brain MRI (n=11) and brain 18-F-fluorodeoxyglucose positron
emission tomography (n=2).
Patients with neoplastic disorders or depression were excluded in order to avoid
confounding effects on serum NT levels.
The control population consisted of 26 healthy age- and sex-matched volunteers.
Patients were excluded if they were pregnant, under the age of 18, or unable to
give valid consent. The “NeuroLED” study received ethical approval from The
Limoges University Hospital Research Ethics Committee (N°I06023), and was
carried out in accordance with the Helsinki Declaration. Written consent was
obtained from all patients and control subjects.
## Clinical features
The clinical features of SLE present at the time of blood sampling were
cutaneous (n=22, 85%), articular (n=21, 81%), renal (n=4, 16%), neurological
(n=2, 8%) and pleural (n=1, 4%), as well as pericarditis (n=1) and vasculitis
(n=9;). The mean SLEDAI score was 7.2 ± 4.1 (range 4-20). Six patients (23%)
presented a severe systemic SLE flare defined by a mean SLEDAI score over 9. Six
patients (23%) presented an associated anti-phospholipid syndrome and 7
presented secondary Sjögren’s syndrome (27%).
Out of the 26 patients, 14 (54 %) were treated with glucocorticoids (mean 18.1 ±
22.2 mg/day), 20 (77%) with anti-malarial drugs, and 4 (16%) with
immunosuppressants (azathioprine n=2, mycophenolate mofetil to maintain
remission of a previous renal flare n=2;).
Treatment was increased directly after the blood sampling for systemic flare in
61% of cases (n=16) with the initiation of hydroxychloroquine (n=5, 19%),
glucocorticoid (n=7, 27%) or immunosuppressants (n=7, 27%), or with an increase
of previous corticosteroid dosages alone (n=3, 11%;).
Another blood sample for serum NT levels was taken one month after the systemic
flare in 9 cases.
## Additional assessment of quality of life
Patients completed the Medical Outcomes Study, a 36-item short form health
survey (SF-36), independently of their visit to the physician. The
questionnaires were returned to the research assistant.
## Measurement of autoantibodies and neurotrophin levels
Antinuclear antibodies were characterized by immunofluorescence in HEp2 cells
(The Binding Site, Saint Egrève, France) and anti-native DNA (nDNA) antibodies
by ELISA (Phadia, Saint-Quentin Yvelines, France). Anti-cardiolipin and anti-β2
Glycoprotein 1 (β2-GP1) antibodies were measured by ELISA (Ingen, Chilly
Mazarin, France).
Serum NGF, BDNF and NT-3 levels were measured using commercial ELISA kits
according to the manufacturer’s instructions (NGF E<sub>max</sub><sup>®</sup>
ELISA, BDNF E<sub>max</sub><sup>®</sup> ELISA, NT-3 ELISA, Promega,
Charbonnières, France). All assays were performed in duplicate and the data are
presented as pg/mL. Detection limits were 15 pg/mL for BDNF and 4 pg/mL for NT-3
and NGF.
## B cell activation analysis and determination of Th1 and Th2 profiles
Serum BAFF, INF-γ (Th1) and IL-10 (Th2) levels were measured with an ELISA kit
according to the manufacturer’s instructions (Quantikine<sup>®</sup> Human
Immunoassay R&D system, Lille, France). All assays were performed in duplicate
and the data are presented as pg/mL. Detection limits were 4 pg/mL for BAFF and
2 pg/mL for INF-γ and IL-10.
## Determination of neurotrophin expression by B and T cells
Expression of intracellular NGF, BDNF and NT-3 in T and B lymphocytes was
assessed by flow cytometry. Whole blood cells were stained with either
phycoerythrin (PE)-cyanin (Cy) 7-conjugated anti-CD3 or anti-CD19 antibodies for
15 min at room temperature. After lysing the red blood cells (Immunoprep,
Beckman Coulter, France), white blood cells were fixed, permeabilized
(Intraprep, Beckman Coulter) and incubated at room temperature for 30 min with
rabbit anti-NGF, anti-BDNF and anti-NT-3 antibodies (all 1/100; Santa Cruz
Biotechnology, France) in Phosphate-Buffered Saline (PBS) containing 1% Bovine
Serum Albumin. After two washes in PBS, antibodies were detected with Alexa
Fluor 488-conjugated goat anti-rabbit IgG antibodies (10 μg/mL; Invitrogen,
France) for 30 min at 4°C. Cells stained with rabbit isotypic immunoglobulins
(Santa Cruz Biotechnology, France) were used as controls to determine background
and positive result thresholds. After washing twice in PBS, cells were suspended
in PBS and analyzed with a flow cytometer (FacsCanto<sup>TM</sup> II, Becton
Dickinson, Le Pont-de Claix, France).
## Quantitative analysis of circulating T regulatory cells
Cells were stained with Cy7-conjugated anti-CD3, FITC-conjugated anti-CD4,
Cy5-anti-CD25 (Beckman Coulter), and PE anti-FOXP3 (eBioscience) antibodies or
isotype controls, and FACS analysis was performed as previously described.
## Statistical analysis
The results were expressed as means ± standard deviation. *P* values ≤ 0.05 were
considered significant. One-way analysis of variance (ANOVA), Chi-square tests
and Mann-Whitney tests were used when appropriate. To detect correlations
between serum NT levels, clinical and other biological data, linear regression
analysis was used and *p*-values were determined by Spearman’s rank correlation
test.
# Results
## Variations in serum NT expression in SLE
### The NT levels in sera of SLE patients, NGF, BDNF and NT-3 were determined by ELISA
Serum NGF levels were higher in SLE patients (426.13 ± 70.85 pg/mL) than in
healthy controls (373.9 ± 52.3 pg/mL, *p*=0.003). BDNF levels were also
increased in SLE patients (598.9 ± 129.8 *vs* 326.1 ± 60.5 pg/mL in controls,
*p*\<0.0001). Average serum NT-3 levels were similar in the SLE group and
controls (2911.7 ± 1248.8 *vs* 2553.7 ± 879.7 pg/mL, NS,).
The increases in serum levels of NGF and BDNF were statistically independent
(r=0.28, NS). Therefore, we examined the correlation between enhanced NGF and
BDNF levels and lupus systemic activity.
## Serum neurotrophins and SLE activation profile
NGF, BDNF and NT-3 serum levels were not correlated with initial SLEDAI score
(NGF: Γ=0.19, *p*=0.34, BDNF: Γ=0.16, *p*=0.42, NT-3: Γ=0.35, *p*=0.07).
However, there were higher NT-3 levels in a subset of patients with severe
systemic flare (SLEDAI ≥10; 4171.6 ± 1013.17 *vs* 2533.7 ± 1062.9 pg/mL,
*p*=0.002). In contrast, concentrations of NGF (426.2 ± 62.6 *vs* 425.9 ± 74.6
pg/mL, NS) and BDNF (612.6 ± 73.6 *vs* 594.7 ± 143.7 pg/mL, NS) were similar in
patients regardless of SLEDAI score.
SLEDAI score reflects the global systemic activity of SLE, which can correspond
to the involvement of various organs. The presence of cutaneous, neurological or
renal SLE manifestations did not influence serum levels of NGF, BDNF and NT-3.
However, NT-3 levels were significantly increased in patients with articular
manifestations (3185.6 ± 1213.3 pg/mL *vs* 1761.7 ± 581.2 pg/mL, *p*=0.02). In
contrast, there was no difference in NGF and BDNF levels between patients with
and without joint involvement.
We then investigated the correlation between NT serum levels and immunological
parameters associated with lupus flare. Serum NT-3 levels alone were
dramatically increased in patients with complement activation (n=8, 3749.1 ±
1433.25 *vs* 2457.9 ± 950.7, *p*=0.01) and these levels correlated negatively
with serum CH 50 levels (Γ=-0.28, *p*=0.032). In contrast, anti-nDNA antibodies
are independent of the serum levels of NGF (Γ=0.09, *p*=0.68), BDNF (Γ=0.29,
*p*=0.18) and NT-3 (Γ=0.35, *p*=0.07).
Considering that SLE may be associated with secondary Sjögren syndrome or anti-
phospholipid syndrome, we examined if there was a correlation between NT serum
levels and SLE-associated autoimmune disease.
## Serum neurotrophins and SLE-associated disease
NGF, BDNF and NT-3 concentrations were similar in SLE patients with or without
secondary Sjögren’s syndrome (NGF 430.7 ± 113.2 *vs* 424.5 ± 51.8 pg/mL, BDNF
624.27 ± 102.31 *vs* 589.4 ± 139.89 pg/mL, NT-3 3094.86 ± 1262.33 *vs* 2860.87 ±
1274.52 pg/mL, all NS). However, the presence of anti-SSB antibodies (n=3) was
associated with higher serum NGF levels (507.2 ± 23.4 *vs* 417.4 ± 69.1,
*p*=0.03).
In patients with anti-phospholipid autoantibodies, lower serum BDNF levels were
observed: anti-cardiolipin (n=9, 529.2 ± 142.9 *vs* 635.7 ± 109.2 pg/mL,
*p*=0.04) or anti-β2GP1 antibodies (n=4, 438.8 ± 185.4 *vs* 627.9 ± 96.87 pg/mL,
*p*=0.004). Furthermore, BDNF levels negatively correlated with the titers of
both IgG and IgM anti-cardiolipin antibodies (Γ=0.46, *p*=0.01 and Γ=0.41,
*p*=0.04, respectively).
In addition, we examined a possible correlation between systemic flare, quality
of life and serum NT profile.
## Serum neurotrophins and quality of life
Scores from both the Physical Component Summary (PCS) and Mental Component
Summary (MCS) were lower in SLE patients than healthy controls (PCS, 36.5 ± 6.9
*vs* 54.1 ± 7.4, *p*\<0.0001; MCS, 38.6 ± 10.1 *vs* 50.2 ± 6.5, *p*\<0.0001) and
did not correlated with SLEDAI score (PCS: Γ=0.03, *p*=0.87, MCS: Γ=0.22,
*p*=0.32). In SLE patients and the controls, neither PCS nor MCS scores were
related to serum NT levels.
*In order to evaluate the influence of immunomodulating drugs on the increased
circulating levels of NT, we examined systemic NT levels before and after SLE
flare treatment.*
## Dynamics of neurotrophin serum profile and SLE treatment
Serum NGF, BDNF, and NT-3 concentrations were similar in SLE patients at the
time of the blood sample whether they were untreated (n=4) or previously treated
by corticosteroid, hydroxychloroquine or immunosuppressants.
Interestingly, serum BDNF concentrations decreased one month after the systemic
flare of the disease (407.7 ± 65.6 pg/mL) but were still higher than those in
healthy volunteers (326.1 ± 60.5 pg/mL, *p*\<0.001). In contrast, serum levels
of NGF (426.12 ± 70.8 *vs* 462.6 ± 47.1 pg/mL, NS) and NT-3 (2986.9 ± 954.5 *vs*
2911.7 ± 1248.8 pg/mL, NS) remained unchanged after treatment of the SLE flare.
NGF, BDNF and NT-3 levels remained uncorrelated with the SLEDAI score (mean 4 ±
1.8) assessed after flare treatment (Γ=0.35, Γ=0.46 and Γ=0.27 respectively, all
NS).
In order to determine if there was a correlation between NT and SLE activity at
the level of immune cells, we tested if there was a relationship between serum
and lymphocytic NT expression, T and B cell activation, and cytokine profiles.
## Lymphocytic neurotrophins and clinical and immunological SLE profile
### NGF and BDNF-producing B cells are increased in SLE
The numbers of B cells producing NGF and BDF were greater in the SLE group than
in healthy controls (NGF, 29.3 ± 31.3 *vs* 11.3 ± 20.9, *p*=0.02; BDNF, 71.2 ±
30.9 *vs* 47.9 ± 27.8, *p*=0.03). Cell numbers were independent of serum NGF,
BDNF and NT-3 concentrations. In contrast, the numbers of NT-3-CD19-positive
cells were similar in the two groups (72.4 ± 29.7 *vs* 64.7 ± 29.1, NS).
The numbers of NGF and BDNF-producing B cells were independent of SLEDAI score
(Γ=0.22 and Γ=0.2 respectively, NS). Although numbers of NGF-producing cells
were independent of clinical SLE profiles, CD19+ BDNF-producing B cells were
dramatically decreased in patients with an associated anti-phospholipid syndrome
(40.2 ± 8.9 *vs* 80.5 ± 22.05, *p*=0.03).
NGF and BDNF-producing B cells were not influenced by corticosteroid (NGF: 36.5
± 33.8 *vs* 20.8 ± 27.11, NS, BDNF: 75.4 ± 23.7 *vs* 88.8 ± 17.5, NS),
hydroxychloroquine (NGF: 33.6 ± 23.6 *vs* 14.7 ± 16.8 NS, BDNF: 79.9 ± 23.3 *vs*
84.3 ± 19.2, NS) or immunosuppressants (NGF: 25.3 ± 19.8 *vs* 30.1 ± 33.3, NS,
BDNF: 76.06 ± 30.2 *vs* 81.6 ± 21.7, NS).
Interestingly, NGF production by B cells tended to be higher in SLE patients
with cryoglobulinemia (mean fluorescence intensity, Xmean, 8.7 ± 7.7) than in
patients without cryoglobulinemia (Xmean 4.1 ± 2.7, *p*=0.06). A similar
relationship was observed between CD19-related NGF production and complement
activation (Xmean 7.1 ± 5.41 *vs* 3.5 ± 1.8, *p*=0.05).
CD19-BDNF-producing B cells were also decreased in patients positive for lupus
anticoagulants (28.5 ± 14.3 *vs* 79.0 ± 23.9, *p*=0.01).
### NGF and BDNF-producing T cells in SLE
The numbers of T lymphocytes expressing NGF (17.2 ± 21.2 *vs* 9.8 ± 21.3, NS),
BDNF (55.1 ± 33.5 *vs* 55.1 ± 33.5, NS) and NT-3 (57.5 ± 33.7 *vs* 52.9 ± 24.2,
NS) were similar in the SLE and control groups and were independent of patients’
clinical and immunological SLE profiles.
### NT and B-cell activation parameters
As expected, serum BAFF levels were increased in the SLE group (1980.7 ± 1315.9
*vs* 1019.9 ± 193.4 pg/mL, *p*=0.01). However, they did not correlate with serum
concentrations of NGF, BDNF, or NT-3 (Γ=0.12, Γ=0.03 and Γ=0.04, respectively)
in the SLE group.
In contrast, in the control group, serum concentrations of NGF positively
correlated with BAFF levels (Γ=0.64, *p*=0.02), which were also independent of
the serum levels of both BDNF and NT-3 (Γ=0.02 and Γ=0.6, respectively).
Serum levels of BAFF were also independent of B and T cell NT expression in both
the SLE and control groups.
### NT and T regulatory cells
Only serum NGF levels and T regulatory cell counts were negatively correlated in
SLE patients (Γ=0.48, *p*=0.01), whereas there was no significant correlation
between BDNF or NT-3 levels and T regulatory cell numbers (Γ=0.06, NS, Γ=0.28,
NS, respectively). T regulatory cells and NTs appeared to be independent in
healthy controls (NGF Γ=0.27, BDNF Γ=0.12, NT3 Γ=0.08, NS).
BDNF-positive CD19 cells and T regulatory cells were negatively correlated
(Γ=0.39, *p*=0.04).
### NT and IL-10 production
IL-10 concentrations were higher in the SLE group (29.4 ± 81.1 *vs* 0.19 ± 3.3
pg/mL, *p*=0.06), especially in the subgroup of patients with severe systemic
flare (91.28 ± 162.6 *vs* 10.9 ± 11.26 pg/mL, *p*=0.03) or positive anti-SSA
antibodies (77.25 ± 152.48 *vs* 12.24 ± 13.27 pg/mL, p=0.07).
In the SLE group, NGF, BDNF and NT-3 serum levels were statistically independent
of serum IL-10 levels (Γ=0.02, Γ=0.12 and Γ=0.12 respectively), which did not
influence NT production in T or B cells. The same results were observed in the
control group.
### NT and Interferon-*γ* production
Serum INF-γ levels were higher in the SLE group (INF-γ 136.7 ± 230.9 *vs* 6.9 ±
10.5 pg/mL, *p*=0.03), particularly in patients with severe systemic flare
(258.9 ± 422.32 *vs* 79.58 ± 98 pg/mL, *p*=0.08). NGF, BDNF and NT-3 levels were
also statistically independent of INF-γ concentrations (Γ=0.21, Γ=0.17 and
Γ=0.35, respectively) in the SLE group. In contrast, serum BDNF levels
negatively correlated with INF-γ concentration in the control group sera
(Γ=0.55, *p*=0.03).
While NT production by B and T cells was independent of INF-γ concentration in
the control group, INF-γ serum levels correlated with NGF production in T
lymphocytes (Γ=0.64, *p*=0.01).
# Discussion
The present study provides new evidence of the significant variation in NT
levels in both serum and circulating B lymphocytes in SLE patients. Previous
studies mainly focused on serum NGF levels, and serum BDNF and NT-3 had not been
studied in a SLE cohort. Furthermore, NT expression in circulating B cells had
not yet been documented. We have identified an increase in NGF and BDNF levels
in both serum and circulating blood CD19-B cells in patients. Interestingly,
NT-3 levels were only increased in severe forms of SLE and correlated with
complement activation.
We have identified a significant increase in sera NGF and BDNF levels in SLE
patients compared to a healthy control group. We confirm that serum NGF levels
are increased in SLE patients, a result previously demonstrated. However, we did
not find any correlation between serum NGF levels, systemic complications, and
SLEDAI scores in this SLE population, characterized by previous treatment with
either corticosteroid or immunosuppressants. These results differ from those of
a previous study reporting a correlation between serum NGF levels and SLE
activity. However, the inclusion criteria of this study was different, involving
only untreated children (and not adults) with an over-representation of renal
involvement (60% of patients *vs* 16% in the present study). Moreover, a link
between renal involvement and enhanced NGF levels has been described in SLE mice
and in patients with SLE glomeronephritis-related renal insufficiency, which
could explain this discrepancy. However, we did not find any correlation between
NGF serum levels and the presence of renal involvement (only 4 adults) or other
systemic complications. Therefore, the effect of immunomodulating drugs on serum
NGF levels in previously treated patients needs to be discussed in regards to
other reports. In fact, NGF serum levels have been found to be independent of
immunomodulating treatments in other diseases such as systemic sclerosis,
rheumatoid arthritis, and primary Sjögren syndrome.
Concerning BDNF levels, this study reports for the first time an increase in
serum BDNF levels in SLE patients, occurring independently of central
neurological involvement (absent in all but two patients). Serum BDNF levels did
not correlate with SLEDAI score. Only one previous case report has been
described of psychotic involvement in SLE with an increase of serum BDNF.
Likewise, in multiple sclerosis and rheumatoid arthritis, a decrease in serum
BDNF levels occurs with clinical improvement, independently of other
inflammatory markers. After treatment, a decrease in BDNF levels is detected in
these SLE patients. The significance of this decrease with treatment is debated
and also appears to be independent of both corticosteroid and immunosuppressive
drugs in psychiatric form of SLE patients, as well as in systemic sclerosis and
rheumatoid arthritis. In contrast, BDNF serum levels are enhanced in primary
Sjögren syndrome patients (pSS) treated with either corticosteroid or
immunosuppressants for severe systemic involvement. Interestingly, we have shown
that BDNF levels decrease after treatment of systemic flares, signifying that
this decrease could be a biological marker for improvement and a therapeutic
response in SLE.
Strikingly, we find an increase of NT-3 serum levels only in severe (SLEDAI ≥
10) and articular forms of SLE. Serum NT-3 has been infrequently studied in
inflammatory diseases, although NT-3 immune function has been demonstrated in
asthma: autocrine NT3 autocrine secretion leads to plasma cell survival. NT-3
involvement in autoimmune articular symptoms is indicated by its presence in the
synovial fluid of patients with spondyloarthritis and by increased levels in the
sera of systemic sclerosis patients with articular complications that require
hydroxychloroquine treatment. Enhanced NT-3 serum levels in severe forms of SLE
could reflect a link between NT-3 and lupus flare, in that complement activation
(decrease of CH 50 levels) correlates with elevated NT-3 levels. This direct
relationship between NT-3 synthesis and complement (C1q) has been shown in
neuronal cells. In another cell model, NT-3 production by endothelial cells is
upregulated by local ischemia. Therefore, NT-3 secretion could be a biological
marker of severe SLE forms, potentially associated with vascular damage related
to an active vasculitis process.
The direct impact of NT on SLE physiopathology is also underlined by the
significant overproduction of both NGF and BDNF by B cells, and could reflect an
activation of circulating B cells in SLE. The activation of circulating B-cells
in SLE patients has been reported during nephritis and SLE activity. Moreover,
in some experimental conditions, a direct link between *in vitro* activation of
B cells and their secretion of NGF and BDNF has been established. Overall, the
overexpression of these NTs in SLE patients’ circulating B cells (compared to
healthy controls) could be a hallmark of activation that correlates with the
disease.
To further evaluate the impact of NTs on SLE pathogenesis, we have looked for
correlations between both serum and lymphocytic NT levels and immune
characteristics, especially antibody production and T regulatory cell profiles.
NGF, BDNF and NT-3 levels did not correlate with anti-nDNA levels. This
correlation has not been previously examined in the 3 published studies on NGF
and BDNF serum levels in SLE. This absence of relationship between NT serum
levels and autoantibody production was also found in pSS and systemic sclerosis.
Concerning rheumatoid arthritis, to our knowledge, the correlation between NGF,
BDNF and anti-cyclic citrullinated peptide has not been previously studied.
Interestingly, serum BDNF levels were the lowest in the group positive for anti-
phospholipid. This negative correlation could be due to anti-phospholipid-
associated vasculopathy and oxidative stress, an association previously
identified in systemic sclerosis, atherosclerosis and diabetes. Similarly, we
identified for the first time a striking decrease of BDNF-producing B cells in
patients with an associated anti-phospholipid syndrome. These results suggest
that a reduction of both circulating BDNF levels and BDNF-producing B cells are
involved in the vascular damage associated with SLE.
In addition, we report a negative correlation between serum NGF levels and T
regulatory cells in SLE patients, which could reflect either SLE activity or a
direct impact of serum NGF level on T regulatory cell survival. Also, NGF is an
inducible survival growth factor for T cells, depending on the cytokine profile;
anti-NGF treatment enhances Foxp3+ regulatory T cells and decreases Th17 cells
in a murine model of asthma. Moreover, IL-17 enhances NGF production in human T
cells. Together, this data suggests that NGF could regulate the balance of
T-regulatory and Th17 cells. Nevertheless, no reports have shown that either
p75<sup>NTR</sup> or TrkA NGF receptors are present on human T regulatory cells,
which are known to produce high levels of BDNF in HIV-associated
neurodegeneration models.
IL-10 serum levels were enhanced in the SLE group, reflecting systemic activity
and anti-SSA production as previously described, whereas NT and IL-10 levels are
were not correlated. Indeed, IL-10 release by immune cells, which is enhanced by
both NGF and BDNF in normal conditions, is dramatically reduced in cases of
allergy.
As expected, INF-γ serum levels were increased in SLE. Interestingly, in our
control group only, we found a negative correlation between serum BDNF level and
INF-γ, which is known to reduce BDNF production in neurons, glia, and bronchial
smooth muscle cells. Moreover, a cytokine balance between INF-γ and BDNF has
been described in bowel mucosa, as a regulating factor of enteric glia cell
apoptosis. The deregulation of the INF-γ and BDNF balance in SLE may reinforce
the importance of BDNF over-secretion in SLE pathogenicity.
In conclusion, the present study finds that the expression of the neurotrophins
NGF and BDNF, overexpressed by circulating B cells, are increased in the sera of
SLE patients independently of Th1/Th2 profile. NT-3 is upregulated only in
severe flares of the disease, and BDNF levels are closely related to anti-
phospholipid syndrome.
## Thus, we hypothesize that evaluating NT in both sera and circulating-B lymphocytes could be a new biological marker of SLE activity and systemic complications
To test this hypothesis, prospective large cohort studies, including naive SLE
patients, need to be performed. In addition, further studies of NT-secreting
subpopulations of B and T cells, in association with their NT receptors (i.e.
TrkA, TrkB, TrkC, p75<sup>NTR</sup> and sortilin), will be conducted to define
their fine-tuning functions during SLE disease. This study supports the idea
that neurotrophins are involved in SLE physiopathology and thus could be a
potential target of systemic treatment.
We thank *Cornelia* Wilson for the English correction of this manuscript.
We especially thank the Limoges Clinical Research Center (DRCI CHU Limoges) for
its help in the completion of this study.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: ALF MOJ PM MCL FL. Performed
the experiments: MCL PM HB FXL CM KL. Analyzed the data: ALF MOJ FL.
Contributed reagents/materials/analysis tools: AS ME EV. Wrote the
manuscript: ALF MOJ EV. In charge of the clinical protocol study: SD. |
# Introduction
Honeybees are key pollinators of crops and wildflowers. They contribute globally
more than €53 billion annually to the economy. Still, honeybees are presently
facing multiple stressors such as habitat destruction, pests, and pathogens.
Across the Northern hemisphere the spread of the ecto-parasitic mite *Varroa
destructor* (*Varroa*) and the key viral pathogen it transmits (Deformed Wing
Virus) is the number one threat faced by beekeepers over past decades, causing
an increase in over-wintering losses. In the UK, this was evidenced in the very
high losses of managed colonies and the near eradication of feral colonies
within a few years of *Varroa* becoming established. Over-wintering colony
losses have been declining in the UK during the past decades since
*Varroa*-treatments became widespread. Although, colony losses remain
consistently high in the USA, which is unexpected due to the high levels of
treatment carried out by beekeepers to control *Varroa* populations in both
countries. Typically, a variety of acaricides are primarily used to control
*Varroa* populations, but with the development of acaricide resistance
alternative commercially based treatments established around thymol and organic
acids (oxalic and formic) have become increasingly popular. More time consuming
and often less effective biomechanical treatment methods can also be used, such
as powdered sugar and drone trapping. The application of Integrated Pest
Management (IPM) techniques is suggested to be the most effective way to treat
mites since it involves a variety of methods applied in rotation throughout the
year determined by mite-infestation levels through routine observations and mite
monitoring carried out by beekeepers.
In contrast, some countries like South Africa and Cuba took a decision not to
treat *Varroa* and allowed mite-resistance to appear naturally. In both
countries 1,000’s of colonies were lost initially, but losses declined after
several years as resistance to the mite developed. Whereas, in Brazil the
evolution of mite-resistance by Africanised bees (*Apis mellifera scutellata*)
was not observed and any losses were probably masked by the losses from
untreated non-resistant European honeybee colonies, although there is no
published evidence to support or contradict this idea. Despite this *Varroa*
resistance rapidly became widespread in these countries, hence treatment-free
beekeeping has already been established for several decades.
More recently in mainland Europe, the UK, and USA it appears an increasing
number of mite-resistant populations are being managed treatment-free. Allowing
honeybees to develop natural resistance will foster a long-term solution to the
*Varroa* problem.
Resistance is defined as any situation in which *Varroa* populations are
maintained at a suitable level for the long-term survival of the honeybee
colonies. Recently, three key traits (cell recapping, mite infertility and brood
removal) have been associated with natural resistance in almost all resistant
populations studied in different countries. In the UK the government advice is
to treat *Varroa* with either biotechnical methods, with registered varroacides
or a combination of them. Despite this, anecdotal evidence from beekeepers
suggests that numbers of non-treating beekeepers are more than expected.
The aim of this study was therefore to conduct an online survey of UK beekeepers
via their associations to assess individual treatment habits, as it was the most
efficient way to reach beekeepers easily. The survey will provide crucial
empirical data to support or refute common perceptions around treatment and non-
treatment beekeeping practices.
# Materials and methods
## The survey
The survey was constructed using Google Forms as it allowed an unlimited number
of responses whilst also allowing the incorporation of the University logo to
add credibility to the survey. The survey consisted of a brief description
outlining the study, its aims, and our definition of treatment, which was, “any
form of external or additional control administered to bees by beekeepers aimed
at reducing *Varroa* numbers”. Then followed six questions: 1) association name,
2) number of colonies, 3) if they treat or not, 4) number of times a year they
treat, 5) number of years since last treatment, 6) type of treatment (see for
survey). Answers were either multiple-choice questions or open questions that
all helped assess the beekeeper’s treatment habits. The survey was kept short in
duration to increase the response rates.
The contact details for 325 beekeeping area associations across the UK were
obtained via the four UK Beekeeper Association websites (British, Scottish,
Welsh and Northern Ireland). Initial contact was made by email with 303 of them;
outlining the study aims to determine general interest. The responding
associations were then sent the survey online link and asked to forward it to
their respective members. Beekeepers were given ten weeks from 23<sup>rd</sup>
Dec 2020 to complete the survey. A small number of beekeepers did not complete
every section of the survey fully, so for each analysis the precise number of
responses obtained from that part are given. Amateur bee-associations were
chosen as they contain around 30,000 members in the UK.
## The survey analysis
After the survey closed, all 2,897 responses from 243 associations were exported
into an excel spreadsheet. The 25 responses from the Isle of Man, which remains
a *Varroa*-free island were excluded along with the 16 beekeepers with zero
colonies leaving 2,856 for further analysis. A total of 67 beekeepers did not
either belong to or failed to put down an association. As over 20 types of
*Varroa* treatment were reported, we grouped them into single methods (Chemical,
Biomechanical and Natural) and mixed methods. To estimate the number of colonies
surveyed we multiply the median colony group size, or 30 for the 30+ group by
the number of beekeepers in that group.
## Spatial analysis
While conducting previous research into *Varroa-*resistance colonies it appeared
that they existed in many parts of the UK. To investigate this maps were created
using ArcGIS Pro (v. 10.8.1) software, showing the location of all UK area
associations and those that participated. To investigate if any spatial patterns
in treatment-free vs treated existed, the proportion of beekeepers falling into
either category were calculated from each association that had at least five
responses to avoid illegible pie diagrams on the figure.
## Statistical analysis
A GLM was conducted in R using treatment status as the response variable,
association as the fixed effect and colony number as the random effect. A
binomial family was used to fit the model due to only two variants of the
response variable (i.e., treating or treatment-free).
The median group size or 30 for the 30+ group were used for colony group size in
the GLM.
# Results
The 2,856 beekeepers who responded represents almost 10% of the estimated 30,000
members belonging to the four UK beekeeping associations and were widely
distributed across the UK. We estimated that we surveyed around 21,200 colonies
that is again around 10% of the 220,000 colonies estimated by the Center for
Ecology and Hydrology to be in the UK.
The majority (67%) of beekeepers that responded managed between 1 to 5 colonies
and only 3% had over 30 colonies. Our data indicated that across the five colony
size groups the proportion of treatment-free beekeepers was greatest in the 1–5
group, but all other groups were within 5% of that group. The GLM showed that
treatment status was not affected by association but did indicate a significant
effect of colony group size on treatment status. However, as the 95% CI
(-19.559–15.493) contains 0 then we can conclude that there is not a strong
significant relationship. Due to such high variability in the data, we conclude
that there is not a statistically significant relationship between colony number
and treatment status.
## Treatment-free beekeeping
A total of 596 (21%) beekeepers stated they were not treating, and 2,260 (79%)
beekeepers were treating their colonies against *Varroa*. When asked about the
duration elapsed since their last treatment was applied, 72% had treated within
the last year and 173 (6%) responders had not treated for 6 years or more. The
spatial distribution indicates Scottish beekeepers were almost all treating and
only four associations had more than 75% of treatment-free beekeepers as
members, otherwise treatment free beekeepers were spread throughout England.
This indicates the widespread distribution of beekeepers attempting treatment
free approaches particularly in England.
## Treatments used in the UK
An estimated 4,093 treatments per year were administered by 2,238 beekeepers
with the aim of reducing *Varroa* numbers. The majority (70%) are treated once
or twice a year using a single chemical method. The most popular chemical
treatment method is oxalic acid, followed by commercially produced thymol, and
amitraz. The current study found 78% of beekeepers use chemical treatments
(oxalic acid, thymol etc), 3% use biomechanical methods (drone brood removal,
sugar dusting etc) and less than 1% use other methods (rhubarb leaves, etc.).
Overall, 80% use a single method of treatment and only 20% use a combination of
treatment methods.
# Discussion
Based on 2,856 beekeeper responses from 243 UK beekeeping areas, the proportion
of beekeepers not-treating ranged between 21–28% e.g., if the beekeeper can only
choose between treatment or treatment-free (21%), or 28% of beekeepers if you
include those that had not treated in the last year. Those not-treating for over
6 years represented 6% of responders. Based on this survey, that would mean
1,800 of the 30,000 estimated UK beekeepers are truly treatment-free having not
treated their colonies for *Varroa* for six years or more. Around 100 of these
beekeepers are in a single region in North Wales with many belonging to the
Lleyn and Eifionydd Beekeepers’ Association. Likewise, in Swindon, a small
beekeeper group have kept treatment-free colonies since 1995. In the UK there
are active and growing “treatment-free” communities. For instance, the Westerham
beekeepers are approaching their 5<sup>th</sup> year of becoming treatment free
and starting to bring in neighboring bee clubs. Finally, the ‘Natural Beekeeping
Trust’ was established in 2009 and now has links to 35 like-minded groups. We
had 18 responses from their members all falling into the ‘never-treated group’,
along with 16 beekeepers that failed to provide an association. These ‘natural
beekeepers’ and those not-treating for *Varroa* have not always been welcomed by
those adopting treatment regimens. The ‘never-treated group’ represent 4% of the
respondents but the length of time they have not been treating is unknow. These
treatment-free beekeepers occur across all colony group sizes.
The annual BBKA (British Beekeepers Association) overwinter survival survey
found that in 2020/21, 27% and 37% of 2,950 randomly selected members from a
pool of 26,407 did not treat from August to September or from October to April
respectively. In 2019/20 the BBKA survey found 25% and 38% of treatment-free
beekeeping during Aug-Sept and Oct-Apr, respectively. In both years the BBKA
survey found treatment-free beekeeping was present in all English regions, as
was found by this study. This is important, since the two surveys used different
sampling methods. The BBKA and this study surveyed similar numbers of
beekeepers, except the BBKA survey targets members randomly selected from their
membership each year, while this study approaches the beekeepers via their
associations using email, so they are self-selecting. Despite this the two
outcomes are similar in many aspects indicating that our survey has not
disproportionally been returned by treatment-free beekeepers that may be more
active online.
Nonetheless, the majority (72–79%) of UK beekeepers are still treating their
colonies to control *Varroa* numbers. We found beekeepers in the UK are
predominantly using a single or bi-annual chemical treatment regime. The order
of popularity starting with the most common is oxalic acid, thymol, amitraz and
formic acid. Only 20% of beekeepers in this study are adopting a combination of
methods approach.
The popular chemical *Varroa* treatments like formic and oxalic acids reported
in this study and other UK surveys are also the preferred methods used in Europe
and the USA. These compounds have a high efficacy but without the stigma of
synthetic compounds or mite resistance, which could explain why many beekeepers
are choosing to adopt these methods. However, the impact of these “natural”
treatments should not be ignored. Whilst thymol is thought to be unproblematic
at temperatures between 5°C-9°C, high mortality levels have been observed when
temperatures exceed 27°C.
The success of any treatment method or treatment-free beekeeping is determined
by their overwinter losses. Over the last 14 years in the UK over-wintering
annual colony losses have ranged from over 30% to 8% with an average of 18%.
This falls to 14% when the three spikes related to unusually cold and wet
winters are removed (data derived from BBKA annual survey data collected by D.
Aston that is based on 2,500–3,500 beekeepers each year \[e.g. 6,25\]).
Principal causes of colony losses reported by beekeepers in 2021 were queen-
related problems (24%), isolation starvation (21%), weather-related 17%, and
*Varroa* was just 4% reflecting the situation that the mite is no longer
considered a major problem by beekeepers currently in the UK. However, *Varroa*
could be contributing to some of the other colony losses indirectly. In
2019–2020 the BBKA annual survey reported both losses in the region as well as
the percentage of colonies not treated in each region.
Beekeeper observations by the Oxford shire Natural Beekeeping Association (ONBA)
and the treatment-free group in Northwest Wales both found their colony survival
rates were on par with those from the BBKA, but independent scientific studies
are required to see if *Varroa*-resistance effects the rate of colony losses.
One of the dangers of switching over to an intervention free-treatment regime
involves a period of high winter losses whilst the bees develop resistance. This
is due to re-invasion from collapsing colonies to surviving surrounding colonies
or by visits from infested non-natal bees. Several strategies have been adopted
at both local and national level to encourage mite-resistant honeybees. Catching
free-living swarms from locations where they appear to have persisted for many
years has been a successful strategy in the UK and Hawaii. Others have reduced
the frequency of mite-treatments by selectively treating colonies with high mite
levels, or to use less efficient biomechanical methods only when required.
Already many countries (South Africa, Brazil, Mexico, Cuba etc), along with
beekeepers in the UK and elsewhere have been able to stop treating as their
honeybees have learnt to detect mite infested cells and remove the pupa to
prevent mite-reproduction, which leads to decreased mite fertility and
population size. However, the majority (72–79%) of UK beekeepers are still
treating and it will be many years before most beekeepers in the UK and
elsewhere can stop treating for *Varroa*. In the USA a recent survey found only
around 63 (3%) of 2275 respondents stated no advantages to *Varroa* management
and 92% of this group did not treat for *Varroa*, a very different situation
than found currently in the UK. Most beekeepers in the Northern hemisphere have
long wished for a silver bullet for the *Varroa* problem, however, it turns out
that the bullet is their own bees. Beekeepers just need to give their bees time
to develop mite resistant as has been done so successfully elsewhere in the
world.
# Supporting information
We thank all the UK beekeepers that submitted a survey, help from their
associations and G. Webb of University of Salford for proofreading the original
submission. The original data is available on dryad at
<https://doi.org/10.5061/dryad.xksn02vkn>.
10.1371/journal.pone.0281130.r001
Decision Letter 0
Chaline
Nicolas
Academic Editor
2023
Nicolas Chaline
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
29 May 2022
PONE-D-22-05221A survey of UK Beekeeper’s Varroa treatment habitsPLOS ONE
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about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: The submitted survey of UK beekeeper´s varroa treatment habits
presents interesting new data and fills a gap in our knowledge on recent
beekeeping practice. The study is well presented with a clear description of the
data sampling and evaluation procedure. The discussion picks up relevant links
to related surveys and the establishment of mite resistance in bee populations
depending on the treatment strategies. The length of the different chapters is
appropriate and well balanced.
Some confusion may arise about the number of respondents for the data in table
3. The table reports on a total of 2,868 responses from 2,897 responses in
general (L116). If 25 responses from the Isle of Man have been excluded, some of
the 16 participants with zero colonies (L146f) seem to be included here.
However, this wouldn´t make any sense for my understanding. The authors should
give a precise description on this.
L102 refers to Fig S1 which is missing in the supplied manuscript.
In L120 Table S1 is mentioned but it should be Table 5 as far as I understand.
In L 264 the second “by” has to be erased.
Reviewer \#2: The paper has the potential to be interesting, but is currently
missing the access to the data and the supplemental information, so difficult to
assess. Also the conclusions are very broad and the data presented don't support
the conclusions the authors make, as the survey did not include any assessment
of colony losses. Without distinguishing what the actual loss rates of the
treatment free beekeepers have, it is not possible to state that the bees are
actually resistant to varroa or have a high tendency for survival. The higher
colony numbers actual suggest the opposite.
Also there are a number of odd sentence structures and grammatical errors. I
have made note of the mores specific comments in a separate document.
Reviewer \#3: The study aims to estimate the number of beekeepers in the UK who
are not treating their colonies for varroa, and to estimate whether the
existence of untreated and resistant colonies in the UK could be assumed.
Information on beekeeping practices are currently limited in the scientific
literature, and this question and the data the study provides are really
relevant and valuable. The article is generally clear and well written. The
methodological choices are globally sound and with a high number of respondents
to the survey, which is important to note. Still some information should be
added in the methodological section, and the limits induced by some
methodological choices should be identified and discussed as they can have
important impacts on the interpretation of the results. Also, the findings
should be discussed regarding the scientific literature on the subject. For
these reasons I recommend major revisions.
Major comments
Some methodological limits should be identified and discussed, especially i) the
possible influence of the sampling through the beekeeping associations (are
these associations involved in prescribing varroa treatments?) and ii) the lack
of information that the survey provides about the colony losses and about the
beginning year of the beekeeping activity. Without this last information, the
share of beekeepers who have “never treated” cannot be interpreted as it is done
currently regarding the possible existence of surviving colonies (those
beekeepers could have started recently and their colonies may not have faced
this absence of treatment for varroa for a long time). It should not be
suggested that these colonies had never known any treatment (it could be the
case, but the data of the survey do not allow to conclude on this question).
The existence of colonies surviving without treatment for varroa and the
possible losses that the absence of treatment may induce should also be
discussed regarding the scientific literature on these questions.
Other comments
L. 57: “due to almost universal treatment by beekeepers”: this statement should
be either nuanced or supported by a reference about American beekeepers’
practices. E.g. see Thoms et al. (2019 -
<https://doi.org/10.1007/s13280-018-1130-z>), which indeed reveals a high
percentage of American backyard beekeepers reporting not to treat for varroa.
L. 100-105: If I understand correctly, there was no question about the colony
survival (cf. L100-105). It would have been a valuable information, as the
emergence of resistant colonies is only possible if the non-treated colonies
survive (in the case of backyard beekeepers replacing regularly their colonies
that died from varroa, these non-treated colonies would not be more resistant
than other).
L. 106-113. As the beekeeping associations who forward the survey to their
adherents play a major role in the sampling, it would be useful to provide more
information about these associations (as such beekeeper associations and their
role can differ from one association or one country to the other). Especially,
can these associations interfere with their adherents’ treatment choices (e.g.
by advising or not advising to treat for varroa)?
L. 123. One of the main analysis choices is the investigation for a possible
spatial pattern. As many other factors could possibly influence beekeepers’
choice about their treatment for varroa (beekeeping experience, age, etc), this
hypothesis of a spatial pattern could be explained and justified.
L. 130. Some biomechanicals and natural methods that were reported by beekeepers
(cf. Table 5) are not really efficient against varroa. As the study focuses on
the possible existence of untreat / resistant colonies and not on the choice of
beekeepers to treat or not to treat (reasons for such choices, etc), these “low-
efficacy” treatments could have been considered jointly with the treatment-free
group, or a third variant for the response variable could have been considered.
So the choice to gather all the treatments together in a single response
variable could be explained.
L. 133. As one of the questions of the study is the existence of untreated and
possibly resistant colonies, it would be useful to know how many colonies in
total these 2,872 beekeepers manage and what percentage of the total number of
colonies in the UK it represents.
L. 141-144. This passage is not very clear, especially what is supposed to
“explained why there was significant increase in treatment-free beekeepers as
the numbers of colonies they managed increased”. It would be useful to rephrase
or to complete it.
L. 146 / Table 1. The third column heading is not very clear: does it represent
the percentage of colonies of the group (1-5,…) which are not treated? So 73% of
the colonies of the 16-30 group are not treated?
An additional column providing the percentage of treatment-free beekeepers in
each of the groups would be welcome. Besides, the final percentage of untreated
colonies (gathering all groups) should be added.
L. 160-162. Grouping the beekeepers who indicated that they have "never treated"
with those indicating a specific number of years since the last treatment (here
10 years and more, or 6 years and more L. 201) is questionable as the date of
installation of the beekeepers was not in the survey and is not known. Some of
them could have started beekeeping recently, and the fact that they have “never
treated” for varroa does not presume that their colonies had to face a
significant period without treatment. This group of beekeepers with an unknown
number of years without treatment should be considered separately and the
confusion with the groups where a long treatment-free period is known should be
avoided.
L. 163-164. The fact than some association gather more than 75% of treatment-
free beekeepers raises questions about the role of these associations, and about
the possible exchanges related to varroa treatments that its members may have.
Even if it was not the objective of the study to understand the determinants of
the absence of treatment, it would be interesting to discuss the possible role
of associations on this point given their central place in the survey sample.
L. 178. “the majority are treated”: the majority of colonies?
L. 180-184. It would be useful to add the global percentage of beekeepers not
using any treatment and to more clearly distinguish if the percentages given for
the types of treatments are exclusive of each other or not. E.g. can the 3% of
beekeepers using biomechanical methods be also in the 78% using chemical? Or are
they only using biomechanicals methods?
L. 201. See comment on L. 160-162
L. 202. The extrapolation of the study results to the 30,000 estimated UK
beekeepers should be supported by a discussion about the representativity of the
sample. That joins the question of giving information about the beekeeping
associations (see comment on L 103-115), and about their representativeness (are
they some apicultural practices which may differ between members of these
associations and beekeepers who are not members, e.g. if the associations are
involved in prescribing treatments for varroa? Are the members of these
associations representing a large share of the UK beekeepers?)
L. 257-260. As varroa is usually considered in scientific and technical
literature as an important factor of colony losses, some scientific references
about the place of varroa in colony losses need to be added and the technical
references which are the only provided references should be discussed regarding
these scientific references.
L. 267. It would be useful to precise “in Hawaii” and not only “in the USA” as
the context of an island can be specific.
Fi. 1. A. A legend with the number of respondents corresponding to the different
pie sizes is needed here.
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10.1371/journal.pone.0281130.r002
Author response to Decision Letter 0
1 Jul 2022
Data is said to be publicly available, but no link included. Same goes for the
supplemental information.
THE DATA WILL BE PLACED ON DRYAD IF ACCEPTED
Please apply the use of commas for numbers consistently. For example, compare
line 35 and line 38. Also Varroa is not consistently italicized. DONE
I don’t understand why the feral population of bees is mentioned in line 43. You
surveyed beekeepers with managed colonies. This has no implications for the
feral bees and no evidence is presented that the UK has truly feral colonies.
DELETED ‘FERAL COLONIES’ AS THE IDEA WAS THAT MORE VARROA-RESISTANT MANAGED
COLONIES WILL LEAD TO MORE VARROA-RESISTANT FERAL COLONIES BUT WE AGREE WITH THE
COMMENT TO DELETED.
Line 56-57, awkward sentence structure. Unclear what remained high. ENTIRE
SECTION REWORDED
Line 60, based used twice REPLACED ONE WITH ESTABLISHED
Line 65-66, not clear why an IPM strategy would be linked to long-term
resistance and low mite levels. IPM, as I understand it, is threshold base and
involves monitoring and then treating appropriately. How does this encourage low
mite levels? And why would this lead to long-term resistance? Needs explanation
and evidence.
THIS IS JUST A REMOTE POSSIBILTY SO THIS STATEMENT HAS BEEN DELETED
Line 102: I have no access to the Figure S1
Line 120: I have no access to Table S1 SORRY THIS HAS OCCURRED
Line 201-203, that assumes that the survey reached and was completed by a
representative wedge of the beekeeping population. Many treatment-free
beekeepers tend to be younger and more active online, so this may have skewed
the responses. These caveats should be mentioned.
THE COMMENT ABOVE ONLINE ACTIVE HAS NOW BEEN ADDED TO THE RELEVENT PART OF THE
DISCUSSION L 239-241.
Line 218-219: This is not surprising at all. Most treatment free beekeepers in
the United States expect annual losses of 50%. Thus, in order to rebuild the
following year, they must keep more colonies in reserve. I am disappointed this
survey didn’t ask for self-reporting of annual loss rates or honey production
per colony. The high level of treatment-free suggests that beekeepers and the
much higher colony numbers suggests that the resistance is probably not enough
for low annual losses.
WE DID NOT INCLUDE COLONY DEATHS IN OUR SURVEY SINCE THIS IS ALREADY COVERED BY
THE LONG RUNNING BBKA SURVEY DATA FROM WHICH WE HAVE NOW INCULDED(see Fig S2)
AND WE HAVE EXPANDED THE PARAGRAPHY COMPAIRING COLONY LOSS RATES FROM UK
TREATMENT-FREE BEEKEEPERS. MY PERSONAL EXPERINCE WORKING WORK WITH SEVERAL
TREATMENT-FREE BEEKEEPERS THEIR LOSSES ARE SIMILAR TO BEEEKEEPERS THAT ARE
TREATING.
WE DID NOT ASK ABOUT HONEY PRODUCTION AS IN THE UK THIS VARIES GREATLY DUE TO
LOCATION AND MICRO-CLIMATE. CLEARLY THERE ARE BIG DIFFERENCES BETWEEN THE
SITUATION IN THE UK AND USA IN NUMBER OF TREATMENT FREE BEEKEEPERS THIS IS NOW
MENTION IN THE LAST SECTION AS WELL AS THE (Thoms et al)
Line 235-240: This makes the assumption that IPM requires multiple treatments
per year. IPM requires monitoring and treating accordingly when a pest level
rises above a certain point. Also, many do not consider drone trapping a
treatment. As I don’t have an actual copy of the survey questions, I cannot
assess if there may have been misunderstandings in the responses.
POOR PLACEMENT OF SENTENCE SO RE-STRUCTED THIS SECTION AND COMMENTED THAT ONLY
VERY BASIC MITE MONTIROING TAKES PLACE.
Line 253-255: This needs more clarification. Is the 4% what beekeepers reported
was specifically due to varroa? This would then be an underrepresentation of
actual winter losses due to varroa. Numerous surveys have shown that when varroa
levels are high, winter losses increase, even though beekeepers blame other
factors for the loss. Many beekeepers with colony winter losses don’t know what
killed their colony, but beekeepers with more experience can often diagnose the
contracted brood nest area and varroa frass.
WE HAVE GIVEN MORE CLARIFICATION ON THE PRINCIPAL CAUSES OF COLONY LOSSES IN THE
UK
Line 254-255: Why is the low winter loss attributed to treatment-free
beekeeping. There could be other explanations, such as very active beekeeping
groups that teach how to monitor and treat effectively. The evidence presented
does not warrant this statement, as there is no segregation of losses by those
treating and those not treating presented.
WE AGREE THAT NOT ENOUGH EVIDENCE IS PRESENT AT THE MOMENT SO THE SENTENCE HAS
BEEN DELETED
Line 256-260: These statements require the reader to go to the original articles
and assess the numbers of colonies surveyed. Important information such as
colonies assessed in the survey should be included with the paraphrased results,
especially as these are not peer-reviewed studies but association results and so
the statistics have not been evaluated. The way they are presented in this
paper, the reader assumes they are peer-reviewed papers, unless the references
are checked in detail.
WE AGREE SO ADDED IN ADDITIONAL DATA/INFO BOTH IN THE TEXT AND SUPP DATA
Line 276-279: This is a very broad statement and is not justified by the results
presented, as no losses were calculated in the survey.
WE HAVE SHOWEN USING BBKA SURVEY BASED ON 2,613 BEEKEEPERS THAT THERE IS NO SIG
RELATIONSHIP BETWEEN %TREATMENT AND %COLONY LOSSES AT A REGIONAL LEVEL IN THE
UK. WE HAVE ALSO ADDED IN MORE REFRENCES TO SUPPORT OUR BROAD STATEMENT.
FINALLY THE PAPER HAS BEEN CAREFULLY PROOFED NOW
5\. Review Comments to the Author
Reviewer \#1: The submitted survey of UK beekeeper´s varroa treatment habits
presents interesting new data and fills a gap in our knowledge on recent
beekeeping practice. The study is well presented with a clear description of the
data sampling and evaluation procedure. The discussion picks up relevant links
to related surveys and the establishment of mite resistance in bee populations
depending on the treatment strategies. The length of the different chapters is
appropriate and well balanced.
Some confusion may arise about the number of respondents for the data in table
3. The table reports on a total of 2,868 responses from 2,897 responses in
general (L116). If 25 responses from the Isle of Man have been excluded, some of
the 16 participants with zero colonies (L146f) seem to be included here.
However, this wouldn´t make any sense for my understanding. The authors should
give a precise description on this.
THE 25 RESPONSES FROM THE ISLE OF MAN WERE EXCLUDED FROM THE 2897 (= 2872) WHICH
IS NOW STATED IN THE METHODS. IN TABLE 1 THE 16 BEEKEEPERS WITH ZERO COLONIES
WHERE EXCLUDED WHICH LEAVES 2856 BEEKEEPERS AS STATED IN TABLE 1. WE HAVE
REWRITTEN THE METODS TO INCLUDE ALL SAMPLES EXCULDED
L102 refers to Fig S1 which is missing in the supplied manuscript. (Sorry our
error, CORRECTED)
In L120 Table S1 is mentioned but it should be Table 5 as far as I understand.
(deleted)
In L 264 the second “by” has to be erased. Deleted
Reviewer \#2: The paper has the potential to be interesting but is currently
missing the access to the data and the supplemental information, so difficult to
assess. Also, the conclusions are very broad, and the data presented don't
support the conclusions the authors make, as the survey did not include any
assessment of colony losses. Without distinguishing what the actual loss rates
of the treatment free beekeepers have, it is not possible to state that the bees
are resistant to varroa or have a high tendency for survival. The higher colony
numbers actual suggests the opposite.
AS MENTIONED ABOVE WE HAVE A PARAGRAPH COMPARING LOSSES IN TREATMENT-FREE
POPULATIONS VS THE NATIONAL AVERAGE. ANOTHER SURVEY COLLECTS LOSS DATA BUT NOT
AIMED AT SEPARATING OUT THE TREATED AND NOT TREATED. WE HAVE ALSO INCLUDED THE
MAIN REASONS IN ENGLAND & WALES FOR COLONY LOSSES AND VARROA ACCOUNTS FOR ONLY
4% ALTOUGH WE AGRREE THIS MAY BEEN UNDERESTIMATED BY THE BEEKEEPERS.
Also there are a number of odd sentence structures and grammatical errors. I
have made note of the mores specific comments in a separate document. ALL SHOULD
BE FIXED NOW
Reviewer \#3: The study aims to estimate the number of beekeepers in the UK who
are not treating their colonies for varroa, and to estimate whether the
existence of untreated and resistant colonies in the UK could be assumed.
Information on beekeeping practices is currently limited in the scientific
literature, and this question and the data the study provides are relevant and
valuable. The article is generally clear and well written. The methodological
choices are globally sound and with a high number of respondents to the survey,
which is important to note. Still some information should be added in the
methodological section, and the limits induced by some methodological choices
should be identified and discussed as they can have important impacts on the
interpretation of the results. Also, the findings should be discussed regarding
the scientific literature on the subject. For these reasons I recommend major
revisions.
Major comments
Some methodological limits should be identified and discussed, especially i) the
possible influence of the sampling through the beekeeping associations (are
these associations involved in prescribing varroa treatments?)
THERE IS NO PRESCRIPTION OF TREATMENTS VIA ASSOCIATIONS SINCE TREATMENT ADVICE
COMES CENTRALLY FROM THE NATIONAL BEE UNIT & THE MAP INDICATES WE HAD A VERY
WIDE SPREAD OF RESPONSE
and ii) the lack of information that the survey provides about the colony losses
and about the beginning year of the beekeeping activity. Without this last
information, the share of beekeepers who have “never treated” cannot be
interpreted as it is done currently regarding the possible existence of
surviving colonies (those beekeepers could have started recently, and their
colonies may not have faced this absence of treatment for varroa for a long
time). It should not be suggested that these colonies had never known any
treatment (it could be the case, but the data of the survey do not allow to
conclude on this question).
THERE ARE A GROWING GROUP OF BEEEKEEPERS IN THE UK THAT NEVER TREAT SUCH AS
OXFORDSHIRE NATURAL BEEKEEPING ASSOCIATION AND THEIR LOSSES FOR TWO WINTERS WERE
SIMILAR TO THE NATIONAL AVERAGE. THIS INFORMATION IS PROVIDED IN THE MS AS WELL
AS A NEW SUPPLEMENTAL FIGURE SHOWING THE NATIONAL AVERAGE COLONY LOSSES FROM
2007 TO 2021.
The existence of colonies surviving without treatment for varroa and the
possible losses that the absence of treatment may induce should also be
discussed regarding the scientific literature on these questions.
WE HAVE EXPANDED THE SECTION ON COLONY LOSSESS IN THE UK AS REQUESTED
Other comments
L. 57: “due to almost universal treatment by beekeepers”: this statement should
be either nuanced or supported by a reference about American beekeepers’
practices. E.g. see Thoms et al. (2019 -
<https://doi.org/10.1007/s13280-018-1130-z>), which indeed reveals a high
percentage of American backyard beekeepers reporting not to treat for varroa.
I CHECKED THOMS ET AL AND ALOUGHT MOST TREATMENT SKEPICS DO NOT TREAT THIS GROUP
MADE UP LESS THAN 3% OF THE SURVEY EG (63 VS 2276). THUS, IT APPEARS THAT THERE
ARE MUCH FEWER TREATMENT-FREE BEEKEEPERS IN THE USA THAN UK. HOWEVER, WE HAVE
CHANGED ‘UNIVERSAL’ TO ‘HIGH’ WHICH IS MORE ACCURATE AND CITED THIS PAPER AT THE
END OF THE DISCUSSION.
L. 100-105: If I understand correctly, there was no question about the colony
survival (cf. L100-105). It would have been a valuable information, as the
emergence of resistant colonies is only possible if the non-treated colonies
survive (in the case of backyard beekeepers replacing regularly their colonies
that died from varroa, these non-treated colonies would not be more resistant
than other).
SEE EALIER COMMENTS ABOUT COLONY LOSSES
L. 106-113. As the beekeeping associations who forward the survey to their
adherents play a major role in the sampling, it would be useful to provide more
information about these associations (as such beekeeper associations and their
role can differ from one association or one country to the other). Especially,
can these associations interfere with their adherents’ treatment choices (e.g.,
by advising or not advising to treat for varroa)?
THIS IS SOMETHING THAT IS NOT DONE THE ASSOICATIONS ONLY FORWARED THE SURVEY
LINK TO ALL ITS MEMBERS USING THERE (CONFEDENTIAL DATABASE). IF THESE TYPE OF
BEHAVIOR DID OCCUR, WE WOULD HAVE PICKED IT UP IN THE ANAYLSIS. FURTHERMORE, THE
ASSOICATIONS THAT SEND OUT THE LINK HAVE NO WAY OF SEEING WHAT THEIR MEMBERS
WORTE.
L. 123. One of the main analysis choices is the investigation for a possible
spatial pattern. As many other factors could possibly influence beekeepers’
choice about their treatment for varroa (beekeeping experience, age, etc), this
hypothesis of a spatial pattern could be explained and justified.
EACH ASSOICATION HAS A WIDE RANGE OF AGES, GENDERS, AND EXPERIENCE TO REQUEST
THIS TYPE OF DATA AND CONSDIER THEM IN THE ANAYLSIS LIES WAY BEYOND THE SCOPE OF
THIS STUDY.
L. 130. Some biomechanical and natural methods that were reported by beekeepers
(cf. Table 5) are not efficient against varroa. As the study focuses on the
possible existence of untreated / resistant colonies and not on the choice of
beekeepers to treat or not to treat (reasons for such choices, etc), these “low-
efficacy” treatments could have been considered jointly with the treatment-free
group, or a third variant for the response variable could have been considered.
So, the choice to gather all the treatments together in a single response
variable could be explained.
THE REVIEWER IS CORRECT IN THAT THE DIFFERENT TREATMENTS RANGE WIDLY IN THEIR
EFFICACY’S. TO ERR ON THE SIDE OF CAUTION WE HAD TO MAKE IT VERY CLEAR TO
UNDERSTAND WHAT ‘TREATMENT-FREE’ MEANT. OTHERWISE, WE COULD HAVE RUN THE PROBLEM
OF BEEKEEPERS DECIDING WHAT THEY CONSIDER IS TREATMENT FREE. WE AGREE THE LOW-
EFFICACY TREEATMENTS COULD BEE ALLOWING NATURAL RESISTANT TO DEVELOP AND THIS
ONE OPTION FOR BEEKEEPERS TO BECOME TREATMENT FREE. IN THE FUTURE IT WOULD BE
INTRESTING TO SEE IF THE USAGE OF THESE LOW-EFFICACY METHODS INCREASED.
L. 133. As one of the questions of the study is the existence of untreated and
possibly resistant colonies, it would be useful to know how many colonies in
total these 2,872 beekeepers manage and what percentage of the total number of
colonies in the UK it represents.
WE HAVE NOW ADDED IN A SECTION AT THE START OF THE RESULTS SHOWING THAT SURVEY
ALSO COVERED ABOUT 10% OF THE UK COLONIES
L. 141-144. This passage is not very clear, especially what is supposed to
“explained why there was significant increase in treatment-free beekeepers as
the numbers of colonies they managed increased”. It would be useful to rephrase
or to complete it.
THIS SECTION HAS BEEEN REEWORDED
L. 146 / Table 1. The third column heading is not very clear: does it represent
the percentage of colonies of the group (1-5,…) which are not treated? So, 73%
of the colonies of the 16-30 group are not treated?
An additional column providing the percentage of treatment-free beekeepers in
each of the groups would be welcome. Besides, the final percentage of untreated
colonies (gathering all groups) should be added.
WE RE-CHECKING THE DATA IN TABLE 1 WE DISCOVER AN ERROR SO THANKS FOR THAT. WE
HAVE NOW RE-DONE TABLE 1 AND TABLE 2 SO SHOULD BE MUCH CLEARER AS REQUESTED BY
THE REVIEWER
SORRY ITS BEEEKEEPERS NOT COLONIES SO WE HAVE REWORDED IT.
L. 160-162. Grouping the beekeepers who indicated that they have "never treated"
with those indicating a specific number of years since the last treatment (here
10 years and more, or 6 years and more L. 201) is questionable as the date of
installation of the beekeepers was not in the survey and is not known. Some of
them could have started beekeeping recently, and the fact that they have “never
treated” for varroa does not presume that their colonies had to face a
significant period without treatment. This group of beekeepers with an unknown
number of years without treatment should be considered separately and the
confusion with the groups where a long treatment-free period is known should be
avoided.
IN TABLE 2 WE HAVE SEPRATED OUT THE NEVER ADMINISTRED GROUP SO THEY ARE CONSIDER
SEPRATLY. THE MANY OF THESE BEEKEPERS BELONG TO THE OXFORD NATURAL BEEKEEPING.
THIS GROUP HAS BEEN AROUND FOR 10 YEARS AND WE PROVIDE COLONY LOSS DATA FROM
THIS GROUP AND COMPARE AGAINST THE NATIONAL AVERGAE AND ITS VERY SIMILAR.
L. 163-164. The fact than some association gather more than 75% of treatment-
free beekeepers raises questions about the role of these associations, and about
the possible exchanges related to varroa treatments that its members may have.
Even if it was not the objective of the study to understand the determinants of
the absence of treatment, it would be interesting to discuss the possible role
of associations on this point given their central place in the survey sample.
AS PREVIOUSLY MENTIONED VARROA TREATMEENTS ARE RECOMMENDED BY THE GOVERMENTS
NATIONAL BEE UNIT. THE TREATMENT-FREE BEEKEEPERS IN MOST ASSOICATIONS DO NOT
MENTION THAT THEY DO NOT TREAT AS THEY ARE IN MINORITY HENCE WHY WE DID THE
SURVEY TO GET A BETTER ESITATMTION OF THEE THE NUMBER OF TREATMENT FREE
BEEKEEPERS.
L. 178. “the majority are treated”: the majority of colonies? YES, SO CHANGED
L. 180-184. It would be useful to add the global percentage of beekeepers not
using any treatment and to more clearly distinguish if the percentages given for
the types of treatments are exclusive of each other or not. E.g., can the 3% of
beekeepers using biomechanical methods be also in the 78% using chemical? Or are
they only using biomechanical methods?
THIS DATA DOES NOT CURRENTLY EXIST THE Thoms et al survey and this study are
some of the first attempts to collect such data.
L. 201. See comment on L. 160-162
L. 202. The extrapolation of the study results to the 30,000 estimated UK
beekeepers should be supported by a discussion about the representativity of the
sample. That joins the question of giving information about the beekeeping
associations (see comment on L 103-115), and about their representativeness (are
they some apicultural practices which may differ between members of these
associations and beekeepers who are not members, e.g., if the associations are
involved in prescribing treatments for varroa? Are the members of these
associations representing a large share of the UK beekeepers?)
IN THE UK THERE ARE AROUND 30,000 AMATEUR BEEKEEPERS BUT ONLY 400 PROFESSIONALS
THAT BELONG TO THE BEE FAMERS SOCIETY. THIS IS NOW MENTIONED IN THE METHODS.
L. 257-260. As varroa is usually considered in scientific and technical
literature as an important factor of colony losses, some scientific references
about the place of varroa in colony losses need to be added and the technical
references which are the only provided references should be discussed regarding
these scientific references.
THIS IS VERY TRUE PARTICULAR IN THE NORTHERN HEMISPHERE BUT IN MANY COUNTRIES
(E.G., AFRICA, BRAZIL-MEXICO, CUBA) VARROA IS NOW NOT CONSIDERED A PROBLEM SINCE
THE BEES ARE ABLE TO KEEP THE INFESTATION LEVEL DOWN BELOW 10-4% DEPENDING ON
THE POPULATION. HENCE IN THE EYES OF WELL-ESTABLISHED (6YRS+) TREATMENT-FREE
BEEKEEPERS VARROA IS NOT CONSIDERED AS A SERIOUS PROBLEM. THE SAME CAN BE SAID
BY BEEKEEPERS THAT REGULAR TREAT THEIR VARROA POPULATIONS. WE HAVE NOW ADDED IN
A SHORT SECTION SHOWING IN ONE SURVEY THE BEEKEEPERS REPORTED THAT ONLY 4% OF
THEIR DEATHS WAS DIRECTLY CAUSED BY VARROA.
L. 267. It would be useful to precise “in Hawaii” and not only “in the USA” as
the context of an island can be specific. CHANGED AS SUGGESTED
Fi. 1. A. A legend with the number of respondents corresponding to the different
pie sizes is needed here. THIS HAS NOW BEEN DONE
10.1371/journal.pone.0281130.r003
Decision Letter 1
Corby-Harris
Vanessa
Academic Editor
2023
Vanessa Corby-Harris
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
7 Dec 2022
PONE-D-22-05221R1A survey of UK Beekeeper’s Varroa treatment habitsPLOS ONE
Dear Dr. Martin,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
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We look forward to receiving your revised manuscript.
Kind regards,
Vanessa Corby-Harris, Ph.D.
Academic Editor
PLOS ONE
Additional Editor Comments:
Dear authors,
(First, I apologize for my lengthy response. This manuscript was re-assigned to
me and so I was reading it for the first time.)
This manuscript summarizes a survey of whether UK beekeepers treat for mites or
not. Overall, I think that this manuscript could be a good fit for PLoS One
based on the journal’s publication criteria, with one exception noted below. I
also think that the general idea of treatment-free beekeeping is interesting and
that it would be a useful addition to the literature. However, I do agree with
reviewer 2 that the manuscript is somewhat narrow in scope and rather
descriptive, and would be better suited for a more specialized or beekeeper-
friendly journal. However, many of those journals are pay walled and so this
could be a better way to more widely disseminate the authors’ results.
This manuscript has been through one round of review and was much improved after
the reviewer comments were addressed. You will see that the reviewers have some
lingering concerns that still need to be addressed. Please address each comment
in your revision. Specifically, reviewers 2 and 3 have multiple concerns, such
as the rationale behind several hypotheses, statements that need supporting
citations, the level of detail in the methods, and concerns about the survey
itself. There are also several instances where the conclusions are not supported
by the data (ex. treatment-free beekeeping is becoming more common). Please
revise those statements so that the manuscript meets the PLoS One publication
criteria, specifically criteria \#4 that “Conclusions are presented in an
appropriate fashion and are supported by the data”.
I also saw several small grammatical errors and had a few minor questions. I
apologize that these are coming up now, but they need to be addressed.
1\) There are several sentences that start with “Although”, “Thus”, “Whereas”.
Please revise these sentences because they are often grammatically incorrect.
L56 – decades
L59 – I wouldn’t say they are highly effective. For example, most or all can’t
get into the brood cell, where mite reproduction happens. Revise?
L61-62 – watch what you capitalize (ex. oxalic?)
L65 – take out the comma after reference 9
L83 – is this resistance or tolerance?
L103 – six questions:
L134 – isn’t “treatment status” the response variable?
L140 – widely or wildly?
L141-143 – this should be in the methods and needs more detail (see reviewer 2
comment)
Figure 1 – it could be helpful to remind the reader that the 158 area
associations in the spatial study had ≥5 respondents, so associations with \<5
respondents were not colored in black. At least that was my interpretation of
what you said in the text.
L160-161 – Is this an important or real trend if you don’t see a difference
between these groups and the larger groups? Can you explain or speculate why you
didn’t see differences between these small and medium colony number groups and
the larger group?
Table 2 – had no effect (see legend text); also should you have a range for the
colony number group estimate since you had four groups?
I like Tables 4 and 5, although I agree with reviewer 3 that there are issues
with the “treatment-free” group. Some of those natural methods were interesting
(rhubarb??). And how does queen trapping differ from a brood break? Just
wondering, really.
L218-219 – This is repeating part of my point above, but I agree that this group
could also include beekeepers that have kept bees for only a few (\<6) years and
so combining them with the beekeepers that have been treatment-free for 6 years
or more could be misleading. It seems that there was no criteria that
respondents have kept bees for 6 or more years.
L275-276 – revise the sentence where it says weather-related and Varroa – should
this be split up somehow?
L281- that’s not a lot of data (N=6) to base a conclusion on. I am also of the
opinion that the discussion should not include too many results/statistics, so
this is something to potentially omit. It is appropriate to talk about it, just
not as something that you tested using data and stats.
L307-310 – revise this sentence, it is too long. I like the general idea of the
sentence though!
\[Note: HTML markup is below. Please do not edit.\]
Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. If the authors have adequately addressed your comments raised in a previous
round of review and you feel that this manuscript is now acceptable for
publication, you may indicate that here to bypass the “Comments to the Author”
section, enter your conflict of interest statement in the “Confidential to
Editor” section, and submit your "Accept" recommendation.
Reviewer \#1: All comments have been addressed
Reviewer \#2: All comments have been addressed
Reviewer \#3: (No Response)
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#1: Yes
Reviewer \#2: Yes
Reviewer \#3: Partly
\*\*\*\*\*\*\*\*\*\*
3\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#1: Yes
Reviewer \#2: Yes
Reviewer \#3: Yes
\*\*\*\*\*\*\*\*\*\*
4\. Have the authors made all data underlying the findings in their manuscript
fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
manuscript fully available without restriction, with rare exception (please
refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
participant privacy or use of data from a third party—those must be specified.
Reviewer \#1: Yes
Reviewer \#2: Yes
Reviewer \#3: No
\*\*\*\*\*\*\*\*\*\*
5\. Is the manuscript presented in an intelligible fashion and written in
standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
articles must be clear, correct, and unambiguous. Any typographical or
grammatical errors should be corrected at revision, so please note any specific
errors here.
Reviewer \#1: Yes
Reviewer \#2: Yes
Reviewer \#3: Yes
\*\*\*\*\*\*\*\*\*\*
6\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: please correct the following typos in the revised document:
L168:...had no effect...
L270: overwinter losses. Over the last...
L307:...than found currently in the UK.
Regarding your statement in L249-251I suggest a more careful wording as the BBKA
report is restricted on the treatment period between August and September while
OA as the most common treatment in UK should be mainly used on broodless
colonies during winter.
Reviewer \#2: The paper is well written, but the survey lacks depth. The authors
wished to investigate how wide spread treatment free beekeeping is within the
UK. They did this by conducting an online survey with limited questions that
could be completed within five minutes. It would have been far more interesting
if they then did a follow up with the beekeepers who qualified as treatment-free
to learn more about their beekeeping practices. I feel the current results
presented are more appropriate for a bee journal than a rigorous scientific
publication. For specific comments regarding the manuscript, see attached PDF.
Reviewer \#3: SPECIFIC COMMENTS
L. 72 References are still missing to support this assertion
L 127-132 The choice to explore a potential spatial pattern (rather than the
other possible explanatory factors for the varroa management) should still be
explained: why is this hypothesis made?
L 233. References or studies on the number of treatment free beekeepers a few
years ago would be appropriate here to support the assertion that the number of
treatment-free beekeepers is growing.
L 240-244 The BBKA studies the authors refer to are about a specific time of the
year: even if some of the beekeepers who do not treat at this time of the year
could be treatment-free beekeepers as the authors underline, some of them could
also treat at another time of the year. Thus, the results of these BBKA studies
should not be directly used to compare with the percentage of treatment-free
beekeepers which was estimated in the study, as they do not estimate the same
thing.
L.279 It is unclear if the percentage of colonies not treated here refers to the
“august to September non treated” (cf. L 241) or to year-long non treated (and
the BBKA study the authors refer to does not seem to be available online to find
this information). It would be appropriate to precise this information.
GENERAL COMMENTS
This study provides useful information about the number of treatment-free
beekeepers in the UK. Some relevant changes have been made to the paper,
considering the reviewers’ feedback on the first manuscript.
Still, the authors did not respond to some of the feedback, in particular
regarding their methodological choices, which were not always explained (e.g.
why explore a spatial pattern?) and regarding the limits of these methodological
choices and therefore of the study.
One of the major methodological issues is the choice to gather beekeepers who
indicated that they have “never treated against varroa” with beekeepers who have
not treated for six years or more. This choice relies on the hypothesis that
beekeepers who have “never treated” manage colonies which have not been treated
for a significant period of time (6 years or more, or a least several years).
This cannot be stated, as the survey lacks a question on the duration of the
colony management by beekeepers: the beekeepers who have “never treated” could
also be beekeepers who just started beekeeping in the year the survey was made.
The number of years in which their colonies would have been treatment-free is
then totally unknown, and could also be less than a year.
Considering this issue, the number of treatment-free beekeepers have to be
estimated without this “never treated” category.
Also, one of the main conclusions of the authors seems to be that treatment-free
beekeeping would be increasing and that it would be a reliable solution as the
colony losses would not be greater: these conclusions are not supported by the
study as the survey was not designed properly to study these questions. It would
have required questions about the colony losses and about the age of the
beekeeping activity. These questions were not asked in the survey and the
references that the authors provide to support these conclusions are clearly not
sufficient to conclude about this. Indeed, the compared category is not the same
for the BBKA study, and the given references e.g. the Oxfordshire Beekeeping
Group are particular cases and not scientific literature. This does not seem to
be strong enough arguments to support such an important conclusion as the
supposed absence of additional colony losses in the case of treatment-free
beekeeping.
There are of course relevant questions and perspectives that this study opens
up. Still, it should be seen as such and not considered as actual and current
results.
To conclude on that new version of the manuscript, some efforts were made by the
authors since the first draft and these efforts must be acknowledged. Still, it
seems that this manuscript still requires some modifications to address properly
the methodological issues and to ensure a better and clear distinction between
what is an actual conclusion of the study and what remains a perspective without
sufficient evidence to conclude here.
\*\*\*\*\*\*\*\*\*\*
7\. PLOS authors have the option to publish the peer review history of their
article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-
and-peer-review-process#loc-peer-review-history)). If published, this will
include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may
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**Do you want your identity to be public for this peer review?** For information
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Policy](https://www.plos.org/privacy-policy).
Reviewer \#1: No
Reviewer \#2: No
Reviewer \#3: No
\*\*\*\*\*\*\*\*\*\*
\[NOTE: If reviewer comments were submitted as an attachment file, they will be
attached to this email and accessible via the submission site. Please log into
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While revising your submission, please upload your figure files to the Preflight
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Please note that Supporting Information files do not need this step.
10.1371/journal.pone.0281130.r004
Author response to Decision Letter 1
20 Dec 2022
1\) There are several sentences that start with “Although”, “Thus”, “Whereas”.
Please revise these sentences because they are often grammatically incorrect.
L56 – decades CORRECTED
L59 – I wouldn’t say they are highly effective. For example, most or all can’t
get into the brood cell, where mite reproduction happens. Revise? REVISED
L61-62 – watch what you capitalize (ex. oxalic?) DONE
L65 – take out the comma after reference 9 DONE
L83 – is this resistance or tolerance? SEVERAL YEARS A GO I USED TOLERANCE BUT
WAS TOLD TO USE RESISTANCE WHEN TALKING ABOUT THIS MECHANSIM. IN FACT BOTH TERMS
CAN APPLY DEPENDING WHAT VIEW YOU TAKE. THEREFORE, I NOW USE RESISTANCE AS THIS
IS THE DOMINANT TERM USED IN THE FIELD AND DEFINE IT AS I HAVE DONE HERE.
L103 – six questions: DONE
L134 – isn’t “treatment status” the response variable? YOU ARE CORRECT SO
CHANGED
L140 – widely or wildly? WIDELY IS THE CORRECT TERM
L141-143 – this should be in the methods and needs more detail (see reviewer 2
comment) DONE AND ADDED NEW DETAIL REQUESTED
Figure 1 – it could be helpful to remind the reader that the 158 area
associations in the spatial study had ≥5 respondents, so associations with \<5
respondents were not colored in black. At least that was my interpretation of
what you said in the text. WE HAVE NOW ADDED THIS INFO INTO THE LEGEND
L160-161 – Is this an important or real trend if you don’t see a difference
between these groups and the larger groups? Can you explain or speculate why you
didn’t see differences between these small and medium colony number groups and
the larger group?
FURTHER ANAYLSIS (by calculating the 95% CI) INDICATED THAT DUE TO A HIGH
VARIABILITY IN THE DATA IN THE DATA WE CAN CONCLUDE THERE IS NO SIGNIFICANT
RELATIONSHIP BETWEEN COLONY NUMBER AND TREATMENT STATUS. THIS IS NOW MENTIONED.
Table 2 – had no effect (see legend text); also should you have a range for the
colony number group estimate since you had four groups? ALEX
I like Tables 4 and 5, although I agree with reviewer 3 that there are issues
with the “treatment-free” group. Some of those natural methods were interesting
(rhubarb??). And how does queen trapping differ from a brood break? Just
wondering, really.
TABLE 4 WE HAVE CHANGED TO ‘NOT TREATING’ AS THIS IS WHAT THEY WERE DOING AT THE
TIME OF THE SURVEY, BUT WE DO AGREE WITH YOU AND REVIEWER 3 THAT ELSEWHERE WE
BREAK THIS GROUP UP INTO ITS VARIOUS GROUPS AS WE HAVE DONE IN TABLE 3.
AS FOR TABLE 5 WE COULD COMBINE BROOD BREAKS WITH QUEEN TRAPPING BUT THESE ARE
THE TERMS THE BEEKEEPERS USED IN THE SURVEY.
RHUBARD LEAVES CONTAIN OXALIC ACID BUT THE LEVEL WILL BE LOW
L218-219 – This is repeating part of my point above, but I agree that this group
could also include beekeepers that have kept bees for only a few (\<6) years and
so combining them with the beekeepers that have been treatment-free for 6 years
or more could be misleading. It seems that there was no criteria that
respondents have kept bees for 6 or more years.
IN HINDSIGHT THIS WAS A OVERSIGHT OF THE QUESTIONS ASKED AND RESPONSE OPTIONS
AVAILABLE. BUT NOW WE HAVE SEPARATED OUT THE GROUP OF NEVER TREATED AND 1-5
YEARS GROUP. THE READER SHOULD NOW BE ABLE TO DISTINGUSH BETWEEN THOSE NOT-
TREATING AND THOSE THAT ARE TREATMENT FREE (I.E. 6+ YEARS)
L275-276 – revise the sentence where it says weather-related and Varroa – should
this be split up somehow?
DONE
L281- that’s not a lot of data (N=6) to base a conclusion on. I am also of the
opinion that the discussion should not include too many results/statistics, so
this is something to potentially omit. It is appropriate to talk about it, just
not as something that you tested using data and stats.
SECTION REMOVED
L307-310 – revise this sentence, it is too long. I like the general idea of the
sentence though!
SPLIT INTO 2 SENTENCES
Reviewer \#1: please correct the following typos in the revised document:
L168:...had no effect... CORRECTED
L270: overwinter losses. Over the last...CORRECTED
L307:...than found currently in the UK…CORRECTLY
Regarding your statement in L249-251I suggest a more careful wording as the BBKA
report is restricted on the treatment period between August and September while
OA as the most common treatment in UK should be mainly used on broodless
colonies during winter.
THE BBKA SURVEYS ALSO COVERS THE OCT TO APRIL PERIOD AS WELL SO THIS DATA IS NOW
GIVEN IN ADDITION TO THE AUG-SEPT DATA.
Reviewer \#2: The paper is well written, but the survey lacks depth. The authors
wished to investigate how wide spread treatment free beekeeping is within the
UK. They did this by conducting an online survey with limited questions that
could be completed within five minutes. It would have been far more interesting
if they then did a follow up with the beekeepers who qualified as treatment-free
to learn more about their beekeeping practices. I feel the current results
presented are more appropriate for a bee journal than a rigorous scientific
publication. For specific comments regarding the manuscript, see attached PDF.
ALL COMMENTS ADRESSED ON ATTACHED PDF (see attached PDF)
Reviewer \#3: SPECIFIC COMMENTS
L. 72 References are still missing to support this assertion
IT IS NOW MADE CLEAR THAT THERE IS NO EVIDENCE TO SUPPORT OR CONTRADICT THIS
IDEA.
L 127-132 The choice to explore a potential spatial pattern (rather than the
other possible explanatory factors for the varroa management) should still be
explained: why is this hypothesis made?
WE HAVE INSERTED THE FOLLOWING TEXT ‘WHILE CONDUCTING PREVIOUS RESEARCH INTO
VARROA-RESISTANCE COLONIES \[14\] IT APPEAR THEY EXISTED IN MANY PARTS OF THE
UK. THEREFORE, MAPS WERE CREATED……’
L 233. References or studies on the number of treatment free beekeepers a few
years ago would be appropriate here to support the assertion that the number of
treatment-free beekeepers is growing.
THIS SECTION HAS BEEN DELETED AS THIS STUDY IS MEANT TO PROVIDE BASE-LINE DATA
FROM WHICH WE CAN SEE IF IN THE FUTURE THINGS INCREASE OR DECREASE.
L 240-244 The BBKA studies the authors refer to are about a specific time of the
year: even if some of the beekeepers who do not treat at this time of the year
could be treatment-free beekeepers as the authors underline, some of them could
also treat at another time of the year. Thus, the results of these BBKA studies
should not be directly used to compare with the percentage of treatment-free
beekeepers which was estimated in the study, as they do not estimate the same
thing.
WE HAVE INCLUDED ADDITIONAL ‘NON-TREATMENT’ DATA (OCT-APRIL) FROM THE BBKA
SURVEY
L.279 It is unclear if the percentage of colonies not treated here refers to the
“august to September non treated” (cf. L 241) or to year-long non treated (and
the BBKA study the authors refer to does not seem to be available online to find
this information). It would be appropriate to precise this information.
TO ACCESS THE DATA YOU MUST FIRST JOIN THE BBKA IN ORDER TO ACCESS THERE
NEWSLETTER INCLUDING THE BRITISH BEE JOURNAL, SINCE THE JOURNAL/NEWSLETTER LIKE
MANY JOURNALS IS BEHIND A PAYWALL.
GENERAL COMMENTS
This study provides useful information about the number of treatment-free
beekeepers in the UK. Some relevant changes have been made to the paper,
considering the reviewers’ feedback on the first manuscript.
Still, the authors did not respond to some of the feedback, in particular
regarding their methodological choices, which were not always explained (e.g.
why explore a spatial pattern?) and regarding the limits of these methodological
choices and therefore of the study.
THE REASON HAS NOW BEEN GIVEN
One of the major methodological issues is the choice to gather beekeepers who
indicated that they have “never treated against varroa” with beekeepers who have
not treated for six years or more. This choice relies on the hypothesis that
beekeepers who have “never treated” manage colonies which have not been treated
for a significant period of time (6 years or more, or a least several years).
This cannot be stated, as the survey lacks a question on the duration of the
colony management by beekeepers: the beekeepers who have “never treated” could
also be beekeepers who just started beekeeping in the year the survey was made.
The number of years in which their colonies would have been treatment-free is
then totally unknown, and could also be less than a year.
THE ‘NEVER TREATED’ GROUP IS NOW TREATED SEPARATLY
Considering this issue, the number of treatment-free beekeepers have to be
estimated without this “never treated” category.
THIS HAS NOW BEEN DONE
Also, one of the main conclusions of the authors seems to be that treatment-free
beekeeping would be increasing and that it would be a reliable solution as the
colony losses would not be greater: these conclusions are not supported by the
study as the survey was not designed properly to study these questions. It would
have required questions about the colony losses and about the age of the
beekeeping activity. These questions were not asked in the survey and the
references that the authors provide to support these conclusions are clearly not
sufficient to conclude about this. Indeed, the compared category is not the same
for the BBKA study, and the given references e.g. the Oxfordshire Beekeeping
Group are particular cases and not scientific literature. This does not seem to
be strong enough arguments to support such an important conclusion as the
supposed absence of additional colony losses in the case of treatment-free
beekeeping.
There are of course relevant questions and perspectives that this study opens
up. Still, it should be seen as such and not considered as actual and current
results.
WE HAVE REDUCED AND RE-WRITTEN THAT SECTION AND ADDED THAT AN ‘independent
scientific studies are required to see if Varroa-resistance effects the rate of
colony losses.’
10.1371/journal.pone.0281130.r005
Decision Letter 2
Corby-Harris
Vanessa
Academic Editor
2023
Vanessa Corby-Harris
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
16 Jan 2023
A survey of UK Beekeeper’s Varroa treatment habits
PONE-D-22-05221R2
Dear Dr. Martin,
We’re pleased to inform you that your manuscript has been judged scientifically
suitable for publication and will be formally accepted for publication once it
meets all outstanding technical requirements.
Within one week, you’ll receive an e-mail detailing the required amendments.
When these have been addressed, you’ll receive a formal acceptance letter and
your manuscript will be scheduled for publication.
An invoice for payment will follow shortly after the formal acceptance. To
ensure an efficient process, please log into Editorial Manager at
<http://www.editorialmanager.com/pone/>, click the 'Update My Information' link
at the top of the page, and double check that your user information is up-to-
date. If you have any billing related questions, please contact our Author
Billing department directly at <authorbilling@plos.org>.
If your institution or institutions have a press office, please notify them
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than 48 hours after receiving the formal acceptance. Your manuscript will remain
under strict press embargo until 2 pm Eastern Time on the date of publication.
For more information, please contact <onepress@plos.org>.
Kind regards,
Vanessa Corby-Harris, Ph.D.
Academic Editor
PLOS ONE
Additional Editor Comments (optional):
After carefully reading through the reviewers' comments on the first revision
and the authors' response to these comments, I feel this article is acceptable.
10.1371/journal.pone.0281130.r006
Acceptance letter
Corby-Harris
Vanessa
Academic Editor
2023
Vanessa Corby-Harris
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
20 Jan 2023
PONE-D-22-05221R2
A survey of UK Beekeeper’s *Varroa* treatment habits
Dear Dr. Martin:
I'm pleased to inform you that your manuscript has been deemed suitable for
publication in PLOS ONE. Congratulations! Your manuscript is now with our
production department.
If your institution or institutions have a press office, please let them know
about your upcoming paper now to help maximize its impact. If they'll be
preparing press materials, please inform our press team within the next 48
hours. Your manuscript will remain under strict press embargo until 2 pm Eastern
Time on the date of publication. For more information please contact
<onepress@plos.org>.
If we can help with anything else, please email us at <plosone@plos.org>.
Thank you for submitting your work to PLOS ONE and supporting open access.
Kind regards,
PLOS ONE Editorial Office Staff
on behalf of
Dr. Vanessa Corby-Harris
Academic Editor
PLOS ONE
[^1]: The authors have declared that no competing interests exist.
[^2]: Current address: School of Natural Sciences, National University of
Ireland Galway, Galway, Ireland |
# Introduction
Plant pathogenic *Xanthomonas* bacteria cause severe losses of crop production
worldwide. Their virulence mainly relies on a type-III-secretion system that
translocates effector proteins into plant cells. Such proteins interfere with
cellular processes and manipulate the plant to the benefit of the pathogen.
Transcription activator-like effectors (TALEs) constitute an important group of
effectors that manipulate the transcriptome of the host plant. After entering
the nucleus TALEs bind to promoter sequences and initiate transcription. TALE-
induced plant genes that support pathogen virulence encode transporters for
sugar or sulfate, factors that stabilize small RNAs, or transcription factors.
DNA association of TALEs is initiated by their N-terminal region whereas the
central part of TALEs provides specific binding to matching sequences. This
central DNA binding domain is composed of tandem repetitions of a nearly
identical 34 amino acid repeat. Each repeat mainly differs in two-amino acids,
the so-called RVD (repeat-variable diresidue), which encodes the specificity for
one or several DNA bases. The TALE repeats form a right handed superhelix that
wraps around the DNA double strand. Although these repeats can be rearranged to
specify practically any given target sequence (called TALE-box or EBE/effector-
binding element), a well-balanced presence of so called "strong" and "weak" RVDs
is required for efficient activation. A nuclear localization signal (NLS) and an
acidic activation domain in the C-terminal part ensure import into the plant
nucleus and gene activation, respectively. This modular structure and designable
DNA-binding specificity has turned TALEs into versatile tools for targeted
genome modification and gene regulation in many organisms.
Computational analysis of genomic target sites of natural TALEs showed a
preferential occurrence in apparent core promoter regions of -300 to +200 bp
around the transcriptional start site (TSS;). Previous studies based on the
TALEs AvrBs3, AvrXa7, and AvrXa27 showed that they shift the natural TSS of
target genes around 40–60 bp downstream of the position at which the TALE is
binding the DNA. Moving the AvrBs3-box in the *Bs3* promoter to a position
further upstream resulted in a concomitant upstream shift of the TSS. These
observations led to the impression that TALEs control the onset and the place of
transcription functionally analogous to the TATA-binding protein. One host
component that is involved in TALE-mediated gene induction has been identified.
A rice line that is homozygous for the *xa5* allele exhibits an increased
resistance to *Xoo* infection due to a loss of efficiency of TALE-mediated
transcriptional initiation. *xa5* represents a recessive point mutation of the
transcription factor IIA small γ subunit. Apparently, this mutation interferes
with the interaction of TALEs with TFIIA and likely accompanying basal
transcription-complex components. Furthermore, interactions with parts of RNA
stabilizing and modifying enzymes have been suggested for TALEs of the PthA
family, but so far a functional link is missing.
In contrast to the already intensively studied DNA-binding mode, the
circumstances for successful TALE-mediated transcriptional initiation *in
planta* are poorly understood. If the presence of a target sequence alone is
sufficient to initiate transcription at any genomic locus or if the target locus
has to satisfy additional requirements is still puzzling. Here, we
systematically analyze TALE-mediated gene activation potential in different
promoter contexts, from different positions or orientations within a promoter,
and in a possible cooperative fashion. To dissect whether an effect is dependent
on the TALE activation domain or the TALE DNA-binding domain, we replaced the
TALE activation domain with a heterologous activation domain and the TALE DNA-
binding domain with the Cas9-DNA-binding domain. Our findings suggest that TALEs
can not dictate transcriptional activation on their own, but rather rely on
further promoter elements. Furthermore, TALEs can support transcription by
binding in either forward or reverse orientation, an observation which has not
been reported, before. We show that this mode of activation is biologically
relevant, because reverse-binding TALEs are able to support virulence of *Xoo*
during an infection. This study is significant for understanding TALE-mediated
gene activation, and will have implications for target site prediction *in
planta*.
# Material and methods
## Bacterial strains and growth conditions
The bacterial strains used in this study were *Escherichia coli* Top10 (New
England Biolabs Frankfurt am Main, Germany), *Agrobacterium tumefaciens* GV3101
pMP90 and *Xanthomonas oryzae* pv. *oryzae* (*Xoo*) strains BAI3 and
BAI3Δ*talC*. *E*. *coli* cells were cultivated at 37°C in LB, *Xoo* strains at
28°C in PSA and *A*. *tumefaciens* GV3101 at 28°C in YEB medium.
## Plant material and plant inoculations
*Oryza sativa* ssp. *japonica* cv. Nipponbare was grown under glasshouse
conditions at 28°C (day) and 25°C (night) at 70% relative humidity (RH). Leaves
of 4-week-old plants were infiltrated with a needleless syringe and a bacterial
suspension of an optical density (OD) at 600 nm of 0.5 as previously described.
Symptoms (watersoaked lesions) were scored 5 days post inoculation (dpi).
*Nicotiana benthamiana* plants were grown under 16 h of light, 40–60% RH, at
23°C (day) 19°C (night) in the growth chamber. Leaves of 4- to 6-week-old plants
were inoculated with *A*. *tumefaciens* strains using a needleless syringe.
## Construction of artificial TALEs and the dCas9 activator
TALEs were constructed by using the Golden TAL technology as described by
Geißler *et al*. (2011). Up to six individual repeats were subcloned in an
assembly vector, resulting in a hexa-repeat module. Three of these hexa-repeat-
modules were then fused to the N- and C-terminal parts of Hax3 and inserted in
an expression vector for *Agrobacterium* or *Xanthomonas*, respectively. This
results in a N-terminally fused GFP for the *Agrobacterium* vector and in a
C-terminally fused FLAG Epitope in the *Xanthomonas* vector. The dCas9 variant
fused to the C-terminal region of Hax3 was used as dCas9 activator and generated
as described before. The activity score of the used sgRNAs was analysed by using
deskgen.
## β-Glucuronidase (GUS) reporter constructs and GUS activity analysis
β-Glucuronidase assays from plant samples were performed as described by Boch
*et al*., 2009. Briefly, the PCR-amplified fragments of the promoters (*Bs4*,
*OsSWEET14*) were cloned into pENTR/D-TOPO (Life Technologies GmbH, Darmstadt,
Germany) and fused to the *uidA* reporter gene by LR recombination into pGWB3.
To analyze reporter activity, *A*. *tumefaciens* strains delivering TALE
constructs and GUS reporter constructs were resuspended in infiltration medium,
resulting in an OD<sub>600</sub> of 0.8, mixed in equal amounts and inoculated
into *N*. *benthamiana* leaves. Two dpi, leaf discs were sampled and GUS
activities were quantified using 4-methylumbelliferyl-β-D-glucuronide (MUG).
Total protein concentrations were determined using Bradford assays. Data were
compiled from triplicate samples originating from different plants. Error bars
represent the standard deviation.
## RNA isolation and qRT- PCR
Leaves of 4-week-old Nipponbare plants were infiltrated with 10 mM
MgCl<sub>2</sub> or with the different *Xoo* strains using an OD<sub>600</sub>
of 0.5. At 1 dpi, 5 cm segments were harvested and rice total RNA was isolated
using the Qiagen RNeasy kit. cDNA was generated from 2 μg RNA using the
Fermentas first-strand cDNA synthesis kit (Thermo Fisher Scientific
Inc.,Waltham, MA, USA) and real-time PCR was performed using the iCycler (Bio-
Rad, München, Germany) as described before. The amplification efficiency for
each primer pair was analyzed using a standard curve plot of a dilution series.
cDNA amounts were normalized using actin as a reference gene. The fold change
induction was calculated in comparison to leaves treated with the BAI3Δ*talC*
mutant by using the ΔΔCt method.
## RNA isolation and 5´RACE
TALE-dependent transcriptional start sites at the *OsSWEET14* promoter were
determined by using 5´RACE. Leaf discs were harvested from the same plant
material that was used for determining GUS-activity in *N*. *benthamiana* and
total RNA was extracted by using the Qiagen RNeasy kit. 1 μg total RNA was used
to produce cDNA for 5´RACE by using the SMARTer RACE cDNA amplification kit
(Takara Bio, Inc., Shiga, Japan). The 5´ends were amplified by using the
universal forward primer and the gene specific primer GUS 5\`RACE, cloned, and
sequenced.
# Results
## TALE-mediated gene activation depends on flanking DNA sequences
So far it is unknown if TALEs that bind to a certain genomic target site (TALE-
box) are sufficient to initiate transcription at that location or if surrounding
DNA sequences influence gene induction. To address this, we designed reporter
constructs in which the Hax3-box, that is bound by Hax3, a natural TALE from
*Xanthomonas campestris* pv. *campestris*, is placed in front of varying
downstream sequences. A 300 bp fragment of the tomato *Bs4* promoter was used.
This promoter fragment has a very low basal activity which makes it well suited
for transcriptional activation assays. We subdivided this fragment into portions
of equal length (75 bp) and placed them downstream of the Hax3-box and upstream
of a promoterless *uidA* reporter gene. This setup ensured a fixed distance
between the TALE-box and the reporter gene open reading frame (ORF;).
Additionally, we questioned whether non-promoter regions can trigger TALE-
mediated gene expression. Hence, we included two random 75 bp fragments from the
*Bs4* ORF in our analysis. The reporter constructs and the constructs containing
*hax3* were co-transformed in *N*. *benthamiana* using *Agrobacterium*-mediated
T-DNA transfer. Quantification of the reporter gene activity showed that the
three *pBs4* derived fragments *pBs4-278*, *pBs4-126* and *pBs4-50* mediated the
strongest Hax3-dependent gene activation. In contrast, the two *Bs4* ORF
sequences *oBs4+550* and *oBs4+2362* and the *pBs4-202* promoter fragment
facilitate only a slight or no gene induction by Hax3. These findings indicate
that TALEs cannot initiate transcription at any genomic location but rely on
specific surrounding DNA sequences for efficient gene induction. To get a
general idea of other putatively involved DNA-binding factors we predicted
potential cis-regulatory motifs by using the program PlantCARE.
To further dissect which sequences and potential promoter elements support TALE
activity, we chose the fragment *pBs4-50*, that was highly responsive to Hax3,
and *pBs4-202*, that was not, and exchanged parts of them. We divided the
promoter fragments into four parts (A1 to A4 and B1 to B4) and generated
promoter-swap constructs fused to the Hax3-box and the *uidA* reporter gene
(75bp). In comparison to the reference *pBs4-50* only the constructs 1 and 10
showed a comparable or even higher activation by Hax3. This already indicates
two findings (i) the reason why *pBs4-50*, but not *pBs4-202* allows TALE-
dependent expression is not due to sequences directly downstream of the Hax3-box
that might interact with C-terminal parts of Hax3 and (ii) the presence of the
5´UTR in *pBs4-50* is not crucial for robust activity by e.g. influencing mRNA
stability. In contrast, all other constructs showed a decreased or no activity
(construct 2–9). Apparently, the predicted CAAT-box, the TATA-box, and the
sequence between them are relevant (, construct 6 vs. 10). In summary, these
results demonstrate that TALE-boxes which are targeted by TALEs are not always
sufficient to initiate transcription. This implies that TALEs, binding to these
sequences, do not initiate gene expression on their own. They likely need to
bind in proximity to supporting sequences—probably core promoter elements—to
ensure efficient gene activation.
## Systematic analysis of TALE-dependent reporter gene activation at the *OsSWEET14* promoter
To systematically investigate from which promoter positions TALEs can initiate
transcription we used a complementary approach in which we kept the promoter
sequence constant, but used designer TALEs targeted to different positions. The
well characterized rice *OsSWEET14* promoter was used, that is activated by a
number of natural and artificial TALEs. *OsSWEET14* encodes a sugar exporter and
represents an important rice virulence target whose TALE-dependent activation
supports bacterial virulence. An *OsSWEET14* promoter fragment 1000 bp upstream
of the ATG was chosen that comprises possible core and distal promoter elements.
The fragment was inserted upstream of a promoterless *uidA* reporter gene which
allows a systematic comparison of TALE activities in *N*. *benthamiana*. 14
artificial TALEs with an equal number of 17,5 repeats were built to target
various positions within this fragment (; ;. By using the TALgetter prediction
program, all TALEs used in this study have been carefully examined to bind
exclusively their desired target sequence and not at other possible locations in
the *OsSWEET14* promoter. As control to compare the impact of the varying RVD
sequences on TALE activity and to exclude an impact of variable flanking
sequences, we first analyzed the corresponding TALE-boxes in the *pBs4-*based
reporter construct and verified protein integrity *in planta*. Next, the TALE
constructs were co-delivered together with the *pOsSWEET14* based reporter
construct in *N*. *benthamiana* via *Agrobacterium*. The two natural *Xoo* TALEs
AvrXa7 and TalC which induce the *OsSWEET14* promoter in the natural *Xoo*-rice
interaction were used as positive controls and the *OsSWEET14* promoter in
combination with a non-matching TALE (Hax3) was used as negative control,
respectively. The majority of the analyzed TALEs activated the reporter gene
irrespective of their position in the *OsSWEET14* promoter. This demonstrated
that TALEs can drive gene expression from highly different positions in a
promoter. TAL7, TAL14, and TAL18 show a particularly low activity (\<35%
compared to TalC) at the *OsSWEET14* promoter although they efficiently induce
expression of the *pBs4*- driven reporter gene. This suggests that their binding
sites are not favorable or not accessible in this particular genomic context.
## Artificial TALEs can activate the *OsSWEET14* promoter in reverse orientation
All known TALE targets are based on TALEs binding a promoter in "forward"
orientation, with the C-terminal activation domain facing the downstream ORF. In
contrast, classical enhancer elements are often orientation-independent.
Therefore, we investigated whether TALEs also function in "reverse" orientation
*in planta*. Nine TALEs were designed to bind in reverse orientation in the
*OsSWEET14* promoter and TALgetter target site predictions verified that
these are the only possible binding-sites within the promoter. The two natural
*Xoo* TALEs AvrXa7 and TalC were used as positive controls. TALE constructs and
the reporter construct were co-delivered in *N*. *benthamiana* via
*Agrobacterium*.
Remarkably, the three reversely placed TALEs, TAL-11, TAL-1, and TAL-2,
activated the reporter highly efficient with an activity of 71%, 104%, and 179%,
respectively, compared to TalC. We used the highly active TAL-2 to analyze
experimentally whether its activation potential was indeed due to its binding in
reverse orientation. We designed two reporter constructs based on the *pBs4*
reporter system to exclude any *pOsSWEET14*-based effects. One reporter covers
the TAL2 binding site on the forward and the TAL-2 binding site on the reverse
strand fused to the minimal *pBs4* promoter and the *uidA* reporter gene. Both
TALEs activated this reporter albeit TAL-2 to a significantly lesser extent
compared to the *pOsSWEET14* context. As control, we mutated all five Guanine
nucleotides in the upper strand to Adenine (construct TAL2/-2mut). TAL2 should
still bind this sequence because its RVD NN recognizes G and A, whereas TAL-2
has HD RVDs matching only to C on the reverse strand. Accordingly, TAL-2 lost
the ability to activate the reporter verifying that TAL-2 in fact binds to the
reverse DNA strand.
The other reverse TALEs resulted in weak activities of 10 to 17% compared to
TalC although some of these (i.e., TAL-AvrXa7, TAL-81, TAL-82, TAL-9) target the
same region as highly active forward TALEs. All TALEs were active in combination
with forward-oriented TALE-boxes upstream of the *pBs4* minimal promoter, and
protein levels were comparable, indicating that the RVD composition is not
causing the observed variability.
In summary, the systematic analysis of differentially positioned forward and
reverse binding TALEs resulted in three important findings: (i) Some TALEs
(TAL11/-11, TAL2/-2 and TAL1/-1) activate transcription in both orientations,
suggesting that their DNA target sequences are accessible and allow a flexible
interaction with the transcriptional machinery. (ii) The activity of some TALEs
(AvrXa7/TAL-AvrXa7, TAL8/-81/-82 and TAL9/-9) is orientation-dependent,
suggesting that the reverse orientation in these cases prevented an efficient
recruitment of the transcriptional machinery. (iii) Some TALEs do not
efficiently activate gene expression irrespective of their orientation,
suggesting that either the TALE-box is not accessible within the promoter
context or the relative position is not suitable to recruit the transcriptional
machinery. The observed activity of reverse binding TALEs breaks the long-
standing assumption that TALEs only activate genes in one-direction.
## TALEs can synergize to induce transcription at the *OsSWEET14* promoter
Reverse-binding TALEs seem to be particularly dependent on further promoter
elements and we wondered whether they can enhance the capacity of other TALEs to
induce transcription. The phenomenon of synergistic gene activation has been
described in human cells using TALEs fused to the heterologous VP64 activation
domain, but it is unknown whether the native TALE has a similar potential in
plant cells. For this, we used low-activity artificial TALEs from our
collection. Individual TALEs or combinations of two or three TALEs and the
*OsSWEET14*- reporter were co-transformed into *N*. *benthamiana* and GUS
activity was measured. In most cases, combinations of TALEs at the promoter
increased transcriptional induction. Even the presence of low activity reverse-
binding TALEs can stimulate other TALEs (e.g., TAL3 and -AvrXa7), but this
requires that their relative position does not hinder each other e.g., TAL3 and
TAL-7 whose activation domains face each other and might spatially block the
simultaneous assembly of the transcriptional machinery. Together, this
demonstrates that multiple TALEs, including reverse TALEs and low-active TALEs
can act synergistically to efficiently induce gene expression in plants.
## The activity of TALEs targeted to reverse oriented sequences is dependent on other promoter elements
Based on our finding that the presence of core promoter regions can influence
TALE-mediated gene activation, we aimed to analyze the influence of the TATA-box
and surrounding DNA sequences on the activity of TALEs. For this, we used an
*OsSWEET14* promoter variant in which 33 bp encompassing the TATA-box were
deleted in rice via genome editing. This *ossweet14-11* variant lacks the
AvrXa7-box and is thus resistant to *Xoo* strains carrying the TALE AvrXa7. The
edited promoter was amplified from the rice mutant and inserted into the GUS
reporter. TALE and reporter constructs were co-transformed into *N*.
*benthamiana* and GUS activity was measured.
The deletion of the TATA-box and the surrounding sequences did not significantly
influence the activity of the majority of the forward binding TALEs, indicating
that an interaction with the transcriptional machinery in these cases is TATA-
box independent. The binding sites of TAL4, TAL5, and Avrxa7 are at least
partially deleted resulting in an expected loss of activity. Surprisingly, the
activities of the TALEs that bind directly upstream of the TATA-box, including
TAL1, TAL2 and TalC, were reduced by 60, 54, and 70%, respectively, if compared
to the activity measured at the wild type promoter. This indicates that the
activity of TALEs binding to a distinct region directly upstream of the TATA-box
is positively supported by this core element and surrounding sequences whereas
other TALEs are not. This effect is even more pronounced if the activity of
reverse binding TALEs is analyzed. The highly active reverse TALEs TAL-11, TAL-1
and TAL-2 show a strong decrease in their activity of 61, 81 and 91% compared to
the WT promoter. This shows that the activity of reverse binding TALEs in
particular is highly connected to the presence of supporting DNA-elements like
the TATA-box.
## The use of alternative activation and DNA-binding domains, respectively, do not change the relative activation pattern at the *OsSWEET14* promoter
TALE derivatives that were fused to the synthetic VP64 transactivation domain
and analyzed in human cell lines showed a highly position and orientation
independent activity and activated genes over long distances. TALE-VP16 fusions
are in principle known to be functional *in planta*, but with a much decreased
activity in comparison to natural TALEs. To analyze if the VP64 AD changes the
activation pattern relative to the binding position of TALEs *in planta* a
subset of our artificial TALEs targeting the *OsSWEET14* promoter was fused to
VP64 instead of the TALE AD. Their gene activation capacity was compared at the
*pOsSWEET14*::*uidA* reporter construct following transformation into *N*.
*benthamiana*. The overall activity of the VP64-fused TALEs was lower than the
activity of the TALEs containing their native AD, likely because VP64 is not
completely compatible with the plant transcriptional machinery. Importantly, the
use of VP64 instead of the TALE AD did not profoundly change the relative
activation level of the analyzed TALEs at their respective positions. This
indicates that the TALE AD functions in a manner that is analogous to the VP64
AD in plant cells.
Although TALEs employ a highly flexible DNA binding domain their access to DNA
can be inhibited by methylated cytosines or nucleosomes occupying the sequences.
The *Streptococcus pyogenes* Cas9 is a non-related DNA-binding protein that is
guided to target sequences by a mechanism that is unrelated to TALEs. DNA-
binding of SpCas9 is directed by a guide RNA that forms base pairing to 20
nucleotides in the target sequence and is therefore not affected by methylation.
To test whether the TALE DNA-binding domain or its activation domain is
responsible for the orientation-independent gene activation we analyzed the
TALE-activation domain in the context of the Cas9-DNA binding platform. We fused
the catalytically inactive "dead Cas9" (dCas9, D10A; N863A; lacking nuclease
activity to the C-terminus of Hax3 to generate a dCas9 activator that is
functional in plants. To compare the activity of TALEs and the dCas9 activators
we designed sgRNAs (single guide RNAs) that bind in proximity to existing TALE-
boxes. T-DNAs producing TALE and Cas9-activator, respectively, were co-
transformed with the reporter construct containing the *OsSWEET14* promoter into
*N*. *benthamiana* and reporter gene activation was determined. Those sgRNAs
that hybridized to the region neighboring the highly active TAL1 and TalC
mediated strongest reporter gene activation. Taken together, the use of an
alternative DNA binding domain identified the same promoter region upstream of
the TATA-box as highly suitable for activation by the TALE-AD.
## TALEs control the transcriptional start site dependent on their position
Previous studies indicated that TALEs can shift the transcriptional start site
(TSS) of a plant gene. Since some of them bound to sequences overlapping the
TATA-box, it is not clear whether the TSS shift in these previous examples is
based on the action of the TALE or by spatially blocking the TATA-box. Hence, we
aimed to systematically analyze TALE dependent TSS shifts in the *OsSWEET14*
promoter by using our collection of differentially positioned artificial TALEs.
The TALE constructs were co-transformed together with the reporter construct
into *N*. *benthamiana* followed by RNA extraction and 5´RACE. The
AvrXa7-dependent TSS at the *OsSWEET14* reporter construct in *N*. *benthamiana*
resembled the one following *Xanthomonas*-mediated delivery in rice indicating
that both systems are comparable.
The TALEs that were located downstream of the TATA-box (TAL3/-3, TAL-9, TAL-10;
) shifted the *OsSWEET14* TSS to positions around 40–60 bp downstream of the
respective TALE-box which is in concordance to previous observations (;.
Interestingly, the TSS for TAL3 and TAL-3 are comparable although the activation
domain which is supposed to be the platform to assemble the initiation complex
is located in a flipped orientation. TALEs binding upstream of the TATA-box
(TAL2/-2, TAL1/-1 and TAL11) on the other hand show a different response. TAL2
and TAL-2 initiate transcription around 50–60 bp downstream of their target site
which mainly resembles the natural *OsSWEET14* TSS. Although the binding site of
TAL1 and TAL-1 is located further upstream it primarily triggers the same TSS as
TAL2 and TAL-2. This suggests that the TSS in presence of TALEs that bind in a
certain region may not primarily be dictated by the position or orientation of
the TALE itself but by the proximity to other active promoter-elements e.g. the
TATA-box. In contrast, TAL11, which binds even further upstream of the TATA-box,
again resulted in a TSS 40-60bp downstream of its binding site and additionally
in the natural TSS. These results show that TALEs can trigger transcription in
two ways. Either they directly influence the TSS, suggesting a direct role in
the assembly of the pre-initiation complex (PIC) or they support other promoter
elements e.g. the TATA-box which recruit the PIC.
## Artificial reverse binding TALEs restore the virulence of the BAI3Δ*talC* mutant in rice
To analyze whether reverse binding TALEs can activate expression of target genes
in a natural infection, a subset of artificial TALEs was introduced into the
*Xoo* mutant BAI3Δ*talC* (. This mutant is deficient in the major virulence
factor *talc* which targets *OsSWEET14*. Consequently, this strain fails to
induce *OsSWEET14* and does not cause disease symptoms. Complementation of this
*Xoo* mutant strain with *talC* or artificial TALEs that target *OsSWEET14*
restored *OsSWEET14* induction and virulence
*Xoo* BAI3Δ*talC* was complemented with artificial TALEs from our collection,
and *talC*, respectively. The strains were inoculated into the rice variety
Nipponbare, and virulence symptoms were documented in comparison to the mutant
strain without any *OsSWEET14*-targeting TALE. To test TALE-dependent activation
of *OsSWEET14*, qRT-PCR was performed in parallel. The forward-binding TALEs
TAL2, TAL8, and TAL10 efficiently induced *OsSWEET14* and supported the
development of disease symptoms. Moreover, also the highly active reverse
binding TALE TAL-2 resulted in a gain of virulence and significant increase in
*OsSWEET14* expression. However none of the forward or the reverse binding TALEs
TAL14, TAL3/-3, TAL7/-7, and TAL-82, which had a low activity in the *N*.
*benthamiana* transient system, restored the virulence of BAI3Δ*talC* or
activated the expression of *OsSWEET14* significantly. One of the TALEs, TAL10,
did induce *OsSWEET14* expression, but did not lead to a gain of virulence.
Instead, it led to the formation of dark brown lesions around the inoculation
spot, suggesting that this TALE triggers a collateral defence reaction in
parallel which blocks the development of virulence symptoms. In summary, TALEs
can activate a target gene from different promoter positions and via binding in
the reverse orientation also in the biologically relevant natural infection
system.
# Discussion
TALEs are versatile virulence factors that have been evolved by the pathogen to
target different plant promoter sequences and induce a variety of target genes.
The unique modular TALE DNA-binding domain has been well studied, but how the
host transcription machinery is recruited and whether further promoter elements
are needed is still largely unknown. Protein interaction screens to identify
host components have yielded only few candidates, and those shed little light on
the gene induction requirements. Instead, we here used a set of functional
studies to gain first insights into the conditions under which TALEs induce gene
expression.
Our reporter gene induction experiments demonstrate that the presence of a TALE
binding site alone can not trigger transcription, instead, a specific DNA
context is required. This indicates that TALEs function analogous to enhancer-
binding proteins that require the basal transcription machinery for their action
and function as control agents for gene expression. Previous studies have
revealed a binding preference for natural TALEs at -300 to +200 bp around the
TSS of a target gene, a region that typically includes core promoter elements.
One of such elements apparently supporting TALE-mediated gene activation in some
of our experiments, is the TATA-box. Interestingly, several natural TALE-boxes
overlap with the TATA-box of their respective target genes. This finding led to
the assumption that TALEs functionally replace the TBP. According to our present
data, this is presumably not the case. Instead TALEs can benefit from the
presence of a TATA-box. In *Oryza sativa* ssp. *japonica* only around 19% of the
promoters contain a TATA-box. Indeed, some TALE-activated promoters (*Xa23*,
*OsSWEET2b*, *OsSWEET4*, *pAGT582-1*, and the fragments *pBs4-278*, *pBs4-126*
and *oBs4+550* used in this study) lack a prototype TATA-box. This shows that
the presence of a TATA-box is not absolutely required for TALE-dependent
induction, but can be substituted for by certain other sequences.
Using the prominent TALE virulence target *OsSWEET14* from rice, we could
further show that TALEs function from many different positions within a given
promoter, but not equally well. Although individual artificial TALEs have been
used before to induce promoters a quantitative comparison of multiple TALE
positions within a native promoter context has not been done, so far. The
variable efficiency of TALEs could be caused by several reasons: sequences are
blocked by other proteins or nucleosomes, TALEs do not bind equally well because
of their RVD composition, methylation of target sequences interferes with RVD-
base recognition, or by the relative position of TALEs to other transcription
factors. We surveyed available data to identify occupied and accessible DNA
regions in the *OsSWEET14* promoter. Although these data do not give a
consistent picture, the region upstream of the TATA-box should in principle be
accessible for TALEs. To exclude factors like RVD composition, DNA methylation,
and other effects specific for the TALE-DNA interaction, we employed designer
activators as fusions between dCas9 and the C-terminal domain of TALEs.
Comparison of a collection of sgRNAs to target the dCas9-activator to different
positions within the *OsSWEET14* promoter revealed that they are also position-
dependent and that it is not the DNA-binding domain, but rather the position of
the TALE activation domain within the promoter context that dictates the
efficiency of the activator. Although classical enhancers are typically regarded
as being highly independent in their positioning relative to a given promoter,
this is not unambiguously the case. In fact, re-positioning of such enhancer
elements within promoters demonstrated that they also show a different activity
at different positions. We postulate that TALEs can in principle function from
different positions within a promoter, thus functionally resemble classical
enhancer-binding proteins.
It has been reported earlier that TALEs shift the transcription start site (TSS)
to a position 40–60 bp downstream of their binding position upon gene
activation, but do not do so in a synthetic promoter with multiple inserted
TALE-boxes (and TATA-boxes). The analysis of our collection of TALEs targeting
the *OsSWEET14* promoter now suggests, that the TALE-dependent TSS shift does
not depend on the TALE alone, but depends on the presence of additional promoter
elements. TALEs positioned in a region closely upstream of the natural TATA-box
supported the natural TSS, whereas TALEs positioned further upstream or
downstream triggered novel TSS. We envision that TALEs are enhancer-binding
proteins that define their own binding position according to their modular DNA-
binding domain, but at the same time, they directly cooperate with and recruit
the transcription machinery.
In a breakthrough study one of these interactions could recently be shown. TALEs
interact with the gamma subunit of the basal transcription factor TFIIA, which
is well known to interact with activation domains of several activators similar
to the one from TALEs, belonging to the family of acidic activation domains
(e.g. VP16, Zta and Gal4). Indeed, when we replaced the native acidic activation
domain of TALEs with the VP64 activation domain, these activators contained a
similar relative activity profile, with their highest activity at comparable
positions to native TALEs at the *OsSWEET14* promoter. This shows again that the
position within the promoter dictates the overall efficiency of the activator
and that the activation domain can functionally be replaced by related ones. Our
TALE-VP64 fusions still contained most of the C-terminal domain of native TALEs
and the portion that interacts with TFIIAy. This domain might therefore have
facilitated the interaction to the basal transcription machinery, and caused the
comparable activity profiles along the different promoter positions. Because the
region upstream of the TATA-box in the *OsSWEET14* promoter is so well suited
for activators, we propose that our TALE-positioning screen actually identified
a region spanning a potential natural enhancer element that regulates the
expression of *OsSWEET14* in rice.
We were highly surprised to realize that TALEs also function when bound in a
reverse orientation relative to the open reading frame. This has, so far, not
been considered for the identification of TALE targets and the reverse binding
mode will be an important implementation to identify novel TALE virulence
targets. It further points out that the orientation, relative to the TSS, of the
TALE activation domain, which is interacting with host components, does not play
a role under certain, but not all, conditions. This observation further suggests
that TALEs may initiate transcription bidirectionally. A bidirectional
transcription describes a situation in which—originating from one promoter—two
divergent transcripts are initiated. This phenomenon has frequently been
observed for many promoters of human and animal origin and increasingly also for
plants. During the review process of our study another manuscript was published
which supported our findings that TALEs function when bound in reverse
orientation and initiate transcription bidirectionally. In this work, the
authors suggest that forward and reverse binding TALEs function equally well. In
contrast, in our experimental setup most reversely placed TALEs did not induce
transcription to the same level as forward placed ones ( an). This suggests
that although the bidirectional transcription is a general feature of TALEs it
is possibly restricted by surrounding promoter sequences. Therefore, we propose
that TALEs only function in reverse orientation if placed in defined promoter
regions that allow bidirectional transcription, e.g., the region upstream of the
TATA-box in the *OsSWEET14* promoter. This indicates that both orientations are
not equally well suited to induce gene expression and that TALEs prefer the
forward orientation. Possibly, in reverse orientation, the TALE protein itself
is blocking the path of the polymerase and an efficient establishment of a novel
TSS can only occur downstream of the TALE. This is supported by our observation
that both, forward- and reverse-binding TALEs upstream of the TATA-box trigger
the same TSS downstream. This further suggests that TALEs bound in either one of
the orientations can activate already present, but paused polymerase II
complexes.
Comparing the activity of our TALE collection at the wild-type and a promoter-
derivative lacking the TATA-box and surrounding sequences revealed that those
TALEs that are located closely upstream of the TATA-box function less well,
whereas further upstream positioned TALEs and TALEs downstream of the TATA-box
are not influenced. This indicates that TALEs can either use existing elements
or use novel downstream promoter sequences if they are suitable. In the latter
case, the TSS is changed. This observation is consistent with our promoter-swap
experiments which revealed that the region downstream of the TALE is crucial for
TALE-dependent gene activation. Intriguingly, the activity of the reverse-
oriented TALEs in particular is negatively impacted by the removal of the TATA-
box. This indicates that transcription initiated by reverse TALEs is especially
dependent on the presence of potent other promoter elements. This restriction is
likely the reason why so far most natural TALEs bind in forward orientation at
their respective target promoters.
Our combination of several weakly active TALEs targeted to either forward or
reverse binding sites in the *OsSWEET14* promoter revealed that TALEs can act
synergistically in plants. This feature was only shown in human cells before,
where TALE derivatives with a truncated C-terminus were fused to the VP64
activation domain. Interestingly, the synergistic effect was only observed if
TALEs do not spatially hinder each other. Our results further suggest that TALEs
*in planta* may not only act synergistically with each other but are further
supported by promoter elements which are possibly occupied by host transcription
factors (e.g. the TATA-box). Whether the synergistic activity of TALEs is based
on increased chromatin remodeling or other effects remains to be elucidated.
Our systematic analysis of variably positioned TALEs shows that they act in
principle as enhancer-binding proteins. When placing designer TALE activators in
promoters to trigger efficient target gene activation one should consider the
presence of existing promoter elements, e.g. the TATA-box, and the expected
possible shift of the TSS. The novel reverse binding mode of TALEs now allows
for the detection of formerly overlooked virulence targets in host plants.
Therefore, we have added the option to search for reverse-binding TALEs in the
TALE-target prediction programs TALgetter, and the feature is also implemented
in the alternative programs TALVEZ and TALE-NT 2.0, respectively. Forward and
reverse binding TALEs will furthermore be excellent tools to tackle fundamental
questions of gene induction in plants.
# Supporting information
We thank Ulla Bonas for support, and the 2013 MSc course *"Plant Genetics"* at
the Martin Luther University for demonstrating that reverse binding TALEs can
activate gene expression.
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** J. Streubel HB JB. **Formal analysis:** JG.
**Funding acquisition:** JB JG. **Investigation:** J. Streubel HB JB.
**Resources:** J. Stuttman. **Writing – original draft:** J. Streubel JB.
**Writing – review & editing:** J. Streubel JB. |
# Introduction
Intermittent hypoxia (IH) is a key pathological feature of obstructive sleep
apnea (OSA), the most common sleep related breathing disorder. Epidemiological
studies show that patients with OSA suffer an accelerated decline in kidney
function. However, it is unclear whether this is due to OSA *per se* or to
confounding factors such as obesity, hypertension, diabetes or other concomitant
disorders. A rodent model of OSA was developed in 2001 that simulates moderate
to severe OSA in clinical settings, and has been used to investigate the
systemic effects of OSA such as insulin resistance, endothelial vascular
dysfunction, and alterations in tumor-associated macrophages function.
Importantly, the pathological pattern of IH is distinct from the pathological
feature of sustained hypoxia, where IH promotes excessive production of reactive
oxygen species and inflammatory mediators, and increases sympathetic activity.
With respect to kidney disease, OSA enhances the activity of the renin
angiotensin-aldosterone system, increases the activity of the sympathetic
nervous system and generates systematic and local reactive oxygen species; these
alterations are known to induce functional and structural kidney damage. For
instance, focal segmental glomerulosclerosis (FSGS) is an important
histopathological component of impaired renal function that can result from
several metabolic and haemodynamic factors including a diabetic milieu,
increased blood pressure and mitochondrial oxidative stress. Mechanistically,
mesangial matrix expansion (MME) is the cornerstone of FSGS. Moreover, excessive
accumulation of glomerular extracellular basement membrane (mesangium) is driven
by an over- production of a number of growth factors including transforming
growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and vascular
endothelial growth factor-A (VEGF-A). However, the histopathological alterations
of the kidney in response to IH have not been reported. We tested the hypothesis
that IH alters glomerular structure and modulates the expression of glomerular
growth factors.
# Materials and methods
## Animals
After approval from the University of British Columbia Animal Care Committee,
ten 8-week old wild-type male CB57BL/6 mice were obtained from JAX Laboratories
and allowed to acclimatize for one week with free access to water and regular
chow before initiating the IH procedure.
## IH protocol
Animals were randomly assigned to receive either IH or intermittent air (IA) for
60 days as we described previously. Briefly, mice assigned to receive IH were
placed in specially designed cages containing oxygen sensors for measuring the
fraction of oxygen inspired (FIO<sub>2</sub>) in the cages. The cages were
connected to a gas regulator that allowed the flow of sufficient amounts of
nitrogen gas to reduce FIO<sub>2</sub> to 8% within 30 seconds, after which the
gas regulator allowed for a rapid replacement of the nitrogen by oxygen and
causing the FIO<sub>2</sub> in the cage to be restored to 21% (room air) within
30 seconds. This 1-minute IH cycle was repeated 60 times per hour for a total of
12 day-light hours per day.
## Animal monitoring and euthanasia
Animals were monitored twice a week for the following parameters: body weight,
activity, appearance, breathing and posture/gait. A score of 1 or 2 in any
category resulted in increased monitoring, including daily weighing, supportive
care (e.g. supplemental heat, SQ fluid replacement, gel food or food
treats/moistened pellets). A score of 3 in any category or a cumulative score of
\>5 after appropriate supportive care resulted in immediate euthanasia. Animals
were euthanized by inhaling an anesthetic followed by carbon dioxide.
## Kidney sectioning
After 60 days of either IH or IA, mice were euthanized and the right kidney from
each animal was fixed in 4% paraformaldehyde (Sigma-Aldrich, Germany) for 24
hours and then sectioned (5 μm) for histopathological and immunohistochemical
studies.
## Histopathological assessment
Two paraffin-embedded kidney samples from each group were sectioned (4 kidney
sections per slide) and stained with hematoxylin and eosin, periodic acid–Schiff
(PAS) and Masson's trichrome stains for pathological evaluation. Sections were
evaluated by a certified pathologist who was blinded to the study protocol (i.e.
blinded to identity of IA or IH samples) for evaluation of glomerular
congestion, matrix expansion and pelvic inflammation.
## Glomerular and mesangial area calculation
Following deparaffinization, kidney sections were stained with PAS for
assessment of the total glomerular tuft area and glomerular basement membrane
(mesangium) area. The mean glomerular tuft area was determined by counting all
available glomeruli (where the glomerular vascular pole is evident) within each
kidney section, where one kidney section was used from each animal. The
mesangial matrix area was calculated as the PAS-positive area compared to the
total glomerular tuft area using 50 randomly selected glomeruli per kidney
section (see below). Images were taken at 400x magnification using an Aperio
ScanScope**®** CS microscope slide scanner, and measurements of glomerular tuft
area were made using Aperio ImageScope (V 12.1.0.5029) software.
## Glomerular selection for area measurements and protein expression
We created a virtual grid with 25 different slots to enable sampling of all
areas of each kidney section. A minimum of two different glomeruli were randomly
selected from each slot for mesangial area measurements and protein semi-
quantification. As some slots did not include any glomeruli (example: slot E1),
the total number of missing glomeruli from such slots was calculated, allowing
for a similar number of glomeruli to be reinvestigated so that we were able to
sample 50 glomeruli per kidney section.
## Immunohistochemistry
Sections of kidney tissues were deparaffinised and rehydrated using xylene and
downgraded concentrations of alcohol, after which slides were immersed in 10 mM
sodium citrate (pH 6) for 20 minutes in a steam bath (95 °C) to allow for
antigen retrieval. Slides were then incubated in 3% hydrogen peroxidase (Sigma-
Aldrich–Germany) for quenching of endogenous peroxidase activity, and non-
specific proteins binding was blocked using 1.5% normal blocking serum for 90
minutes. Kidney sections slides were then incubated in goat anti-mouse TGF-β1
(1:50 sc-146-G), CTGF (1:50 sc-14939) and VEGF-A (1:50 sc-152-G) primary
antibodies (Santa Cruz Biotechnology–USA) for 24 hours at 4°C. After several
washes, slides were incubated with avidin biotinylated horseradish peroxidase-
labeled secondary antibody as per manufacturer instructions (ImmunoCruz™ goat
ABC Staining System: sc-2023). Finally, slides were stained with
3,3′-diaminobenzidine (DAB) chromogen for protein detection and quantification,
and counterstained with hematoxylin for detection of nuclei. Images were taken
at 400x magnification using an Olympus BX61 electron microscope. Only 50 images
of randomly selected glomeruli were considered per kidney section, and images
were analyzed using IHC Profiler plugin within ImageJ free software, which
measures the intensity of the brown (DAB) color caused by the antigen-antibody
reaction. The percentage of only “highly-positive” brown staining in each
glomerulus was measured, so reducing the possibility of other colors influencing
the brown stain and thus enhancing the accuracy of protein quantification. The %
of highly-positive staining was averaged from 50 different glomeruli per kidney
section (to give n = 1).
## Western blotting
Upon euthanasia, 20 mg of the left kidney cortex of each animal was harvested
and homogenized in RIPA lysis buffer (sc-24948), sonicated thoroughly and then
kept frozen (-80 degrees) for protein analysis. A total of 50 μg total protein
from each tissue homogenate was used for protein electrophoresis separation on
12% precast polyacrylamide gel (Bio-Rad: 4568044) as per manufacturer
instructions. Proteins were then transferred to nitrocellulose membranes (Bio-
Rad: 1620115) and incubated with rabbit anti-mouse 1:1000 β-actin (cell
signaling: 4967), 1:1000 GAPDH (Cell signaling: 2118), 1:100 TGF-β1 (sc-130348),
1:100 CTGF (sc-365970), 1:100 VEGF (sc-7269), 1:100 hypoxia inducible factor-1α
(HIF-1α) (sc-13515), 1:100 Bax (sc-7480) and 1:100 Bcl-2 (sc-7382) primary
antibodies overnight at 4 degrees. After several washes, the membrane was
incubated for 1 hour in horseradish peroxidase-conjugated anti-rabbit secondary
IgG (cell signaling: 7074S) for β-actin and GAPDH proteins detection, and with
horseradish peroxidase-conjugated mouse IgG kappa binding protein (sc-516102)
for detection of other mouse monoclonal antibodies. Finally, 1 ml of Clarity™
Western ECL Blotting Substrates (Bio-Rad: 1705060) was added to the membrane for
chemiluminescent signaling detection, and images were taken by ChemiDoc™ XRS+
System device (Bio-Rad: 1708265). Images were analyzed by Imagej software and
reported as the pixels’ intensity of a target protein relative to either β-actin
or GAPDH housekeeping proteins.
## Fluorometric *In situ* cell death detection
Paraffin-embedded kidney sections were deparaffinized and rehydrated as
described earlier. Slides were then incubated in phosphate-buffered saline with
tween-20 (PBST) solution for 10 minutes for the purpose of permeabilization.
After washing with PBS, 50 μl of TUNEL reaction mixture (Roche Applied Science–
12156792910, USA) was added to each kidney section, and sections were then
incubated at 37 °C for 1 hour in a humidified chamber. After several washes,
slides were counterstained with 4',6-diamidino-2-phenylindole (DAPI) stain for
detection of nuclei and finally mounted. Fluorescence images were taken with
Olympus BX61 electron microscope at 200x. Four random images were taken from
each kidney section to give n = 1, with one kidney section per animal. The
number of TUNEL staining-positive cells was normalized to the number of
available cells in each image, and averaged from 4 images per kidney section.
Cell counting was performed using cell counter plugin within ImageJ free
software.
## Renal function index
At the end of study protocol, each mouse was kept separately in a metabolic cage
for 24-hour urine collection. Urine samples were kept at -20 degrees for total
urinary albumin excretion analysis. Total urinary albumin was quantified using a
mouse albumin ELISA kit (41-ALBMS-E01) as per manufacturer instructions. Serum
creatinine was measured by a creatinine enzymatic assay (Crystal Chem: 80350) as
per manufacturer instructions.
## Statistical analysis
Data are presented as mean ± SD. The normal distribution for each set of the
results was tested using the Shapiro-Wilk test of normality; an unpaired t-test
was used to calculate differences between groups using GraphPad Prism (Version
6) statistical software, and p values less than 0.05 were considered
significant.
# Results
## Animal characteristics
Some animals showed signs of minor distress during the initial phase of exposure
to IH, but no signs of illness were observed in any animals during the study
period. There were no differences in kidney weight, body weight or fasting blood
sugar for IH and IA groups. Details of animal characteristics at the end of the
study are shown in.
## Histopathological assessment
A subjective histological assessment by an independent pathologist indicated no
obvious differences between groups in PAS and trichrome staining, except for a
variation in the amount of mesangial matrix in the glomeruli of the IH group
that required further non-descriptive analysis. On the other hand, differences
were evident in hematoxylin and eosin stained slides. Kidney sections from the
IH group had a mild degree of glomerular congestion, mesangial expansion and
pelvic inflammation. shows examples of kidney sections stained with hematoxylin
and eosin, PAS and Masson's trichrome stains.
## Glomerular area and MME
A minimum of 145 different glomeruli per kidney section was included in the
analysis of glomerular tuft area. The glomerular area in mice exposed to IH was
increased by 13.4%; the average glomerular tuft area in the IH group was 2782.84
± 72.68 μm<sup>2</sup> (95% CI: 2692–2872 μm<sup>2</sup>) compared to an average
glomerular tuft area of 2454.98 ± 73.59 μm<sup>2</sup> (95% CI: 2362–2546
μm<sup>2</sup>) (p\< 0.001) in the IA control group. Further examination of PAS
stained kidney sections indicated an expansion of mesangial matrix in the IH
group by 1.8-fold compared to controls. The mean fraction of mesangial matrix to
glomerular tuft area in IH exposed mice was 0.087 ± 0.015 (95% CI: 0.032–0.065),
compared to 0.049 ± 0.012 (95% CI: 0.064–0.108) in controls (p\< 0.01).
## Immunohistochemistry
We examined a number of profibrotic proteins to explore their role in MME
secondary to IH. The glomerular expression of TGF-β1 and one of its essential
downstream proteins, CTGF, were significantly increased by 2.7 (p\< 0.05) and
2.2 (p\< 0.01) fold respectively in response to IH. Moreover, the glomerular
expression of VEGF-A was significantly increased by 3.7-fold (p\< 0.05) in the
IH group compared to control IA group. summarizes the differences in glomerular
protein expression between IH and IA groups.
## Western blotting
Cortical TGF-β1, CTGF and VEGF proteins were semi quantified by western blotting
analysis. Compared to the IA group, the expression of TGF-β1, CTGF and VEGF-A
proteins in IH-exposed mice was greater by 1.9, 4.0 and 2.6-fold respectively
(p\< 0.05 for all). The cortical expression of HIF-1α protein was also higher in
the IH group by 2.9 times (p\< 0.05). Finally, the ratio of apoptotic
(Bax)/antiapoptotic (Bcl-2) proteins was 2.4 fold higher in IH-exposed mice (p\<
0.01). summarizes cortical protein expression in IH and IA groups.
## Fluorometric *in situ* cell death detection
Five paraffin-embedded kidney sections from each group underwent TUNEL-staining
for detection of cells with DNA damage, and counterstained with DAPI for
detection of nuclei. The percentage of TUNEL-positive nuclei in the IH group was
3.2 ± 0.67% (95% CI: 2.7–3.8%) compared to 0.38 ± 0.27% (95% CI: 0.14–0.61%) in
the IA control group (p\< 0.001).
## Renal function index
An average of 0.9 ± 0.13 ml and 0.86 ± 0.22 ml of urine was collected from IH-
exposed and control mice respectively (p = 0.45). The average 24-hour urinary
albumin excretion in the IH-exposed mice was 43.4 ± 16 μg (95% CI: 29.3–57.5
μg), while in control mice urinary albumin excretion averaged 9.7 ± 4.6 μg (95%
CI: 5.7–13.8 mg) in 24-hours (p\< 0.01). In contrast, the average serum
creatinine level in IH-exposed mice was 108.02 ± 56.1μmol/L, compared to 86.1 ±
46.4 μmol/L in the IA control mice (p = 0.51).
# Discussion
Our study shows for the first time that IH causes glomerular hypertrophy and
expansion of the glomerular mesangial matrix in a mouse model of OSA. We also
show that mice exposed to IH have increased expression of glomerular TGF-β1,
CTGF and VEGF-A proteins. In addition, there was a significant increase in renal
cellular apoptosis after 2-months of IH. Our data also shows that IH-exposed
mice excrete higher amounts of urinary albumin compared to control mice,
suggesting that there was mild disruption of glomerular filtration in these
mice, possibly related to glomerular hypertrophy. There was no evidence of
severe renal functional damage secondary to IH, at least based on serum
creatinine data.
Glomerular hypertrophy is associated with glomerulosclerosis, where the
interplay of hemodynamic and various growth factors determines the balance
between glomerular matrix accumulation and degradation. Although MME occurs in
diabetes, other diseases such as hypertension also cause glomerular hypertrophy
in humans. Nevertheless, factors such as obesity and hyperuricemia are also
associated with glomerular hypertrophy. Increases in glomerular profibrotic
cytokines is suggested to be the principal pathway in the pathogenesis of MME or
glomerular hypertrophy. With respect to renal pathophysiology, TGF-β1 is a
pleiotropic cytokine secreted by glomerular mesangial cells and podocytes in
response to common fibrogenic stimuli, and is considered the primary fibrogenic
growth factor in the pathogenesis of renal fibrosis. In addition, TGF-β1 is also
a major inducer of CTGF in renal fibrosis, where excessive production of
glomerular CTGF is implicated in glomerulosclerosis and thickening of glomerular
basement membrane.
Several epidemiological studies suggest a possible relationship between sleep
apnea and declining kidney function. A retrospective cohort study reported that
about 30% of patients with sleep-related breathing disorders were also diagnosed
with chronic kidney disease (CKD), which was significantly greater than the
prevalence of CKD in the healthy population. However, the apnea-hypopnea index
was not a significant determinant of CKD in sleep-related breathing disorder
patients. Another retrospective cross-sectional study detected significant
reductions in glomerular filtration rates as the severity of OSA increased. The
prevalence of diabetes and hypertension is also significantly higher in patients
with severe OSA. On the other hand, sleep apnea is a significant comorbidity in
patients with CKD as shown in a prospective study where the prevalence of sleep
apnea (predominantly obstructive) increased significantly with declining kidney
function. This study reported that 57% of patients with end-stage renal disease
had sleep apnea, compared to 41% of patients with CKD not on dialysis and 27% of
patients with glomerular filtration rates of ≥60 ml/min.
Suggested mechanisms linking OSA and CKD include, but is not limited to,
activation of the renin-angiotensin-aldosterone system (RAAS), increased
sympathetic regulation and elevations in systemic and local reactive oxygen
species. The effect of nocturnal hypoxia is common in OSA patients and cannot be
overlooked in the context of OSA-related CKD. A recent cohort study reported
that OSA patients with nocturnal hypoxia (defined by oxygen saturation\< 90%
for\> 12% during night-time monitoring) were at significant risk for accelerated
loss of kidney function, with an adjusted odds ratio of 2.89 (95% CI:
1.25–6.67). In addition, another study reports that RAAS can be influenced by
nocturnal hypoxemia in OSA patients, where patients with severe hypoxemia (mean
SaO<sub>2</sub>≤ 90% during overnight) have significantly greater renal RAAS
activity compared to moderate hypoxemia patients (SaO<sub>2</sub>≥ 90%) and
control subjects. Interestingly, continuous positive airway pressure (CPAP)
therapy reverses the increased activity of RAS in patients with OSA, as shown by
a recent clinical trial where CPAP therapy increased renal plasma flow and
significantly reduced plasma aldosterone and urinary protein excretion in non-
diabetic normotensive OSA patients.
Although our study focusses on glomerular signs of injury secondary to IH, we
have not examined specific markers of tubular structural or functional injury.
The chronic hypoxia hypothesis states that hypoxia is a key player in inducing
primary glomerular injury and that it creates a hypoxic tissue environment that
eventually triggers tubular injury. In fact, renal tissue hypoxia is a major
pathological mechanism in triggering several renal pathologies such as
hypertensive and diabetic nephropathies. Renal tissue hypoxia induced in rats by
administering dinitrophenol (a mitochondrial uncoupler that increases oxygen
consumption) increases markers of renal injury similar to those occurring in
hypertensive and diabetic nephropathy, e.g. increased renal tissue oxygen
consumption and urinary protein excretion.
Despite growing clinical evidence of OSA-related CKD, the direct effect of OSA
on kidney structure and function is largely unexplored, leaving the pathological
association between OSA and CKD unclear. A possible explanation for this is that
the majority of OSA patients frequently present with multiple comorbidities,
making it difficult to establish the direct effects of OSA on CKD. Studies on
animal models may therefore be a useful approach to examine the direct effects
of OSA on kidney pathology. For example, a study in mice using similar study
design as in our study reported that 8 weeks of IH upregulated a number of
inflammatory and profibrotic proteins (including CTGF) in kidney tissues,
suggesting a direct destructive effect of IH on kidneys. However, unlike the
urinary protein analysis data reported in our study, Sun et al reported no
differences in 24-hour excretion of urinary proteins. Others report that
metallothionein (potent antioxidant) knock-out mice were more prone to renal
damage when exposed to 8 weeks of IH. Another recent study reported that 2 weeks
of IH significantly increased plasma VEGF in mice; interestingly, mouse
macrophages exposed to IH *in vitro* also have significant increases in VEGF
expression. Such studies not only strengthen a clear pathological linkage
between OSA and the renal system, but also illustrate potential treatment
strategies to avoid OSA-related renal consequences.
The animal model of IH we use has also been used to investigate other sleep
apnea-related comorbidities such as cancer. It is important to note variations
in the severity and duration of IH in this model; for instance, Almendros and
colleagues reported a significant increase in lung epithelial TC1 cell tumors in
mice exposed to levels of IH that are close in its severity to the hypoxic
levels in our study (FIO<sub>2</sub> = 6%), but with a duration of only 28 days.
Another study by the same group reported increased melanoma lung metastasis in
mice after being exposed to IH for 30 days using FIO<sub>2</sub> of 5%, but for
only 6 hours per day.
Our study has a number of strengths; the use of an animal model excludes the
effects confounding variables such as pre-existing hypertension, diabetes, and
obesity that are potential confounders in human studies. However, our study also
has a number of limitations. First, the number of mice studied was relatively
small. Second, our mouse model is not a perfect representation of human OSA. For
example, patients with OSA tend not to have such severe desaturation, and suffer
from hypercapnia during events as opposed to hypocapnia. However, IH is one of
the key components of OSA, and this model has been used extensively in the
literature especially in terms of elucidating cardiometabolic complications
associated with OSA. Another important limitation of this model is that it does
not allow for easy recording of factors usually accompany sleep apnea, such as
sleep fragmentation. The recurrent arousals combined with
hypoxemia/reoxygenation can activate the sympathetic nervous system, oxidative
stress, and inflammation; these may represent important components in the
increased risk of cardiovascular diseases including myocardial infarction and
stroke in OSA patients. Although mice exposed to IH in our study did not show
any signs of diabetes and obesity, we were unable to reliably monitor blood
pressure during the wake and sleep cycles of mice. Others have reported
increases in the mean arterial pressure (by \~14 mmHg) in a rat model of IH.
Therefore, we are unable to rule out the effects of secondary hypertension due
to IH on glomerular alterations.
In summary, we show that kidneys are an important target for the harmful effects
of IH. We provide evidence for glomerular hypertrophy and MME in response to IH,
with simultaneous increases in glomerular TGF-β1, CTGF and VEGF-A proteins.
Taken together, these findings allow for a better understanding of the
mechanisms by which IH induces renal damage, and suggest potential targets for
mitigating the potential kidney damage related to OSA.
# Supporting information
The support of the Saudi Arabian Cultural Bureau in Canada (to BA) is gratefully
acknowledged.
[^1]: The authors have declared that no competing interests exist.
[^2]: ‡ These authors also contributed equally to this work. |
# Introduction
Secondary metabolites are often bioactive and are thus an attractive source of
lead compounds in drug discovery efforts. In many cases, secondary metabolites
isolated from higher organisms such as marine invertebrates are thought to
ultimately derive from symbiotic bacteria residing in these hosts. In the
majority of cases, little is known about the symbionts that produce secondary
metabolites due to difficulties in culturing and/or sequencing their genomes
directly from complex microbiomes. However, understanding these symbionts'
lifestyle is of critical importance, both to natural products discovery and
chemical ecology. In our own efforts, we have used the tunicate *Lissoclinum
patella* as a model system to understand the interactions between microbial
symbionts, host animals and secondary metabolite chemistry.
*L. patella* is a colonial tunicate in the family Didemnidae, with a wide
distribution across much of the Western Pacific. Like many didemnid species, *L.
patella* harbors a photosynthetic symbiont, the cyanobacterium *Prochloron
didemni*. *P. didemni* carries out several metabolic functions for the host, and
has been shown to synthesize a series of highly modified cyclic ribosomal
peptides, termed cyanobactins. The biosynthetic pathways that make cyanobactins
are all related to the prototypical patellamide pathway. One or several
precursor peptides are expressed, containing the residues that are incorporated
into the finished compounds, flanked by recognition sequences and a leader
peptide on the N-terminus. A heterocyclase may act on the immature precursor
peptide, to produce methyloxazoline, thiazoline and oxazoline from threonine,
cysteine and serine, respectively. Optionally, these heterocycles can be
oxidized by an oxidase in the pathway (i.e. to methyloxazole, thiazole,
oxazole). The leader peptide and the 5′ recognition sequence are then cleaved by
a protease homologous to PatA. The last step in the process is the cleavage of
the 3′ recognition sequence and macrocylization of the precursor peptide by a
PatG homolog. In some pathways, the macrocycle can be further prenylated, if
suitable side chains remain. Remarkably, the patellamide pathway and relatives
are extremely tolerant to altered precursor cassettes, and are capable of
processing precursor sequences quite unlike the naturally occuring compounds. It
is clear that the currently known cyanobactins account for a miniscule portion
of the chemical diversity that is biosynthetically possible, suggesting strong
evolutionary or other influences on natural systems.
Beyond *P. didemni*, *L. patella* has a complex microbiome of resident
microflora, which contribute to secondary metabolite production and which vary
according to microhabitat within the animal. We recently described another
symbiont, *Candidatus* Endolissoclinum faulkneri, which is found only in a
subset of *L. patella* animals and is associated with the presence of the highly
cytotoxic patellazoles, that may serve a protective function for the host
animal. Our analysis of *Ca.* E. faulkneri genomes indicates that the bacterium
is a long-term symbiont that is exclusively vertically transmitted. Strains of
*Ca.* E. faulkneri from phylogenetically distant hosts are correspondingly
divergent in genomic sequence, indicating genetic isolation. The patellazoles
biosynthetic pathway is a large polyketide synthase (PKS) system, which has been
maintained for the ∼6–31 million years that *Ca.* E. faulkneri has been
associated with *L. patella*.
In contrast to *Ca.* E. faulkneri, several lines of evidence suggest that *P.
didemni* can be transmitted between hosts both horizontally and vertically. *P.
didemni* genomes obtained from geographically distant animals are remarkably
similar (above 97% nucleotide sequence identity across the whole genome),
indicating that these strains are not genetically isolated. This strongly
suggests there is at least a transient free-living fraction of the *P. didemni*
population that can move between hosts. Consistent with this notion, although
*P. didemni* has never been cultured outside of its host, genome analysis
suggests that independent life may be possible. In fact, it has been found that
the microenvironment inhabited by *P. didemni* varies significantly in terms of
O<sub>2</sub> saturation and pH during dark/light cycles, indicating that in
contrast to reduced-genome intracellular symbionts, *P. didemni* must maintain
the ability to adapt to different environmental conditions. Stable *P. didemni*
symbiosis is limited to the didemnids, but unstable associations have been
reported in other host groups, such as holothurians and sponges. Amongst the
didemnids, *P. didemni* phylogeny has been found to be independent of host
species, and the distribution of photobionts amongst this group suggests
multiple symbiosis establishment events. The presence of a specialized vertical
transmission apparatus only in *Diplosoma* likewise suggests parallel evolution
of such mechanisms corresponding to multiple origins for this symbiosis.
Consistent with findings in *P. didemni*–didemnid relationships, we previously
found a random distribution of *P. didemni* secondary metabolite pathways across
and within host species. However, we observed that in some cases, different
animal species collected in the same vicinity contained different secondary
metabolites, suggesting a degree of symbiont selection. We also observed that
chemistry is not consistent across all *L. patella* samples. Many ascidian
species have been found to encompass cryptic species, due to their similar or
identical morphologies. We hypothesized that similarly, the designation *L.
patella* may contain several divergent populations. In the present work, we show
that *L. patella* encompasses at least three distinct phylogenetic groups, and
that these groups contain different secondary metabolites. In the case of
symbionts that can be horizontally-acquired, such as *P. didemni*, this suggests
the host is involved in secondary metabolite selection from a free-living pool.
# Results and Discussion
As a result of our long-standing interest in didemnid tunicates, *P. didemni*
and the cyanobactins, we have collected specimens of *L. patella* spanning a
large geographic area from Fiji to Palau between 2002 and 2011. We were able to
amplify host 18S rRNA genes from a number of these samples, and all show \>98%
nucleotide identity to an 18S rRNA sequence in the NCBI database identified as
*L. patella* (accession no. AB211085, see). We constructed a phylogenetic tree
based on these nucleotide sequences plus other members of Didemnidae from the
NCBI database and the *Ciona intestinalis* 18S rRNA gene to act as an outgroup.
All of our sequences formed a clade along with the type *L. patella* sequence.
We then turned our attention to the mitochondrial cytochrome *c* oxidase I
(COXI) gene, because this marker has been used for fine phylogenetic
distinctions and identifying cryptic species, including many types of tunicate.
Using a variety of primer sets, we were able to amplify COXI genes from 15
animals. The resulting sequences were pooled with other Didemnidae COXI
sequences from NCBI along with a *Ciona savigyni* COXI sequence to act as an
outgroup in a tree based on the translated protein sequences. Unfortunately, we
found that amongst these NCBI sequences, two non-overlapping sections of the
COXI gene were present, and thus we could not make one complete COXI tree with
all sequences. In particular, the only reported *L. patella* COXI sequence could
not be included in the tree containing our sequences, although we were able to
compare it to full length COXI sequences assembled from shotgun Illumina data
obtained from three animals (vide infra).
Our collections of *L. patella* fall into at least three separate clades. We
already observed that animals containing *Ca.* E. faulkneri and patellazoles
were restricted to a divergent clade we termed group ‘B’. Detailed analysis of
the COXI nucleotide identities of this clade reveal it contains three highly
divergent cryptic populations which could be different species. We now see that
the animals of group ‘A’ are also somewhat divergent. When we examined in detail
the pairwise nucleotide identities of COXI sequence, we saw that animals
collected in 2005 from Southeastern Papua New Guinea have significantly diverged
from other group ‘A’ animals, with identities ranging from 91–95%. This
divergence is on the order of other cases of cryptic speciations claimed in
tunicates based on COXI divergences between 2 and 16.5%. In a survey assessing
the use of COXI for phylogenetic distinctions, it was found that conspecific
samples rarely diverged more than 2% in nucleotide identity. Therefore it is
possible that animals from the ′05 collection are a distinct cryptic species
from other group ‘A’ animals. We found evidence that there may be further
cryptic populations in the remaining group ‘A’ animals, as animal E11-097
exhibited an intermediate divergence between both the 2005 collection and the
remainder of group ‘A’ (90–95% identity). We therefore term these groups as A1
(05–033, 05–039, 05–027, 05–044), A2 (E11-097) and A3 (L4, 07-103, 03-005,
07-002B, 07-005, and 07-110). Interestingly, we were able to compare the *L.
patella* COXI sequence from NCBI (AB602781.1) with full length COXI sequences
assembled from Illumina shotgun sequencing data of L2, L5 and L6. This revealed
that AB602781.1 was as closely related to L5 and L6 as it was to L2. The
pairwise sequence identities between all three groups (L5/L6, L2 and AB602781.1)
were similarly and significantly different. This suggests that AB602781.1,
collected in Sanur, Bali, may be another member of group B and could contain
*Ca.* E. faulkneri and the patellazoles.
The divergent group B contains individuals collected over a wide swath of the
Pacific, from the Eastern Fields region south of Papua New Guinea to Fiji.
Conversely, group A1 contains only individuals collected off Southeastern shores
of Papua New Guinea and is minimally divergent. These results alone would
suggest that a primary influence on *L. patella* phylogeny is geography along
with founder effects, similar to some populations of sponges. However, group A3
is phylogenetically quite uniform and yet covers a large area encompassing the
Bismarck Sea and Palau. Further sampling is required to determine whether A1 and
A2 are truly more geographically restricted than A3, and this will likely reveal
the full extent of these groups' ranges and also determine whether any coexist
in the same locale. Coexistence might suggest that there is little genetic
exchange between the groups; for instance there are several color morphotypes of
the didemnid tunicate *Didemnum molle* that are phylogenetically distinct.
Sometimes several morphotypes occupy the same area and thus there may be
mechanisms to maintain reproductive isolation of these forms. From analysis of
the aligned COXI sequences in our tree set, the different *D. molle* morphotypes
shared between 89 and 97% nucleotide identity. Another didemnid tunicate, *D.
vexillum*, was recently found to be composed of two phylogenetic clades. The two
clades share ∼96% COXI nucleotide identity, and the authors concluded that these
were not separate species. Nevertheless, colony fusion experiments showed
significantly higher success rates amongst the genetically similar invasive form
from New Zealand versus the genetically diverse population from Japan.
We then examined the secondary metabolite chemistry of *L. patella* animals by
LCMS, identifying known cyanobactins and patellazoles previously found in *L.
patella* based on their mass. We used skiff, a Perl script used in the Clovr-16S
pipeline, to analyze the tabulated peak areas. This script's intended purpose is
to take tables of 16S abundances, binned at an arbitrary taxonomic level, and
create a heatmap and dendrograms that reflect the Euclidean distance between
normalized samples and bins. Because skiff is agnostic as to the type of data it
receives, we were able to use it to visualize the Euclidean distance between
samples based on *L. patella* chemistry as shown by a dendrogram. With this
method, we observe that the secondary metabolites present mirror the clades we
determined in our COXI phylogenetic tree, except that E11-097 is closer
chemically to the A1 clade than phylogeny would suggest. The A1 members are
classified by the presence of lissoclinamides 5–8, ulicyclamide and isomers, as
well as ulithiacyclamide. The A3 group lack lissoclinamides 5–8 and in addition
can contain lissoclinamide 1, 9 and patellamides. The B group are quite
chemically distinct. As well as containing the patellazoles, they also contained
different cyanobactins compared to group A animals, including the prenylated
patellins and trunkamide A.
It is not surprising that the patellazoles are limited to group B animals,
because these compounds are produced by an exclusively vertically transmitted
symbiont that is not found in other clades (vide supra). However, it is notable
that even though *P. didemni* populations are highly uniform and do not
correlate with host phylogeny, there is a strong host phylogenetic signal for
their secondary metabolites. We previously found a high sequence identity across
three whole *P. didemni* genomes (\>97%), in both patellazole containing and
patellazole-negative animals. This suggests that *P. didemni* strains are not
genetically isolated, and that there must be significant horizontal exchange
between strains in different hosts, in addition to well-established vertical
transmission mechanisms. The P2 and P3 *P. didemni* genomes come from animals we
now know are significantly divergent (L2 and L3, respectively). The P1 genome
comes from animal L1. The majority of the extracted DNA from this animal was
used in the extensive sequencing of the P1 genome, and therefore we were not
able to obtain a COXI sequence, but chemically it aligns to members of group A3
and was collected from the same geographic area as other members of this group.
We show that although P1 and P2/P3 have highly similar genomes, they produce
different secondary metabolites. Together, our results suggest that there is
some degree of selection for *P. didemni* strains based on secondary metabolism,
and that this correlates with host phylogeny. The patellazoles are highly toxic
and therefore likely to be defensive in function. This may also be the case for
the cyanobactins, although they are not generally as cytotoxic as the
patellazoles. Some have suggested that cyanobactins may have metal binding
capabilities, and some have moderate cytotoxicity, but their true ecological
function remains obscure. We have found, however, that their distribution is not
random, and this may be used as a basis for further exploration of their
function. Previous reports of natural product isolation may indicate the
potential ranges for the different *L. patella* clades. For instance,
patellazoles were previously isolated from an animal collected in Guam, and
compounds related to both trunkamides and patellamides have been isolated from
animals collected in the Great Barrier Reef.
The mitochondrion is thought to be the result of an ancient endosymbiosis event
in the early evolution of eukaryotes, and its tiny genome is therefore likely
the end result of the process of genome degradation and erosion observed in
endosymbiotic bacteria. Like more recent endosymbionts, mitochondia exhibit
accelerated evolution because of their population structure and lack of DNA
repair pathways, and their genome sequences can be used to infer a great deal
about the hosts' evolutionary history. Additionally, in tunicates it has been
shown that gene order in mitochondria is hypervariable, potentially providing an
additional phylogenetic signal. We had previously obtained shotgun metagenomic
sequence in Illumina HiSeq runs for three group B animals (L2, L5 and L6, see
Materials and Methods), and set out to assemble mitochondrial genomes from them.
In all cases, contigs that appeared mitochondrial were high coverage (several
hundred × or more), and could be separated from other genomes on this basis. In
all cases, mitochondrial assemblies were resolved to single contigs, ranging in
size from 12,562 bp (L6) to 14,403 bp (L2), and all were ∼21% GC. Annotated ORFs
all correspond to genes previously found in other tunicate genomes, but some
genes commonly found were missing from the assemblies (NADH dehydrogenase
subunit 4L was missing from L2 and L6, while NADH dehydrogenase subunit 6 and
ATPase F0 subunit 8 were not found in any of the assemblies). This may be due to
general difficulties in assembling such high-AT sequence. The L2 mitochondrion
assembly contained an unannotated section, roughly equivalent in size to the
small and large subunit rRNA genes in other tunicate mitochondria. This section
may include the L2 mitochondrial rRNA genes, but only small parts of the
sequence showed any homology to tunicate mitochondrial rRNA genes in BLASTN
searches against the NCBI database. Independent assembly efforts in L5 and L6
were syntenic with the L2 assembly, but they appear to lack the putative rRNA
region in L2. The consistent synteny across three samples suggests that there
are no misassembled portions of the L2 assembly. As with previously reported
tunicate mitochondrial genomes, all genes are on the same strand, but the gene
order exhibited is unique. We previously determined that the members of group B
may represent several cryptic species, with COXI identities suggesting that
animals L2 and L5 diverged somewhere between 6 and 31 million years ago. As
expected, L5 and L6 share greater gene identities with each other than either
one has with L2. Interestingly, our analysis shows that L5 and L6 are not
clones. These animals were collected in the same vicinity (within ∼100 m); while
L5 contains *Ca.* E. faulkneri and patellazoles, L6 has lost *Ca.* E. faulkneri
and contains a potentially pathogenic bacterium in its place that we termed
*Ca.* Xenolissoclinum pacificiensis. Because L5 and L6 have highly similar
mitochondrial genomes, colony fusion may be possible, and therefore we cannot
exclude the possibility that the loss of *Ca.* E. faulkneri in L6 is reversible
through this mechanism. Further studies will be needed in order to investigate
the structure of this population, and whether the loss of *Ca.* E. faulkneri in
L6 is recent and/or stable. Such studies might be a unique opportunity to
investigate the influence of symbionts and natural products on host population
structure and speciation.
Variation in the distribution and abundance of natural products is a significant
problem that affects the utility of natural compounds in drug discovery efforts.
Often vanishingly small amounts of a compound are isolated in an initial
collection. Although only small amounts are required for the characterization of
structure and *in vitro* activity, recollection for further development is often
challenging. Our efforts here show that one of the factors that can control
chemical variation in symbiotic systems is unappreciated cryptic speciation of
the host, even when symbionts may be obtained from the environment. Similar
issues are beginning to be recognized in other secondary metabolite producers.
For instance cyanobacteria designated as *Lyngbya majuscula* have been credited
with several hundred natural products in the literature. Recent genomic
sequencing of a strain that fell under this classification (now *Moorea
producta*) suggests *Lyngbya majuscula* could be a morphologically identical
species complex whose natural products may be a marker of phylogeny.
Our mitochondrial genome sequences hint at the complexity of symbiotic
interactions in the environment, where individual hosts can be found that have
lost even stable symbionts. Loss and gain of strictly vertical symbionts may
play a major role in host speciation if these events affect fitness and
reproductive compatibility, consistent with previous models of symbiosis. We
observed one such local extinction in animal L6, which lacks *Ca.* E. faulkneri
and is likely unable to regain this symbiont, potentially driving host
speciation and adaptation in L6's descendents.
This study shows that the previously supposed random distribution of *P.
didemni*–produced cyanobactins is in fact based on host phylogeny. Genomic data
indicate that this symbiont is highly uniform, and therefore populations within
individual hosts are *not* genetically isolated and must undergo frequent
horizontal exchange between didemnid hosts. Thus, our results implicate host-
dependent recruitment of *P. didemni* based on secondary metabolite production.
Because cyanobactin pathways are highly tolerant to precursor peptide mutations,
the host may play a major role in maintaining ecologically important precursor
sequences. These results have implications for biodiversity and drug discovery.
In tandem with previous results showing new compounds can be isolated by
surveying individual ascidian colonies, it is now clear that cryptic populations
of ascidians are an untapped source of new potential pharmaceuticals. In turn,
if a local, cryptic population goes extinct due to habitat loss, there is a
likelihood that potential pharmaceuticals will be permanently lost to science.
While extinction of cryptic species is often proposed to negatively impact drug
discovery, there is a prevalent contrary view that, at least among bacteria,
‘everything is everywhere’. Here we provide a concrete example of how important
bacterial compounds might be lost through destruction of local habitats.
# Materials and Methods
## Extraction of DNA and sequencing of phylogenetic markers
Permission to perform field research was granted by the Papua New Guinea
Department of Environment and Conservation, the governments of Palau, Fiji and
Solomon Islands. These efforts were facilitated by the University of Papua New
Guinea, the Coral Reef Research Foundation and the University of South Pacific,
respectively. Samples of *L. patella* were collected from the sites indicated in
and preserved in RNAlater. Portions of each sample were set aside as frozen
specimens for chemical analysis. DNA was extracted from tunicate samples
preserved in RNAlater either using an established tunicate method, the Qiagen
DNeasy kit, or by direct pulverizing of tissue in DMSO. Tunicate 18S rRNA and
mitochondrial COXI genes were amplified using primers shown in, with Platinum
Taq High Fidelity (Invitrogen). In all cases, PCR reactions were 10 µL volume,
containing 0.1 µL Taq, 1× of the supplied buffer, 2 µM each primer, 0.2 mM each
dNTP (Invitrogen) and 2.0 mM MgSO<sub>4</sub>. Reactions consisted of hot start
(94°C, 2 min), followed by 35 cycles of \[94°C/30 s, variable annealing
temperature/30 s, 68°C/1 min per kb extension (minimum 45 s)\], then a final
extension step of 68°C/10 min. PCR products were either Sanger sequenced
directly with the relevant primers, or else cloned using the Topo-TA cloning kit
(Invitrogen) before sequencing.
## Construction of phylogenetic trees and identity matrices
Marker sequences (both 18S rRNA and COXI nucleotide sequences) for members of
the family Didemnidae and *Ciona intestinalis* or *Ciona savignyi* were
downloaded from the NCBI database. The *Ciona* sequences acted as outgroups for
rooting the trees. Database 18S rRNA sequences, along with experimental
sequences from *L. patella* samples were aligned with Clustal Omega. The
alignment was inspected manually in ClustalX, and sequences that were extremely
short or unilaterally introduced large inserts were discarded. The alignment was
trimmed using a Perl script (tim_aligned_fasta.pl), and used to construct a
phylogenetic tree with FastTreeMP with the parameters -slow -spr 5 -mlacc 3
-gamma -gtr -nt. All trees were viewed and manipulated using the Interactive
Tree of Life server.
COXI sequences were translated into protein sequences using the ascidian
mitochondrial translation table (NCBI translation table 13), then aligned with
Clustal Omega. It was found that COXI sequences from NCBI aligned to two
distinct, non-overlapping regions of the *Ciona savignyi* sequence (accession
no. BAC57000.1), and so two distinct trees were constructed, one of which
included sections of COXI sequence obtained from the assembled mitochondrial
genomes of L2, L5 and L6 (vide infra). In each case the alignments were manually
inspected and trimmed as with the 18S rRNA alignment, before the trees were
constructed with FastTreeMP using the parameters -slow -spr 10 -mlacc 3 -bionj
-gamma. In order to determine the nucleotide identities of the sequences in the
trees, the original nucleotide sequences were aligned to the protein alignments
with a Perl script (nucleotide_translation_alignment_2.pl). Pairwise identities
were calculated from this alignment with another Perl script (identity_matrix.pl
).
## UHPLC/HRMS analysis
Frozen chemistry voucher samples of each animal were freeze-dried, then
sequentially extracted with chloroform and methanol. The combined extracts were
dried down and passed over a small C<sub>18</sub> plug, eluting with methanol.
LC/MS data were acquired using a Bruker MaXis ESI-Q-TOF mass spectrometer
coupled with a Waters Acquity UPLC system operated by Bruker Hystar software. A
gradient of MeOH and H<sub>2</sub>O (containing 0.1% formic acid) was used with
a flow rate of 0.3 mL/min on a RP C18 column (Phenomenex Kinetex 2.6 µm, 2.1 mm
× 100 mm). The gradient went from 10% MeOH/90% H<sub>2</sub>O to 97% MeOH/3%
H<sub>2</sub>O in 12 mins, followed by 97% MeOH/3% H<sub>2</sub>O held for 3.5
mins. Full scan mass spectra (*m*/*z* 150-1550) were measured in positive ESI
mode. The mass spectrometer was operated using previously published parameters.
Tune mix (Agilent, ESI-L low concentration) was introduced through a divert
valve at the end of each chromatographic run for automatic internal calibration.
## Construction of secondary metabolite heatmap and dendrogram
Raw data files from LCMS runs were converted to mzXML format and processed in
MZMine according to the following procedure: 1. peak detection in centroid mode
with a noise level cutoff of 5.0 × 103; 2. chromatogram building with a minimum
time span of 0.05 min, minimum peak height of 2.5, and 5.0 ppm *m*/*z*
tolerance; 3. chromatogram deconvolution using the local minimum search
algorithm, a chromatogram threshold of 65.0%, a search minimum retention time
range of 0.05 min, a minimum relative height of 5.0%, a minimum absolute height
of 5.0 × 103, and a minimum ratio of peak top/edge of 2; 5. isotopic peak
grouping with a *m*/*z* tolerance of 5.0 ppm, retention time tolerance of 0.01
min, maximum charge of +2, assuming monoisotopic shape with the lowest *m*/*z*
being representative; 6. peaks list row filtering with a minimum of 1 peak in a
row, a minimum of 1 peak in an isotopic pattern, a peak duration range of
0.0–2.0 min and auto *m*/*z* and retention time. Compounds were identified in
MZMine using a custom database containing compounds previously found in *L.
patella*, with a tolerance of 5 ppm error. The peak areas reported in MZMine
were tabulated, and compounds arising from the same biosynthetic precursor
peptides or pathways were added together (including both \[M + H\]+ and \[M +
Na\]+ ions). The table was used as an input for skiff
(<http://clovr.org/docs/skiff/>), a Perl script that is a component of the
CloVR-16S pipeline. This script normally takes tables of 16S abundances for
groups of samples, binned according to a certain level of phylogenetic
classification. In this case, the peak areas were expressed as a fraction of the
sum of assigned peak areas for each sample, and the log<sub>10</sub> of each
fraction was used to plot the heatmap. Clustering of samples (i.e. the
dendrogram portion of) was achieved by calculating the Euclidean distance
between samples based on these transformed values.
## Sequencing, assembly, annotation and comparison of draft ascidian mitochondrial genomes
Assemblies of the mitochondrial genomes of *L. patella* animals L2, L5 and L6
were constructed from Illumina HiSeq 2000 datasets previously reported. In each
case, 10% of the full dataset was used (8.0 M paired reads for L2, 14.8 M paired
reads for L5 and 20.0 M paired reads for L6). A script was used to screen out
PCR duplicates
(<https://github.com/ibest/GRC_Scripts/blob/master/screen_duplicates_PE.py>),
then the reads were filtered for length \> 40 bp and quality \> 30 with
Seqyclean (<https://bitbucket.org/izhbannikov/seqyclean>). Only the first 4.8 M
filtered reads were used for subsequent processing in L6, due to high
mitochondial genome coverage. Overlapping paired reads were then joined with
FLASH, then both paired and overlapping reads were subjected to assembly with
SPAdes in -careful mode. All three mitochondrial genomes were resolved into a
single contig, using K-mer values of 67,73,77,83,87 (L2); 41,45,51,55,61 (L5);
and 77 (L6). The genomes were annotated manually in Artemis, and found to all be
syntenic (although with different break points in the respective contigs).
Sequence comparisons were carried out by first aligning gene protein sequences
with ClustalX and then constructing nucleotide alignments from these as
described above. The hive plots that form part of were created using the D3JS
javascript framework (<http://d3js.org>), by adaptation of an example plot
(<http://bl.ocks.org/mbostock/2066415>).
## Accession Numbers
The ascidian marker sequences and mitochondrial assemblies have been submitted
to the National Center for Biotechnology Information (NCBI)
(<http://www.ncbi.nlm.nih.gov>). The accession numbers are as follows. **18S
rRNA genes**: L3, KJ009375; L2, KJ009376; L5, KJ009377; L6, KJ009378; E11-097,
KJ009379; 05-027, KJ009380; 05-033, KJ009381; 05-039, KJ009382; 05-044,
KJ009383; 07-110, KJ009384; 07-103, KJ009385; 03-005, KJ009386; L1, KJ009387;
L4, KJ009388; 07-005, KJ009389; 07-002B, KJ009390. **COXI genes**: L3, KJ009363;
05-033, KJ009364; 05-039, KJ009365; 05-027, KJ009366; 05-044, KJ009367; E11-097,
KJ009368; L4, KJ009369; 07-103, KJ009370; 03-005, KJ009371; 07-002B, KJ009372;
07-005, KJ009373; 07-110, KJ009374. **Mitochondrial genome assemblies**: L2,
KJ596321; L5, KJ596322; L6, KJ596323.
# Supporting Information
Permission to perform field research was granted by the Papua New Guinea
Department of Environment and Conservation and facilitated by the University of
Papua New Guinea. Additional permission for field collections was obtained from
the governments of Solomon Islands, Fiji and Palau. We thank C. M. Ireland for
help with collections, M. K. Harper for many helpful discussions on this paper
(both University of Utah), Sam Hunter (Institute of Bioinformatics and
Evolutionary Biology, University of Idaho) for helpful discussions on
mitochondrial assembly, Anton Korobeynikov (St. Petersberg Academic University)
for discussions on the use of the SPAdes assembler, and James Robert White for
advice on the use of the skiff algorithm.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JCK TSB EWS. Performed the
experiments: JCK MSD TPW. Analyzed the data: JCK MDBT TPW. Wrote the paper:
JCK EWS.
[^3]: Current address: Pharmaceutical Sciences Division, University of
Wisconsin–Madison, Madison, Wisconsin, United States of America
[^4]: Current address: Department of Bioengineering and Therapeutic Sciences
and California Institute for Quantitative Biosciences, University of
California, San Francisco, California, United States of America |
# Introduction
Organ and tissue donation is essential for fundamental and applied research. A
state as close as in-vivo is equally important to ensure reliability of the
results and clinical relevance. Needs are steadily increasing while availability
is decreasing. Legal and cultural differences between nations regarding donation
in general and those for research complicate comparisons. However, quantitative
data are lacking as well as concrete solutions to correct imbalance.
In ophthalmology, cornea is no exception. A growing shortage of tissue for
research is well documented in the USA. Worldwide, shortage also concerns
donation for transplantation. Cornea is by far the most transplanted tissue, but
in 2015 only 1/70 patients waiting worldwide could benefit from a corneal graft
each year.
In France, corneal procurement is governed by the Bioethics Laws revised in
2018, based on the European standards. Cornea is procured from a deceased donor,
then necessarily stored in an authorized eyebank which carries out tissue
quality and microbiological safety tests. Donation chain is an opt-out system
with an online national refusal register (NRR). In absence of refusal expressed
during his/her lifetime, any deceased person may legally be procured. Routinely,
the hospital coordination team systematically seeks to ask relatives (without
precise guidelines on degree of kinship) about the intentions of their deceased
if no instruction (written or oral) is left by deceased and the relatives
themselves refuse, the coordinators trust them and the procurement is never
imposed.
The scientific human corneas used in France have two different origins: they
come mainly from waste products of transplantation donation and a minority from
body’s donation to science. The first are corneas discarded by quality controls
in eyebanks. They represent about 50% of 11,000 corneas procured, but infected
corneas must be withdrawn and only those that don’t have refusal for scientific
use can be used. They are mostly poorer quality corneas to those required for
transplantation. Besides these corneas are only available after several days of
storage and are no longer fresh tissue. The second, resulting from donation in
anatomy laboratories of medicine faculties, represents only a few hundred per
year in France. They are the only fresh scientific corneas currently available,
provided that death-to-procurement time is \<24hours when body arrives in
laboratory. According to legislative framework, distribution of these corneas
for research are free of charge in France.
Among our research axes aims we developed an active storage machine for corneal
grafts, which restores equivalent of intraocular pressure and renews storage
medium. After testing it on porcine corneas, preclinical validation of this
device was performed on a large series of human fresh corneas. To avoid all
biases related to discarded corneas from eyebanks, we requested from the Agence
de la Biomédecine (ABM, whose mission is to supervise, evaluate, promote
procurement and transplantation) opportunity to procure fresh scientific human
corneas, for this preclinical study and all other protocols using our device,
and for protocols aiming at make evolve eyebanking, corneal imaging and
bioengineering. We were concerned that donation for research could raise
concerns because of its novelty and specificity. We therefore conducted a-year
prospective study comparing characteristics of targeted donation for research
and usual donation for transplantation.
# Material and methods
## Ethical considerations
All procedures conformed to the tenets of the Declaration of Helsinki for
biomedical research involving human subjects. The ABM specifically authorized
corneas procurement for research (PFS15-008 & PFS16-010) and study was approved
by local Institutional Review Board “Ethics Committee of the CHU de Saint-
Etienne, Research Commission of Terre d’Ethique” (IORG0007394,
N°IRBN272016/CHUSTE). The ABM asked us to select for research only persons with
medical contraindication to transplantation, based on the European standards, so
as not to reduce corneas number for waiting recipients. All other inclusion
criteria didn’t differ from those usually used for corneal transplantation
donation.
## Study design
We collected prospectively during a-year, all data concerning corneal donation
for research and transplantation in the Saint-Etienne university hospital. Main
objective was to compare acceptance rates between both groups.
Secondary objectives were to analyze whether differences exist between both
groups in donor profiles or corneas procured. We compared: 1/ consent seeking
process data: obtaining consent methods (face-to-face or telephone), answer
delay (immediate or delayed to think about); 2/ reasons for refusal; 3/ methods
of expressing opposition to donation (NRR, instructions left to relatives); 4/
donors and corneas characteristics: endothelial cell density (ECD) measured
48hours after procurement (main quantitative quality criterion in eyebanks: done
by the Saint-Etienne eyebank technicians for transplantation, and a unique
skilled operator (TG) for research) and cataract operated eye (potentially with
lower corneal quality); 5/ acceptance rate according to hospital coordinators
experience to potentially optimize interview protocol.
Finally, to verify if this new research task impacted transplantation activity,
we compared this year’s study to previous 13 years' activity carried out in the
same hospital environment with the same eyebank connection.
## Hospital coordination team missions
Three nurses of the hospital coordination team were involved. One was very
experienced (20 years of experience and about 2000 interviews), one was
experienced (15 years of experience and about 1500 interviews), the third
(devoting 50% of her time to administrative-regulatory tasks) had 5 years of
experience and about 500 interviews.
Regular mission consisted in obtaining consent to organ and tissue donation for
transplantation. In France donation chain follows an opt-out system. More
specifically for the corneas, they daily screened all intra-hospital deaths.
Following French guidelines health authorities, maximum death-to-procurement
time was 24 hours. Were recommended but not mandatory: death-to-body-
refrigeration time \<4hours, if not death-to-procurement time should be
\<12hours. In parallel, coordinators screened deceased persons with medical
contraindication to donation for transplantation. Interviews were conducted
according to standard written procedures, either face-to-face or by telephone,
as we reported in 2002. For the research group, coordinators explained to the
relatives of the eligible deceased that corneal donation wasn’t possible for
transplantation due to medical contraindication, but for local medical research:
so the coordinators asked the relatives to consent to cornea donation directly
for research. General purpose of the corneal grafts research axes conducted by
same team of surgeons and researchers was explained, if the relatives so wished.
When clarifications were required, a detailed explanatory letter from
department’s head (PG) could be provided to the relatives, just as follow-up
support if needed. The type of the consent depended of each eligible deceased:
written or oral refusal during lifetime for deceased, or oral consent or refusal
(by telephone or face-to-face) from relatives contacted and informed.
## Corneas procurement
All corneas were procured by 3 trained ophthalmology residents. Fresh human
corneas were procured by in situ excision with the same settings whatever the
target, transferred to our laboratory within 20 minutes (for research) or to
eyebank as usual (for transplantation). Costs of sterile single-use instruments
and organoculture medium for the research group were covered by our research
laboratory funding. The ABM authorized us to procure only fresh scientific
corneas: the remaining eye tissue was left on the deceased as procurements for
transplantation, with tegumentary reconstruction ad integrum.
## Statistics
Normality of continuous data distribution was analyzed with Shapiro-Wilk test
with non-normality threshold of 5%. Normal distribution data were described by
mean±SD, \[min-max\]. When the variable followed normal distribution, an
unpaired Student t-test was used to compare donors’ characteristics of both
groups. Chi<sup>2</sup> test was used to compare percentages. Statistical
significance was set at P\<0.05, with two-tailed tests (unless specified one-
tailed) and adjusted with Tukey technique when multiple tests were performed.
Analyses were performed with SPSS 25.0 (IBM Corp, Armonk, NY).
# Results
## Medical wards of pre-selected deceased
In 12 months, on 1442 intra-hospital deaths, coordinators sought absence of
opposition to donation for 371 deceased eligible to potential corneal donation:
127 for research, 244 (232 in circulatory arrest, 12 from multi-organ donor) for
transplantation. For each group, deceased were mainly from 3 services: emergency
(21%), geriatrics (20%) and intensive care (16%) for research, and intensive
care (28%), emergency (12%) and pneumology (9%) for transplantation.
## Methods of interviewing the relatives and answer delay
Seventy-one percent of interviews were conducted by telephone, without
difference between both groups (72% for research, 70% for transplantation P =
0.389). Answer was immediate in 69% (257/371) of cases and didn’t differ between
both groups (64% for research, 72% for transplantation P = 0.540). Relatives
answered more often immediately during face-to-face interview (79% of cases)
than by telephone (66% of cases) (P = 0.016), without difference between both
groups (immediate answer was done: a/during face-to-face interview 74% for
research, 81% for transplantation P = 0.393; b/ by telephone 61% for research,
68% for transplantation P = 0.218).
## Acceptance rates
Consent was obtained in 62% (79/127) of cases for research and 54% (132/244) for
transplantation (P = 0.135), providing 158 fresh corneas for research and 264
for eyebank respectively. Acceptance rates weren’t influenced by gender of
eligible donors, with 57% (123/216) of acceptance among males and 57% (88/155)
among females (P = 0.974) respectively for overall series, without difference
between both groups: 62% of females gave their consent for research versus 52%
for transplantation (P = 0.212); respectively 62% of males for research versus
55% for transplantation (P = 0.357). details acceptance rates within and between
both groups, according to interview type and answer delay by relatives: no
significant differences were found. Telephone made it possible to procure 110
corneas for research and 172 for transplantation (i.e. 67% of all corneas).
Acceptance rates varied significantly with coordinator's experience: the most
experienced obtained 71% and 62% of consent, respectively for research and
transplantation, the one with intermediate experience 55% and 52%, the 3rd less
experienced 43% and 39% (P = 0.003 for overall comparison between coordinators,
both groups combined). None of them had more success for research than for
transplantation (P = 0.253, P = 0.730, P = 0.824 respectively for the 3
coordinators). Coordinator's experience was directly correlated to acceptance
rates whatever target: r = 0.9966 for research (P = 0.026), r = 0.9972 for
transplantation (P = 0.024) (unilateral tests).
## Analysis of opposition to donation
No opposition was found on NRR. Clear (oral or written) opposition from subject
prior to death, left to the relatives, was found in 19% (9/48) of cases for
research versus 37.5% (42/112) for transplantation (P = 0.045). In other cases,
refusal was a relatives’ decision (from one person if alone, or from a collegial
decision). There wasn’t any influence of the gender or relationship of the
person(s) providing consent (P = 0.342 and P = 0.254 respectively). Regardless
target (79% for research, 80% for transplantation), the relatives mainly didn’t
provide any justification (P = 0.036). When refusal was mentioned, it was:
"Donation isn’t priority" in 10% of cases for research and 11% for
transplantation (P = 0.999); "Our relative suffered too much, was too sick or
too old" in 8% of cases for research and 6% for transplantation (P = 0.999);
relatives didn’t call coordinators back in 3% of cases for research and 3% for
transplantation (P = 0.999).
## Donors characteristics and corneal cells quality
Medical contraindications that allowed selection in the research group were
mainly the cognitive disorders in 66% (52/79) of cases, followed by the blood
cancers (8%). The details were provided in. Donors in the research group were
significantly more likely to be females (P = 0.004) and older by an average of
10 years (P\<0.001); their bodies were also refrigerated quicker (P\<0.001).
However, there wasn’t significant difference in death-to-procurement time (P =
0.911), percentage of eyes with cataract surgery (P = 0.120) and ECD (P =
0.071). Twelve pairs of corneas (15%) in the research group were available for
experimentation within 6 hours or less after death, and 24 (30%) within 12 hours
or less, with an average death-to-experimentation in laboratory time
15h15±6hours \[0h55;24h08\]. Further details of these characteristics were noted
in.
## Comparison of transplantation activity with previous years
With 244 families contacted for transplantation during this study year, activity
wasn’t different from that of previous 13 years, which had concerned an average
of 227±24 \[181 to 264\] families (P = 0.994). Similarly, number of corneas
procured for transplantation (264) didn’t differ from the average of previous 13
years (284±24 \[from 223 to 330\] (P = 0.998)).
# Discussion
Corneal donation for research is still too little-known to public and must be
encouraged as much as that for transplantation, both of which are essential to
fight against blindness. We showed that it is possible to specifically target
deceased to procure fresh corneas for research, in parallel with transplantation
activity, without putting them in competition.
Acceptance rate didn’t differ between the research and the transplantation
groups, whereas we feared that donation for research might have raised more
reluctance. Several mutual and non-exclusive explanations can be formulated: 1/
people who accept idea of corneal donation don’t oppose research and
transplantation; 2/ some may think that they help even more people via research
than transplantation, which treats traditionally only two patients; 3/ older age
(10 years) in the research group can make higher consent rate; 4/ great
experience of the coordinators in presenting legislation and research issues
with empathy plays a key role; 5/ longstanding involvement of the coordinators
in research projects, directly related here to improve corneal grafts storage
keeps motivation over time; 6/ general public’s knowledge of our health region
and team's research works (dedicated mainly but not only to corneal eyebanking
and transplantation) through regular follow-up support for the relatives may
help to make meaning of donation. But in practice relatives didn’t ask the
coordinators for so much details about research aims.
Our high acceptance rate for transplantation was significantly higher than those
reported for transplantation in literature where rates exceeding two-thirds
acceptance are exceptional. Comparison between the teams is highly complex
because it questions donation regulation, overall organization of the teams,
donors screening and experience of the coordinators evolving over time. Our
findings result certainly from combination of an opt-out system and a small
well-structure, highly motivated experienced team. Key role of "human factor" in
success of this deeply personal approach was reported before.
Our study further confirms crucial role of telephone interview, reported
regularly. In 2002, we showed that for transplantation, telephone accounted for
58% of interviews and 48% of the corneas procured. Here for transplantation, its
role is even more important with 71% of interviews and 67% of corneas procured.
While in 2002 telephone was less efficient than face-to-face interviews, this
difference wasn’t significant in 2017: communication resources evolution with
the smartphones pervasive in daily life, is probably involved.
Refusals analysis shows that NRR is probably widely underused. The donation
opponents tell probably rarely their relatives why, with a potential memory
bias, as the justifications collected by our coordinators seem to indicate.
Whatever target, donation legislative framework like presence of NRR aren’t well
known to general public. Generally speaking, question of donation isn’t still
sufficiently addressed in families, even for favorable people with only 50% of
them having informed their relatives. At time of death, when relatives have to
decide in place of their deceased, fear of doing wrong or assuming
responsibility can lead to refusal.
The medical contraindications to corneal donation for transplantation are
numerous, based mainly on precautionary principle solely and deprive recipients
of potentially great intrinsic quality corneas: only rabies (constantly fatal),
herpes simplex virus, bacteria, fungi and retinoblastoma have been formally
demonstrated to be transmissible via the cornea. So, targeting these
contraindications allows researchers to benefit from them. Some sporadic cases
of prion disease were reported in recipients, and circumstantial evidence has
implicated corneal transplantation as a mechanism of transmission of iatrogenic
prion disease: transplantation is presumed (but not formal) to be the source of
prion disease in recipients. Although we could be concern about disease
transmission or research bias induced by these contraindications, it clearly
depends on the research aim(s). In this study, every cornea was procured by in
situ excision with single-use sterile instrument, without any contact with
retina or optic nerve, which are be considered as specific risk factors to
possible iatrogenic spread of sporadic and variant prion disease. We did risk
analysis before the use of those corneas to develop our active storage machine
to make evolve eyebanking, with assessment of their baseline intrinsic quality
(endothelial cell density, transparency, presence of scar or not, presence of
previous refractive surgery or not) and safety (no infection), following tests
used in daily routine in eyebanks. Furthermore, with these common medical
contraindications people may wish to give but feel excluded from donation:
donation for research can be a way to give them back this opportunity. However
many people are interested in donating their corneas for research but aren’t
aware that it was possible to do so. Thus, they leave more often no instruction
to their relatives concerning procurement for research.
Targeted donation for research has another advantage: it allows the researchers
to dispose the fresh tissues immediately after procurement, without passing
through the eyebanks. Several positive consequences can be stated: it provides
short circuit, without extra-work for the eyebanks and less charges for the
researchers at the end of the chain (variations occur in different countries,
such as the USA where procurements are done by paid technicians); it removes the
damaging processes on cornea quality induced by storage itself.
Our specific approach imposed by the ABM made it possible to procure a large
quantity of fresh scientific corneas over a short period of time, without
difference quality from those grafted, ensuring optimal clinical relevance for
the research protocols in eyebanking. Through people giving their bodies to
science, we found that we could just procure 41±17 fresh scientific corneas per
year over last 3 years in our faculty of medicine: it would have taken almost 4
times longer to obtain same number of fresh corneas versus our study with 158
corneas a-year. Without these fresh tissues, preclinical validation of our
active storage machine couldn’t have been performed under same conditions as its
future use, like the other following protocols to improve corneal storage using
our device with the extensions of authorizations by the ABM. Death-to-
refrigeration time difference between groups was explained by difference in the
services from which deceased came, not involving same post-death logistics.
Donors age was logically higher, with more females in the research group, since
we were asked to select potential donors for research on the diseases that were
more frequent with aging and females live classically longer. It didn’t impact
research protocols, since donor age isn’t a significant factor in survival of
corneal grafts.
Concerning coordinators, despite their additional workload to obtain targeted
donation for research, there wasn’t negative impact on already important
transplantation activity, neither on number of the families interviewed nor on
number of corneas procured. That underlines possibility to have clinical and
research processes coordinated without penalizing each other. The coordinators
increased their efficiency without additional working time, didn’t receive
bonus, made and have pursued it with belief: whatever research and clinical
endeavors, they did both tasks without any conflict. Donation chain may vary in
different countries. Regarding cost effectiveness of targeted corneal donation,
we believe it may be applied whatever medico-economic system. In France, our
laboratory had to pay only materials. But if needed, extra-cost from people in
charge of obtaining consent or/and procurements, must be implemented at chain’s
end for researchers. Anyway, without passing through the eyebanks, researchers
will have less fees and better-quality fresh corneas, immediately available for
experimentations, with this targeted corneal donation.
Despite being prospective and designed to minimize potential bias, our study
presents some limitations: 1/ center-effect with highly motivated coordinators
that have strong and long-lasting links with medical-research team for two
decades. However, we believe that extrapolation to the other research teams
motivated to obtain exceptional quality tissues, could have similar results. 2/
net inter-coordinator effect. Despite using standard protocol, some variations
were intrinsically linked to age-related experience: more experienced
coordinator probably adapts better to each family profile whatever target
donation.
Several proposals have recently been made to improve acceptance of corneal
donation for research. In the USA, Williams *et al*. advocates for scientific
donation, by proposing creation of an eye donation registry for research;
collaborations between the eyebanks and the research institutes to recover
corneas unsuitable for transplantation, as we have implemented since almost 20
years to procure discarded grafts from our eyebank. Besides, proposed the
creation of an online portal, under the aegis of the Association for Research in
Vision and Ophthalmology, to specifically link the eyebanks to researchers in
need of eye tissue.
To make this work useful to other teams, we may suggest some ways to increase
number of donation for research: 1/ to develop privileged relationships between
the research laboratories and the team in charge of donation in local hospital.
Exposing her regularly aims, advances and results of researches, is probably
crucial to induce lasting links; 2/ to encourage companionship between the
novice and the experienced coordinators; 3/ to integrate in donation promotion
campaigns, awareness raising to donation legislative framework and possibility
to donate for research; 4/ to increase the resources allocated to the
coordinators and ensure that all eligible relatives of deceased can be contacted
in a timely manner: pressure comes from legislative framework and bodies
transportation from hospital mortuary to private funeral homes, explaining
mainly gap between number of intrahospital deaths and deceased eligible
contacted in this study. Respectively 413 families were not contacted due to
legislative framework with death-to-procurement time \>24h (which discards
deceased from eligibility to for corneal donation for research or
transplantation); 411 families were not contacted due to fast transport of the
deceased body from hospital mortuary to private funeral homes or conservative
care (which prevent from interviewing families for research or transplantation).
The third cause is that sometimes the coordinators didn’t have enough time to
treat every dossier of eligible deceased (n = 243 families) for research or for
transplantation in their daily multi-tasks: they share time between corneas,
other tissues (vessels, bone), organs (mainly kidney).
In summary, in parallel with transplantation activity, by targeting donors with
medical contraindication to corneal donation for transplantation, we can obtain
many fresh scientific corneas of similar quality to those grafted, immediately
available for the researchers, without increasing shortage for waiting
recipients or incurring extra-fees by passing through the eyebanks. This
targeted corneal donation could be a potential solution to make research advance
better and faster.
# Supporting information
We are grateful to those who donated their corneas to science, and to their
families. We also thank the Agence de la Biomédecine for its institutional
support and the authorizations.
10.1371/journal.pone.0233392.r001
Decision Letter 0
Liu
Yu-Chi
Academic Editor
2020
Yu-Chi Liu
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
13 Feb 2020
PONE-D-19-35417
Corneal donation for research versus for transplantation: a-year prospective
study of acceptance rates in a French University Hospital
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Reviewer \#1: The study is technically sound with a valid scientific question.
The results were interesting and surprising. We note that the paper did not
mention whether the research cornea donors were tested for relevant communicable
diseases and what the disposition of the corneas would be should the results
come back positive. Was there any concern that some of the cognitive disorders
were due to prion disease?
Our other query would be the consent taking process for biomedical research,
perhaps this can be mentioned in the paper? Will this be different from the
consent for clinical transplant purposes? Or can we assume that ABM has already
provided the necessary authorization and therefore no further consent is
required?
The manuscript is easily understood but there is room for improvement with
regard to overall sentence construction, grammatical flow and word choices.
Agree with the author’s view that the high consent rates may be a single centre
effect and may not represent the situation in most other institutions around the
world. However, it echoes the need for a consistent and sustained messaging to
the general public about cornea donation, whether it be for therapy, research or
education.
Reviewer \#2: In this prospective study, the authors compared the donation
acceptance rate for cornea transplantation (deceased patients with no medical
contraindication for corneal transplantation) and for research (deceased
patients with contraindication for corneal transplantation) and evaluated if the
acceptance rate for transplantation donation remained stable over a year.
General comments
Obtaining fresh corneas for research is highly challenging and a main concern
for worldwide researchers. While it is highly interesting to see that acceptance
rates for research and for transplant were comparable, this paper opens several
questions regarding research regulations of corneas. Indeed, in France,
historically, ineligible corneas for transplant were being used for research, in
consequence, research corneas came from patients without medical
contraindication for transplant.
In the present study, corneas from deceased patients with medical
contraindication for transplant (including cognitive disorders, blood cancers,
neurologic disorder, uncontrolled infection, tumor of CNS, severe influenza and
hemodilution) were used for research to increase the number of fresh corneas
needed for research purpose.
The main question here might be “should we use tissues from cognitive disorders,
blood cancers, neurologic disorder, tumor of CNS, uncontrolled infections… for
our research?” Indeed, the authors explained that one of the main focus of their
lab was to develop an active storage machine for corneal graft. We could be
concern about both disease transmission and induced research bias which is not
at all discussed in the paper.
Thus, the discussion should address those points which can’t be only resumed in
one sentence (Line 279-281) “The medical contraindications to corneal donation
for transplantation are numerous, based mainly on precautionary principle solely
and deprive recipients of potentially great intrinsic quality corneas”.
Was any risk analysis performed before the use of those corneas?
Point by point comments
Some additional points need to be clarified and are listed below.
Results
Line 153 the authors reported 1442 intra-hospital deaths but only 371 deceased
eligible. Can the authors explain the reasons of the gap since cornea for
research are based on medical contraindications meaning that families of all
deceased patients should be interviewed. What were the limitations?
Table 1 is nonreadable, please correct. In addition, in the table, the total of
research interviews is 126 but number written in results line 154 is 127. Please
review and correct.
Line 212 “medical contraindications that allowed selection in the research
group” can you please clarify the French rules about research on tissues with
medical contraindications for transplant?
End of comments
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Reviewer \#1: Yes: Howard Cajucom -Uy
Reviewer \#2: No
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10.1371/journal.pone.0233392.r002
Author response to Decision Letter 0
9 Mar 2020
PONE-D-19-35417
Corneal donation for research versus for transplantation: a-year prospective
study of acceptance rates in a French University Hospital
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First, we would like to thank the Academic Editor, reviewer \#1 and \#2 for
her/his reviewing and advises. We have chosen to respond to the comments and to
resubmit this manuscript. We deeply apologize for grammar or language mistakes.
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Consent was informed. In France we have opt out system. The type of the consent
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contacted.
We precised it in Methods section.
3\. Thank you for including your ethics statement: The Agence de la Biomedecine
(ABM) specifically authorized corneas procurement for research (PFS15-008 &
PFS16-010) and study was approved by local Institutional Review Board
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We have amended the full name of our institutional review board that approved
our specific study Ethics Committee of the CHU de Saint-Etienne, Research
Commission of Terre d’Ethique
We precised it in the Methods Section and in the “Ethics statement” field of the
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5\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
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your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: The study is technically sound with a valid scientific question.
The results were interesting and surprising. We note that the paper did not
mention whether the research cornea donors were tested for relevant communicable
diseases and what the disposition of the corneas would be should the results
come back positive. Was there any concern that some of the cognitive disorders
were due to prion disease?
Thank you for your comment, we have added the following sentences in the
discussion section (lines 291 to 307)
“only rabies (constantly fatal), herpes simplex virus, bacteria, fungi and
retinoblastoma have been formally demonstrated to be transmissible via the
cornea. So, targeting these contraindications allows researchers to benefit from
them. Some sporadic cases of prion disease were reported in recipients, and
circumstantial evidence has implicated corneal transplantation as a mechanism of
transmission of iatrogenic prion disease : transplantation is presumed (but not
formal) to be the source of prion disease in recipients \[31-34\]. Although we
could be concern about disease transmission or research bias induced by these
contraindications, it clearly depends on the research aim(s). In this study,
every cornea was procured by in situ excision with single-use sterile
instrument, without any contact with retina or optic nerve, which are be
considered as specific risk factors to possible iatrogenic spread of sporadic
and variant prion disease \[35, 36\].”
Our other query would be the consent taking process for biomedical research,
perhaps this can be mentioned in the paper? Will this be different from the
consent for clinical transplant purposes? Or can we assume that ABM has already
provided the necessary authorization and therefore no further consent is
required?
Thank you for your comment. We clarified this point lines 122 to 139.
The manuscript is easily understood but there is room for improvement with
regard to overall sentence construction, grammatical flow and word choices. We
deeply apologize for grammar or language mistakes. We updated this point.
Agree with the author’s view that the high consent rates may be a single centre
effect and may not represent the situation in most other institutions around the
world. However, it echoes the need for a consistent and sustained messaging to
the general public about cornea donation, whether it be for therapy, research or
education.
Reviewer \#2: In this prospective study, the authors compared the donation
acceptance rate for cornea transplantation (deceased patients with no medical
contraindication for corneal transplantation) and for research (deceased
patients with contraindication for corneal transplantation) and evaluated if the
acceptance rate for transplantation donation remained stable over a year.
General comments
Obtaining fresh corneas for research is highly challenging and a main concern
for worldwide researchers. While it is highly interesting to see that acceptance
rates for research and for transplant were comparable, this paper opens several
questions regarding research regulations of corneas. Indeed, in France,
historically, ineligible corneas for transplant were being used for research, in
consequence, research corneas came from patients without medical
contraindication for transplant.
In the present study, corneas from deceased patients with medical
contraindication for transplant (including cognitive disorders, blood cancers,
neurologic disorder, uncontrolled infection, tumor of CNS, severe influenza and
hemodilution) were used for research to increase the number of fresh corneas
needed for research purpose.
The main question here might be “should we use tissues from cognitive disorders,
blood cancers, neurologic disorder, tumor of CNS, uncontrolled infections… for
our research?” Indeed, the authors explained that one of the main focus of their
lab was to develop an active storage machine for corneal graft. We could be
concern about both disease transmission and induced research bias which is not
at all discussed in the paper. Thanks for this comment. We added new comment
concerning this point. (cf. response to comment of reviewer \#1 about prion
disease =\> line 291 to 307)
Thus, the discussion should address those points which can’t be only resumed in
one sentence (Line 279-281) “The medical contraindications to corneal donation
for transplantation are numerous, based mainly on precautionary principle solely
and deprive recipients of potentially great intrinsic quality corneas”. We added
new comment concerning this point (line 291 to 307).
Was any risk analysis performed before the use of those corneas? Yes, We added
new comment concerning this point (line 303 to 307).
“We did risk analysis before the use of those corneas to develop our active
storage machine to make evolve eyebanking, with assessment of their baseline
intrinsic quality (endothelial cell density, transparency, presence of scar or
not, presence of previous refractive surgery or not) and safety (no infection),
following tests used in daily routine in eyebanks.”
Point by point comments
Some additional points need to be clarified and are listed below.
Results
Line 153 the authors reported 1442 intra-hospital deaths but only 371 deceased
eligible. Can the authors explain the reasons of the gap since cornea for
research are based on medical contraindications meaning that families of all
deceased patients should be interviewed. What were the limitations?, We added
new comment concerning this point (line 371 to 380).
Gap can be explained by several causes :
1/ legislative framework with death-to-procurement time \>24h, which discards
deceased from eligibility to for corneal donation for research or
transplantation (n= 413)
2/ fast transport of the deceased body from hospital mortuary to private funeral
homes or conservative care, which prevent from interviewing families for
research or transplantation (n=411)
3/ Only 3 nurses of the coordination team are present to do all the work about
corneas but also for other tissues (vessels, bone) and organs (mainly kidney).
So, sometimes they do not have enough time to treat every dossier of eligible
deceased for research or for transplantation in their daily tasks (between
corneas, other tissues, organs) (n=243) ; so increase the resources allocated to
the coordinators is crucial.
4/ medico-legal impediment with prosecutor objection (n=4).
Table 1 is nonreadable, please correct. In addition, in the table, the total of
research interviews is 126 but number written in results line 154 is 127. Please
review and correct. We apologize for that ; we reviewed and corrected on revised
manuscript.
Line 212 “medical contraindications that allowed selection in the research
group” can you please clarify the French rules about research on tissues with
medical contraindications for transplant?
The Rules are edited and updated regularly by the Health Authorities, and this
is The Agence de la biomédecine provides the rules : for each tissue medical
contraindications exist for transplantation. Ineligibility for transplantation
makes tissue discarded and so destructed. But some of this discarded tissue
which does not represent infectious risks, may be used by specific, certified
and authorized laboratories. For ocular tissue, distribution of tissue are free
of charge in France.
End of comments
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Reviewer \#1: Yes: Howard Cajucom -Uy
Reviewer \#2: No
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10.1371/journal.pone.0233392.r003
Decision Letter 1
Liu
Yu-Chi
Academic Editor
2020
Yu-Chi Liu
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
30 Apr 2020
PONE-D-19-35417R1
Corneal donation for research versus for transplantation: a-year prospective
study of acceptance rates in a French University Hospital
PLOS ONE
Dear Dr. GARCIN,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that the manuscript has significantly improved but the
reviewers still have some minor concerns. Therefore, we invite you to submit a
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We look forward to receiving your revised manuscript.
Kind regards,
Yu-Chi Liu, M.D
Academic Editor
PLOS ONE
\[Note: HTML markup is below. Please do not edit.\]
Reviewers' comments:
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**Comments to the Author**
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Reviewer \#1: All comments have been addressed
Reviewer \#2: (No Response)
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
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conclusions must be drawn appropriately based on the data presented.
Reviewer \#1: Yes
Reviewer \#2: Partly
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
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fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
manuscript fully available without restriction, with rare exception (please
refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
participant privacy or use of data from a third party—those must be specified.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
5\. Is the manuscript presented in an intelligible fashion and written in
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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
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Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
6\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: (No Response)
Reviewer \#2: Discussion
Line 268 misspelling of “iin” please replace by “in”
The authors may want to add some additional information regarding the CoVid
current situation which will probably change their future practice. In their
study, all deceased patients were eligible for research donation, will they now
consider testing all deceased patients before harvesting corneas? Are French
rules currently changing regarding cornea donation for research?
\*\*\*\*\*\*\*\*\*\*
7\. PLOS authors have the option to publish the peer review history of their
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Reviewer \#1: Yes: Howard Cajucom-Uy
Reviewer \#2: No
\[NOTE: If reviewer comments were submitted as an attachment file, they will be
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that Supporting Information files do not need this step.
10.1371/journal.pone.0233392.r004
Author response to Decision Letter 1
1 May 2020
PONE-D-19-35417R1
Corneal donation for research versus for transplantation: a-year prospective
study of acceptance rates in a French University Hospital
PLOS ONE
Saint-Etienne, May 1st 2020
Dear Professor Yu-Chi Liu,
Please find attached a revised version of our article titled “Corneal donation
for research versus for transplantation: a-year prospective study of acceptance
rates in a French University Hospital”.
We have carefully considered and responded to all the points addressed by the
reviewers.
Per your instructions, all substantive amendments in the revised version are
stated in our point-by-point response, and are marked in red in the article.
We greatly hope that this new version will meet the reviewers' expectations and
comply with your editorial policy.
Yours sincerely,
Dr. Thibaud GARCIN, M.D., Ph.D., FEBO
Saint-Etienne University Hospital
"Corneal Graft Biology, Engineering, and Imaging" Laboratory EA 2521
Faculty of Medicine
Saint-Etienne
France
Dear Dr. GARCIN,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that the manuscript has significantly improved but the
reviewers still have some minor concerns. Therefore, we invite you to submit a
revised version of the manuscript that addresses the points raised during the
review process.
We would appreciate receiving your revised manuscript by Jun 14 2020 11:59PM.
When you are ready to submit your revision, log on to
<https://www.editorialmanager.com/pone/> and select the 'Submissions Needing
Revision' folder to locate your manuscript file.
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your updated statement in your cover letter.
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you deposit your laboratory protocols in protocols.io, where a protocol can be
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Please include the following items when submitting your revised manuscript:
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Reviewer \#2: Discussion
Line 268 misspelling of “iin” please replace by “in”
This has been corrected
The authors may want to add some additional information regarding the CoVid
current situation which will probably change their future practice. In their
study, all deceased patients were eligible for research donation, will they now
consider testing all deceased patients before harvesting corneas? Are French
rules currently changing regarding cornea donation for research?
Thank you for the suggestion. The continuation of corneal procurement as early
as possible is indeed crucial both for patients waiting for transplants and for
laboratories like ours that work on these irreplaceable human tissues. The SARS-
Cov-2 epidemic has effectively stopped all but multi-organ donation. It appears
that we may soon be able to resume therapeutic procurement from symptomatic,
non-at-risk individuals, but there are many unanswered questions.
In order to be able to answer the question of the risk of donor-recipient
transmission, we have already started a new research work and obtained the
authorization from our health authority (Biomedicine Agency, PFS-2020-011) to
resume retrieval for scientific purposes for the next 150 donors. Thus, we will
systematically collect and test all donors (nasopharynx, conjunctiva and cornea)
and carry out serologies. This will provide us with robust data to make good
decisions about what new tests to perform or not to perform on donors, for
transplantation and for research. We hope that the new recommendations will be
based on evidence and not on an unfounded precautionary principle.
Of course, during this new study we are recording the acceptance rate for
corneal donation among all potential donors, and possible reasons for refusal.
We will know whether the current epidemic is changing behavior and we will
submit a letter to report the results, if you wish.
For this article, as suggested, we would like to add the following paragraph as
the end of discussion:
“By the time this article is accepted, the global SARS-Cov-2 epidemic has
stopped corneal procurement for therapeutic and scientific purposes altogether.
Scientific knowledge is sorely lacking to establish the risk of transmission of
this virus via ocular tissues and therefore to make new recommendations on which
virological tests should be added or not to ensure total safety. Our French
health authority has authorized us in emergency (PFS-2020-011) to perform a
large series of scientific procurements from any potential donor (COVID+ or -)
in order to objectively analyze the risks. During this particular study we will
also analyze whether the epidemic has modified the acceptance rate and the
reasons for a possible refusal.”
Respectfully
Dr T Garcin, MD, PhD, FEBO
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Reviewer \#1: Yes: Howard Cajucom-Uy
Reviewer \#2: No
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10.1371/journal.pone.0233392.r005
Decision Letter 2
Liu
Yu-Chi
Academic Editor
2020
Yu-Chi Liu
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
5 May 2020
Corneal donation for research versus for transplantation: a-year prospective
study of acceptance rates in a French University Hospital
PONE-D-19-35417R2
Dear Dr. GARCIN,
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10.1371/journal.pone.0233392.r006
Acceptance letter
Liu
Yu-Chi
Academic Editor
2020
Yu-Chi Liu
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
6 May 2020
PONE-D-19-35417R2
Corneal donation for research versus for transplantation: a-year prospective
study of acceptance rates in a French University Hospital
Dear Dr. Garcin:
I am pleased to inform you that your manuscript has been deemed suitable for
publication in PLOS ONE. Congratulations! Your manuscript is now with our
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Dr. Yu-Chi Liu
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PLOS ONE
[^1]: GAIN, S ACQUART and G THURET are inventors on “patent US
20160029618A1” submitted by University Jean Monnet that covers “Medical
device intended for long-term storage of a cornea, or for ex vivo
experimentation on a human or animal cornea.” The patent does not alter our
adherence to Plos One policies on sharing data and materials. |
# 1. Introduction
At present, water injection into a coal seam is the most effective way to reduce
dust but is not an ideal solution in the field, mainly because the pore-fracture
structure of coal seams is very complex, and its morphological and structural
characteristics determine the physical and chemical properties of the coal. The
occurrence and flow characteristics of fluid in different coal seam structures
vary. How to reconstruct the pore-fracture structure in coal seams and determine
the relationship between the connectivities of the pores and fractures is an
important means to determine the seepage flow capacity of the coal. Recently, CT
technology has been used to study the pore-fracture structure of coal seams.
This method adopts fault imaging technology, which has many advantages, such as
nondestructive 3D digitization and refinement. Nondestructive visual measurement
of the pore-fracture structure in a coal seam can be realized, and a 3D network
model of real pores can be obtained.
Many scholars have carried out a considerable amount of research on the process
of CT scanning restoration and reconstruction. Researchers R.SH.MIKHALL and
others have calculated the volume surface and hydraulic radius of a micropore
group by analysing a kind of silica gel pore. J. T. Fredrich et al revealed the
geometric complexity of pore spaces by imaging the pore structures of 3D
geological materials using laser scanning confocal microscopy and 3D
reconstruction. C. R. Clarkson carried out low-pressure nitrogen adsorption and
high-pressure mercury intrusion measurements on shale reservoir coal samples.
The results showed that the specific surface area and pore volume results were
quite different among the coal samples. Hiroshi Okabe et al used two-dimensional
rock flakes to describe the three-dimensional pore space of a rock, assuming
isotropy in the reconstruction process, and obtained three-dimensional
stereograms. Li et al used CT scanning technology and electron microscope
scanning technology, the fracture pore structure at different scales is studied,
and the influence of pore characteristics on coal seam gas storage capacity is
analyzed. Alexandra Roslin et al used CT scanning technology, the influence of
confining pressure on coal seam fracture is analyzed. The results show that
confining pressure can inhibit coal structure to some extent. Zhang et al
results showed that the effect of fracturing on the horizontal and vertical
direction of coal body is obtained by the method of fractal dimension counting.
After analyzing the pore volume and roar length before and after fracturing, it
is concluded that liquefaction fracturing can improve the permeability of coal
body. S.M.Shah et al. used lattice Boltzmann and pore network models to simulate
single- and two-phase flow, analysed porosity results, predicted porosity,
unidirectional permeability and multiple physical properties at different length
scales, and improved the flow law from the pore scale to the core scale.
At present, most scholars mainly focus on CT scanning to restore the
distribution of pore fissures in coal rock. Few studies have been performed on
the multiscale characterization and modelling of coal, the seepage flow
law of water in real pores and the dynamic seepage evolution mechanism. Based on
this work, the dynamic seepage process of coal seam water injection has been
accurately described. This paper uses the high-precision CT scanner by Dandong
Aolong Ray Co., Ltd., to scan the coal body, filter the interference noise in
the original image by a median filter, and reconstruct the three-dimensional
pore network model of the coal body by using Avizo numerical simulation
software. The pore and fissure structures at different scales are extracted. The
maximum sphere algorithm and coordination number are used to analyse the
relationship between the connectivities of pore and fissure groups at different
scales, and the effects of different quantitative relationships between the pore
size and pore throat channel permeability are studied. Avizo software is used to
simulate the flow path of a fluid in the seepage channel, and the trend of the
variation in fluid velocity between different seepage channels is discussed to
explore the mechanism of the microseepage of fluid in a coal body and provide
guidance for coal seam water injection and dust control measures.
# 2. Material and methods
## 2.1. Coal sample collection
The coal was taken from the No. 4 coal seam in Leijia District of Fuxin City,
and this coal is long-flame coal. According to the GB/T 482–2008 Chinese
national standards for sampling, the coal specimen was cut into 5 cm×5 cm×10 cm
cuboids by a standard coal sample cutting machine in the laboratory, and three
standard coal samples were obtained. An SDLA618 proximate analysis of the coal
was carried out by using an industrial analyser. The results are shown in.
Experimental coal samples are shown in.
## 2.2. Experimental equipment and methods
The basic principle of CT detection technology is that the cross-section of the
penetrating object absorbs the X-ray, and the detector receives the information
of the X-ray passing through the section of the layer. When the uniform object
is irradiated by X-ray penetration, the attenuation coefficient x of the object
is exponential. The black and white colourscale in the grey image from the CT
scanning process clearly characterize the density distribution of the object
under detection, including the fissures, texture and other defects inside the
object. The absorption ability of the object to the X-ray is also different,
which is reflected by its attenuation coefficient. Thus, the pore structure of
the coal body can be revealed without damage, and the coal sample can be further
tested after CT scanning.
As shown in, using a Dandong Oron Ray Instrument Co., Ltd., AL-CT-225 industrial
X-ray CT detection system, the coal sample was placed in the centre of the
platform. A variety of detection methods, such as cone beam scanning and DR
real-time imaging, were used. During 360° coal sample rotation, scan information
about the defect locations and porosity was obtained. A total of 720 projection
images were collected. The coal sample scan was performed at a radiation source
voltage of 60 kV at a high temperature and constant pressure and a current of
240 μA. The minimum CT spatial resolution of the tomography was 6 μm, and the
density resolution was 0.5, a maximum of 1024×1024 pixels.
## 2.3. Analysis of scan results
An initial image of the coal sample is obtained by CT scanning technology, as
shown in. A group of slices in the yz, xz, and xy planes are selected. The black
linear feature is a fissure in the coal sample, and the yz plane has a large
number of pore-fracture groups. There are clearly fracture channels and pores
visible in the coal matrix in the xz and xy planes. However, there is a
considerable amount of noise in the original image, so it is necessary to select
the appropriate filtering function to further process the image.
## 2.4. Median filtering of CT image noise
There is system noise in the original image obtained by X-ray CT scanning. The
system noise is mainly due to the noise that inevitably arises in the process of
data transmission. The noise is randomly distributed in the image. These noise
points will increase or decrease the real pixel value of the image. Noise is
often represented as an additional pixel position or block on the image.
Therefore, the quality of the image is reduced, and the restoration, feature
extraction and interpretation of the image are affected. In this paper, a median
filter is selected to process the noise in the image. Processing the noise in
the image can not only eliminate the noise in the image but also maintain the
data of the original image. The grey value of the original image is not changed.
is the original image of a slice of the CT scan result, and is the image
filtered by the median filter. The median filter is used to filter the noise
points in the image, to protect the data of the original pore structure and
clarify the edge position of the image clearly so that it is not blurred. The
processed image can be used as the basis image for reconstructing the three-
dimensional coal body structure.
# 3. Results and discussion
## 3.1. Comprehensive characterization of pore-fissure spaces at different scales
The coal images from the CT scanning reflect the pore-fissure characteristics of
each layer. To analyse the three-dimensional pore-fissure distribution
characteristics of the coal samples, a three-dimensional reconstruction of the
coal samples is carried out. The internal structure at different scales is
extracted and characterized. The two-dimensional CT slice after median filtering
is used to construct the three-dimensional model of the coal sample by using the
volume rending module in Avizo, as shown in. The pore-fracture characteristics
in the coal samples are extracted with the threshold segmentation module, The
data parameters of pore volume 3d, pore radius and roar length of coal sample
can be analyzed by using Label Ansysaily function in Avizo, and the pore model
after segmentation of the coal matrix is accurate undamaged, as shown in.
shows the comprehensive spatial characterization pattern of the pore fissures,
in which the pore fissures of the coal sample result in massive, isolated pore
blocks with a zonal vertical distribution. These blocks in the coal are
distinguished by different colours, and the connected fissures are displayed in
the same colour. The results of the quantitative analysis of the coal samples
show that the total pore volume imaged is 3.6735×10<sup>13</sup> μm<sup>3</sup>,
the total pore area is 9.098×10<sup>10</sup> μm<sup>2</sup>, there are a large
number of micron-sized pores in the coal samples, and the coal samples show good
three-dimensional uniformity in terms of the pore distribution.
As shown in the, the three-dimensional spatial distribution of the coal pores at
different scales is extracted. shows that there are a large number of very small
pores; larger pores are added in, and some connected pores are added in. As the
scale increases, the pore connectivity is gradually revealed. By extracting coal
bodies at different scales, the distribution of different components at each
scale can be clearly seen. The diagram shows that the pores of a sample will
disform pore-fracture groups with increasing scale. This part of the connected
pores will provide the main seepage channels for the coal body. In the process
of seepage flow, hydraulic fractures communicate with connected pore groups.
## 3.2. Quantitative analysis of pores and pore throats
The diversity of the pore system of coal and rock depends on the pore structure
distribution, which is reflected in the quantitative relationship among the pore
size, pore volume area, pore throat channel radius and pore throat channel
length. The distribution law of the pores and pore throat channels in coal
samples is the key to determining the permeability of coal seams. The pore
radius determines the connectivity between pore throat channels, and the radius
and length of the pore throat channels directly determine the permeability of
the coal seam.
As shown in, the distributions of pore radius, volume contribution rate and pore
throat radius and length of coal samples are given. Analysis of the data shows
that the pore radii below 500 μm account for 85% of all the pores and that the
pore radii greater than 500 μm account for 15% of all the pores. The coal
samples have pores with sizes of 200–300 μm, and the pore throats in coal are
well developed. The pores in a coal sample are well connected by the pore
throats. The micron-sized pores in the coal samples are divided into micropores,
transition pores and mesopores, defined in pore radius ranges of 0–500 μm,
500–2000 μm and 2000 μm, respectively. The three-dimensional spatial
distribution constants of the micropores, transition pores and mesopores in the
coal samples are shown in. The pore size analysis of the coal sample data shows
that the number of micropores, transition pores and mesopores is 2763, 598 and
96, respectively, the contribution of micropores and transition pores to the
total pore volume is 0.78% and 11.21%, and mesopores account for 88.01% of the
total pore volume. Despite the number of micropores and transition pores, due to
their small sizes, micropores and transition pores contribute little to the
connectivity of the fluid channels. The mesopores, due to their larger pore
size, mainly provide the seepage passages. also show good connectivity in 3d
visualization. Based on quantitative pore space analysis of coal samples
containing micropores (79.92%) and transition pores (17.29%), the pore volume
ratio is 11.99%, and the pore volume ratio also considering the moderately
developed mesopores (2.79%) is 88.01%.
shows the scatter plot of the pore throat radius and pore throat length results
of the coal samples and the corresponding law determined by exponential function
fitting. The pore throat radii range from 46.29 to 4286.89 μm, and the lengths
of the pore throats range from 4206.28 to 48073.5 μm. The volumes of the pore
throats range from 0.4 to 121 mm<sup>3</sup>. From the diagram, most of the pore
throat radii in the coal sample are in the range of 100–2000 μm, and the pore
throat lengths are concentrated in the range of 5000–25000 μm. The results show
that the distribution of the pore throat radius and length parameters in the
coal samples are fairly uniform, and the pores are connected through the pore
throat channels. Compared with the results of the complex 3D pore morphology of
coal-like porous media, micron-scale pores are more developed in these coal
samples. The three-dimensional pores are intricate, indicating that the
macroscopic pores and microscopic cracks in coal samples have a complex spatial
distribution and interconnectivity.
## 3.3. Connectivity analysis and network characteristics of a porous rock
To further explore the permeability of the coal samples, it is necessary to
further extract the pore-fracture structure of the coal body, identify the pore
position and form an equivalent pore network. The maximum sphere algorithm is
generated by uniformly distributing spheres at the pore voxels in the pore
space, one for every two voxels in each direction. The maximum ball algorithm is
as follows: $$EqDiameter = \sqrt[3]{\frac{6 \times Volume3d}{\pi}}$$
EqDdiameter is the equivalent pore size, μm, and Volume3d is a single pore
volume, μm<sup>3</sup>.
In, the spheres represent the radii of the pore throat channels. The coloured
cylindrical connection represents the connectivity between the pore throat
channels, that is, the permeability of the coal sample. The pore throat channel
connectivity, pore throat channel length and pore throat channel radius directly
affect the coal permeability. The coal-like pores and pore throats are extracted
using the maximum sphere algorithm. The extracted pore network model has a total
of 3458 pores, 151 pore throats and 93 connected pore channels (representing the
coordination number). The branches and endpoints of the network in the figure
are called pores, and the lines connecting pores are called pore throats. The
maximum pore volume is 3.51×10<sup>12</sup> μm<sup>3</sup>, and the
corresponding pore area is 5.85×10<sup>9</sup> μm<sup>2</sup>. The maximum
capacity of the pore throat channel is 5.77×107 μm<sup>2</sup>, and the maximum
length is 4286.89 μm. As shown in the pore network model in, the coal samples
contain a large number of very small pores, which are basically not connected
with other pores. With increasing pore radius, the connected channels increase,
and the permeability increases gradually. That is, the small cracks in the coal
often appear as isolated points. Because they are not closely connected with the
internal pores, the very small pores cannot be connected with the pore throat
channels or contribute to the seepage. These isolated pore structures often do
not have seepage characteristics. Because pores with larger pore sizes are fully
developed and connected in the coal body, several complete seepage channels are
formed with other pore channel tributaries, which plays a decisive role in the
seepage flow capacity of the coal seam.
## 3.4. Matching analysis
The connectivity between pores directly determines the permeability of the coal
seam. The coordination number reflects the connectivity between pores and pore
throats, that is, if a pore is interlinked with pore throats. The pore structure
parameters represented by the coordination numbers can reflect the spatial
characteristics of the whole pore system and be used to analyse the pore system
more accurately. shows the frequency histogram of the coordination number
distribution.
Pores with a coordination number of zero can be regarded as isolated pores. This
part of the pore characteristics often shows that the pore volume area is small,
it is basically not connected with other fissures, and the contribution to the
mass flow rate of the seepage channel is very small. The flow trajectory of the
fluid in the coal body runs along the seepage channel, that is, the connected
pores form the seepage zone region. In reality, this part of the pore needs
other seepage channels to extend the pressure-driven seepage to the isolated
pore area to form a complete seepage zone. For pores with more than one
coordination number, a complete seepage channel is formed because of the
connectivity with other pores. The seepage water moves along the seepage path,
and the larger the coordination number is, the closer the connection and the
greater the number of seepage channels in the coal. thus, the larger the
coordination number is, the more seepage occurs in the coal, and the better the
wettability of the coal samples.
## 3.5. Permeability simulation principle
The ability of porous media to allow single-phase fluid passage under a certain
pressure gradient is defined in square metres, but square micrometres are more
widely used in practice because 1 square micrometre is closer to 1 Darcy (d) and
1 d = 0.9869233 is the constant coefficient of the absolute permeability of a
material according to Darcy’s law of fluid flow.
<img src="info:doi/10.1371/journal.pone.0252277.e002" id="pone.0252277.e002g" />
Q
S
=
−
k
μ
△
P
L
Q is the flow (m<sup>3</sup>/s) of fluid through the porous medium in a unit of
time; S is the cross-sectional area of the porous medium (m<sup>2</sup>); is the
absolute permeability (Pa.s); k is the dynamic viscosity coefficient of the
fluid flow; △P is the pressure differential of the fluid in the medium (Pa); L
is the length of the fluid passing through a porous medium (m).
$\frac{Q}{S}$ usually represents the flow velocity of fluid flowing through the
surface of a porous medium and is also known as the Darcy velocity. When a
porous medium is saturated with single-phase fluid, the permeability is called
the absolute permeability, and when a porous medium is filled with a multiphase
fluid, the permeability is called the relative permeability.
## 3.6. Permeability simulation results
For a further analysis of the percolation ability of the pore-connected channel,
seepage simulation of the real pore network model is carried out, and seepage
simulation of the coal sample is carried out in the x and z directions. The flow
characteristics and transportation capacity of the fluid can be obtained by
analysing the flow trajectory. The simulation software adopts AVIZO software,
the model size is 5 cm×5cm×10cm, the inlet parameter is set to 1 MPa pressure,
the outlet parameter is set to 0 MPa, the dynamic viscosity parameter of the
fluid is set to 1.01×10<sup>−3</sup> Pa·s.
is a seepage trajectory simulation diagram for the z direction; the fluid
direction is set to flow from left to right, a purple streamline represents the
medium-velocity region of the fluid, and a yellow streamline represents the
high-speed region of the fluid. The diagram shows that the high-speed region is
the connected region among the pore throat channels, that is, the region in
which each tributary connects to the others. The convergence of each branch
leads to momentum superposition, which makes the velocity increase, and the
fluid velocity is in the high-speed region. The medium-velocity region of fluid
is mainly due to the poor development of pore fractures and the decrease in the
pore throat radius in the coal sample, which leads to weak connectivity between
the pore throat channels and the loss of some momentum when the fluid passes
through the seepage channel. The fluid velocity is a medium velocity. To better
observe the influence of pore connectivity and the length of the pore throat
channel on the percolation ability, represents the pore throat channel
connectivity and the percolation trajectory superposition diagram. The fluid
velocity is faster in the superposition region of the large pores and seepage
channels. In the narrow aperture area, the velocity is relatively slow. shows
the trajectory map of the seepage in the x direction, and the grey streamlines
correspond to the low-velocity zone of seepage. It can be clearly seen that the
high-speed zone in the diagram is characterized by tight and large pores and
that the region of smaller pores corresponds to the medium velocity region. The
main reason for the low-speed region is that the pore throat channels are close
to the outer wall of coal, and the pore connectivity is not good there. shows
that when several seepage channels converge, the seepage channels are relatively
flat, and the velocity is high.
In the flow of microscale pore structure, the whole structure of pore system is
connected, but there are some isolated pore channels, which are basically zero.
In the process of fluid migration in connected channels, the velocity change of
fluid trajectory is more complex, which does not follow the linear and nonlinear
relationship. First of all, the size of the roar volume is the most important
factor to determine the seepage capacity, that is, the degree of pore
connectivity. Secondly, the size of pore roar radius is a secondary factor to
reflect the seepage capacity of coal seam. Finally, the distribution of pore
roar structure is the final factor.
# 4. Conclusions
\(1\) The pore-fissure structures in coal samples are analysed qualitatively at
different scales. The reconstructed 3D pore network models of the coal samples
clearly divides the pores, fissures and matrix parts of the coal samples. The
pores in the coal samples are nonuniformly distributed and vertically connected,
and very small pores exist in isolation. An accurate pore model is of great
significance for the analysis of fluid seepage in coal samples.
\(2\) The pore radius, pore throat length and pore connectivity parameters of
the coal samples are analysed by using the maximum sphere method. The coal
samples contain 3458 pore points, 151 pore throat channels, a volume of
3.6735×1013 μm<sup>3</sup>, and a pore capacity of 9.098×1010 μm<sup>2</sup>. A
pore space analysis indicates that the coal samples contain micropores (79.92%)
and transition pores (17.29%), the pore volume ratio of which is 11.99%; when
including the moderately developed mesopores (2.79%), the pore volume ratio is
88.01%. The coal samples exhibit favourable three-dimensional space constants.
The mesopores that connect the pores provide the main seepage channels, and the
contribution of isolated pores to the percolation capacity is negligible.
\(3\) The seepage simulation results based on the real pore-fracture network
model show that the seepage region of fluid in the coal seam is divided into
low-, medium- and high-speed regions. The morphological characteristics and
connectivity of the seepage channels determine the permeability. The more
connectivity there is, the larger the pore radii and the more uniform the
morphological properties; thus, the more connectivity there is, the higher the
permeability. The fluid seepage in the coal seam is driven by the following
factors: pore connectivity compactness \> pore throat channel dimensions \> pore
throat channel structure distribution characteristics.
\(4\) The pore distribution characteristics of the studied coal seam are
analysed qualitatively, and the three-dimensional pore-fracture network
structure is characterized quantitatively. From the microscopic point of view,
the migration trajectory of fluid in the microscale seepage channels is
analysed. The results suggest that this proposed method for evaluating the
physical properties and flow capacity of coal seams is accurate.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Background
India accounted for 19% of the globally estimated 287 000 maternal deaths in
2010. Although the level of maternal mortality in India has shown a definite
decline over the last decade nationally, the MMR declined by 35% from 327 deaths
per 100 000 births in 1999–2001 to 212 in 2007–2009 ; the current number of
maternal deaths is still unacceptably high. The national MMR in India is an
aggregate that conceals wide regional variations. Three large states viz.
Kerala, Tamil Nadu, and Maharashtra with MMRs of 81, 97, and 104 per 100 000
births respectively, have already achieved the Millennium Development Goal 5
(MDG 5) target. However, in nine other large states, MMR estimates still range
between 258 and 390. These nine states account for 62% of maternal deaths in
India, and 12% of the global burden of maternal mortality. The world’s progress
towards the achievement of MDG 5 is largely dependent on maternal mortality
reductions in India, more specifically, in these nine Indian states.
Skilled attendance at all births is considered to be the most critical
intervention for ensuring safe motherhood, and has been accepted as one of the
indicators for measuring progress to achieving MDG 5. Institutional delivery is
expected to improve maternal and neonatal outcomes through timely intervention
by skilled birth attendants backed by essential infrastructure and strong
referral services when needed.
To reduce maternal mortality, the Government of India commissioned nationwide
programmes, including the Child Survival and Safe Motherhood Program (1992–1997)
that was followed by Phase-1 of the Reproductive and Child Health Programme
(RCH-1) (1997–2004). These programmes aimed at increasing the availability of
emergency obstetric care (EmOC) services by enhancing institutional capacities.
However during this period (1992–2004) of focus on strengthening institutions
and investing in the supply side, maternal health care indicators in India were
slow to improve, despite it being a period of substantial economic growth in the
country. A major bottleneck identified was the low demand for and uptake of
institutional deliveries – the proportion of institutional deliveries during
this period showed increase from 26% to 41%; skilled birth attendance increased
from 33% to 47%, yet more than half of women continued to deliver at home.
Demand–side financing programmes, particularly cash transfer programmes, have
emerged recently as newer ways of addressing the chronic problem of
underutilisation of health and social services, particularly among vulnerable
groups. The PROGRESA programme in Mexico provided cash to families in return for
accessing children’s education, health, and nutrition services. Evaluation of
the PROGRESA programme showed a significant positive impact on school enrolment
and health outcomes. Similarly, the conditional cash transfer (CCT) in Honduras
showed that conditional payments to households increased the use and coverage of
preventive health care interventions. Evaluation of cash transfer programmes in
Nicaragua, Colombia, and Brazil also demonstrated a similar positive effect. The
most common mechanisms that have been employed in these regions to stimulate
demand have been CCTs and voucher schemes. The CCT provides monetary incentive
to households/individuals on the condition that they utilise specific services.
Given the limited success with supply–side interventions under RCH-1 (1999–2004)
in raising the proportions of skilled attendance at births and the growing
evidence of the effectiveness of demand–side financing schemes on the
utilisation of health services, the Indian government, in 2005, launched a
nationwide CCT programme known as Janani Suraksha Yojana (JSY) focussed on
maternal health. The JSY aims to reduce maternal and neonatal mortality through
the promotion of institutional births by providing cash incentives to mothers on
giving birth in a health institution. While the outline of the JSY scheme is the
same across the country, it has different eligibility criteria and differential
cash transfer size in different states, based on provincial proportions of
institutional birth at the time the scheme was designed. In the states with high
levels of maternal mortality and low levels of institutional delivery(low
performing states), the JSY scheme provides a cash incentive of \$31 and \$22 to
rural and urban women respectively, irrespective of socioeconomic status, age,
or parity if they give birth in a public or accredited private health facility.
In other socioeconomically better-developed states (high-performing states), the
cash incentive is about half that paid out in the low performing states and is
restricted to the first two live births of women from below the poverty line
(BPL) and from scheduled castes (SC) and tribes (ST). All health facilities pay
incentives into the mother’s bank account at the time of discharge from health
facility after delivery. To provide mothers more options to choose the place for
delivery, in some districts, private health facilities are accredited by
district-level authorities based on broad guidelines issued by the health
ministry. These guidelines include criteria such as infrastructure and human
resources required. Women delivering at private facilities receive JSY benefits
only on producing official certification of belonging to a vulnerable group. The
Indian CCT scheme is the largest CCT in the world, with 52 million beneficiaries
since inception.
The CCT is underpinned by two major assumptions: (1) Financial barriers exist to
access institutional care for childbirth. The cash incentive will enable women
to overcome these financial barriers to access institutional care for delivery,
and (2) increasing institutional births will provide more women access to
skilled birth attendance and, therefore, will reduce maternal and neonatal
deaths. Thus, the CCT is envisaged to result in (a) increased institutional
births and (b) reduced MMR and neonatal mortality in regions with high
proportions of institutional births.
Previous evaluations of the Indian CCT, i.e., the JSY, were limited to small
geographic areas focussed on processes and/or based on data from early years of
the JSY and have limitations owing to unavailability of maternal mortality data
at district levels. The Government of India recently set up the Annual Health
Survey (AHS) (2010–2011) to capture population-based data on health indicators
in these nine states with poor health indicators including high levels of
maternal mortality.
In this paper, we (i) report on trends in uptake of institutional births after
the initiation of the CCT, and (ii) study the association between institutional
birth proportions and the MMR in these nine states using AHS data. There have
been calls in the literature to investigate the success of such CCT programmes
in low-income settings, with more limited health system capacity. This paper
contributes to this body of knowledge; - lessons from this study of the Indian
CCT will be valuable in informing policies around demand-side financing in other
similar settings.
# Method
## Study Setting
India is federal union of 35 states with distinctly different levels of
socioeconomic development. States are further subdivided into smaller
administrative units called districts, each with a population of approximately
1.5 million, which is divided into five to ten units called blocks.
This study includes nine large states - Bihar, Uttar Pradesh(UP), Uttarakhand,
Madhya Pradesh(MP), Orissa, Rajasthan, Jharkhand, Chhattisgarh, and Assam – that
constitute about half of India’s population and account for 62% of her maternal
deaths. These nine states are subdivided into 284 districts. They have
relatively poor socioeconomic indicators; 34%–57% of their populations live
below poverty line (based on a defined degree of deprivation) as per national
surveys carried out by the Indian government. These nine states have relatively
higher MMRs, infant mortality rates (IMR), and birth rates than the national
averages of 212/100,000 live births, 50/1000 births, and 22.5/1000 population,
respectively. The Government of India has classified these states as ‘high focus
states’, implying more focussed attention to and greater allocation of resources
towards strengthening the health system in these states.
## Study Design
This study is an analysis of secondary data from two large population-based
national surveys viz. Sample Registration Survey and Annual Health Survey
conducted by the Government of India.
## Data Sources: The Sample Registration System (SRS)
The SRS is a large demographic survey carried out periodically in India to
generate reliable annual estimates of birth rate, death rate and other fertility
and mortality indicators at the national and state levels. At present, SRS is
operational in all states of India and covers about 7.27 million people in 1.5
million households. The sample unit in rural areas is a village and in urban
areas; the sampling unit is a census enumeration block (population from 750 to
1000). The SRS comprises continuous enumeration of births and deaths in selected
sample units by resident part-time enumerators, and an independent survey every
six months by SRS supervisors. The data obtained by these two independent
functionaries are matched. While recording details of every outcome of
pregnancy, the enumerators and supervisors are required to enquire about the
type of medical attention received by the mother at the time of delivery,
including place of delivery.
Proportions of institutional births reported by the SRS between 2005 and 2010
(years) have been used for the analysis in this paper.
### Annual Health Survey (AHS)
: The AHS is the Government of India’s recent initiative at recording district
level health outcomes in the nine ‘high focus’ states. The rationale for the
survey was to identify districts requiring special attention as these often miss
detection when studying average statistics at the state level. A special feature
of the AHS is that it is the first survey in India to provide estimates of the
district level mortality.
The first AHS reported in 2010–2011 covered all the districts in the nine
states. The AHS adopted a uni-stage stratified simple random sample without
replacement except in case of larger villages and surveyed 18 million people in
3.6 million households. The survey collected background information of selected
households and information from ever married women aged 15–49 years from these
households regarding pregnancy outcome, place of delivery, child immunisation,
and breastfeeding, to mention a few, that took place during the reference period
(Jan. 2007 to Dec. 2009). Further details of data collection and management
procedures are available on the survey website.
Data reported by AHS on district-level MMR, proportion of institutional births,
caesarean rate, total fertility rate, and proportion of literate and poor
population were used in this paper.
### Census of india
The Indian national census is conducted every ten years across all (35) states
in the country. Each household is visited to collect information on a wide range
of demographic and socioeconomic indicators of the household and the
individuals. The district level information on level of urbanisation, vulnerable
population, and total population from Census 2001 were used as covariates in the
analysis presented here.
## Variables
For state-level analysis, the proportion of institutional births, i.e., number
of births that took place in government or private health institutions out of
the total births, before and during the implementation of the JSY (2005–2010)
was used. These data were sourced from the SRS during the period.
For district-level analysis, the main outcome variable was district MMR. Given
that maternal mortality is a rare event, the AHS estimated the MMR for a group
of three to five geographically contiguous districts. In this analysis, we
attributed the estimated MMR to each district in the group.
The main explanatory variable of interest was district level proportion of all
institutional births. Other socio-demographic covariates that influence maternal
mortality included district-level caesarean rate, total fertility rate,
literacy, proportion of households in the lowest 20% quintile of wealth index,
proportion of vulnerable (Scheduled cast/tribes) and urban population. A brief
description of these variables is given below:
### Caesarean rate
Percentage of caesarean deliveries out of total deliveries that occurred at
government and private institutions in the given period. Caesarean rate is the
proxy indicator for availability of comprehensive EmOC services.
### Total fertility rate
The total fertility rate (TFR) in a specific year is the number of children that
would be born to each woman if she were to live to the end of her childbearing
years and if the likelihood of her giving birth to children at each age was the
currently prevailing age-specific fertility rates.
### Literacy
The proportion of population in a district with the ability to read and write in
any language, expressed as a percentage.
### Proportion of poor households
Household wealth index was constructed by the AHS at the state level for each of
the nine study states using the assets possessed (such as ownership and status
of the house) and the facilities availed (such as electricity, toilet) by the
households to determine a household’s relative economic status. Thereafter, the
households were ranked according to their individual household asset score and
then divided into five quintiles with the same number of households in each. In
this paper, we used proportion of households in the lowest-income quintile in
each district based on assets possessed as an indicator of level of deprivation
(poverty) of the respective districts.
### Proportion of vulnerable (Scheduled cast/tribes) population
The proportion of scheduled caste and tribe persons in the population of each
district. Scheduled castes and tribes are those communities that were
historically subject to social disadvantage and exclusion. They are accorded
special status by the Constitution of India and are recipients of special social
benefits as part of a programme of positive affirmation.
### Urban population
The proportion of the total population in urban areas for every district.
Data for district-level MMR, institutional births, caesarean rate, TFR,
literacy, and poor population were sourced from the AHS and while that for
vulnerable, urban and total population of district was sourced from the national
census 2001.
## Ethics Statement
The study is based on the data available in the public domain for use.
## Analysis
State-level data on proportion of institutional births between the years 2005
and 2010 from the SRS were analysed to describe trends in proportions
institutional births before (2005) and during (2006–2010) the implementation of
the JSY. A statistical comparison of mean institutional delivery proportions
before (2005) and during (2010) the JSY programme was done.
Socio-demographic characteristics of the 284 districts in the nine states are
presented. The association of district characteristics and institutional births
was first examined separately by simple correlation analysis.
To study the relationship between the proportion of institutional births and
MMR, TFR, literacy, and the proportions of poor, urban and vulnerable
populations we first used a simple correlation analysis. Subsequently, multiple
regression models were developed to assess the effect of change in the district-
level proportion of institutional births on the district MMR when other relevant
socio- demographic variables were kept constant.
STATA 10 was used for statistical analysis.
# Results
1. Change in the proportion of institutional births in the nine states
since the inception of the JSY programme: The proportion of institutional
births increased in the nine states from a pre-programme average of 20% to
49% in the five years (p\<0.05). While institutional birth proportions
increased across all nine states, the magnitude of the increases varied
across states.
2. Association between institutional birth proportions and MMR in the
district:
- 2.1 *Characteristics of 284 study districts*: District
characteristics for the 284 districts are presented in. On average, each
district had a population of 1.7 million with varying proportions of
poverty, literacy, and urbanisation. The proportion of institutional
births ranged from 16.8% to 92.5% (mean 56.2%), demonstrating wide
variations in utilisation of this service. The MMR ranges from a minimum
of 183 to a maximum of 451.
- *2.2 Correlation of district characteristics with proportion of
institutional births and with MMR:* shows the estimated correlation of
district characteristics with institutional birth proportions and MMR.
Districts with higher fertility rates and higher levels of deprivation had
lower institutional births proportions (r = −0.37 and −0.28,
respectively); conversely, higher literacy and urbanisation in a district
correlated positively with institutional births proportions (r = 0.38 and
0.32, respectively). The proportion of vulnerable population in a district
did not show much influence on the uptake of institutional births
(r = 0.07). There was no correlation between the proportion of SC/ST
populations in the district and institutional birth proportions when these
groups were analysed separately (data not shown).
- Simple correlation between district characteristics and MMR showed
that the fertility rate and the proportion of the poor in the population
were positively correlated with MMR (r = 0.40 and 0.25, respectively). On
the contrary, higher literacy and urbanisation were negatively correlated
to MMR (r = −0.34 and −0.18, respectively). The proportion of births in an
institution and births by caesarean section (CS), each had a small
negative correlation with district MMR (r = −0.11 and −0.19,
respectively). A scatter plot of institutional birth proportion and MMR
does not show any strong relationship between institutional birth
proportions and MMR in the districts.
- *2.3 Regression analysis:* We built a regression model to explore
the association between the proportion of institutional births and MMR.
Covariates included are shown in. This model was unable to detect a
significant association between institutional birth proportion and MMR
\[CI: −0.10, 0.68\] adjusting for other confounders as shown in. Districts
with higher fertility rates or higher proportions of poor population were
significantly associated with higher MMR. Conversely districts with high
literacy and high urbanisation were associated with lower MMR. Districts
with high C-section rates were associated with higher MMR.
# Discussion
This report is based on district level estimates of MMR from the AHS from India.
The results are important given that the nine states included in the study
contribute to 12% of global maternal deaths. Efforts made to reduce maternal
mortality in this area will impact global achievement of MDG 5. These results
are also relevant for policy makers planning to initiate or expand cash
transfers for promotion institutional births in other similar settings.
Our analysis of the nine states indicates a steep rise in institutional birth
proportions since the inception of the JSY programme. Although available data do
not allow segregation of institutional births into JSY and non-JSY births, a
large part of this increase in institutional births is fuelled by the JSY.
Despite the steep rise in institutional births, our analysis was unable to
detect a significant association between institutional birth proportions and MMR
in the districts.
##
### Increase in institutional births
When compared with pre-programme levels, the proportion of institutional births
at state level increased two to three times over a period of five years since
the programme began. Reports from other large-scale household surveys such as
the District Level Household Survey and periodic reports from the health system
also show increases in the institutional birth proportions after the
implementation of the CCT scheme. A slow rise in institutional births during the
RCH-I (1997–2004) and a sharp increase after initiation of the JSY scheme
indicates its success in converting a significant proportion of home births into
institutional births. While some of these institutional births have definitely
occurred outside the JSY i.e., in non- accredited private institutions however
this proportion remains marginal. Calculations made from the AHS reports show
that an average of 13% of all births and 25% of all institutional births occur
in private institutions in the study districts. As women delivering at private
institutions accredited for the JSY receive the cash benefit, a proportion of
institutional births in the private sector (25%) become JSY beneficiaries,
leaving only a small proportion of non-JSY institutional births as a whole.
Another recent survey report also indicated that in the ‘high focus’ states of
MP, Rajasthan, UP, Orissa, and Bihar, the vast majority of all institutional
births do occur under the JSY programme: on average, only 12.9% of all
institutional births do not occur within the JSY programme.
Experience from Nepal has been similar; a cash incentive to women on delivering
in a health facility, increased utilisation in maternity services. A review of
evidence on demand-side financing for sexual and reproductive health services in
low and middle-income countries reports increased utilisation of services as an
effect of demand-side financing strategy.
Despite the overall increase in institutional births at the state level, within
each state there were wide district-level variations that were associated with
background socioeconomic characteristics of the districts. Districts with higher
literacy and larger urban populations tended to have higher institutional birth
proportions, whereas poverty and high fertility rates adversely affected the
utilisation of institutional delivery services. As it is known that poor women
bear the highest burden of maternal death, the programme still needs to develop
mechanisms to reach this most vulnerable group.
### Association between institutional birth and maternal mortality
Our analysis was not able to detect a significant association between district
institutional birth proportion and MMR. While it is possible that we were unable
to detect a significant association given the wide confidence intervals around
our estimates, it is also possible that there is a limited influence of
institutional birth proportions on MMR. Lim et al, in their evaluation of the
JSY scheme in India, also reported an inability to detect its effect on maternal
mortality at the district level, possibly because of a lack of programme effect
or an inadequate sample size to detect the effect.
This finding of a lack of association between institutional birth proportions
and MMR could suggest the possibility that it is likely that the CCT
disproportionately attracts pregnant women without complications to
institutions, i.e., women most vulnerable to maternal death are not entering the
programme. The mortality among women with complications is likely to be higher
if they deliver at home, than if they deliver at institutions, as the latter
should have access to a skilled person and EmOC services. There are currently no
estimates of the proportion of mothers with complications among home or
institutional births. The lack of association could also be related to the poor
quality of care offered at institutions to the mothers with complications.
Despite the focus on supply-side strengthening in the earlier RCH programmes,
there have been recent reports documenting inadequacies in skilled human
resources, infrastructure and supplies, which are critical for provision of
good-quality care.Moreover, as reported by a programme evaluation report of the
JSY, although all public sector facilities are designated as programme
facilities, it is only a rather small number of higher-level facilities that
actually have the ability to handle complications. Therefore, women in rural
areas reach lower level facilities which are ill-equipped to handle
complications. These inadequacies present a challenge to a mother having a safe
delivery, even if she reached a facility. Paxton et al found that correlation
between proportion of skilled attendance at birth and MMR becomes weaker for
developing countries alone than when both developed and developing countries are
included together. The correlation further drops for countries having MMR of
more than 200. From this analysis the authors conclude that skilled attendance
alone is not accountable for higher correlation of skilled attendance and MMR.
Countries with low MMR have high proportion of skilled birth attendance and they
have high proportions of maternal complications managed with high quality EmOC
services.
### The quality of care issues
While our analysis does not deal directly with quality of care under the
programme, it is possibly an important explanation for the lack of association
between institutional birth proportions and MMR. The available literature is
summarised below.
### Need to ensure skilled attendance in an enabling environment
In promoting institutional births, it was hoped that pregnant women would get
skilled attendance at births, and access to appropriate EmOC in the event of
complications. The Safe Motherhood Inter-Agency Group has defined *skilled
attendance* as a process through which a woman is provided with adequate care
during labour, delivery, and the postpartum period. Studies exploring links
between skilled attendance at births and maternal mortality suggest a need of 1)
a partnership of skilled attendants (health professionals with the skills to
provide care for normal and/or complicated deliveries), and 2) an enabling
environment of equipment, supplies, drugs and transport for referral.
Investigation by Sri B et al on high number of maternal deaths in 2010 in the
Barwani district of Madhya Pradesh found a lack of skilled birth attendance,
failure to carry out emergency obstetric care in obvious cases of need, and
referrals that never resulted in treatment. This report questions the policy of
giving cash to pregnant women to deliver in poor quality facilities without
first ensuring quality of care and strengthening the facilities to cope with the
increased patient loads.
Programme evaluation of the JSY reports that the programme has increased access
to delivery by an Auxiliary Nurse Midwife (ANM), nurse, or doctor, but not
necessarily to skilled birth attendant (SBA), because most nurses and ANMs who
are actually providing services were not trained in the SBA training.
The conceptualisation of the JSY programme in India has led to the substitution
of the critical component of skilled attendance with a notion of ‘institutional
births’ as being equivalent to skilled birth attendance, and, therefore, this as
a condition to be met in order to receive the cash benefit from the JSY.
Evidence seems to now indicate that the assumption that institutional birth is
the same as skilled birth attendance in an enabling environment does not hold.
This has resulted in pregnant women arriving in institutions, but this in itself
is not necessarily giving them access to skilled attendance.
### Need to address other non-financial access barriers
The JSY has raised the uptake of institutional births, yet, in some districts,
more than half of women continue to deliver at home. This suggests that even if
a cash incentive is able to attract more women to facilities, there are still
many for whom; other non-financial barriers operate to reduce the likelihood of
an institutional birth. Some of these barriers include the inability to access
transportation or the costs involved in doing so, non-perception of birth as a
risk event, the social status accorded to women, and poor levels of trust in the
public health facilities. Some states initiated emergency transport arrangement,
which reduce the transportation barrier; however, the other barriers are more
complex and require more structural and social change. An analytical framework
by Bart Jacob et al provides guidance on interventions to support structural
changes in the health system that can reduce some barriers. Significant positive
association of literacy, urbanisation and inverse association of fertility rates
and poverty in our regression model seem to suggest that overall socioeconomic
development contributes significantly in maternal mortality reduction. Although
socioeconomic development is merited, reduction in maternal mortality can be
achieved even in countries with poor development indicators. For instance, low
income-countries such as Sri Lanka, Thailand, and Maldives have shown that
universal access to skilled attendance at birth and EmOC services could reduce
maternal mortality drastically.
### Negligence of antenatal and postnatal care
The JSY scheme incentivises pregnant women for utilisation of health facility
for intra-partum care, whereas antenatal (ANC) and postnatal care (PNC) is not a
prerequisite for cash benefit. While splitting the cash benefit can imply large
administrative burdens on the programme, a narrow focus on institutionalising
intra-partum care restricts opportunities of averting deaths by early detection
of risk pregnancies and treatment of common postnatal complications like
puerperal sepsis.
## Limitations
AHS estimates MMR for groups of three to five geographically contiguous
districts. These pooled estimates were attributed to individual districts during
our analysis. Institutional birth proportions are assumed to be JSY deliveries
in this analysis, although a small proportion as reported above are non-JSY
institutional births. MMR estimates are based on community-level surveys; no
information is available on what proportion of deaths occurred within the
programme or outside of it.
### Residual confounding
The results of the study should be interpreted with caution because the
association between institutional birth proportions and maternal mortality can
be confounded by other known and unknown confounders. Examples of these could be
road network in the district, a measure of overall quality of care in the
district, emergency transport services available in the district, etc. Some
confounders such as cultural practices, awareness about need of and availability
of health care services, etc. will influence maternal mortality via access to
institutional birth. Variables used in the analysis (e.g. poverty, literacy,
urbanisation) serve as proxies for these factors. Factors that determine the
quality of care provided in the facilities could have contributed to varying
levels of maternal mortality, but in the absence of precise data on quality of
care provided in study districts, this study is unable to control the
confounding effect of district-level variation in quality of care.
We acknowledge that residual confounding is likely to be present. However,
despite the limitation of this ecological study, it explores the association
between district-level variation in uptake of institutional births and maternal
mortality using available data at the smallest unit of analysis (district)
during the implementation of the JSY; it makes an important contribution.
The counter intuitive finding of an association between caesarean rates and MMR
could possibly be because confounding factors were not taken into account in the
model. It also suggests further exploration of appropriateness and quality of
caesarean and post-operative care provided. It could be possible that the sudden
rise in institutional deliveries resulted into overcrowding in facilities and
quality of care in operation theatres or in postnatal wards was compromised
resulting in more deaths. Another possibility could be the higher rates of
caesarean in private hospitals, exposing more women to higher risk. In the 284
study districts, the median caesarean rates for public and private sectors
reported by the AHS were 5% and 28%, respectively. When we conducted a
regression analysis using a stratified caesarean rate in public and private
facilities it showed that caesarean rate in private facilities, but not in
public facilities, were significantly associated with higher mortality. This
needs to be explored more. Bertan et al, in their analysis of global and
regional estimates of caesarean rate show that although caesarean rates below
15% are associated with lower maternal mortality; higher rates are predominantly
correlated with higher maternal mortality.
## Conclusions
We were unable to detect a significant association between the proportion of
institutional births and the MMR at the district level, though other indicators
of overall development such as literacy showed a significant association with
reduction in the MMR. Although the JSY succeeded in raising institutional birth
proportions significantly; the same has not translated into significant
reduction in the MMR. It is likely that a weak supply side has led to a
situation in which increased access to institutional birth has not resulted in
reduction in maternal deaths, as mothers are not receiving appropriate or
adequate care. It is also possible that the JSY failed to draw mothers with
life-threatening complications into institutions, resulting in most of such
women continuing to deliver at home, contributing to persistent maternal
mortality. Further studies are required to examine the extent to which the JSY
increased access to institutional care among mothers with complications.
Moreover, to translate the JSY gains in institutional delivery coverage into
reduced mortality outcomes, it is important to ensure that all women accessing
an institution for delivery receive good quality obstetric care.
We acknowledge the peer reviewers – Dr Abhijit Das and two anonymous reviewers,
for critical comments on an earlier version of this paper.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: BR. Performed the experiments:
BR VD ADC. Analyzed the data: BR ADC. Contributed
reagents/materials/analysis tools: BR VD. Wrote the paper: BR ADC. |
# Introduction
Epithelial-mesenchymal transition (EMT), a developmental process by which
epithelial cells reduce cell-cell adhesion and lose apical-basal cell polarity,
plays a critical role in the embryogenesis and conversion of early stage tumors
into aggressive malignancies. EMT promotes multiple physiological processes that
increase the invasiveness and metastasis potentials of human tumors. EMT is
typically characterized by loss of E-cadherin, gain of N-cadherin and vimentin,
and translocation of β-catenin from membrane to the nuclear compartment. The
impairment of E-cadherin is a hallmark of EMT, and E-cadherin expression is
often inversely correlated with tumor malignancy and patient survival. The
E-cadherin expression level is down-regulated by gene silencing with CpG
methylation on promoter in hepatocellular carcinoma (HCC), and may be associated
with tumor grade and poor prognosis of HCC. Several transcriptional regulators
that act as E-cadherin repressors are mediated by recognizing the E-box motif on
the E-cadherin promoter region. Factors of Snail zinc finger, Zeb and bHLH
families are known to suppress E-cadherin, thereby promoting the EMT process and
tumor metastasis. In addition, increased Zeb-1, Snail, SIP1, and Twist
expressions are reportedly associated with the clinicopathological significances
of HCC malignant progression, including cancer invasion and poor patient
survival.
14-3-3 proteins are a family of regulatory molecules with highly conserved
homology among all eukaryotic cells. 14-3-3 modulates physiological functions
*via* binding intracellular proteins with Ser/Thr phosphorylation-dependent
domains, thereby influencing conformation, activity, subcellular localization,
and protein complex stability. 14-3-3 proteins comprise seven isoforms (β, ε, γ,
η, σ, τ/θ and ζ), which play crucial roles in regulating multiple cellular
processes, including cell cycle regulation, DNA repair, apoptosis, cell
adhesion, and motility. 14-3-3 proteins have been implicated in various types of
human malignancies. Among the variety of 14-3-3 isoforms, increased expression
of 14-3-3ε has been demonstrated in breast cancer, lung cancer, vulvar squamous
cell carcinoma, follicular and papillary thyroid tumors, meningioma, and HCC,,
although reduced expression of 14-3-3ε in gastric cancer has been reported. In
addition, previous studies have demonstrated that up-regulation of 14-3-3ε
expression protects colorectal cancer cells and endothelial cells from
oxidative-stress induced apoptosis, while suppression of 14-3-3ε by nonsteroidal
anti-inflammatory drugs induces cancer and endothelial cell death. Furthermore,
elevated expression of 14-3-3ε is significantly associated with increased
metastatic risk, shortened overall survival, and progression-free survival of
HCC. Enhanced expression of 14-3-3ε was suggested to induce and be associated
with focal adhesion kinase (FAK) expression *via* activation of NFκB signaling.
These results implied that 14-3-3ε was involved in the modulation of cell
polarization and migration, which may potentially regulate HCC tumor development
and metastasis. In this study, we show for the first time that 14-3-3ε induces
HCC cell migration and EMT *via* regulation of Zeb-1/E-cadherin expression. Our
results reveal that E-cadherin is a downstream modulator for 14-3-3ε during HCC
tumor progression.
# Materials and Methods
## Cell Culture and Stable Cells
Huh-7 (Japanese Collection of Research Bioresources, JCRB-0403), HepG2 (American
Type Culture Collection, ATCC-HB-8065), Hep3B (ATCC-HB-8064), PLC-5 (ATCC-
CRL-8024) and SK-Hep1 (ATCC-HTB-52) human hepatocellular carcinoma cells were
maintained in DMEM (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine
serum (FBS; Hyclone Thermo Fisher Scientific, Waltham, MA), 100 units/ml
penicillin, and 100 units/ml streptomycin, in a humidified incubator (Forma)
with 5% CO<sub>2</sub> at 37°C. For stable transfection, 14-3-3ε cDNA was
amplified by PCR and then subcloned into the p3XFlag-CMV vector. Huh-7 cells
were transfected with p3XFlag-CMV (Control) or p3XFlag-14-3-3ε (14-3-3ε) by use
of Polyjet™ transfection reagent (SignaGen Laboratories, Rockville, MD)
according to the manufacturer’s instructions. The transfected cells were
selected with G418 (500 µg/ml) for 4 weeks. Single colonies of stable clones (at
least 3 in each cell line) were maintained in DMEM with 10% FBS and 200 µg/ml of
G418.
## Transient Transfection
Huh-7 and HepG2 cells were transiently transfected with control and 14-3-3ε by
use of Polyjet™ transfection reagent (Signa-Gen Laboratories, Ijamsville, MD).
Cells were transfected with control or 0.5 to 1.5 µg of 14-3-3ε vectors per
6-well plate followed by incubation with Polyjet™/DNA complex-containing medium
and replaced with complete medium for 24 hours. Transfected cells were incubated
for additional 24 hours before performing cell migration assays or protein
expression analysis.
## Knockdown Studies
Gene silencing was performed using 14-3-3ε, Snail (Stealth RNAi, Invitrogen,
Carlsbad, CA), Zeb-1 siRNAs (Santa Cruz, Heidelberg, Germany) and Stealth RNA
Negative Control (Invitrogen, Carlsbad, CA) with reported sequences. Transient
transfection of siRNA was carried out using Lipofectamine™ RNAiMAX (Invitrogen,
Grand Island, NY) according to the manufacturer’s guidelines.
## Western Blot Analysis
Cells were lysed in ice-cold RIPA buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl,
2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, Millipore, Temecula, CA)
containing cocktail protease inhibitors (Roche, IN, USA). Cell lysates were
centrifuged at 15,000 rpm for 20 minutes at 4°C, and protein concentrations were
determined by a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).
Each sample of 20 µg protein was applied to a gradient SDS-PAGE gel and
immunoblotted onto PVDF membranes. The membranes were blocked and probed with
indicated primary antibodies of Flag and actin (Sigma-Aldrich, St. Louis, MO),
E-cadherin and N-cadherin (BD Biosciences, San Jose, CA), 14-3-3ε, Zeb-1, Twist
and Slug (Santa Cruz Biotechnologies, Heidelberg, Germany), Snail (Cell
Signaling Technology, Beverly, MA), Zeb-2 (Abcam PLC, Cambridge, UK), and
vimentin (Millipore, Temecula, CA). The membranes were immersed in PBST
containing horseradish peroxidase-conjugated secondary antibody, and protein
levels were determined by use of enhanced chemiluminescence reagents.
## Immunofluorescence Staining
Immunofluorescence staining was performed as described previously. Briefly,
14-3-3ε and control cells were fixed with 2% paraformaldehyde for 15 minutes at
4°C. After washing, cells were permeabilized with 0.1% Triton X-100 in PBS for 5
minutes and blocked with PBS containing 10% FBS at room temperature for 1 hour.
For the immunofluorescence staining, cells were incubated with the primary
antibodies of anti-E-cadherin and anti-N-cadherin (BD Biosciences, San Jose,
CA), and anti-vimentin (Millipore, Temecula, CA) in PBS containing 1% FBS at 4°C
overnight, followed by incubation with Alexa Fluor® 488 secondary antibody
(Invitrogen, Grand Island, NY) in PBS containing 5% bovine serum albumin at room
temperature for 2 hours. Samples were mounted and images were analyzed by use of
the Leica TCS SP5 Confocal Imaging System (Leica, Germany).
## Migration Assay
Bio-coat cell migration Boyden chambers were used for cell migration assay
(Becton Dickinson, Pont-de-Claix, France). Briefly, cells were trypsinized and
suspended in 0.1% BSA-DMEM and cells (1×10<sup>4</sup> for SK-Hep1,
6×10<sup>4</sup> for Huh-7 and 2×10<sup>5</sup> for HepG2) were added to the
upper wells with 8-µm pores. Cells were allowed to migrate toward the bottom
wells containing 100 µg/ml fibronectin (Becton Dickinson, Pont-de-Claix,
France), epithelial growth factor (EGF, 20 ng/ml, Sigma-Aldrich, St. Louis, MO)
and 10% BSA-DMEM for 20 hours. Cells remaining on the upper side were removed,
and migrated cells on the bottom side were fixed and stained with 0.1% crystal
violet containing 20% ethanol and 1% formadehyde for 20 minutes. Cell migration
was quantified by counting the total number of migrated cells.
## Quantitative Real-time PCR
As described previously, total RNA was extracted by use of the RNAspin Mini Kit
(GE Healthcare, Freiburg, Germany). cDNA was synthesized from 2–5 µg RNA by use
of the oligo(dT)<sub>18</sub> primers and RevertAid™ First Strand cDNA Synthesis
Kit (Fermentas, Thermo Fisher Scientific, Waltham, MA). Quantitative real-time
PCR involved use of SYBR Green (Kapa biosystem, Woburn, MA) with specific
oligonucleotide primers from the AB 7900HT system (Applied Biosystems, USA).
Applied Biosystems Relative Quantification (RQ) Manager Software version 1.2 was
used to analyze the relative gene expression in each sample by the comparative
Ct method. Gene expression was normalized to that of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH).
## Clinical Specimens
Tissue samples were obtained from 113 HCC patients who had undergone surgery for
tumor resection or biopsy at Taichung Veterans General Hospital from January
1999 to December 2001. The mean follow-up time was 51.5±28.7 months. Thirty-
three patients (29.2%) developed tissue-proved metastasis in 3 to 87 months
after the resection of primary HCC. Slides from paraffin-embedded surgical
specimens of primary tumors with surrounding non-cancerous liver parenchyma were
subjected to immunohistochemical (IHC) staining. The pathological features, IHC
staining results, clinical parameters, including Barcelona-Clinic Liver Cancer
(BCLC) staging, and disease outcomes were collected for analysis. This study was
approved by the Institutional Review Board of Taichung Veterans General
Hospital. The policy that no informed consents are required for using these de-
linked samples for retrospective analysis was also approved by the Institutional
Review Board.
## Immunohistochemical Analysis
For immunohistochemistry analysis, an automatic immunostaining device and
ultraView detection kit (Ventana XT Medical System, Tucson, AZ) were used to
detect 14-3-3ε expression in paraffin-embedded tissues by use of a primary
antibody against 14-3-3ε (1∶800; Santa Cruz Biotechnology, Santa Cruz, CA) and
E-cadherin (1∶800; BD Bioscience). A negative control was prepared by the same
staining procedure without primary antibodies. The intensity of 14-3-3ε and
E-cadherin protein staining was semiquantitatively scored by a Quick-score
(Q-score) method based on intensity and heterogeneity,. Staining intensity was
scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). For
heterogeneity, the proportions of tumor cells positively stained with 14-3-3ε
and E-cadherin were scored as 0 (0%); 1 (1–25%); 2 (26–50%); 3 (51–75%) or 4
(76–100%). The Q-score of a given tissue sample was the sum of the intensity and
heterogeneity scores and ranged from 0 to 7. A Q-score ≥2 was considered
overexpressed, or positive expression, and a Q-score \<2 was considered normal,
or negative expression. Cases with \<5% weakly stained specimens were considered
as negative expression.
## Statistical Analysis
The Student’s *t*-test was used to analyze differences between 2 groups. Kaplan-
Meier curves were plotted and the log rank test was used to analyze time-related
variables of probabilities for metastasis and overall survival. A *P* value
\<0.05 was considered statistically significant.
# Results
## 14-3-3ε Promotes HCC Cell Migration
To explore the potential role of 14-3-3ε in HCC tumor metastasis, we examined
the expression of 14-3-3ε in distinct HCC cell lines. 14-3-3ε was detected in
all tested HCC cells. The well-differentiated HCC cells, including Huh-7, HepG2
and PLC-5, expressed lower levels of 14-3-3ε, while the poorly differentiated
SK-Hep1 cells expressed higher levels. We next established a stable cell line
with 14-3-3ε overexpression. Huh-7 cells were transfected with p3XFlag-CMV
(Control) or p3XFlag-14-3-3ε (14-3-3ε) vectors and selected by G418 for 4 weeks.
Individual colonies were picked and 14-3-3ε expression was confirmed by Western
blot analysis. At least 3 clones were selected, and the representative clone was
used for further experiments. To investigate whether 14-3-3ε regulates cell
migration, we performed the migration assay with Boyden chamber experiments. We
found that 14-3-3ε (stable clones 1–4) significantly induced cell migration. In
addition, the induction of cell migration accessed by 14-3-3ε overexpression was
confirmed by transient transfection in both of Huh-7 and HepG2 cells.
Transiently 14-3-3ε overexpression dose-dependently increased Huh-7 and HepG2
cell migration. To further confirm the effect of 14-3-3ε on modulating cell
migration, control or 14-3-3ε stable cells (clone 1) were transfected with
scramble or 14-3-3ε siRNAs and the efficiency of 14-3-3ε knockdown was
determined by Western blotting analysis (upper panel). Knockdown with siRNA
significantly abolished 14-3-3ε-induced cell migration (lower panel).
Additionally, knockdown of 14-3-3ε with siRNA significantly suppressed SK-Hep1
cell migration with a dose-dependent manner. These results suggest that 14-3-3ε
plays an important role in promoting HCC cell migration.
## 14-3-3ε Promotes Epithelial-mesenchymal Transition of HCC
To investigate whether 14-3-3ε expression regulates EMT of HCC cells, we
determined the expression of EMT markers, E-cadherin, N-cadherin and vimentin,
by Western blotting analysis. We found that 14-3-3ε overexpression significantly
reduced E-cadherin expression, but it induced N-cadherin and vimentin
expression. The expression levels and subcellular localizations of E-cadherin,
N-cadherin, and vimentin were further examined by immunofluorescent confocal
microscopy. E-cadherin expression was detected at the cell-cell contacts in
control cells, while it was dramatically reduced in 14-3-3ε overexpression
cells. Slight expression of N-cadherin and vimentin was detected in control
cells, but such expression was significantly induced by 14-3-3ε overexpression
(middle and right panels). Furthermore, reduction of E-cadherin expression and
the induction of N-cadherin and vimentin expression in 14-3-3ε overexpression
cells were abrogated by transfection with 14-3-3ε siRNA, as determined by
Western blotting analysis. The regulation of these expressions of E-cadherin,
N-cadherin and vimentin by 14-3-3ε knockdown were further confirmed by confocal
microscopy. 14-3-3ε siRNA restored E-cadherin expression, which localized at the
cell junctions. In addition, knockdown with siRNA suppressed the N-cadherin and
vimentin expression induced by 14-3-3ε. These results indicate that 14-3-3ε
overexpression promotes EMT of HCC.
## 14-3-3ε Promotes HCC Cell Migration via Upregulation of Zeb-1 and Snail
To understand the molecular regulation of how 14-3-3ε induces EMT and reduces
E-cadherin expression in HCC, we examined the expression levels of distinct
E-box transcriptional suppressors. We found that 14-3-3ε overexpression
selectively induced Zeb-1 and Snail expression but had no significant effect on
Zeb-2, Twist or Slug. The induced expression of Zeb-1 and Snail by 14-3-3ε was
further confirmed by quantitative real-time PCR analysis. In addition, results
of transient transfection indicated that overexpression of 14-3-3ε dose-
dependently induced Zeb-1/Snail and reduced E-cadherin expression in Huh-7 and
HepG2 cells. Furthermore, 14-3-3ε-induced expression of Zeb-1 and Snail was
abrogated by knockdown of 14-3-3ε with siRNA. These findings suggest that Zeb-1
and Snail may be involved in 14-3-3ε-induced HCC cell migration and EMT. We next
determined the role of 14-3-3ε-induced Zeb-1 and Snail on cell migration. We
found that knockdown of either Zeb-1 or Snail expression by siRNA significantly
abolished 14-3-3ε induced cell migration. These results indicate that Zeb-1 and
Snail play important roles in regulating 14-3-3ε-induced HCC cell migration.
## 14-3-3ε Suppresses E-cadherin Expression Selectively Mediated by Induction of Zeb-1
To further explore the role of Zeb-1 and Snail on 14-3-3ε-induced cell migration
or EMT, we knockdown Zeb-1 and Snail with siRNAs and examined E-cadherin
expression by Western blotting analysis. Interestingly, 14-3-3ε-reduced
E-cadherin expression was specifically restored by Zeb-1 siRNA, but not by Snail
siRNA. This specific effect of Zeb-1 on regulating 14-3-3ε-reduced E-cadherin
was validated by quantitative real-time PCR. In addition, the selective effect
of Zeb-1 knockdown restoring E-cadherin expression in 14-3-3ε overexpression
cells was further confirmed by confocal microscopy. SK-Hep1 cells expressed
higher levels of 14-3-3ε and lower levels of E-cadherin than other HCC cell
lines. We next transfected SK-Hep1 cells with 14-3-3ε siRNA and determined the
expression of Zeb-1, Snail and E-cadherin by Western blotting analysis.
Knockdown of 14-3-3ε reduced the expression of Zeb-1/Snail and induced that of
E-cadherin in SK-Hep1 cells with a concentration-dependent manner. These results
demonstrate that the reduction of E-cadherin expression by 14-3-3ε is
selectively mediated by regulation of Zeb-1. Thus, Zeb-1/E-cadherin expression
is a downstream factor of 14-3-3ε for promoting EMT in HCC.
## Correlation and Impact of Positive 14-3-3ε with Negative E-cadherin Expression in HCC
To further support the likelihood that 14-3-3ε suppresses E-cadherin and
regulates EMT as well as tumor progression, we examined the expression of
14-3-3ε and E-cadherin by immunohistochemical analysis in HCC tumors. Expression
of 14-3-3ε was higher in HCC primary tumors than in the surrounding non-
cancerous liver tissues. We next determined the expression of E-cadherin and
found that E-cadherin expression was reduced in HCC tumors. Positive 14-3-3ε
expression was significantly correlated with negative E-cadherin in HCC tumors
(*p* = 0.043). In addition, expression of 14-3-3ε was correlated with Zeb-1 in
HCC tumors.
## Association of 14-3-3ε/E-cadherin Expression with Extrahepatic Metastasis and Patient Survival of HCC
We have previously shown that 14-3-3ε overexpression in HCC primary tumors was
significantly associated with subsequent extrahepatic metastasis and reduced
5-year overall survival. To evaluate whether E-cadherin plays an important role
as a downstream effector of 14-3-3ε in promoting tumor progression, the
associations of E-cadherin with clinicopathological characteristics and with
14-3-3ε expression were compared. In addition to 14-3-3ε positivity, expression
of E-cadherin is significantly correlated with gender (*p* = 0.011), histology
grade (*p* = 0.001), BCLC staging (*p* = 0.030), tumor size (*p* = 0.003), and
subsequent extrahepatic metastasis (*p* = 0.004). Patients with positive
E-cadherin expression exhibit a lower risk of metastasis (, *p* = 0.013) and
better overall survival rate (*p* = 0.047) than do those with negative
E-cadherin expression in 14-3-3ε positive HCC tumors. These results provide
clinical evidence to support the hypothesis that E-cadherin is one of the
crucial downstream regulators of 14-3-3ε that modulate HCC tumor progression.
# Discussion
We previously demonstrated that 14-3-3ε expression is increased in primary and
metastatic HCC. Elevated 14-3-3ε expression is correlated with higher risk of
extrahepatic metastasis and lower survival rates of HCC patients. In this study,
we investigated the molecular mechanism to determine how 14-3-3ε regulates tumor
progression. Attenuated expression of E-cadherin has been recognized as an
important determinant and biomarker of tumor progression, one especially
indicative of EMT in various tumors. In addition, gene silencing and loss of
E-cadherin expression in the malignant progression of HCC have been
demonstrated, , and it is suggested that E-cadherin is associated with reduced
survival of HCC patients. Our current investigation indicates that 14-3-3ε
promotes HCC EMT and cell migration and also suppresses E-cadherin expression
*via* upregulation of Zeb-1. We found that the expression of Zeb-1 was increased
(14 of 113) in HCC primary tissues, although the increase is not as significant
as in a previous report. This difference may due to the sensitivity of reagents,
sample size or differences in the cohort. It has been suggested that TGF-β and
downstream signals of Smad2/3 activation regulate Zeb expression and EMT.
Although we performed the experiments of Smad2 knockdown in 14-3-3ε
overexpression cells, we did not observe significant restoration of E-cadherin.
In addition, increased 14-3-3ζ expression has been shown to promote EMT *via*
associating with TGF-β receptor signaling and PI3-K subunit p85 in breast cancer
cells. However, treating cells with TGF-β receptor or PI3-K inhibitors
(SB-431542 or LY-294002) did not abolish E-cadherin-suppression induced by
14-3-3ε. These results suggest the effect of 14-3-3ε-suppressed E-cadherin
expression may not be regulated through TGF-β/Smad2/3 or PI3-K signal pathways.
Thus, 14-3-3ε contributes to EMT via induction of Zeb-1 may be mediated by a
novel mechanism. Further work is currently ongoing to investigate how 14-3-3ε
regulates Zeb-1 expression.
A functional motif for 14-3-3 binding in Snail was demonstrated, and the ternary
protein complex comprised of 14-3-3, Ajuba and Snail plays an important role in
transcriptional repression and EMT. Snail contains two potential phosphorylated
residues at Ser11 and Thr177 in putative motifs for 14-3-3 protein binding.
Phosphorylated Snail selectively interacts with 14-3-3γ, 14-3-3ε, 14-3-3θ/τ,
14-3-3η and 14-3-3β, but not with isoforms of 14-3-3σ and 14-3-3ζ. This
interaction has been demonstrated to be required for E-cadherin suppression in
MCF breast cancer cells and 293 cells. To find out whether 14-3-3ε suppresses
E-cadherin *via* a similar mechanism, we co-immunoprecipitated 14-3-3ε stable
cells with anti-Flag antibody, followed by Western blotting analysis of Zeb-1.
However, no significant interaction of 14-3-3ε with Zeb-1 was detected. Further
studies using proteomic approaches are currently underway to investigate the
potential 14-3-3ε interaction partners in regulating Zeb-1 and EMT of HCC.
Expression of transcriptional repressors for E-cadherin, including Zeb-1, Snail,
SIP1 and Twist, is associated with cell invasion, EMT, metastasis and poor
patient survival of HCC. However, no previous studies have shown that 14-3-3ε
promotes HCC tumor progression *via* modulating E-cadherin transcriptional
repressors. Our study shows for the first time that 14-3-3ε induces Zeb-1
expression, thereby repressing E-cadherin expression and promoting EMT. The
14-3-3ε regulation of E-cadherin reduction occurs through Zeb-1, and not through
Snail or other E-cadherin repressors, as supported by. To further clarify the
regulation of 14-3-3ε-reduced E-cadherin expression by Zeb-1, 14-3-3ε
overexpression cells were transfected with Zeb-1 siRNA or control scramble
siRNA, and the gene expression profile was analyzed by use of microarray
analysis. Altered gene expression (fold change \>2) was identified of 557
transcripts in 14-3-3ε overexpression *vs.* the control cells and 160
transcripts in Zeb-1 siRNA *vs.* scramble siRNA cells. Among them, *CDH1*
(E-cadherin), *SMAD2*, and *PLA2G2A* were regulated in 14-3-3ε overexpression
cells but had a reversed expression pattern in Zeb-1 knockdown cells (Data not
shown). These results provide additional evidence to support our findings.
In addition to Zeb-1, our results indicated that 14-3-3ε induces Snail
expression and promotes HCC cell migration. However, knockdown of Snail did not
restore 14-3-3ε-reduced E-cadherin expression. Interestingly, partially
increased of Snail expression was found by treatment with Zeb-1 siRNA. As Snail
and Zeb-1 regulate EMT of HCC may be mediated by separate and complicated
pathways, a compensative effect is possibly involved. Further investigation is
needed to elucidate this finding. Additionally, our results indicated that
14-3-3ε overexpression-induced EMT (increase of N-cadherin, Vimentin, Zeb-1 and
Snail as well as decrease of E-cadherin expression) was impaired by 14-3-3ε
siRNA. However, knockdown of 14-3-3ε has no significant effect on affecting EMT
markers in control cells. We therefore postulate that other endogenous house-
keeping regulators may be involved in maintaining basal level of Snail/Zeb-1
expression. Endogenous level of Snail/Zeb-1 modulates expression of EMT markers
which is independent of 14-3-3ε expression in HCC. Moreover, 14-3-3ε may
upregulate FAK expression *via* activation of NFκB to enhance HCC cell
migration. These results reveal the complicated signal mechanisms that are
involved in 14-3-3ε induced HCC cell migration, EMT, and metastasis. Uncovering
the complex role of 14-3-3ε in tumor progression could contribute to the
development of therapeutic strategies for treatment of aggressive and advanced
HCC.
# Supporting Information
We thank the Comprehensive Cancer Center of Taichung Veterans General Hospital
for providing information concerning the outcomes of patients. We also thank the
core laboratory of National Health Research Institutes for the helpful
assistance.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JYL YJJ BSK TAL. Performed the
experiments: TAL YJJ BSK SML SCC. Analyzed the data: JYL YJJ BSK TAL YMW CH
JW. Wrote the paper: JYL. |
# Introduction
Tuberculosis (TB) continues to be a major global health problem including in
Saudi Arabia. High proportion of immigrants living in the country and annual
massive influx of pilgrims mostly from TB endemic areas constitute favorable
conditions for TB transmission. One of the most important challenges in global
TB control is the early detection and treatment of latent tuberculosis infection
(LTBI). About 10% of individuals infected with *M*. *tuberculosis* develop
pulmonary TB, and the remaining 90% suppress the bacterial invasion through
their immune systems and persist with LTBI. Unfortunately there is very limited
data on the prevalence of LTBI among health care workers in Saudi Arabia, except
reports from few institutions.
Health care workers (HCW’s) in general are considered as a high-risk group of
LTBI because of the increased risk of exposure. A recent systematic review
showed, among HCW’s of low and middle income countries, the LTBI prevalence is
ranged between 33–79% with a median of 5.8% annual LTBI incidence. Usually,
HCW’s may come into contact with patients with undiagnosed cases or
unknown/unsuspected cases of active TB, which elevates the acquisition of LTBI.
HCW’s employed in sections like emergency, intensive care, internal medicine,
radiology, are at greater risk of acquiring *M*. *tuberculosis*. Due to the
epidemiological evidence of TB as a consequential occupational disease, HCW’s
with negative TST results must undergo annual LTBI screening.
Surveillance study on LTBI among HCW’s vaccinated with BCG has been hampered by
the non- specificity of TST. False-positive results secondary to cross-reactions
caused by BCG vaccination and/or exposure to non tuberculous mycobacteria,
booster phenomenon and technical issues, like interpretation of the result may
all lead to unnecessary treatment of presumed LTBI thereby, rendering TST
unsuitable as a surveillance tool in TB risk groups. The advent of Interferon
Gamma Release Assays (IGRA) and their increasing availability show promise for
more accurate LTBI detection in HCW’s. The higher specificity of QuantiFERON TB
gold assay (QFT) compared with TST can reduce unnecessary treatment, follow-ups
and thereby, treatment cost of LTBI.
Most IGRA studies have been done in low TB-endemic regions whereas; data from
low to middle-income settings, with high background of TB infection rates have
been fairly scarce. We are not aware of any report describing the use of IGRA’s
among HCWs in Saudi Arabia or in Gulf Cooperation Council (GCC) countries.
In Saudi Arabia, TB screening is recommended for all HCW’s. Annual LTBI
screening is mandatory for all employees at King Faisal Specialist Hospital and
Research Centre (KFSHRC) as part of their job contract renewals. The TST is the
currently following LTBI diagnostic method at KFSHRC. However due to the
limitations of the TST and to promote a more sensitive technique, IGRA was
considered. The cosmopolitan nature of the employee population (citizens of more
than 60 countries) has not been considered for optimizing the LTBI diagnosis.
Moreover, the majority of the employee populations are from countries where BCG
vaccination is mandatory. In addition, there was an increasing prevalence of NTM
diseases also reported in the country recently. Thus a new LTBI diagnostic test
was badly needed for the institution.
Therefore, a cross sectional study has been designed with two major set
objectives. The key objectives were to analyze the prevalence of LTBI among
HCW’s of highly diverse origin at KFSHRC and to compare the feasibility of using
TST and QFT to screen the LTBI among this diverse population.
# Materials and Methods
This study was carried out for a period of 36 month (February 2012 to January
2015) in KFSHRC, Riyadh. The protocol has been reviewed and approved by the
Research Advisory Council (RAC) of KFSHRC. All the candidates enrolled were
subjected to a short interview and completed the consent form before withdrawing
the blood samples.
## Study population
The inclusion criteria for study subjects was any new (undergoing pre-employment
checkup) or existing employee (annual re-contracting checkup), who can consent
for the study. The exclusion criteria included candidates with previous history
of active TB or undergoing TB medication. The study subjects were recruited from
both clinical and non-clinical health care workers designated in various
departments at KFSHRC. Clinical health-care workers (those included in any
direct patient contact or giving direct care) are doctors, nurses and allied
health professionals. Non-clinical health-care workers included those who did
not involve in direct patient care, such as administrative staffs, researchers,
housekeepers, and hospital technical maintenance staffs.
According to the job profile of the candidate, the degree of TB exposure level
was defined into low, medium and high. Doctors, nurses and allied health
professionals were classified into high risk group. The medium risk group
consisted of candidates who are not directly giving patient care but in contact
with patient samples (ex; medical lab technicians) or technicians managing
respiratory units or equipments. The low risk group included researchers,
maintenance staffs and administrative staffs respectively.
A standard questionnaire was used to collect the information on key variables,
such as age, gender, nationality, previous exposure or treatment details of TB,
previous chest x-ray details, BCG vaccination and BCG scar, prior TST (date and
result), job category, service and years in the health profession. The
questionnaire has been completed by face to face interview and reference to
medical records.
All the TST positive converted staffs were offered with a follow up of Chest
X-ray and a 9 months isoniazid treatment according to the standard guidelines.
The decision of treatment was kept optional to the employee after the discussion
with the physician. However a continuous follow-up was mandatory.
## Performing TST and QFT
All new HCW’s, who did not have a documented TST result, were subjected to TST
during the routine examination at the time of employment in the family medicine
department of KFSHRC. TST was performed by administrating 2-TU of PPD RT23
(Staten’s Serum Institute, Copenhagen, Denmark). Induration was measured 48–72 h
after the application. Well trained nurses performed and interpreted the results
in 48–72 hours according to the American Thoracic Society (ATS) and Centers for
Disease Control and Prevention guidelines. The positive interpretation followed
was a TST with an area of induration ≥10 mm. If HCW’s had a previous positive
TST, we took note of the place and the year. If HCW’s had negative TST, it is
repeated annually or after an exposure as part of KFSHRC’s infection control
practice.
The QFT was carried out by using the commercially available QuantiFERON TB gold
In-Tube Assay (Cellestis, Australia) according to the manufacturer’s protocol.
The raw optical density was measured and interpreted with the software
QuantiFERON TB Gold analysis Software v1.51 (Cellestis, Australia).
### Statistical analysis
The candidates were classified according to the WHO’s geographical
classification into 6 major groups; American, African, European, Eastern
Mediterranean, South East Asian, and Western Pacific. The statistical analysis
has been carried out by using the SPSS v-20 software package. A chi squared test
was utilized for categorical data. All the putative predictive variables were
subjected to calculate the odds ratio and 95% confidence interval.
# Results
Overall 1603 candidates were enrolled during the study period. Of the total, 8
HCWs (1.2%) were excluded from the analysis due to indeterminate QFT results and
1595 were included for final analysis. The study population included candidates
from 33 countries. The native Saudi population was only 17%. The geographical
classification on enrolled candidates showed, 52.4% were from Western-Pacific
followed by 22.9% Eastern Mediterranean and 11.3% South East Asians. The median
of age in the study was 35.5 years with a predominance of female candidates
(67.9%). Clinical staffs represented the major study group by71.5% and among
them nurses were predominant (53.2%). Overall, 185(11.6%) candidates reported
with a previous exposure to known pulmonary TB cases. Majority of the candidates
(90.6%) received at least one shot of BCG vaccination before enrollment.
Candidates holding a healthcare experience of 2–5 years were comparatively more
(37.6%), though the mean duration of experience in the study was 8.4 years.
Positive TST was noticed in 31.5% of the enrolled candidates. Among the TST
positive cases 45.2% were QFT positive while 54.8% were negative. On the other
hand, among TST negative cases, 15.7% were QFT positive. The disagreements
between the two tests were relatively high (kappa co-efficient-0.312±0.026, p
value- \<0.00001). Among 170 TST converted participants only 46 opted for INH
prophylaxis and none of these cases progressed to active TB during the study
period. The regression analysis of the putative predictable factors showed a
significant association of younger age groups (*p* -0.030, 0.049), BCG
vaccination (*p*- 0.028), European (*p* -\<0.001) and south East Asian
(*p*-0.009) origin with TST positivity. QFT positivity was associated with
profession of candidates and occupational TB exposure risk. In addition, the
origin of the candidates (*p*-\<0.001) was highly significant in QFT positivity.
Analysis of agreement of results of TST and QFT showed 14.23% concordant
positive results while 57.7% is concordant negative. However, 10.8% cases were
TST negative and QFT positive. On the other hand 275(17.2%) cases were TST
positive and QFT negative. Analyzing the association of various putative factors
showed the origin of the patient (*p* \<0.001), occupational TB exposure level
(*p*-\<0.001), profession (*p-* 0.001) and BCG vaccination history (*p-* 0.001)
have a statistical significance on the agreement of both the tests. Among the
discordant results, candidates with Western Pacific origin showed a predominant
QFT positivity (65.7%) and negativity (46.9%). Interestingly, among the
different professions, nurses have the highly discordant results.
# Discussion
This is the largest Saudi Arabian study, which evaluated the performance of QFT
and TST among highly diverse HCW’s population. The study has targeted onto the
putative risk factors with an emphasis on the impact of geographic origin of
study subjects. It is also the only cross sectional study in the country that
looked at the possibilities of developing active TB in recently identified LTBI
in HCW’s. This study has been carried out in a tertiary care center which has
been listed among the top five medical facilities in the country employing
staffs from more than 60 countries. The impact of this huge diversity in the
origin of the staffs has been highly considered on the LTBI prevalence, as it
influence the agreement between TST and IGRA. The overall agreement between TST
and QFT was 72%, that can be considered as fair, when many previous
international studies reported moderate or fair agreement only between both the
tests. Supportively, recent studies from Saudi Arabia on dialysis patients also
showed 75.5% and 90.9% agreement between TST and QFT/TSPOT Assay respectively.
The prevalence of LTBI measured in this study by TST (31.5%) and QFT (25.0%) was
relatively high. This elevated rate may be typical for a moderate TB burden
country like Saudi Arabia. Furthermore, the previous estimates showed that,
Saudi Arabia has only an intermediate prevalence of LTBI (2–14%). In Saudi
Arabia, only limited information is available on prevalence of LTBI among HCW’s
particularly screened with IGRA testing. A multicenter study utilizing TST alone
in HCW’s showed 11% LTBI prevalence, while the current study showed 31.5%.
However, the elevated prevalence level of LTBI in the current study may largely
depend on the origin of the candidates, that particularly from TB endemic
regions.
The current study population consisted only 17% Saudi nationals. The results of
TST and QFT stratified by the Saudi and non-Saudi origin showed a prevalence of
18.1% and 17.3% among Saudis. This finding highlights the massive role of
immigrant HCW’s in the institution and mostly their origin from TB endemic
countries. After applying the geographical region classification of WHO into the
study, showed the Western Pacific origin candidates (mainly Philippines) as the
largest group in the study followed by the Eastern Mediterranean and south East
Asians. Interestingly, there are 970(60.8%) candidates truly from high TB burden
countries namely, Philippines, India, Bangladesh, Pakistan, Sudan, South Africa,
Eritrea, Ethiopia and Kenya. Analysis of the TST results against the nationality
of the candidates, showed 305(60.6%) of the total 503 TST positive and 234
(58.6%) of the total 399 QFT positive candidates are from this group. The
agreement between two results was really narrow (only 2%). Among the candidates
from high TB burden countries only 94(9.7%) had TST conversion during their
employment in the study center. This speculates the scope of a remote infection
among those remaining 876(90.3%) candidates from their previous destination that
is mostly their mother country. This finding has a support from molecular
studies by Varghese et al., which showed remote TB infection among immigrants is
very common Saudi Arabia.
In the current study the maximum discordance was observed in TST positive and
QFT negative results. Among the TST positive cases 54.7% were negative for QFT.
This finding corroborates with published studies which reported positive TST and
negative IGRA is the most common discordance. Majority of the study candidates
were from a country where BCG vaccination is mandatory, thus 90.6% of the
enrolled candidates were BCG vaccinated. This could probably affect the higher
level prevalence of TST positivity. Supportively, a statistically significant
association (OR 1.039, 95% CI-0.664–1.625, P value 0.028) was noticed between
BCG vaccination and TST positivity while no significant association with QFT.
This finding is in concordance with previous studies, which showed BCG
vaccination significantly elevates the likelihood of TST positivity. When
compared with age \>60 years, the younger age has significant association with
TST positivity in concordance with various previous studies. The rate of TST
positivity among different professionals showed no significant association. In
contrast, the TB exposure level based on profession showed a high significance
towards QFT positivity, when low risk group was kept as a reference as seen in
previous studies. The origin of the candidates also has significant association
with both TST and QFT positivity.
The maximum discordant results were noticed among candidates from Western
Pacific region. Overall 65.7% of the TST negative QFT positive candidates and
46.9% of TST positive and QFT negative candidates were from mainly Philippines
and Malaysia, while candidates from Eastern Mediterranean region showed the
rates of 17.4% and 30.9% respectively. The massive enrollment of Filipino
candidates (45.2%) has an impact on the overall findings. The two main reasons
are the mandatory BCG vaccine administration and high TB burden of Philippines.
Perhaps, the higher discordant results of TST negative and QFT positive could
not be explained in detail. No significant association could be established
between the discordant results against age of the candidates, gender, and years
of work in the medical field. However, strong associations could be noticed on
origin of the candidates (*p* \<0.001), profession (*p* 0.001), TB exposure
level (*p* \<0.001) and BCG vaccination (*p* 0.001). Nurses and support service
staffs showed majority of the discordant results. This finding was supported by
the rate of TB exposure level as the high TB exposure group has highest
discordance between results. This study has certain limitations; candidates
reported as TST negative and QFT positive were not retested for confirmation and
quantitative analysis of QFT testing was not considered.
# Conclusion
In conclusion, the prevalence of LTBI estimated by QFT is high among Saudi
Arabian HCW’s. The disagreements between TST and IGRA results were relatively
high and thus QFT alone cannot be recommended to screen LTBI. The origin of the
candidates has significant role in both TST and IGRA positivity. However due to
the high level of LTBI prevalence, the screening and management of LTBI in HCW’s
in the country must be immediately streamlined.
# Supporting Information
We deeply acknowledge, Maribel Carbonel, Diana Oliveira, Florence Saguid, Lenora
Reyes, Tracy Lynn Alsarhani, Rania Omar Albadawi and Maria Minette Valesteros
for their immense support for recruiting the candidates.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: SAH A. Alrajhi A. Alkhenizan.
Performed the experiments: BV AD GF. Analyzed the data: BV MS. Contributed
reagents/materials/analysis tools: SA A. AlSaif A. Alzahrani. Wrote the
paper: SAH BV MS A. Alzahrani A. Alrajhi GF. |
# Introduction
Sleep plays an important role in maintaining health. Sleep disorders have been
shown to negatively affect lifestyle-related diseases, such as metabolic
syndrome, hypertension, diabetes, and cardiovascular disease. The following
underlying mechanisms have been suggested for this association: neurobiological
and physiological stressors; the inhibition of glycemic control, which increases
dietary intake through the secretion of ghrelin; and the promotion of insulin
resistance by increasing cortisol, IL-6, and TNFα levels. Therefore, preventing
sleep disorders is important for maintaining health.
Exercise has been noted to have a positive impact on sleep disorders.
Epidemiological studies on sleep disorders and exercise/physical activity have
examined non-restorative sleep in middle-aged and elderly individuals and sleep
quality in nursing homes. Intervention studies have reported improvements in
sleep quality in adults and elderly individuals with depression. Exercise
affects sleep via the following mechanisms: its thermoregulatory effects reduce
wake times during the night, it facilitates sleep onset by activating a heat
dissipation mechanism controlled by the hypothalamus to increase central body
temperature, and it improves mood due to its antidepressant/anxiety effects. In
animal studies, exercise was shown to increase the levels of adenosine, which
activates the sleep center in the hypothalamus, and serotonin, which synthesizes
the sleep hormone melatonin.
Nutrition has been investigated as another factor related to sleep disorders.
Epidemiological studies have reported a relationship between sleep quality and
micronutrients such as carotenoids, vitamin B<sub>12</sub>, calcium, and
selenium. However, the relationships between sleep and macronutrients remain
unclear. A previous study indicated the presence of a relationship between sleep
and protein intake, but another study reported that no such relationship
existed. The latter study demonstrated that sleep was associated with lipid and
carbohydrate intake. Thus, the findings obtained from previous epidemiological
studies are inconsistent. These discrepancies may exist because of the lack of a
uniform method for evaluations using questionnaires. Therefore, we conducted an
epidemiological study to investigate the factors affecting sleep using the
Pittsburgh Sleep Quality Index (PSQI), which is one of the most frequently used
indices for evaluating self-rated sleep quality in sleep medicine.
The role of environmental factors must be considering when examining the
association between nutrient intake and sleep. However, previous studies have
not yet investigated this triangular relationship in detail. Two previous
studies that reported different findings on the relationship between protein
intake and sleep did not perform an analysis adjusted for environmental factors
such as exercise. Therefore, the effects of interactions between environmental
factors and nutrient intake on sleep remain unclear. The present cross-sectional
study examined the effects of regular exercise and nutrient intake on sleep
quality.
# Methods
## Data collection
In this cross-sectional study, comprehensive health survey data were collected
from the residents of Shika Town, Ishikawa Prefecture, Japan, between November
2017 and February 2018. As of November 2017, there were 21,007 residents in
Shika Town, and 15,012 were older than 40 years. The Shika study
epidemiologically investigates the causes of lifestyle-related diseases through
interviews, self-administered questionnaires, and comprehensive medical
examinations. Previous studies have also examined the relationship between
nutrition and health.
## Participants
This study was conducted on participants recruited from those who underwent a
medical examination in Shika Town. For details, a total of 378 people aged 40
years and older who live in four model districts (Horimatsu, Tsuchida,
Higashimatsudo, and Togi) provided their consent to participate in this sleep
study. Of these individuals, 193 were excluded because they did not meet the
survey criteria \[169 participants did not complete the brief-type self-
administered diet history questionnaire (BDHQ), 1 participant did not have
energy records within 600 – 4000Kcal/day, and 23 participants did not complete
the smoking, drinking, or exercise questionnaire\]. shows the inclusion
criteria. In total, 185 participants (95 males and 90 females; mean age ±
standard deviation: 60.5 ± 9.7 years, range 41–83 years) who answered all
relevant questions in the questionnaires and did not withdraw their consent were
included in the analysis.
## Questionnaire and measurements
The participants completed a self-administered questionnaire on lifestyle and
underlying diseases. Lifestyle items included the number of exercise days per
week and the mean exercise time during each session, whether they were current
smokers (1. yes, 2. no) and/or current drinkers (1. yes, 2. no), and education
(1. junior high school, 2. high school 3. junior college, 4. university or
higher). Underlying disease items included metabolic syndrome (1. yes, 2. no),
hypertension (1. yes, 2. no), diabetes (1. yes, 2. no), angina (1. yes, 2. no),
myocardial infarction (1. yes, 2. no), and depression (1. yes, 2. no). Body mass
index (BMI) was measured using health survey data from the Shika study.
Nutrient intake was assessed using the BDHQ. The BDHQ is a four-page structured
questionnaire that assesses the consumption frequency of 58 foods and beverages
that are commonly consumed by the general Japanese population. The BDHQ
estimates dietary intake in the last month using an *ad hoc* computer algorithm.
The validity of the BDHQ has been demonstrated in previous studies in Japanese
populations. To analyze nutrient data, the density method was used to estimate
intake per 1000 Kcal. The following formula was used to calculate the energy
intake ratio (% energy) of energy-producing nutrients (proteins, lipids,
carbohydrates, and alcohol): energy intake from each nutrient/energy intake (EN)
× 100, adjusted intake of non-energy-producing nutrients: crude intake of
various nutrients/EN × 1000 Kcal.
Sleep status was assessed using the PSQI. The PSQI assesses sleep quality and
disturbances over a one-month period and consists of the following components:
subjective sleep quality, sleep latency, sleep duration, habitual sleep
efficiency, sleep disturbance, the use of sleeping medication, and daytime
dysfunction. Each component of the PSQI is scored from 0 to 3. The PSQI global
score is a sum of these components that ranges between 0 and 21, with higher
scores indicating poorer sleep quality. The validity of the PSQI has been
demonstrated in previous studies in Japanese populations.
## Statistical analysis
Regular exercise was defined as exercise for at least 30 minutes at a time and
twice a week. Buysse *et al*. defined a PSQI score \>5 as poor sleep quality;
however, their study population included adolescents and differed from that in
our Shika study, which included those aged 40 years and older. In the present
study, the median PSQI of the participants was 10; thus, they were classified
into PSQI ≤10 and ≥11 groups. The age distribution of the participants in the
present study did not differ from that of the Shika Town inhabitants.
The distribution of variables was checked by the Kolmogorov–Smirnov, and
Shapiro–Wilk normality tests, or the normal distribution curve in the histogram
was confirmed before using other statistical tests. The Student’s *t*-test was
used to compare the means of continuous variables and the Chi-square test was
performed to compare the proportions of categorical variables. All participants
were stratified into two groups based on their PSQI scores (PSQI ≤10 and ≥11)
and whether they participated in regular exercise (regular exercise group and
non-regular exercise group). A two-way analysis of covariance (ANCOVA) was used
to examine the effects of the interaction between regular exercise and PSQI on
nutrient intake. The following confounding factors were adjusted for: age, sex,
BMI, current smoker, current drinker, education, hypertension, and diabetes. A
multiple logistic regression analysis was conducted to examine the effects of
regular exercise and nutrient intake on sleep quality. The dependent variable
was the PSQI (≤10 and ≥11). We used three models in the logistic regression
analysis. Model 1 included the individual factors of age, gender, and BMI. Model
2 included the environmental factors of current smoker and current drinker
together with individual factors. Model 3 included the disease factors of
hypertension and diabetes together with individual factors. Additionally, the
analyses were stratified by whether the participants performed regular exercise.
Pearson’s correlation coefficient was used to confirm multicollinearity.
Specifically, there was no value of \| r \| \>0.9 in the correlation matrix
table between independent variables. The forced input method was used for
variable selection. The significance level was set at 5%. IBM SPSS Statistics
version 25 for Windows (IBM, Armonk, NY, USA) was used for the statistical
analysis.
## Ethics statement
The present study was conducted with the approval of the Ethics Committee of
Kanazawa University (No. 1491). Written informed consent was obtained from all
participants.
# Results
## Participant characteristics
The participants’ sleep quality and nutrient intakes are shown in. Among the 185
participants, there were 95 males with a mean age of 60.9 years (*SD* = 9.6) and
90 females with a mean age of 60.3 years (*SD* = 10.1); there was no significant
difference between genders. BMI (*p* \< 0.001) was significantly higher in males
than in females. Significantly more males were current smokers (*p* \< 0.000),
current drinkers (*p* \< 0.001), and had metabolic syndrome (*p* \< 0.001),
diabetes (*p* = 0.049), and angina (*p* = 0.019) than females. The mean PSQI was
10.7 (*SD* = 2.7) in males and 10.7 (*SD* = 2.8) in females, with no significant
difference between genders. When comparing nutrients, the total energy (*p* \<
0.001) was significantly higher in males. Conversely, the intakes of other
nutrients, excluding carbohydrates, sodium, vitamin D, and vitamin
B<sub>12</sub>, were significantly higher in females.
## Comparison with the PSQI
The mean age of the 106 participants in the PSQI ≤10 group was 59.2 years, which
was significantly younger than that of the 79 participants in the PSQI ≥11group
(62.4 years, *p* = 0.026). The PSQI ≤10 group reported significantly more
exercise days per week (*p* = 0.004) and a significantly longer mean exercise
time per session (*p* = 0.008). Furthermore, these factors were significantly
greater in the PSQI ≤10 group even after adjusting for age, sex, BMI, current
smoker, current drinker, education, hypertension, and diabetes (exercise days
and exercise time: *p* \< 0.001 and *p* = 0.004, respectively). Therefore,
regular exercise was beneficial for sleep quality. The proportion of
participants with metabolic syndrome (*p* = 0.026) was significantly higher in
the PSQI ≤10 group. When comparing each nutrient, the intakes of retinol
equivalent (*p* = 0.044) and vitamin B<sub>2</sub> (*p* = 0.024) were
significantly higher in the PSQI ≤10 group than in the PSQI ≥11 group.
## Comparisons with regular exercise
The mean age of the 59 participants in the regular exercise group (64.2 years)
was significantly older than that of the 126 participants in the non-regular
exercise group (58.8 years, *p* \< 0.001). The mean PSQI (*p* = 0.003) was
significantly higher in the non-regular exercise group. When comparing each
nutrient, the intakes of protein (p \<0.001), minerals (p \< 0.001), and 12
kinds of vitamins were significantly higher in the regular exercise group than
that in the non-regular exercise group.
## Effects of the interaction between regular exercise and the PSQI on nutrient intake
The regular exercise group was divided into two groups based on PSQI scores;
there were 43 participants in the PSQI ≤10 group and 16 in the PSQI ≥11 group.
The non-regular exercise group was similarly divided into two groups based on
the PSQI scores; there were 63 participants in the PSQI ≤10 group and 63 in the
PSQI ≥11 group. A two-way ANCOVA adjusting for age, sex, BMI, current smoker,
current drinker, education, hypertension, and diabetes was used to examine the
effects of interactions between regular exercise and the PSQI on nutrient
intake. Interactions were observed for age (*p* = 0.006), education (*p* =
0.002), protein (*p* = 0.002), carbohydrate (*p* = 0.045), phosphorus (*p* =
0.008), zinc (*p* = 0.031), vitamin D (*p* = 0.015), vitamin B<sub>12</sub> (*p*
= 0.007), and pantothenic acid (*p* = 0.008). A post hoc Bonferroni analysis
indicated that there was significantly higher protein intake in the PSQI ≤10
group than in the PSQI ≥11 group with regular exercise (*p* = 0.001); however,
there was no difference between the two PSQI groups without regular exercise.
## Effects of regular exercise and protein intake on sleep quality
shows the results of a multiple logistic regression analysis with PSQI (≤ 10 and
≥11) stratified by regular exercise. Protein intake was a significant
independent variable in any models that were adjusted for individual factors
(age, sex, and BMI; OR: 1.260; 95% CI: 1.037, 1.531; *p* = 0.020), individual
and environmental factors (current smoker and current drinker; OR: 1.357; 95%
CI: 1.081, 1.704; *p* = 0.009), and individual and disease factors (hypertension
and diabetes; OR: 1.675; 95% CI: 1.206, 2.326; *p* = 0.002) in the regular
exercise group but not in the non-regular exercise group. This result implies
that sleep quality is better with a high protein intake, even after adjusting
for different confounding factors only in the regular exercise group.
# Discussion
In the present study, the PSQI was selected as the most frequently used index
for sleep evaluation. Epidemiological studies on sleep have been performed using
sleep times and questionnaires. However, evaluating sleep by time alone lacks
objectivity because sleep times and measurement items differ among studies. For
example, one study considered the appropriate sleep time to be 7–8 hours, but
another considered it to be 7–9 hours; other studies have evaluated sleep-
related time based on sleep latency (difficulty falling asleep) or sleep
efficiency (maintaining sleep). Therefore, comprehensively evaluating sleep
quality using a questionnaire may provide more objective findings. A previous
study that compared the diagnostic screening characteristics of the Insomnia
Severity Index, the Athens Insomnia Scale, and the PSQI reported similar
sensitivities and specificities. Therefore, the PSQI in the present study was
confirmed to be a valid and comparable questionnaire to those used in other
studies. Buysse *et al*. defined a PSQI score \>5 as poor sleep quality.
Conversely, Das *et al*. demonstrated that the mean PSQI was 8.59 ± 5.35 in a
community-based study among a geriatric population, and they described that the
difference in the PSQI may be due to the different cultures and lifestyles of
people in different countries. The mean PSQI of all the participants in the
present study was 10.7 ± 2.7, which seemed to reflect the current average
Japanese lifestyle.
Comparisons between the PSQI ≤10 and ≥11 groups in the present study revealed
that regular exercise was beneficial for sleep quality, which is consistent with
previous findings. However, other studies did not observe a relationship between
exercise and sleep. Briefly, the lowest or highest levels of exercise were not
associated with sleep disorders, and the effects of short-term resistance
exercise on sleep were inconsistent. By contrast, previous studies reported a
positive relationship between exercise intensity and sleep with 30 minutes or
more of exercise each time, moderate to intense physical activity of 150 minutes
or more per week, or 500 to 1500 metabolic equivalents of task minutes/week of
physical activity. Exercise intensity in the regular exercise group in the
present study was considered intermediate because the mean days of exercise per
week was 4.3 (*SD* = 1.7) and the mean exercise time per session was 80.3
minutes (*SD* = 64.2). Accordingly, the relationship observed between exercise
intensity and sleep in the present study appears to support previous findings
showing that intermediate exercise intensity has a positive effect on sleep
quality.
The multiple logistic regression analysis in the present study revealed a
positive correlation between good sleep quality and protein intake only in the
regular exercise group. These results seem to indicate that there is a mechanism
by which regular exercise promotes protein absorption. In addition to exercise,
the ingestion of protein just before sleep has been reported to improve
nighttime protein synthesis by enhancing its digestion and absorption.
Tryptophan is a constituent amino acid of protein that competes with the other
larger neutral amino acids to gain access to the transport system to cross the
blood-brain barrier. Dietary carbohydrates pull larger amino acids into the
muscle tissue, allowing tryptophan to access the transport system, cross the
blood-brain barrier, and contribute to the synthesis of serotonin and melatonin.
Tryptophan has been shown to affect the serotonin-melatonin pathway because an
intraperitoneal injection in rats increased serotonin levels; likewise, its
administration to patients with moderate insomnia significantly reduced sleep
latency. The reason for good sleep quality among participants who reported high
protein intake in their daily diet and regular exercise was thought to be a
result of the pathogenesis in which the tryptophan-serotonin-melatonin pathway
was activated due to the enhanced protein absorption.
In the present study, the following micronutrients were associated with sleep
quality after adjusting for confounding factors (age, sex, BMI, current smoker,
current drinker, education, hypertension, and diabetes): phosphorus, zinc,
vitamin D, vitamin B<sub>12</sub>, and pantothenic acid. Many of these
micronutrients showed similar results as previous studies. Frank *et al*.
reported that lower intakes of phosphorus and zinc were associated with a
shorter sleep duration. Komada *et al*. reported that vitamin D and vitamin
B<sub>12</sub> in adult males were associated with sleep duration. Grandner *et
al*. reported that vitamin D was associated with sleep maintenance difficulties.
Since the results for many of the micronutrients examined in the present study
agreed with those of previous studies, the relationships observed between
micronutrient intakes and sleep in the present study were considered reliable.
One limitation of the present study is that the number of inputs for the
independent variables was restricted in the multivariate analysis because of the
small number of participants. This study might include selection bias because
the subjects participated voluntarily in this study. Since the PSQI was
evaluated only with a questionnaire, using a more objective method such as a
polysomnogram to assess sleep quality is necessary. Since we have not examined
the effects of obstructive sleep apnea, future studies should be performed that
incorporate a design to evaluate obstructive sleep apnea. Moreover, since this
was a cross-sectional study, interventions analyzing regular exercise and
protein intake could not be conducted. Further multicenter randomized controlled
trials with target values for exercise intensity and protein intake are
necessary to clarify the effects of regular exercise and nutrient intake on
sleep quality.
# Conclusions
We conducted this cross-sectional study on Japanese participants to investigate
the relationship between regular exercise and nutrient intake as factors
affecting sleep quality. Protein intake was higher among the participants with a
PSQI ≤10 in the regular exercise group (mean, 17.13% of energy consumption) than
in those in the non-regular exercise or PSQI ≥11 groups. Furthermore, the
results of the multiple logistic regression analysis showed that sleep quality
was better in the regular exercise group when protein intake was high; this
relationship was not observed in the non-regular exercise group.
# Supporting information
We would like to thank the officials of Shika Town, Ishikawa prefecture and the
staff of the Department of Environmental and Preventive Medicine, Kanazawa
University Graduate School of Medical Sciences, Kanazawa University Graduate
School of Advanced Preventive Medical Sciences, Department of Bioinformatics and
Genomics, University of Tsukuba and Keio University.
10.1371/journal.pone.0247926.r001
Decision Letter 0
Moran
Jose M.
Academic Editor
2021
Jose M. Moran
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
24 Nov 2020
PONE-D-20-33985
Protein intake in inhabitants with regular exercise is associated with sleep
quality: Result of the Shika study
PLOS ONE
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Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. Is the manuscript technically sound, and do the data support the
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The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
2\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
3\. Have the authors made all data underlying the findings in their manuscript
fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
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refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
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Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
4\. Is the manuscript presented in an intelligible fashion and written in
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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
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Reviewer \#1: Yes
Reviewer \#2: Yes
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5\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: I have read with great interest the study of Fumihiko Suzuki and
collaborators and I want to comment the following:
1\. I am struck by the division of two study groups based on a cut-off point of
10 in the PSQI without a clinical justification.
The original PSQI study concluded that a value greater than 5 indicates a
negative impact on sleep. This data is mentioned by the authors in the
methodology. However, it makes no mention of the Japanese validation study that
found similar data. That is, values \>5.5 are indicative of poor sleep quality.
By the way, in the study the control group had an average 38 years old and a
cut-off point of 7.5, a specificity of 97% was found for those patients with
primary insomnia. Then, an average of 10 points seems to be high and difficult
to be considered normal. (Doi Y, Minowa M, Uchiyama M, Okawa M, Kim K, Shibui K,
et al. Psychometric assessment of subjective sleep quality using the Japanese
version of the Pittsburgh Sleep Quality Index (PSQI-J) in psychiatric disordered
and control subjects. Psychiatry Res. 2000;97: 165–172.)
The authors could use the cutoff points that best fit their purposes, but as a
post-frame analysis. In strict adherence to the above, it would be desirable for
the researchers to add an analysis considering the cut-off point of\> 5.5 and
continue with the groups they consider.
2\. The discussion should mention the possible causes for which the average
population has poor sleep quality (mean PSQI of 10) and be careful to consider
the group with \<10 as having a good quality of sleep.
Reviewer \#2: the manuscript is well written with novel findings
comments:
methodology:
the author needs to define what is PSQI and BDHQ and did you use Japanese
version and if so, any validation study done
Results:
good analysis was done
Discussion:
PSQI has its own drawback for sleep quality assessment. furthermore, people with
metabolic syndrome are at greater risk of developing obstructive sleep apnea and
that may hinder their sleep quality
please add these comments in the discussion
\*\*\*\*\*\*\*\*\*\*
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Reviewer \#1: No
Reviewer \#2: No
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10.1371/journal.pone.0247926.r002
Author response to Decision Letter 0
14 Jan 2021
I want my identity to be published for this peer review.
10.1371/journal.pone.0247926.r003
Decision Letter 1
Moran
Jose M.
Academic Editor
2021
Jose M. Moran
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
11 Feb 2021
PONE-D-20-33985R1
Protein intake in inhabitants with regular exercise is associated with sleep
quality: Result of the Shika study
PLOS ONE
Dear Dr. Suzuki,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
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Although authors have appropriately addressed the requirements of the reviewers,
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included in their analysis the t-Student, ANCOVA, multiple linear regression and
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Jose M. Moran
Academic Editor
PLOS ONE
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Reviewers' comments:
Reviewer's Responses to Questions
**Comments to the Author**
1\. If the authors have adequately addressed your comments raised in a previous
round of review and you feel that this manuscript is now acceptable for
publication, you may indicate that here to bypass the “Comments to the Author”
section, enter your conflict of interest statement in the “Confidential to
Editor” section, and submit your "Accept" recommendation.
Reviewer \#1: All comments have been addressed
Reviewer \#2: All comments have been addressed
\*\*\*\*\*\*\*\*\*\*
2\. Is the manuscript technically sound, and do the data support the
conclusions?
The manuscript must describe a technically sound piece of scientific research
with data that supports the conclusions. Experiments must have been conducted
rigorously, with appropriate controls, replication, and sample sizes. The
conclusions must be drawn appropriately based on the data presented.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
3\. Has the statistical analysis been performed appropriately and rigorously?
Reviewer \#1: Yes
Reviewer \#2: I Don't Know
\*\*\*\*\*\*\*\*\*\*
4\. Have the authors made all data underlying the findings in their manuscript
fully available?
The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
manuscript fully available without restriction, with rare exception (please
refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
participant privacy or use of data from a third party—those must be specified.
Reviewer \#1: Yes
Reviewer \#2: Yes
\*\*\*\*\*\*\*\*\*\*
5\. Is the manuscript presented in an intelligible fashion and written in
standard English?
PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
articles must be clear, correct, and unambiguous. Any typographical or
grammatical errors should be corrected at revision, so please note any specific
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Reviewer \#1: Yes
Reviewer \#2: Yes
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6\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: The authors' response to the observation of the cut-off points is
valid because they are supported by a recent report on the geriatric population
and therefore responds favorably to the question about the high value of the
Pittsburgh sleep quality scale in the population of this study.
Reviewer \#2: minor linguistic errors,
i would suggest writing a review linking, sleep, exercise fitness and diet. the
focus on sleep not only from OSA prospective but rather descriping the link
between sleep duration, timing and physical fitness in addition to healthy diet
\*\*\*\*\*\*\*\*\*\*
7\. PLOS authors have the option to publish the peer review history of their
article ([what does this mean?](https://journals.plos.org/plosone/s/editorial-
and-peer-review-process#loc-peer-review-history)). If published, this will
include your full peer review and any attached files.
If you choose “no”, your identity will remain anonymous but your review may
still be made public.
**Do you want your identity to be public for this peer review?** For information
about this choice, including consent withdrawal, please see our [Privacy
Policy](https://www.plos.org/privacy-policy).
Reviewer \#1: No
Reviewer \#2: **Yes: **Mohammed A. Al-Abri
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Please note that Supporting Information files do not need this step.
10.1371/journal.pone.0247926.r004
Author response to Decision Letter 1
16 Feb 2021
Feb 16th, 2021.
Jose M. Moran, Ph. D
Academic Editor
PLOS ONE
Dear Dr. Moran,
Thank you for inviting us to submit a revised draft of our manuscript entitled,
“Protein intake in inhabitants with regular exercise is associated with sleep
quality: Results of the Shika study” to PLOS ONE. We also appreciate the time
and effort you and each of the reviewers have dedicated to providing insightful
feedback on ways to strengthen our paper. Thus, it is with great pleasure that
we resubmit our article for further consideration. We have incorporated changes
that reflect the detailed suggestions you have graciously provided. We also hope
that our edits and the responses we provide below satisfactorily address all the
issues and concerns you and the reviewers have noted.
To facilitate your review of our revisions, the following are our point-by-point
responses to the questions and comments delivered in your letter dated Feb 11th,
2020.
Major points.
Q1. Although authors have appropriately addressed the requirements of the
reviewers, before recommending publication of the article, and given that the
authors have included in their analysis the t-Student, ANCOVA, multiple linear
regression and Pearson correlations, the authors should state in the methodology
section that all the variables involved fulfilled the assumptions required by
parametric methods and that these were tested by the appropriate tests (indicate
which ones the authors have used).
A1. We have added the following phrase to the revised manuscript: “The
distribution of variables was checked by the Kolmogorov–Smirnov, and
Shapiro–Wilk normality tests, or the normal distribution curve in the histogram
was confirmed before using other statistical tests.” (P9 L180–182).
Supplementary explanation: Although some variables had a p-value of 0.05 or less
in the Kolmogorov–Smirnov, and Shapiro–Wilk normality test, it was confirmed
that a normal distribution curve was drawn in the histogram. Therefore, we
believe that the variables used in this study can be expected to have a
multivariate normal distribution.
Reviewer \#1:
There were no additional comments.
Reviewer \#2
Q1. Has the statistical analysis been performed appropriately and rigorously?
(Reviewer \#2: I Don't Know)
A1. We have added the following phrase to the revised manuscript: “The
distribution of variables was checked by the Kolmogorov–Smirnov, and
Shapiro–Wilk normality tests, or the normal distribution curve in the histogram
was confirmed before using other statistical tests.” (P9 L180–182).
Supplementary explanation: Although some variables had a p-value of 0.05 or less
in the Kolmogorov–Smirnov, and Shapiro–Wilk normality test, it was confirmed
that a normal distribution curve was drawn in the histogram. Therefore, we
believe that the variables used in this study can be expected to have a
multivariate normal distribution.
Q2. minor linguistic errors,
i would suggest writing a review linking, sleep, exercise fitness and diet. the
focus on sleep not only from OSA prospective but rather descriping the link
between sleep duration, timing and physical fitness in addition to healthy diet.
A2. Since we were conducting a cross-sectional analysis, we cannot examine the
sleep duration and timing in detail, but the mechanism by which regular exercise
and nutrition on sleep quality were described as follows: "The reason for good
sleep quality among participants who reported high protein intake in their daily
diet and regular exercise was thought to be a result of the pathogenesis in
which the tryptophan-serotonin-melatonin pathway was activated due to the
enhanced protein absorption" (P20 L348-351).
Again, thank you for giving us the opportunity to strengthen our manuscript with
your valuable comments and queries. We have worked hard to incorporate your
feedback and hope that these revisions persuade you to accept our submission.
Sincerely,
Fumihiko Suzuki
Department of Environmental and Preventive Medicine, Graduate School of Medical
Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8640,
Japan.
Tel: +81-76-265-2218
Email address: <f-suzuki@stu.kanazawa-u.ac.jp>
10.1371/journal.pone.0247926.r005
Decision Letter 2
Moran
Jose M.
Academic Editor
2021
Jose M. Moran
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
17 Feb 2021
Protein intake in inhabitants with regular exercise is associated with sleep
quality: Result of the Shika study
PONE-D-20-33985R2
Dear Dr. Suzuki,
We’re pleased to inform you that your manuscript has been judged scientifically
suitable for publication and will be formally accepted for publication once it
meets all outstanding technical requirements.
Within one week, you’ll receive an e-mail detailing the required amendments.
When these have been addressed, you’ll receive a formal acceptance letter and
your manuscript will be scheduled for publication.
An invoice for payment will follow shortly after the formal acceptance. To
ensure an efficient process, please log into Editorial Manager at
<http://www.editorialmanager.com/pone/>, click the 'Update My Information' link
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date. If you have any billing related questions, please contact our Author
Billing department directly at <authorbilling@plos.org>.
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For more information, please contact <onepress@plos.org>.
Kind regards,
Jose M. Moran
Academic Editor
PLOS ONE
Additional Editor Comments (optional):
Reviewers' comments:
10.1371/journal.pone.0247926.r006
Acceptance letter
Moran
Jose M.
Academic Editor
2021
Jose M. Moran
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
19 Feb 2021
PONE-D-20-33985R2
Protein intake in inhabitants with regular exercise is associated with sleep
quality: Results of the Shika study
Dear Dr. Suzuki:
I'm pleased to inform you that your manuscript has been deemed suitable for
publication in PLOS ONE. Congratulations! Your manuscript is now with our
production department.
If your institution or institutions have a press office, please let them know
about your upcoming paper now to help maximize its impact. If they'll be
preparing press materials, please inform our press team within the next 48
hours. Your manuscript will remain under strict press embargo until 2 pm Eastern
Time on the date of publication. For more information please contact
<onepress@plos.org>.
If we can help with anything else, please email us at <plosone@plos.org>.
Thank you for submitting your work to PLOS ONE and supporting open access.
Kind regards,
PLOS ONE Editorial Office Staff
on behalf of
Dr. Jose M. Moran
Academic Editor
PLOS ONE
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Gene targeting (e.g., targeted gene replacement) is one of the main molecular
tools used in yeast science, which helps in understanding gene functions and
interactions as well as cellular and molecular processes in yeasts. Model
yeasts, such as *Saccharymyces cerevisiae* and *Schizosaccharomyces pombe*, have
very efficient gene targeting systems. For instance, disruption cassettes with
short flanking regions that range from 30 bp to 50 bp can integrate with high
efficiencies via homologous integration at the correct genomic locus (routinely
\>70%) in *S. cerevisiae*. In contrast, the gene targeting efficiencies of
various “non-conventional” yeasts, such as *Pichia pastoris*, *Hansenula
polymorpha, Yarrowia lipolytica, Pichia stipitis* and *Kluyveromyces lactis* can
be extremely low (usually \<1%) with the same length of flanking regions.
Accordingly, homologous arms varying from 200 bp to approximately 2000 bp are
usually required to ensure efficient gene replacement in non-conventional
yeasts. Nevertheless, for some “stubborn” genes, the probability of obtaining
the desired gene replacement event is so low that transformation and/or
screening procedures have to be iteratively performed, which is laborious and
time-consuming. Therefore, the low targeting efficiencies of non-conventional
yeasts greatly limits the researches and applications of these industrially
important microorganisms.
To improve the targeting efficiencies of non-conventional yeasts, molecular
mechanisms of gene targeting should be understood. Yeasts have the homologous
recombination (HR) pathway and the non-homologous end-joining (NHEJ) pathway.
The HR pathway, which depends on the Rad52 epistatic group, is responsible for
the targeted integration of DNA. Integration can also be mediated randomly via
the NHEJ pathway, which depends on the Ku70/Ku80 protein complex. When foreign
DNAs are transformed into cells, they are competed by these two recombinant
pathways. Therefore, efficient gene targeting is determined by the relative
strength of the HR pathway compared with that of the NHEJ pathway. The bias,
which favors the NHEJ pathway in non-conventional yeasts, determines their low
targeting efficiencies.
However, NHEJ pathway bias is not the only factor that contributes to the low
targeting efficiency of non-conventional yeasts. The efficiency of homologous
integration in strains with the same genetic background can be very locus
specific. For example, the disruption of *ARG1, ARG2, ARG3, HIS1, HIS2, HIS5,*
and *HIS6* in *P. pastoris* GS115 strain with flanking arms that range from 200
bp to 900 bp is considerably efficient at frequencies of 44% to 90%. However,
the deletion of *PIM* and *OCH1* from the same strain that contains both
flanking arms that were longer than 1 kb occurred at a frequency of \<1%. To
date, this locus specific phenomenon is not well understood. One explanation is
the presence of “hotspot” regions for yeast genome, in which the underlying
mechanism still remains unclear. Another, which is often overlooked in previous
studies, may be the loss of function effect. Several non-conventional yeasts
such as *P. pastoris*, *H. polymorpha*, *Y. lipolytica*, and *P. stipitis* are
predominantly haploid. Thus, the knockout of genes with important physiological
functions often means great loss of cellular fitness, which would lead to a
delayed or failed appearance of correct disruptants. Therefore, the calculated
frequency of correct gene targeting by colony counting is likely lower than the
actual frequency of gene targeting events that happen genetically because of the
unformed colonies.
To validate this assumption as well as provide an efficient solution to delete
“stubborn” genes in haploid non-conventional yeasts, in this study, we proposed
a new strategy for enhancement of gene targeting efficiency by improving
cellular fitness of mutant cells, specifically by increasing the genetic
redundancy of host cells. To achieve this goal, the targeted gene was cloned
into an expression vector (helper plasmid), and transformed into yeast cells to
generate the transition host. Targeted gene disruption was then applied in the
transition host by transformation with the disruption plasmid. After gene
deletion in the genome was successfully validated, the helper plasmid can be
easily removed. *P. pastoris*, a methylotrophic yeast, was used as a model in
this study for two reasons: 1) the efficiency of targeted gene replacement in
*P. pastoris* is very low, which is estimated to occur at a frequency of 0.1%
when the total length of the target fragments is 500 bp ; and 2) *P. pastoris*
is of great industrial importance. It is by far one of the most often used yeast
species in the production of recombinant proteins. The *OCH1* gene of *P.
pastoris* was chosen as an illustrative example because its deletion procedure
by double homologous recombination is notoriously inefficient. To demonstrate
the effectiveness of this strategy, the deletion of two other genes, namely,
*SGS1* and *KU70*, was also assessed using the same method.
# Materials and Methods
## Strains, Plasmids, and Oligonucleotides
A complete list of strains and plasmids is presented in. Oligonucleotide
synthesis and DNA sequencing were performed at the Shanghai Sangon Biological
Engineering Technology and Service Co. (Beijing, China).
## Media and Growth Conditions
*Escherichia coli* DH5α strain was grown at 37°C in LLB broth (10 g
L<sup>–1</sup> of tryptone, 5 g L<sup>–1</sup> of yeast extract, 5 g
L<sup>–1</sup> of NaCl) or LB medium (10 g L<sup>–1</sup> of tryptone, 5 g
L<sup>–1</sup> of yeast extract, 10 g L<sup>–1</sup> of NaCl). *P. pastoris* was
cultivated aerobically at 30°C in yeast extract peptone dextrose (YPD) medium
(10 g L<sup>–1</sup> of yeast extract, 20 g L<sup>–1</sup> of peptone, 20 g
L<sup>–1</sup> of glucose). Antibiotics were added at the following
concentrations: 100 mg L<sup>–1</sup> of ampicillin, 50 mg L<sup>–1</sup> of
kanamycin, and 25 mg L<sup>–1</sup> of Zeocin (Invitrogen) for *E. coli*; 500 mg
L<sup>–1</sup> of geneticin (G418, Invitrogen) and 50 mg L<sup>–1</sup> of
Zeocin for *P. pastoris*.
## Construction of Disruption Plasmids
pZeoloxp, the base vector used to construct all of the other disruption
plasmids, was constructed based on the following procedures. Zeocin resistance
cassette was amplified from pPICZA with P<sub>zeo1</sub>/P<sub>zeo2</sub>,
digested with *Sac*I and *Bgl*II, and cloned into pUG6 to replace the kanMX
cassette between the loxP sites, thereby producing the pUG-zeoloxp plasmid. The
origin of replication (ori), which was amplified from pPICZA by polymerase chain
reaction (PCR) with P<sub>ori1</sub>/P<sub>ori2</sub> primer pair and digested
with *Sal*I, was inserted into the *Xho*I site of pUG-zeoloxp. The DNA fragment
with ori and ampicillin resistance gene was then excised through *Not*I
digestion and religation.
To construct the disruption plasmid for *OCH1* deletion, the upstream and
downstream homologous regions of *OCH1* were initially amplified through PCR
from *P. pastoris* GS115 strain by using two primer pairs, namely,
OCH1-N<sub>5</sub>/OCH1-N<sub>3</sub> and OCH1-C<sub>5</sub>/OCH1-C<sub>3</sub>.
The two fragments were cut with *Spe*I, *Not*I, and *Pst*I, ligated into
pZeoloxp which was cut with *Spe*I and *Pst*I, thereby generating pZeoloxp-OCH1.
The disruption plasmids used to delete the *SGS1* and *KU70* genes, namely,
pZeoloxp-SGS1 and pZeoloxp-KU70 were constructed following exactly the same
procedure with their corresponding primer pairs.
## Construction of the Episomal Plasmids
pGAPZB and the kanamycin resistance gene, amplified from pPIC9K with
P<sub>kan1</sub>/P<sub>kan2</sub>, were cut using *Nco*I/*Stu*I and ligated by
the T4 DNA ligase to replace the original Zeocin-resistant gene, thereby
generating the pGAPKB plasmid. PARS2, an autonomous replication sequence of *P.
pastoris*, was amplified from the genomic DNA of GS115 strain with
P<sub>pars2-F</sub>/P<sub>pars2-R</sub>, cut with *Bam*HI and *Bgl*II, and
inserted into *Bgl*II site of pGAPKB to generate the episomal expression
plasmid, pGKARS. To express *OCH1*, *SGS1*, and *KU70* in *P. pastoris* with
pGKARS, their encoding genes were amplified from the GS115 genome by using their
respective primer pairs. PCR fragments were cut using *Eco*RI and *Nde*I, and
then ligated into the corresponding pGKARS sites to produce the episomal
expression plasmids, namely, pGKARS-OCH1, pGKARS-SGS1 and pGKARS-KU70,
respectively.
An inducible *mazf* cassette was inserted into pGKARS-OCH1 to facilitate the
removal of pGKARS-OCH1 as necessary. To construct the *mazf* cassette, the
*mazf* gene was amplified from *E. coli* by using the
P<sub>mazf1</sub>/P<sub>mazf2</sub> primer pair, digested with *Eco*RI and
*Not*I, and ligated into the corresponding pPICZA sites to place the *mazf* gene
under the control of the alcohol oxidase (AOX1) promoter. The *mazf* cassette
was then excised from the resulting plasmid by *Bgl*II and *Bam*HI digestion and
inserted into the *Bgl*II site of pGKARS-OCH1, thereby generating the
pGKARSmazf-OCH1 plasmid.
## Competent *P. pastoris* Cell Preparation and Transformation
The competent cells were prepared based on a revised version of a previously
described method to achieve a highly efficient transformation of *P. pastoris*.
Briefly, a fresh single clone was inoculated into 100 ml YPD medium and grown
overnight at 30°C by shaking at 200 rpm until the cell density reached an
OD<sub>600</sub> of 1∼2. The cells were pelleted and resuspended at room
temperature for 30 min in 8 ml of 100 mM LiAc, 10 mM dithiothreitol, 0.6 M
sorbitol, and 10 mM Tris-hydrochloride at pH 7.5. The resulting cells were then
washed thrice with 2 ml to 3 ml of 1 M ice-cold sorbitol. Finally, the cells
were suspended in 1 M ice-cold sorbitol and transferred to 1.5 ml
microcentrifuge tubes with an aliquot of 80 µl. *P. pastoris* GS115 strain was
transformed by electroporation according to the protocols outlined by
Invitrogen.
## Direct Gene Disruption and Verification
The disruption plasmids (pZeoloxp-OCH1, pZeoloxp-SGS1, or pZeoloxp-KU70) were
linearized by *Not*I and transformed into *P. pastoris* GS115 strain. The cells
were then spread on YPD plate that contains 50 mg L<sup>–1</sup> of Zeocin to
screen the Zeocin-resistant transformants.
The transformants were further analyzed by colony PCR to verify whether they
contained the correct chromosomal integrations of the gene disruption cassette.
The parental strains were used as the control group. Two primer pairs,
P<sub>1f</sub> (located upstream of the 5′ homologous region in the
genome)/P<sub>1r</sub> (located within pZeoloxp) and P<sub>2f</sub> (located
within pZeoloxp)/P<sub>2r</sub> (located downstream of the 3′ homologous region
in the genome), were used to verify each gene. The successful amplification of
both bands with the expected size indicated that the chromosomal integrations
were correct. The amplification of one band with either primer pairs
corresponded to single crossover recombination. The
P<sub>3f</sub>/P<sub>3r</sub> primer pair, located within the open reading
frame, was used for further verification, and no band should be amplified for
the correct disruptants.
## New Disruption Method and Verification
A typical process involves three steps. First, the helper plasmid, such as
pGKARSmazf-OCH1, was introduced into the GS115 strain to generate the transition
host, GS-tranOCH1. The cells were screened on the YPD medium with 500 mg
L<sup>–1</sup> of G418. The transformants that harbor the episomal expression
plasmids were confirmed by performing colony PCR with the
P<sub>5′GAP</sub>/P<sub>3′AOX1</sub> and P<sub>5′AOX1</sub>/P<sub>3′AOX1</sub>
primer pairs. Second, the transition host was subjected to a one-step disruption
method. Third, the helper plasmid was removed by streaking on MM agar plates
\[13.4 g L<sup>–1</sup> of YNB, 0.5% (v/v) methanol, 0.1 g L<sup>–1</sup> of
histidine, 0.04 g L<sup>–1</sup> of Zeocin, and 0.0004 g L<sup>–1</sup> of
biotin\]. The P<sub>5′GAP</sub>/P<sub>3′AOX1</sub> and
P<sub>5′AOX1</sub>/P<sub>3′AOX1</sub> primer pairs were used to verify the
removal of episomal expression plasmid.
# Results
## Direct Disruption of *OCH1* Gene
α-1,6-Mannosyltransferase, the product of *OCH1* gene, adds the first
α-1,6-mannose to the Man9ClcNAc2 core oligosaccharide and initiates several
subsequent high mannose-type N-glycosylations. Accordingly, *OCH1* is often
chosen as the deletion target to avoid the hyperglycosylation of the expressed
heterologous proteins in yeasts. However, *OCH1* deletion is a very inefficient
process according to previous studies. Choi and his colleagues obtained only one
*P. pastoris* strain with the inactivated *OCH1* gene from approximately 1000
clones using double homologous strategy with 2878/1011 bp length of homologous
flanks. Using homologous flanks as long as 3 kb, Vervecken et al. failed to
delete the *OCH1* gene following the same strategy. To test the efficiency of
*OCH1* deletion, we first used the direct one-step knockout method, and the
results were used as a control group for the succeeding experiment.
We initially constructed a disruption vector for the *OCH1* gene inactivation.
This vector was generated by inserting two flanking regions, which were
amplified from the *P. pastoris* GS115 genome, into the pZeoloxp module plasmid
by performing a three-fragment ligation. The two homologous flanks, 5′ and 3′
regions of the disruption target, were 874 and 852 bp, respectively. The
disruption plasmid, designated as pZeoloxp-OCH1, was then linearized at the
*Not*I site and transformed into *P. pastoris*. More than 1000 clones were
initially analyzed by PCR using the P<sub>1f</sub>/P<sub>1r</sub> and
P<sub>2f</sub>/P<sub>2r</sub> primer pairs. Only one clone, designated as KO24#,
showed one expected amplified fragment (1.42 kb). However, the other expected
1.25 kb fragment was not amplified from KO24#, suggesting that KO24# may be the
result of a single crossover recombinant event at the 5′ homologous region.
## Construction of Plasmid pGKARSmazf-OCH1 for Modification of *P. pastoris*
To apply our proposed strategy, we initially provided a backup *OCH1* gene for
*P. pastoris* before deletion to avoid compromising the fitness of the yeast
cells because of function loss. This process was performed by cloning *OCH1*
into a carefully designed episomal pGKARSmazf-OCH1 vector, which is
characterized by three elements: 1) *Pichia* ARS2 (PARS2) fragment, which kept
the plasmid inside the cells for a considerable time and allowed the easy
removal when needed; 2) the strong constitutive glyceraldehyde-3-phosphate
dehydrogenase (GAP) promoter; and 3) the *mazf* expression cassette regulated by
promoter AOX1, whose presence is essential for plasmid removal (as explained
later).
The pGKARSmazf-OCH1 plasmid was introduced into the GS115 strain by
electroporation without linearization. The transformants were screened on YPD
plate that contains 500 mg L<sup>–1</sup> of G418. Approximately 195 colonies
were formed per µg of DNA. Colony PCR was performed using the two primer pairs,
P<sub>5′GAP</sub>/P<sub>3′AOX1</sub> and P<sub>5′AOX1</sub>/P<sub>3′AOX1</sub>,
to verify the positive clones. Among the 16 clones, 15 showed the two expected
bands, particularly 618 and 1837 bp for *mazf* and *OCH1* cassettes,
respectively (data not shown).
## Disruption of the Chromosomal *OCH1* Gene and Elimination of the pGKARSmazf-OCH1 Plasmid
The transition host that carries the redundant *OCH1* copies (designated as GS-
tranOCH1) was used as the host for the *OCH1* gene disruption. GS-tranOCH1 was
transformed using the linearized pZeoloxp-OCH1 disruption plasmid based on the
same procedure used in the direct one-step method. Approximately 60
transformants per µg of DNA were obtained, randomly picked, and analyzed by
colony PCR using the P<sub>1f</sub>/P<sub>1r</sub> and
P<sub>2f</sub>/P<sub>2r</sub> primer pairs. The results showed that the expected
1.42 kb band was amplified by the P<sub>1f</sub>/P<sub>1r</sub> primer pair from
six clones, which were further analyzed using the P<sub>2f</sub>/P<sub>2r</sub>
primer pair. All of the clones also revealed the expected 1.25 kb band (data not
shown), suggesting that the chromosomal *OCH1* was successfully deleted by gene
replacement. The results were also confirmed by sequencing the 1.42 and 1.25 kb
PCR products.
To remove pGKARSmazf-OCH1, *och1* mutant transition strain (designated as GS-
tranOCH1-ΔOCH1) was streaked on a MM plate, in which the *mazf* could be
expressed with the AOX1 promoter induced by methanol in the medium. The *mazf*
gene, which was obtained from *E. coli*, encodes the MazF toxin that functions
as an mRNA interferase and inhibits the growth of prokaryotes and eukaryotes.
Therefore, *mazf* expression likely causes a strong selection pressure on
streaked strains and forces them to lose the obtained plasmids. The colonies,
which were much smaller than that of the GS115 strain, appeared after the strain
was cultured for 3 d at 30°C. A change in the colony morphology from smooth to
rough appearance was also observed, which was consistent with the previous
characterizations of *och1* mutant strains. Ten randomly selected colonies on
the MM plate were determined by colony PCR with the
P<sub>1f</sub>/P<sub>1r</sub>, P<sub>2f</sub>/P<sub>2r</sub> and
P<sub>3f</sub>/P<sub>3r</sub> primer pairs to verify *OCH1* deletion and plasmid
clearance. All of the 10 clones exhibited the desired amplification pattern, in
contrast to control, suggesting the efficient removal of helper pGKARSmazf-OCH1
plasmid from *och1* mutant strains (resultant strains were named GS-ΔOCH1).
The growth profiles of GS115, GS-tranOCH1-ΔOCH1, and GS-ΔOCH1 were then
compared. The results showed that the duration of the lag phase of GS-ΔOCH1 was
approximately doubled compared with that of GS115 strain, indicating that the
loss of the *OCH1* gene is severely detrimental to yeast growth. By contrast,
the lag phase of GS-tranOCH1-ΔOCH1 was slightly longer than that of GS115,
suggesting that the *och1* phenotype can be rescued by the presence of the
redundant *OCH1* gene in the episomal expression plasmid to a large extent.
## Disruption of the Chromosomal *SGS1* and *KU70* Genes
To assess the effectiveness of our strategy, two *P. pastoris* genes, namely,
*SGS1* and *KU70* were selected for gene-targeted deletion. The conventional
one-step strategy and the new strategy were both applied. The disruption
plasmids for *SGS1* and *KU70*, namely, pZeoloxp-SGS1 and pZeoloxp-KU70, as well
as two transition strains with the redundant copies of the corresponding genes,
were generated based on the same procedure used in the *OCH1* gene. shows that
the *KU70* deletion by the one-step strategy resulted in five positive clones
among the 34 selected transformants, whereas ten positive clones were obtained
by performing the new strategy, indicating a 1-fold increase in the frequency of
*KU70* gene targeting. For *SGS1*, the frequency of obtaining positive clones
was increased from 1% to 24%, which is a 23-fold increase, compared with that in
one-step strategy.
# Discussion
For non-conventional yeasts, efficient gene targeting remains a challenge, which
largely limits the study and the application of these industrially important
strains. To address this issue, two strategies were most commonly employed: 1)
increasing the HR efficiency, usually by increasing the homologous arm length.
However, longer flanks are not always sufficient for a high percentage of
homologous integration, ; 2) suppressing the NHEJ pathway by deleting important
functional proteins, such as KU70 and KU80, involved in the NHEJ pathway. For
instance, the deletion of the *KU70* gene in *K. marxianus* yields 80%
homologous gene targeting efficiency by using homologous sequences with at least
40 bp in length. The integration at *HIS4* and *ADE1* loci results in \>90%
targeting efficiencies with only 250 bp of flanking homologous DNA when the
*KU70* homolog of *P. pastoris* is knocked out. Nevertheless, the stability and
robustness of these strains should be further evaluated.
While previous studies have mainly focused on increasing the HR efficiency or
decreasing the competition from the NHEJ pathway, one fact that is overlooked is
that the strains after gene deletion on molecular and cellular basis, require a
recovery and proliferation process to form colonies. Unfortunately, several non-
conventional yeasts, such as *P. pastoris*, *H. polymorpha*, and *K. lactis*,
are predominantly haploid. The deletion of functionally important genes,
particularly when these genes do not have paralogs in the genome, often results
in the loss of fitness, which inhibits the proliferation into sizeable colonies.
This condition may be more evident under unfavorable conditions, such as
cellular recovery from electroporation and growth on solid media with poor
nutrition. For instance, the *acs2* mutant of *K. lactis* requires three weeks
to form large colonies. Therefore, the possibility that the actual gene
targeting efficiency can be reduced is high because of the unformed colonies.
Based on this rationale, we aimed to improve the fitness of haploid yeast cells
by providing backup genes presented in a well-designed episomal vector. The
fitness of mutant cells was improved effectively by this strategy. The growth
defect of *och1* mutant cells was restored to a large extent. As a result, the
improvement of gene targeting efficiencies with this strategy is significant.
The *och1* disruptants, which cannot otherwise be acquired in practice by direct
one-step deletion, can be obtained at a frequency of approximately 10% with
mediate length of homologous flanks. The efficiencies of the other two genes,
*KU70* and *SGS1*, were also increased by 1- and 23-fold, respectively. These
results validated our assumption that cellular fitness is an important factor
that limits the efficiency of gene targeting in non-conventional yeasts.
In summary, we provided an efficient gene targeting strategy for non-
conventional yeasts. The targeted gene was initially amplified by cloning into a
helper expression plasmid. After the gene was successfully deleted from the
genome, the helper plasmid was removed. Thus, the *och1* disruptants were
obtained at a frequency of 10%. The gene targeting efficiencies of *SGS1* and
*KU70* were increased by 1- and 23-fold, respectively.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: CZ TZ. Performed the
experiments: CZ HS PL. Analyzed the data: CZ TZ YL. Wrote the paper: CZ TZ
NH YL. |
# Introduction
In the Near East, cradle of the domestication process, goats were among the
first to be domesticated ca. 10,500 years ago. Several thousands years later,
domestic goats (*Capra hircus*) were dispersed beyond the natural distribution
of its wild ancestor (*Capra aegagrus*). They spread in Anatolia and Europe
(starting from 8,800 calBP) throughout the Neolithic dispersal, along with pigs,
cattle and sheep. Today, goats are present all over the world with more than 867
million of individuals. In order to better assess the historical processes of
domestication, goat mitochondrial genetic diversity has been largely studied
across the old world (Europe, Asia, and more recently Africa). It is structured
in six different haplogroups A, B, C, D, F and G,, with more than 90% of goats
solely from the A haplogroup. Moreover, a very weak phylogeographic structure is
observed at the continent scale contrary to other domestic species, such as
sheep. Thus, A and C haplogroups have a worldwide distribution although B is
mostly present in Asia. Some genetic structure is suspected however at a more
restricted geographical scale and some haplogroups, such as G and F, are now
restricted to small regions (Middle-East and Sicily respectively).
If many studies have been dedicated to the characterization of the mainland
diversity few were pursued on islands. However, large Mediterranean islands are
of particular importance in the description of the genetic diversity of domestic
species since they are considered as biodiversity hot spots, have a high degree
of endemism and present a reservoir of cultural practices that have disappeared
from the Mainland. In the case of goats, the study of genetic diversity on large
Mediterranean islands is highly relevant for several reasons. First, goats are
found on most of these islands from the beginning of the Neolithic diffusion and
can serve as testimony of this spread. Second, imported domestic goats, are
physically isolated from their Mainland relatives. Third, breeding and husbandry
practices on the Mediterranean islands are usually different to those on the
continent because islands present large but restricted areas and preserve
traditional practices mentioned previously. From these observations, we expect
that mitochondrial diversity observed on islands can present evidence of
historical events or ancient diffusion that would be lost elsewhere, such as the
presence of the F haplogroup in Sicily that is unique outside the wild
ancestor's area of distribution.
Here we characterize goat mitochondrial DNA (mtDNA) diversity through time by
studying modern, but above all historical, goat populations from Corsica. We
compared them with current continental or island breeds of the Mediterranean
Basin to document the microevolution and the influence of insularity and
husbandry practices on their genetic diversity. Corsica is an 183 km long and 83
km wide island located in the Northwestern part of the Mediterranean Basin. It
formed a unique block with another island, Sardinia, during most of the
Pleistocene until the land masses split approximately around 11,000 years ago.
The presence of domestic Caprinae is attested in Corsica from the beginning of
the Neolithic, ca. 7,600 calBP. The herding system in Corsica, although close to
other free-ranging and seasonal transhumance characteristic of other
Mediterranean islands, displays a very interesting peculiarity namely the
“wandering” of flocks in the mountains for weeks, between the end of the
lactation period and the beginning of the births, under very loose surveillance
from the herder. These free-ranging practices in Corsica are not recent as
Polybius had already mentioned them in his book XII during the II<sup>nd</sup>
century Before Christ. Nonetheless, being able to ascertain that transhumance
and free ranging were common herding practices in Corsica since the Neolithic is
a difficult task for zooarchaeologists. Goats are known to be hardy, tough and
able to adapt to very difficult habitats compared to other livestock. More than
200,000 goats were still present in Corsica less than 80 years ago but this
number has decreased to 30,000 individuals during the last decades.
Nevertheless, goats have retained a particular status in Corsica where
pastoralism is still strongly established. Besides, a Corsican breed has been
recently recognized by the French CNAG (Commission Nationale d'Amélioration
Génétique depending on Ministry of Agriculture) in 2003 and by decree of the
French Ministry of Agriculture in 2007. It is a dairy breed, with relatively
long hairs, either of uniform colour or multicoloured, characterized by its
rustic character and ability to adapt to difficult grounds. This was possible
because efforts were made to protect and promote local breeds by avoiding
mixings with commercial breeds. However, the traditional Corsican breed is now
endangered because the traditional husbandry system seems difficult to maintain.
The strains on this traditional system are not only economic but also social.
Traditional goat husbandry practices implicate daily mobility from the herder,
which most of the new generation does not wish to pursue. More recently, the
occurrence of Johne's disease (i.e., paratuberculosis) presents another very
serious threat and has decimated flocks.
This study aims to (i) better characterize the current and past (medieval)
mitochondrial genetic diversity of Corsican goats using a Control Region (CR)
fragment; (ii) bring information about goat dispersal in the Neolithic by
testing the congruence between scenario proposed and data observed on this
island; (iii) explain the maintenance of endemic variability in Corsica; (iv)
discuss implications for conservation of the Corsican breed. For these purposes,
we gathered samples of 28 present-day individuals and 29 bones dated from the
Middle Ages (XII<sup>th</sup> and XIV<sup>th</sup> centuries).
# Methods
## The archeological site of Rostino
The castrum (strong hold) of Rostino is situated in the North East of Corsica.
Occupied between the XII<sup>th</sup> and XIV<sup>th</sup> centuries AD, this
late medieval site has yielded the largest assemblage of Caprinae in Corsica in
a good state of preservation, which is rare in the acidic soil of Corsica. Among
the domestic species of the XIV<sup>th</sup> century deposit, Caprinae represent
more than 70% of the identified mammal remains. The economy of the castrum
relies on specialized caprine exploitation where the production of sheep and
goat complement each other: sheep for meat production and goat for milk and hair
production. During the XIV<sup>th</sup> century, the caprine exploitation
specialized in goats with *Capra hircus* representing more than 70% of the total
caprine remains. This large amount of late medieval Corsican *Capra hircus*
represents a great opportunity to investigate the consequence of the secular
herding practices and the selective choices made by herders to renew their flock
given the genetic diversity of goats in large Mediterranean islands.
## Archeological samples
We analysed 29 bone fragments from Rostino: 17 were excavated from a
XII<sup>th</sup> century deposit and 12 from a deposit dated to the
XIV<sup>th</sup> century. These bones have been identified as *Capra hircus*
using morphoscopic criteria on both dental, and appendicular – characters. There
is no ambiguity about the origin of bones from domestic animals as the wild
ancestor (*Capra aegagrus*) has never been present in Europe. According to the
type of bone (mandible, radius, humerus …), the laterality (left, right) and
detailed information from the excavation, we were able to clearly identify 25
different individuals from the 29 fragments. The four other fragments very
probably came from previously identified individuals. Two samples were
subsequently identified as sheep by the molecular analyses. This is not
surprising as the inter-specific distinction between sheep and goat on
fragmented bones cannot be ascertained with a 100% reliability. Molecular
analyses have precisely proven to be useful in this case as has, more recently,
the analysis of collagen by mass spectrometry.
## Ancient DNA extracts and PCR amplifications
Retrieval of the DNA preserved in bones was performed in ultra clean rooms
dedicated to ancient DNA experiments (French National Platform of Paleogenetics
PALGENE, CNRS, ENS Lyon). No more than 4 *capra* samples were treated in the
same session of DNA extraction along with a bone from another species (cervids,
ursids) and a blank control. 100 to 500 milligrams of each bone fragments were
reduced in powder and suspended in 5 or 10 ml of EDTA buffer as described in. We
extracted the DNA using one, or both, of the two following protocols: a
classical phenol-chloroform approach, or direct purification using Qiaquick
column (Qiagen kit). 25 out of the 29 samples were independently re-extracted in
a second laboratory dedicated to ancient DNA in another city, Grenoble, using
the Qiaquick protocol.
A 130 bp fragment of the CR (HVI) was amplified with the CapFII and CapRII
primers with conditions identical to those described in. At least 2 or 3
independent positive amplifications per sample were obtained, cloned and
sequenced following protocols described in. The final sequence of one individual
was obtained by making the majority-rule consensus of all consensus of all
different clones obtained from each of the independent amplification. More than
80% of the differences observed between clones were G to A or C to T punctual
substitutions. This result is consistent with ancient DNA degradation profiles
where deamination of cytosines is known as the major factor of artifactual
substitutions.
## Present-day Corsican goat sequences
We sampled 28 goats in 5 different localities in Corsica (7 from Moltifao, 6
from Tralonca, 11 from Corte, 3 from Altiani, 1 from Quenza;). 14 sequences
were already published and 14 were produced for this study. To amplify the CR
fragment we used the same primers (CapFII and CapRII) than for medieval
sequences with slightly modified conditions: 40 cycles were performed instead of
50–60 for ancient DNA and products were directly sequenced on both DNA strands.
## Sequence analyses
All the sequences obtained were aligned (Seaview v4) and the primers removed
leading to a fragment of 130 base pairs. Four data sets were constituted: (i)
all medieval sequences, (ii) the XII<sup>th</sup> century sequences, (iii) the
XIV<sup>th</sup> century sequences and (iv) the present-day sequences.
Firstly, the mitochondrial haplogroup of each Corsican sequence found was
determined by performing phylogenetic analyses. The different Corsican
haplotypes were analyzed together with 20 haplotypes of known haplogroups. These
20 haplotypes corresponded to the 20 reference sequences that were different for
the 130 bp fragment under study, in the dataset selected by Naderi et al. to
represent the worldwide variability of the whole HVI-control region (558 bp).
Identical sequences, or haplotypes, were identified in the Corsican dataset by
using Fabox. After estimating the better model of evolution using jModeltest
program and the Akaike Information Criterion (AIC), we performed Bayesian
analyses (BA) with MrBayes v3.1.2, (independently confirmed by Maximum
Likelihood analyses, not shown). The parameters used for BA were the following:
GTR+I+G (nst = 6 and rates = invgamma), 5,000,000 generations sampled every
1000<sup>th</sup> generation, 4 chains, a burn-in period of 500 trees (i.e. 10%
of generations) visually confirmed using Tracer v1.4.1 (developed by Rambaut A
and Drummond A; available from <http://beast.bio.ed.ac.uk/Tracer>), allcompat
option. Two independent runs were performed with an average standard deviation
of split frequencies at completion of 0.006598. The average values obtained for
alpha and proportion of invariable sites parameters were 0.287 and 0.323
respectively.
Secondly, we assessed the relationships between only medieval or all Corsican
sequences, by performing median-joining networks, using the Network software
(available at fluxus-engineering.com) with default parameters. Network Publisher
was used to manipulate the networks. To compare the Corsican mitochondrial
genetic diversity with the Mediterranean or worldwide diversity, we defined
supplementary datasets, one by haplogroup observed, gathering all the sequences
published and covering the 130 bp fragment. These datasets combined with the
Corsican sequences were used to draw median-joining networks (references are
given in the legends of).
Thirdly, to assess the past demographic history of the Corsican goats we
performed a Bayesian Skyline Plot (BSP) using BEAST v.1.5.4 software. All the
Corsican sequences were used and average tip dates were given for all medieval
sequences: 850 years BP for the XII<sup>th</sup> century and 650 years BP for
those of the XIV<sup>th</sup> century. BEAUti v1.5.3. was used to build the xml
file by using the following parameters: HKY+I+G4 (best model for this dataset
assessed by jModeltest and AIC criteria); uncorrelated lognormal relaxed clock;
5 groups and 100 millions of iterations with parameters saved every 10 000
iterations; Burn-in: the first 10% were discarded. The results of 4 independent
runs were analysed and the Bayesian Skyline Plot reconstructed with Tracer
v.1.4.1.
Finally, to describe the genetic diversity observed for the Corsican goats, we
computed different classical parameters using either DnaSP v5 or Arlequin
v3.5.1.2 including haplotype diversity and frequencies, sequence diversity,
pairwise comparisons, and population comparisons (*F<sub>ST</sub>*, Fu tests).
We compared the different Corsican datasets with each other but also with other
datasets corresponding to mainland or island populations (see for accession
numbers and geographical origin). We focused in particular on Sardinia's island
(75 sequences, accession numbers FJ571522 to FJ571596, ; Corsica's closest
island) and Portugal (the biggest dataset generated for mainland goats, 288
sequences, accession numbers AY961629 to AY961916). We also considered 4
different datasets (see Supporting Information for details and references)
corresponding to the southern or the northern area of the Mediterranean Sea, the
Mediterranean islands (Sicily and Sardinia), and finally 8 Neolithic goats of
Baume d'Oullens. To reduce possible biases due to large differences in the size
of the datasets (low number of Corsican sequences), we randomly sampled 49
sequences of the non-Corsican sequences and repeated this operation at least
three times. The genetic parameters estimated by DnaSP v5 or Arlequin v3.5.1.2
were then computed on these resampled datasets of equal size.
# Results
## Medieval goat samples
We analyzed 29 bones and obtained reproducible and congruent sequences of *Capra
hircus* for 25 of them. We are confident that these results are authentic as we
obtained the same results in both laboratories. We also took the ancient DNA
precautions recommended by the community as we are used to do. When different
bones were supposed to be from a single individual (Ro-5, Ro-10 and Ro-22),
systematically we obtained the same sequence confirming the first assessments.
Finally, we determined 21 sequences coming from different individuals: 10 dated
to the XII<sup>th</sup> century and 11 dated to the first half of the
XIV<sup>th</sup> century. All medieval sequences were from the A haplogroup as
shown by the phylogenetic analysis. The six haplogroups appeared monophyletic
and were supported by posterior probabilities (pp) higher than 0.9 for 3 of them
B, C, G (F is not concerned as only one sequence is used). The A haplogroup that
had the highest number of sequences and that was the more diverse received the
lowest support (pp\<0.5). Nevertheless, the clustering of the newly determined
sequences inside the A haplogroup raises no doubt as confirmed by subsequent
network analyses (see also mismatch distributions). Substantial diversity is
observed among medieval haplotypes as seen on the network. Among the 21
sequences, we detected 14 unique haplotypes with haplotype Ha 04 being the most
frequent (5 individuals). Six out of the 14 haplotypes have never been described
before. On average, the mean number of pairwise differences observed between
sequences reaches 4.65±2.37. The diversity appeared not significantly different
between the XII<sup>th</sup> century with 9 haplotypes for 10 sequences (13
polymorphic sites) and the XIV<sup>th</sup> century with 8 haplotypes for 11
sequences (15 polymorphic sites). According to the network performed on all
medieval sequences (14 haplotypes, 19 polymorphic sites), only 3 haplotypes were
shared between both periods. Two of them had a central position in the network
(Ha 04 and 09;).
## Present-day goat samples
28 individuals from 5 different localities were studied ( for details). Two
different mitochondrial haplogroups were observed (A and C;) with a higher
proportion of A sequences (92.8% i.e. 26 out of 28 sequences). Considering all A
and C sequences, the mean number of pairwise differences reaches 7.01±3.39 (29
polymorphic sites;). However, when only sequences of the A haplogroup were
considered, this value drops to 4.90±2.47 (19 polymorphic sites) which is close
to the one observed for A medieval goats. The two sequences from the C
haplogroup came from the same locality, Tralonca, and shared the same haplotype
Ha 26 that has not been described elsewhere. 15 haplotypes were obtained for the
26 sequences of the A haplogroup among which, 10 were only observed in Corsica.
The most frequent haplotypes were, like for the late medieval goats, Ha 04 and
Ha 09, with 4 individuals from 3 different localities in both cases.
## Comparison of Corsican goat diversity through time
No significant difference was observed between the goats of the XII<sup>th</sup>
and the XIV<sup>th</sup> centuries (non significant *F<sub>ST</sub>-*value;).
The difference became significant when medieval and present goats were compared
(0.036, p-value 0.027). This was due to the presence of the C haplogroup in the
present-day sequences since the test was no more significant when only A
sequences were taken into account (0.027, p-value 0.099). Finally, there were no
significant differences between the medieval and present-day sequences, with or
without considering the C sequences. Medieval and present-day goats shared 4
haplotypes (Ha 04, 06, 07 and 09) but only one was specific to Corsica (Ha 06).
The 3 remaining haplotypes were generally frequent in other populations.
No significant changes were observed in the demographic history of the Corsican
population using the Bayesian Skyline Plot. Neither a sign of expansion nor of a
crash were observed from the medieval times to date, as rather a constant
population size pattern was obtained. This is congruent with the Tajima's D
values obtained but not with all the Fu's Fs values computed. However, this
latter parameter can reflect other factors than population growth (selection,
bottleneck, …).
## Comparison of Corsican goat diversity with other geographical places
We compared the diversity observed in Corsica with other Mediterranean island
populations and mainland breeds. The 75 sequences of Sardinia were clustered in
46 haplotypes that were all from the A haplogroup. Similarly, the 288 Portuguese
sequences were represented by 104 haplotypes, from which only one was from the C
haplogroup and all the others from the A haplogroup. By expanding the comparison
to larger or other areas, we observed that the C haplogroup is described only in
Europe (in present-day Northern Mediterranean area and already at the Neolithic
time, and not shown). Both the medieval and present-day Corsican groups appeared
significantly different from all other groups whatever the resampled datasets
taken into account. Similarly, all the non-Corsican groups of goats also
appeared significantly different from each other (data not shown). However, four
different haplotypes (Ha 05, 08, 09 and 10) were shared between Corsica and
Sardinia islands and six (Ha 07, 08, 09, 10, 12 and 21) between Corsican and
Portuguese goats.
The median-joining network performed on 39 worldwide sequences of the C
haplogroup revealed a classical expansion structure with European sequences on
one side and Asian sequences on the other (with the single exception of one
Swiss haplotype;). As expected, the sequences obtained from Neolithic goats of
the archeological site of Baume d'Oullens in France, among the first goats to
have been diffused in Europe, appeared in the central part of the European
cluster. The Corsican haplotype showed the highest number of substitutions with
this central node (3 substitutions). The same analysis performed on the
sequences from the A haplogroup restricted to the Mediterranean area, showed a
more complex history with no clear emerging pattern.
# Discussion
## DNA preservation in medieval samples
From the 29 archeological bones dated back to the XII<sup>th</sup> and
XIV<sup>th</sup> centuries, we obtained 21 sequences from different ancient goat
individuals: 10 from the XII<sup>th</sup> century deposit and 11 from the
XIV<sup>th</sup> century deposit, which represents a surprisingly high DNA
preservation for remains in Corsica. Such a good preservation of the DNA, which
is here correlated to the good preservation of the bones themselves like in most
late Medieval Corsican sites, is probably due to the recent age of the site.
## Characterization through time of mitochondrial genetic diversity in Corsican goats
The comparison of Middle Ages and present mitochondrial diversity was carried
out using 28 present-day goats from five different localities. This comparison
may be slightly biased by differences existing in the time span and geographic
distribution for either ancient or modern samples. Indeed, the sampled
geographic area is larger for the modern goats whereas the time span is longer
for the ancient samples.
Our results tend to prove that the mitochondrial diversity of Corsican goats has
remained relatively constant since the Middle Ages. Moreover, we detect no
significant demographic changes (*F<sub>ST</sub>*, BSP and) or decrease of
genetic diversity. The only difference between both periods is the occurrence of
two C haplotypes in the present-day samples, all other goats belonging to the A
haplogroup. However, given a dataset of 21 medieval individuals and assuming a
constant frequency of the C haplogroup in Corsica (2/28 = 0.0714), we have a 21%
of chance of having missed this haplogroup in the Middle Ages sampling.
## The occurrence of the C haplogroup in Corsican goats in the context of the Neolithic diffusion
The presence of the A and C haplogroups in Corsica is in agreement with the goat
mitochondrial variability observed in European countries. Most of European goats
are from the A haplogroup, with C haplotypes found at a rare frequency in
Switzerland, Portugal, Spain and Slovenia. This is also consistent with previous
paleogenetic studies that already detected both haplogroups in goats from
Southern France in the early Neolithic period. The median-joining network
performed on all the C haplotypes found worldwide gave results congruent with
the diffusion of goats in the Neolithic. Indeed we observed: i) a clear
separation of the European haplotypes from the Asian ones with only one
exception, a shared haplotype between China and Switzerland; ii) a star-like
pattern for European haplotypes suggesting a population expansion with a central
position for the Neolithic haplotypes ; iii) a divergent haplotype for Corsica
compatible with a subsequent isolation.
Without a doubt, the origin of the C haplogroup in Europe can be traced back to
the Neolithic spread where its frequency was probably higher than the one
observed now. Today, the C haplotypes are relatively scarce in goats and appear
more like reminiscent testimonies of this first diffusion. Interestingly, not
one has been detected yet in the Southern Mediterranean area or in other
Mediterranean islands than Corsica (see Supporting Information). According to
the few data we have, it is difficult to conclude on the ancient origin of the
Corsican C haplotype or a more recent origin linked to subsequent exchanges with
the Northern Mediterranean mainland. However, its position in the network is
compatible with the first explanation. Further ancient DNA studies, for a larger
area and older period, would be very interesting to highlight this question.
## Variability in Corsica vs other Mediterranean areas
The relative stability through time of goat diversity in Corsica could be
explained by regular importation of goats from other continental areas or
islands, e.g. for commercial trade, as many contacts by sea have been reported
during the last centuries in the Mediterranean area. However, a striking point
is that the diversity observed in Corsica is substantial for both periods
(Middle Ages and present-day, and respectively) and differs from that of other
places (data not shown); only about half of the Corsican haplotypes (14/26) are
shared with goats from other geographical regions despite our study focused on a
short CR fragment (130 bp). This result is not unexpected however. Previous
studies - usually targeting the 480 bp fragment first described in and covering
our shorter fragment - have already shown that goats were more polymorphic than
other livestock (cattle, sheep, pig) on the CR. Moreover, the specific analysis
of the A haplogroup (more than 90% of the modern goats) confirmed high haplotype
diversities for 20 populations/countries with values close to one. Pereira et
al. showed a strong correspondence between mitochondrial genetic and geographic
distances suggesting that after the initial expansion, differentiation among
regions has been established and maintained. A similar conclusion was obtained
with large-scale nuclear SNP analyses obtained for 16 breeds of goats and from
microsatellites analyses including Corsican and other European goat populations.
This seems in agreement with what we observed in Corsica when compared with
other Mediterranean populations (*F<sub>ST</sub>* comparisons, data not shown).
## Implication of the traditional Corsican husbandry in the maintenance of the variability
Except for some haplotypes that are common in many countries and probably
constitute traces of the initial diffusion, 46% of the Corsican haplotypes found
have not been previously described elsewhere. The preservation through time of
this endemic genetic diversity and its constant level since medieval time could
suggest that relatively large effective population sizes have been maintained in
domestic goats through exchanges of animals. But ethnographic insights into the
herding practices carried out in the Niolu provide another possible or
complementary explanation for the preserved genetic diversity of the Corsican
goat breed.
Typically in the past for the Mediterranean area, goats were usually moved
according to the seasonal changes (transhumance) to gain access to more reliable
food. Because fodder resources fluctuate in the wild according to different
factors (e.g. annual climatic conditions), the system developed in Corsica has
been extensive with goats left free-ranging most of the times although under the
careful control of the herders. This particular system led to the
characteristics observed in the Corsican goats, : i) the high diversity of coat
colours is encouraged as individuals can be more easily identified by sight; ii)
only the strongest and toughest goats can generally survive in this relatively
hostile environment, explaining why the introduction of goats from industrial
breeds usually failed; iii) large herds are usually managed to maximize the
herd's productivity overall instead of individual productivity. Indeed,
selection is not performed to optimize for instance, either milk productivity or
the fecundity, but instead to obtain a constant productivity of the herd by year
whatever the weather or difficulties encountered. All these points lead to a
strategy where the phenotypic diversity, and the underlying genetic diversity,
is maximized in order to obtain herds that are highly adaptable, rustic and
robust.
In order to do that, herders exert strong selection pressure while forming their
herds to gain in productivity but also to favour behavioural traits. Indeed,
along with the search for constant productivity, one of the main objectives of
herders is to design flocks that will maintain “families” from the same maternal
lineage. A young descendant female is usually chosen according to its mother's
and grandmother's family, taking into account its desirable productive traits as
well as its abilities to endure the tough conditions. Animals coming from the
same family/pool will live together and move together more easily and naturally.
Such “familial” behaviour is extremely beneficial to the cohesion of herds under
free-ranging exploitation and especially during the movements on the
pasturelands (transhumance). On the contrary, introduction of new animals from
other herds can induce significant disturbance in the herd movements by breaking
the cohesion of the group. This practice of herders of course has the effect of
reducing the genetic diversity. Hence, to insure the persistence of the genetic
diversity and to “change the blood”, “cambià u sangui” in the language of
traditional Corsican herders, selected males of these different maternal
lineages or “families” will be exchanged between flocks to avoid inbreeding and,
without control, renew the pool of the mating males within a seasonal time span.
As mitochondria are inherited maternally, this system will naturally lead to
maintaining high mitochondrial genetic diversity between herds or “families”.
So, the diversity we observed would be not promoted by large herds with many
exchanges of females between them but on the contrary by traditional practices.
These ones rely on very few introductions of females coming from other herds to
keep the kinship within the herd as tight as possible to reinforce cohesive
behaviour during the ranging but also on a mixing of the genetic diversity
through the exchanges of males only.
Such entangled practices in the Corsican husbandry system can explain why we do
not observe change of the mitochondrial diversity in Corsican goats since the
late Medieval period, and probably earlier if we had been able to investigate
the genetic diversity of goats from earlier periods. It's thus highly probable
that sustainable husbandry practices in today's Corsica that are so well adapted
to their environment are, at least partly, the result of practices over
millenaries contributing to the maintenance of a relatively high level of
genetic diversity. Finally, analyses of additional samples and genetic markers
coupled with population simulations using varying population genetic parameters
should help to test between both hypotheses, regular importation of goats or
millenaries husbandry practices.
## Towards a protection of the Corsican goat breed
In this study, we observed that the mitochondrial diversity of goats in Corsica
Island has been maintained since the Middle Ages to date. In a time where rustic
breeds are endangered and industrial breeds tend to reduce the genetic
resources, Corsican goats, by the model of husbandry used and the high diversity
observed constitute an interesting pool to preserve for the future management of
domestic genetic resources. All the actors concerned by the Corsican goat
(regional association in charge of its management, public authorities,
researchers and extension services) should so pay attention to make their
preservation successful all the more because paratuberculosis has started to
decimate flocks.
## Ethic statement
The medieval bones of goats were excavated from the Rostino archeological site.
Daniel Istria, in charge of the excavation, authorized their analyses. Tissue
samples were collected in Corsica in the framework of the ECONOGEN project
(European Union contract QLK5-CT-2001-02461), following the European ethical
rules implemented in all European projects.
# Supporting Information
We thank Hamid Reza Rezaei and Saeid Naderi for having given us details about
samples of Corsican goats they have published. We also thank members of the
paleogenetics lab and the Palgene Platform (CNRS, ENS Lyon) for help during the
experiments. We are also grateful to Javier Oliver and Benjamin Gillet for
critical reading of the manuscript. Finally, we express gratitude to Joanne
Burden who kindly proofread the English.
[^1]: Conceived and designed the experiments: PT HF TC SH CH. Performed the
experiments: MD HF. Analyzed the data: SH TC. Contributed
reagents/materials/analysis tools: DI J-DV PT CH. Wrote the paper: SH FP TC.
Provided expertise for Corsican husbandry, ancient DNA and archeozoological
data: FC CH J-DV.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
HCV is a worldwide health concern with severe consequences. Globally, HCV is
estimated to affect around 3% of the world's population, counting to
approximately 170 million people. While it may remain asymptomatic for years, it
can lead to serious liver diseases, which include cirrhosis or hepatocellular
carcinoma. As with all viruses, HCV is prone to genetic mutations that lead to
multiple reproducible variants. Seven genotypes of HCV with various subtypes
have been discovered around the world.
The genotype HCV-1 is common in America, Europe, and Japan. The subtype HCV-1a
is predominant in North American and Northern Europe whereas HCV-1b is the most
common subtype in Japan and Eastern Europe. Additional countries where HCV
infection rates are very high are Egypt (15% of population, 18 million people)
and Pakistan (4.8%, 8.5 million). Approximately 90% of those infected in Egypt
carry the genotype 4, with subtype 4a (HCV-4a) predominating. In Pakistan,
around 67% of the HCV infections are due to genotype 3, with subtype 3a (HCV-3a)
being the most common.
Genotype 1 has been the focus of intensive investigations over decades and a
variety of effective antiviral drugs and/or inhibitors have been developed.
Conversely, variants that are predominant in developing countries have not
received much attention.
As a result of the crucial role of the nonstructural protein 3 (NS3) in the
replication cycle of HCV, the protease domain of NS3 has been an attractive
target for direct-acting antiviral agents. The NS3 protease cleaves four
downstream sites in the HCV polyprotein and is characterized as a serine
protease with a chymotrypsin-like fold, which is activated by the NS4A cofactor.
Similar to chymotrypsin, the catalytic triad of the HCV NS3 protease is made of
three essential residues, histidine-57, aspartic acid-81, and serine-139. These
three residues are collectively known as the catalytic triad and will perform
general acid-base catalysis on target peptides. In summary, a charge relay
system is formed in which the carboxylic group of D81 forms a hydrogen bond with
N*δ*1 of H57. This event increases the p*K*a of the histidine side chain from 7
to about 12. Consequently, H57 deprotonates the hydroxyl group of the S139 side
chain and a proton shuttles to Nε2 of H57. The Oγ of S139 then nucleophilically
attacks the carbonyl carbon of a substrate's scissile bond resulting in the
formation of an oxyanion-containing tetrahedral intermediate,. At this point,
the protonated H57 acts as a general acid assisting in the collapse of the
tetrahedral intermediate and the cleavage of the substrate. Zinc, which is a
part of NS3 protease, plays an important role in the structural stability of the
protease by enthalpically disfavoring protein denaturation. Additionally, a
bound peptide cofactor (NS4A) increases the protease activity by nearly
1000-fold. It is noteworthy to mention that HCV-3a and HCV-4a NS3 proteases
exhibit a several fold decrease in catalytic efficiency relative to that of
HCV-1b. This implies a possible correlation between the catalytic efficiency of
the NS3 protease and its responsiveness to inhibition, at least by the linear
inhibitor Telaprevir. The differential susceptibilities of different drugs to
protease variants in relation to their enzymatic activities have been
investigated.
The commercially available linear NS3 protease inhibitors Telaprevir and
Boceprevir are shown to be effective against HCV-1. Furthermore, HCV-3a and
HCV-4a NS3 proteases show several fold resistivity to inhibition to Telaprevir
compared to HCV-1b, with HCV-4a being the most resistant. Additionally, the
newly developed macrocyclic NS3 protease inhibitor Danoprevir has been shown to
be effective against genotypes 1b and 4a, but not as effective against genotype
3a. Another macrocylic drug, Simeprevir, has been shown to be somewhat effective
in all HCV genotypes, but is most effective against HCV 1a and b. Very recently,
the drug Sofosbuvir, which inhibits a different HCV target protein (NS5B
polymerase), has been approved by the FDA to treat genotypes 1 through 4.
Although Simeprevir and Sofosbuvir provide a way to treat multiple genotypes of
HCV, the cost of the typical 3 month treatment (\$66,000 and \$84,000
respectively), is too expensive for use in developing countries. In addition,
due to the high mutability of the virus, a sub-type can emerge during the course
of treatment, resisting the administered antiviral drug and complicating the
treatment regimen.
In our earlier work investigating the drug resistivity in HCV-4a, we developed a
computational methodology to analyze, in 4D, the active site geometry in HCV NS3
protease. The results showed that both proteases share very similar rigid and
overall dynamics features. Conversely, both exhibit significantly different
local dynamics and distance distribution profiles, in peak values and broadness,
at the catalytic triad.
Here we have improved the methodology further and extended our investigation to
include the drug resistant genotype HCV-3a. Our data, consistent with our
previous report, suggest that genotype-dependent structural dynamics could play
a significant role in the stability of the catalytic triad, and possibly, in
drug response among HCV genotypes. The results show that the divergent dynamics
behavior of the catalytic triad in the NS3 protease of genotype 3a, represent an
intermediate state between that of genotype 1b (most stable) and genotype 4a
(most unstable). This correlates well with their reported catalytic activities
and drug susceptibilities to the linear inhibitor Telaprevir. Therefore, our
comparative investigation reported here, illuminates a possible variant-
dependent pathway from an active/drug responsive protease to a weakly
active/drug resistive one, an understanding with implications in catalysis and
drug design.
# Results and Discussion
HCV-3a and HCV-4a NS3 proteases share 80% and 83% sequence identities
respectively, with HCV-1b protease. The HCV-3a and HCV-4a protease structure
models superpose very well on the threading template structure (HCV-1b, PDB-
ID:1dy8), along with nearly identical structural features. When the three
structures superpose, the RMSD in back-bone positions is about 0.3 Å and none of
the 174-threaded amino acids fall within the disallowed Ramachandran area and no
steric clashes or stereochemical outliers were detected.
The three catalytic residues H57, D81, and S139 are located in a crevice between
the two protease β-barrels as shown in. The active site is nonpolar and shallow.
The central region of NS4A is buried almost completely inside the NS3 protease
and serves as a cofactor for proper folding of the protease. The rigid
structures indicate that access to the active site is nearly identical in the
structural models (HCV-3a and HCV-4a) and template (HCV-1b). Molecular dynamics
simulations predict that both HCV-4a and HCV-1b proteases share more or less
similar average RMSD in the Cα positions, around ∼1.2 Å at equilibrium. However,
the average RMSD in the Cα positions for HCV-3a protease is higher (∼1.6 Å).
This implies that the main chain of HCV-3a protease, as a whole, experiences
more fluctuations compared to that of the two other genotypes (1b and 4a).
Interestingly, as will be shown later, this malleability in HCV-3a protease,
which is even greater than the barely functioning genotype 4a, does not
propagate fully to the catalytic triad region. In this regard, HCV-3a represents
an interesting case where the conformational stability of the catalytic region
of the enzyme is somehow “shielded” against an overall positional instability of
the protein.
Locally, molecular dynamics simulations revealed a strain-dependent, gradually
divergent dynamics behavior within the catalytic triad region, with HCV-1b being
the most stable, the HCV-4a the most divergent and HCV-3a representing an
intermediate state. These dynamic differences seem to correlate well with the
differences in catalytic activities and drug susceptibilities to Telaprevir seen
in the three genotypes. This result strongly suggests that the local dynamics
within the triad region in the NS3 protease could be used as a direct predictive
measure for HCV pan-genotype drug susceptibilities.
## 4D simulation of the interactions between the catalytic residues
Following the same methodology we reported on previously, we have investigated
the local positional dynamics of the catalytic triad residues during the course
of simulation and used the distance distribution profiles of catalytically
relevant distances as indicators of the 4D variations.
The alpha carbons (Cα) of the catalytic residues D81 and S139 exhibit somewhat
similar dynamics throughout the simulations for the three genotypes (1b, 3a and
4a). However, the Cα of the catalytic residue H57 of the HCV-4a and HCV-3a shows
a slight to moderate increase in RMSD of ∼0.2 Å and 0.7 Å respectively, relative
to that of HCV-1b.
As entire residues, H57 and D81 in both HCV-4a and HCV-3a models, demonstrate
dynamics behavior divergent from that of the template HCV-1b. The RMSD of H57
in the HCV-3a model varies from that predicted in the HCV-1b template by up to
1.5 Å at certain points during the simulation. A very similar trend is observed
for HCV-4a. A slight increase in the Cα dynamics of H57 in HCV-4a is associated
with a significant increase in the dynamics of the entire residue. Therefore,
one should expect a much higher RMSD for H57 (as an entire residue) in HCV-3a,
if only the corresponding Cα instability is considered. The observation that H57
(as an entire residue) shows similar RMSD pattern in both HCV-3a and HCV-4a (in
spite of the relative stability of the Cα in HCV-4a) points to additional
stabilizing interactions acting on H57 in HCV-3a. This is evident in HCV-3a from
the intermediate RMSD dynamics of D81 residue, which is known to fulfill a
stabilizing role for H57. The RMSD of D81 in the HCV-3a and HCV-4a models
gradually diverge from that of HCV-1b template by nearly 0.5 Å and 1 Å
respectively. S139 exhibits similar dynamics behavior in the three genotypes,
which is expected for a relatively small side chain whose movement is sterically
encumbered by the nearby residues.
The distance distribution profiles between Nε2 of H57 and Oγ of S139, as well as
between Nδ1 of H57 and Oδ2 of D81, of the template (HCV-1b) and models (HCV-3a
and HCV-4a) vary widely in both peak value and breadth. In the template
structure (HCV-1b), the distance between Oδ2 of D81 and Nδ1 of H57 exhibits a
sharp distribution with a peak value around 3 Å. In the model HCV-4a, the
corresponding distance distribution is bimodal, much broader, and distributed
around 4.5 and 7.5 Å. It is noteworthy to mention that in our previous report,
the 4.5 Å peak was not conspicuous. With the improved methodology in the current
analysis , this peak becomes clearer. In the model HCV-3a a broader bimodality
(∼70% of the distribution) still dominates, with the recovery of a sharp peak
around 3 Å, overlapping with that of HCV-1b peak. It is interesting that the
distribution for the HCV-3a model is almost a mixture of that of HCV-1b and
HCV-4a. Furthermore, the distance distribution between Oγ of S139 and Nε2 of H57
in the template HCV-1b shows a peak value at around 4 Å, while the corresponding
distribution in the model HCV-4a is broader and bimodal. Again, the distribution
in HCV-3a is a mixture of HCV-1b and HCV-4a shifted by about 0.5 Å.
The collective dynamic behavior of the three catalytic residues as vertices of a
triangle is analyzed. This is to gain an insight into the respective relative
positions of the three residues simultaneously. The atoms chosen were Oδ2 of
D81, Nδ1 of H57 and Oγ of S139. The choice of Nδ1 over Nε2 of H57 was to include
conformations in which the H57 has rotated in such a way that Oδ2 of D81 and Nδ1
of H57 can no longer hydrogen bond. While more than three atoms are involved in
the hydrogen bonding network, the vertices of the triangle collectively probe
the hydrogen bonding distances. Thus, any other choice of vertices would only
shift the values, not the distribution. The distribution profiles of the area of
the triangle during the course of simulation indicate a uni-modal sharp peak in
HCV-1b, a broad bimodal distribution in HCV-4a and a hybrid behavior in HCV-3a.
The area of the triangle somehow represents a “catalytic plane” whose
distribution profile could be predictive of distortions of optimal catalytic
geometries. In this sense, HCV-1b is predicted to be the most stable (most
active) and HCV-4a is the least stable (least active) while HCV-3a represents an
intermediate state. This is consistent with the observation that the catalytic
activity of HCV-4a NS3 protease is several orders of magnitude less than that of
HCV-1b, while the catalytic activity of HCV-3a is still less than that of
HCV-1b, but not as hampered as that of HCV-4a.
The same trend was observed in the genotype-dependent drug susceptibility seen
in HCV against the linear inhibitor Telaprevir; HCV-1b is the most responsive
(least resistive), HCV-4a the least responsive (most resistive) and HCV-3a
representing an intermediate state. Telaprevir is a linear inhibitor that fits
within the natural substrate' binding site “envelope”. Therefore, it is expected
that variations (whether rigid or dynamic) altering inhibitor binding will
simultaneously interfere with the binding of substrate; thus, impact the
enzymatic activity. However, other inhibitors protrude from the substrate
binding envelope, interacting with sites remote from the substrate binding site.
Variations occurring at these sites incur drug resistivity, with little effect
on the catalytic activity,. In general, drug susceptibilities to protease
variants depend on both the 3D location of the variation (mutation) sites, and
the stereochemical structure and conformation of the inhibitor. However,
dissecting the molecular and structural basis of the differential
susceptibilities of different drugs to protease variants in relation to their
enzymatic activities is rather extensive, beyond the scope of this work.
Together, these data indicate that in the model HCV-3a, H57 spends less time
positioned within a probable hydrogen bonding distance to both S139 and D81
compared to HCV-1b, but more time compared to HCV-4a. Thus, H57 is less likely
to act as an efficient general acid–base in case of HCV-3a. The effect is more
severe in case of HCV-4a where H57 is not positioned within hydrogen bonding
distance with D81 and barely with S139. As mentioned before, it seems that the
mode of Telaprevir binding somehow allows the protease drug responsiveness to
follow the enzymatic activity trend.
It is important to note that the predicted divergent dynamics behavior in HCV-3a
and HCV-4a is completely hidden by the apparent similarity seen in the catalytic
site in the rigid structures. Further, the global instability of the protein's
backbone fails to accurately account for the stability of the triad as does the
stability of the Cα of the H57. These results highlight the importance of
utilizing molecular dynamics as a method of future investigations into protease
activity. Furthermore, the correlation between the divergent conformational
stability of the catalytic triad region with both the catalytic activity and
drug resistivity seen in HCV-proteases cross genotypes, opens an interesting
avenue for inquiry with potential predictive applications.
# Methods
## DNA sequencing
For HCV-4a (strain ED43), we used the amino acids sequence, as described
previously. To obtain nucleotide/amino acid sequence of NS3 protease from HCV
3a, RNA was extracted from blood sample of a Pakistani patient and cDNA was
synthesized using first strand cDNA synthesis kit (Fermentas, cat no. K1612).
Freshly synthesized cDNA was used to amplify the full length NS3 gene using
forward primer 5' TATAGGATCCATGCACCATCACCATCATCACGCCCCGATCACAGCATAC3' and
reverse primer 5' GAGCAAGCTTTTAGGTGGTTACTTCCAGATCAG 3' containing the *Bam* H1
and *Hind* III sites, respectively, through a gradient PCR reaction. The
amplified product was cloned in pET 11a vector and sequenced. The sequence was
submitted to NCBI GenBank under the accession number JQ676838. The Research
Ethics Review Committee of National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad, Pakistan has approved the protocols and
procedures used to collect the blood samples from HCV patients. A written
informed consent (as outlined in PLOS consent form) to participate in this study
and publish the case details was taken from every donor.
## 3D structure prediction and validation
The 3D structure of HCV-3a and HCV-4a NS3 proteases were predicted by threading
its amino acid sequence through the X-ray crystal structure of HCV-1b NS3
protease (1dy8) via the threading program LOOPP. LOOPP is a fold recognition
program that generates atomic coordinates of a sample molecule based on an
alignment with a homologous template structure. By integrating the results from
direct sequence alignment, sequence profile, threading, secondary structure, and
exposed surface area prediction, the LOOPP builds main-chain and all-atom
models. Nearly identical models were also obtained via homology modeling using
the SWISS-MODEL Workspace. The RMSD values between models obtained using LOOP
and SWISS-MODEL were about 0.2 Å and 0.16 Å for HCV-3a and 4a respectively.
To build the NS4A cofactor, we superposed the model structures onto the template
structure (1dy8, RMSD 0.3 Å) and built the sequence of the NS4A cofactor for the
model based on the corresponding coordinates found in the template crystal
structure. Similarly, a single zinc ion was manually docked at the cysteine
triad C97, C99, and C145 into the model guided by the corresponding position in
another structure of the template protein (1dxp) in which zinc is present. With
the cofactor and zinc bound, the model was energy minimized using the CCP4
program suite – and the GROMOS96 program, an implementation of the Swiss-pdb
viewer.
The final models were validated using the NIH MBI Laboratory for Structural
Genomics and Proteomics Structural Analysis and Verification Server. This server
utilizes five programs (Procheck, What_Check, ERRAT, Verify_3D, and Prove) to
analyze the stereochemical parameters and the quality of the model.
Additionally, CCP4 programs suite 6.0 was used for the calculation of a
Ramachandran plot, structure superposition, and RMSD value calculation in
addition to the evaluation of the stereochemistry.
## Molecular dynamics simulation
The molecular dynamics simulation (MD) was performed using NAMD 2.9 under the
CHARMM27 force field for proteins. Initially, the 3D structures were solvated
using the solvation tool in VMD. The TIP3P model was used for the water
molecules. Lengevin dynamics for all nonhydrogen atoms with a damping
coefficient of 1 ps<sup>−1</sup> was used in maintaining a constant temperature
of 310 K throughout the system. A constant pressure of 1 atm was maintained
using a Nosé–Hoover Langevin piston with a period of 100 fs and damping
timescale of 50 fs.
Periodic boundary conditions were used on a 61 Å cubic box with the long-range
electrostatics calculated using the particle-mesh Ewald method with a grid point
density of 0.92 Å<sup>−1</sup>. This process ensured that adjacent copies of the
protease were never close enough for short-range interaction. A cut-off of 10 Å
for van der Waals interactions and a switching distance of 8 Å were found to
give convergent results, thus used for production runs. The solvation box was
neutralized, using VMD's Autoionize plugin version 1.3, with sodium chloride
placed at distances greater than 5 Å from the protease.
A time step of 1 fs was used in order to resolve the hydrogen motion of water.
The initial structure was first subjected to three rounds of an 800 cycle
conjugate gradient energy minimization flanked by 100 ps of MD simulation at 278
K. The system was then heated up in increments of 5 K with 100 ps of MD
simulation at each temperature increment until the desired temperature of 310 K
was established (the last heating increment was 2 K). This is an overly cautious
stochastic heating scheme to ensure that the models explore wider space around a
local minimum, given that HCV-3a and 4a proteases are predicted models, not
crystal structures like HCV-1b.
The system was then simulated for 25 ns. Time frames used for the measurements
within the protease were only done for frames where the protease had
equilibrated: 15–25 ns. The equilibrium state of the protease was determined by
the RMSD of the entire protein's backbone. For HCV-3a, longer runs (40 ns) were
performed to ensure equilibration beyond 25 ns.
Multiple copies of each protease, which included the cofactor and a zinc ion
(nonbonded), were run with different initial conditions to ensure that the
results were well converged. All data presented are averaged over six distinct
runs in order to ensure a representative sample of the parameter space the
protease explores.
In order to quantify the relative positions of the three catalytic residues
(H57, D81 and S139) simultaneously, three atoms were used as vertices of a
triangle. The atoms chosen were Oδ2 of D81, Nδ1 of H57 and Oγ of S139. The area
is calculated by where is the vector from Oγ of S139 to Nδ1 of H57 and is the
vector from Oγ of S139 to Oδ2 of D81. The distribution of the area of the
triangle was monitored during the course of the simulation.
We thank Tom Teague for his technical assistance.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: MY EA. Performed the
experiments: MY EA SS MR MI MIA MK DH. Analyzed the data: MY EA MK DH.
Contributed reagents/materials/analysis tools: MY EA. Contributed to the
writing of the manuscript: MY EA SS MR MI MIA MK DH. |
# Introduction
In 1983 Gouras et al. reported an unusual type of retinal dystrophy, which was
associated with characteristic alterations in the rod electroretinogram (ERG).
This rare, autosomal recessive condition has been reported in several further
studies, and was named “cone dystrophy with supernormal rod responses (CDSRR)”.
CDSRR is characterized by an early markedly reduced central visual acuity with
central scotoma, photophobia, severe color disturbances, and occasionally
nystagmus. In contrast to other cone dystrophies, a disease-typical alteration
of the rod system could be observed: while rod sensitivity to weak flashes was
reduced, an augmented responsiveness to higher levels of flash stimuli could be
detected, and implicit times were considerably prolonged. These characteristics
were unique for CDSRR, however, the underlying disease mechanism could not be
elucidated at that time.
In 2006 Wu et al successfully linked the disorder to chromosome 9p24 and the
*KCNV2* gene, which is predominantly expressed in retinal rod and cone
photoreceptors. It encodes a member of voltage gated potassium channels (Kv
channels), representing a silent subunit (Kv8.2) that is able to assemble with
Kv2.1 to form functional heteromeric channels. This results in a shift in the
steady-state activation curve of the Kv2.1 channel towards more negative
potentials due to a permanent outward K<sup>+</sup> current, a lower threshold
potential for activation, a shortened activation time and slower inactivation
kinetics. A mutation in *KCNV2* may thus alter important characteristics of the
I<sub>kx</sub> current that influences the photoreceptor membrane potential.
However, the dysfunction and mechanisms that link *KCNV2* mutations with the
clinical picture still remain to be elucidated.
Over 50 different mutations in *KCNV2* have been reported so far, mainly small
indel mutations or point mutations that constitute protein truncation mutations
and amino acid substitutions. Recently, several large deletions within or of the
*KCNV2* gene of up to 237 kb in size have been described. Although the
genetically detected patients did show altered rod responsiveness, the term
“supernormal rod response” was in many cases deceptive, as previously shown. The
term “supernormal rod ERG” is a misnomer and most recently, the disorder has
been referred to as “KCNV2 retinopathy”.
This study employs detailed psychophysical and electrophysiological testing as
well as spectral domain optical coherence tomography (OCT) and fundus
autofluorescence (FAF) to reveal novel insights into disease-specific functional
changes in KCNV2 retinopathy. Additionally, we explore differences of disease
specific functional aspects in the phenotype that correlate with the underlying
*KCNV2* gene alterations. The genotype of three patients has already been
published elsewhere, the remaining three patients' genetic findings are
presented here for the first time.
# Methods
## Patients
Six otherwise healthy patients of German origin (3 female and 3 male; 2 simplex
cases and 2 sibling pairs; mean age: 39 years, range 28–60 years) with
previously diagnosed stationary retinal disorder and known mutations in the
*KCNV2* gene were examined.
All examinations were carried out after written informed consent and in
accordance with the Declaration of Helsinki. The study was approved by the
Ethics Committee of the Medical Faculty of University of Tübingen.
## Molecular Genetics
Genomic DNA was extracted from venous EDTA-blood samples according to standard
procedures. Genetic testing for point mutations was performed by PCR
amplification and subsequent Sanger sequencing of both coding exons and flanking
intronic sequences of the *KCNV2* gene, as described previously.
Analysis for genomic deletion was investigated by quantitative copy number
analyses of the *KCNV2* gene, with realtime PCR employing TaqMan technology or
SYBR Green detection assays, as reported earlier.
Comparative genome hybridizations (CGH) using a predesigned chromosome 9
specific 385k oligonucleotide array (HG18 CHR9 FT; Roche NimbleGen Inc.,
Madison, WI) was performed for subject CHRO8.I who had suspected deletions at
the *KCNV2* locus (Roche NimbleGen).
The deletion junctions in patients CHRO8.I and RCD307 were determined by long
distance PCR amplifications and subsequent Sanger sequencing to define the
precise breakpoints.
Independent segregation of the mutations within the families were conducted by
Sanger sequencing of PCR amplified genomic DNA for point mutations, and by qPCR
in the two families segregating the *KCNV2* gene deletion.
## Clinical Examination
A complete ophthalmological examination was performed including psychophysical
tests (Snellen visual acuity, Lanthony Panel D-15 and Nagel anomaloscope color
vision tests, visual field and dark adaptation) and an extended
electrophysiological protocol (Ganzfeld and multifocal ERG).
## Psychophysical testing
Kinetic 90° and static 30° visual field tests were carried out with an Octopus
900 perimeter (Haag-Streit International, Germany). Dark adaptation curves were
measured with a dark adaptometer (Roland Consult GmbH, Brandenburg, Germany)
after pupil dilation with Tropicamid. After 3 minutes of bleaching with bright
white light (intensity 5.5 log photopic trolands), a staircase procedure was
used to estimate detection thresholds over a period of 40 minutes. Thresholds
were alternately measured for red (635 nm) and green (565 nm) circular targets,
presented 20° nasal of the fovea. Cone and rod thresholds were then determined
by a model fit using the equation:with *t<sub>k</sub>* describing the time to
the rod-cone break and *I*1, *R*1 the exponential decay of the cone, *I*2, *R*2
of the rod thresholds. Cone and rod parameters are derived from red and green
target functions respectively. For group analysis and for comparison to normals
the model was fitted to the raw data of each group of subjects.
## Electrophysiological testing
Ganzfeld and multifocal electroretinograms ERGs were recorded according to the
standards of the International Society for Clinical Electrophysiology of Vision
(ISCEV). All tests were performed using DTL electrodes with an Espion
E<sup>2</sup> (Diagnosys LLC) recording device coupled with a ColorDome
(Diagnosys LLC) as light source. After 30 minutes of dark adaptation a series of
responses to increasing flash intensities (4 ms–0.0001 cd.s/m<sup>2</sup> to 10
cd.s/m<sup>2</sup> in 0.5 log unit steps) were recorded and the stimulus-
response (S-R) functions modelled using the equation:with the saturated b-wave
amplitude *V<sub>max</sub>*, the flash intensity *K* required for semi-
saturation as a measure of retinal sensitivity and the slope related exponent
*n*.
Rod response characteristics were estimated from the a-wave by the Hood and
Birch (1994) formulation of the Lamb and Pugh model of the biochemical processes
involved in the activation of rod phototransduction. The a-wave ensemble was
fitted with a computational model describing the response (*P<sub>III</sub>*) as
a function of time (*t*) and intensity (*I*):where *Rm<sub>pIII</sub>* is the
maximum amplitude, *S* is a sensitivity variable and *t<sub>d</sub>* is a brief
delay before the response onset.
*P<sub>III</sub>* was then subtracted from the original ERG waveform to give the
*P<sub>II</sub>* response, which is thought to represent mainly the ON-bipolar
cell response, but also the postreceptoral activity in other second- and third-
order retinal neurons. The relation between flash intensity and the delay
between stimulus onset and reaching a given arbitrary criterion voltage of the
*P<sub>II</sub>* component was then plotted on a log-log coordinate and the
slope of this function was calculated. The voltage criterion chosen in this
study was 50 µV.
Finally, dark-adapted responses to a series of blue flicker (LED 470 nm) with an
intensity of 0.03 cd.s/m<sup>2</sup> and frequencies between 5 and 30 Hz were
recorded to isolate temporal retinal characteristics of the rod system.
The light-adapted protocol (10 min of light adaptation to a background luminance
of 30 cd/m<sup>2</sup>) included a single flash cone stimulus and a 30 Hz
flicker (both: 4 ms, 3.0 cd.s/m<sup>2</sup>). In addition, responses to a series
of flicker white stimuli of 3.0 cd.s/m<sup>2</sup> with increasing frequency
from 5 to 45 Hz were included to investigate possible alterations in the
temporal resolution of the cone retinal pathway.
Multifocal ERG (mfERG) was performed with a VERIS System (Version 5.1) using a
Grass amplifier (model 12, Quincy, USA). The stimulus, consisting of 61 scaled
hexagonal elements covering a central visual field of 60×55°, was presented on a
19″ monitor at a frame rate of 75 Hz at a distance of 32 cm from the subject's
eyes. The same DTL electrodes as those for the Ganzfeld recordings were used.
Responses were amplified (200 000×), bandpass-filtered (10–100 Hz), and analysed
according to ring averages.
## Morphological testing
Color and infrared fundus photography, autofluorescence (FAF) and spectral
domain OCT recordings (Heidelberg Engineering GmbH, Germany) were performed.
# Results
## Molecular Genetic Findings
Mutation screening and segregation analysis led to the identification of
mutations in the *KCNV2* gene in our patients. The genotypes of the six patients
are listed in. and the mutation localization is shown in. We observed compound
heterozygous mutations in the two sib pairs (CHRO8.I and CHRO8.II, and BD27.I
and BD27.II): both patients from family CHRO8 carried two compound heterozygous
nonsense mutations p.Cys113stop and p.Glu148stop, while both patients of family
BD27 were compound heterozygous for a complete deletion of the *KCNV2* gene and
a missense mutation p.Leu404Pro located in the linker between transmembrane
domains S4 and S5. The simplex subject BCM5 harboured two compound heterozygous
small deletions: c.8_11del and c.447_449del. The c.8_11del mutation created a
frame-shift at the very beginning of the *KCNV2* polypeptide, resulting in a
premature stop codon and a severely altered and truncated protein
(p.Lys3ArgfsX95). The other deletion only resulted in the loss of a single
phenylalanine at position 150 (p.Phe150del) within the NAB domain. The last
patient RCD307 was homozygous for another large deletion spanning from exon 1
into the 3′UTR. All mutations, except for the missense mutation p.Leu404Pro and
the single amino acid deletion p.Phe150del (see.), are expected to result in the
complete loss of Kv8.2. Consequently the siblings BD27.I and BD27.II, and
patient RCD307 most likely did not express any KV8.2 gene product, causing an
altered subunit composition of the respective Kv-channel and an altered or lost
potassium channel function. Previous clinical data have indicated that both the
complete absence of Kv8.2 (where Kv2.1 was unaffected) and its altered forms
result in CDSRR, which suggests that the special constellation of Kv2.1/Kv8.2
heteromeric channels are essential for functionality in the photoreceptor cells.
To examine whether these genotype differences are also evident in the phenotype,
we divided the six patients into two groups: group 1, NOP (no protein), included
the three patients with a complete absence of Kv8.2, and group 2, ALP (altered
protein) with three patients with mutant Kv8.2 subunits.
## Clinical Findings
Clinical findings are summarized in. All patients reported an early onset of
their visual symptoms without any progression or change over the years. Only the
oldest patient (RCD307) reported a slight reduction of his visual acuity in the
last three years. Marked photophobia and a prolonged light adaptation time were
evident in every case. Only the oldest patient complained of nyctalopia, other
subjects denied having difficulty with night vision. All patients were myopic
with variable degrees of astigmatism; patients of the ALP group showed a slight
tendency for higher myopia and astigmatism. Four of six patients had undergone
strabological surgery in childhood and two of them had suffered additionally
from infantile nystagmus.
## Psychophysics
All subjects presented with reduced central visual acuity (mean VA (logMAR):
0.97±0.2 SD). There was a slight tendency to poorer VA in the NOP group
(1.06±0.23 SD) compared the ALP group (0.88±0.13 SD). Color vision testing using
the Lanthony D-15 Panel desaturated and saturated tests and with fixation with
the preferred retinal locus (PRL) showed severe color confusions in all patients
predominantly along the scotopic or red-green axis with relative sparing of the
tritan axis. The Rayleigh anomaloscope matches, presented eccentrically at the
PRL, were consistent with a rather rod dominated function in five of six
patients. Only one patient's (RCD307) results suggested protanopia. Perimetric
results showed nearly normal outer boundaries of the visual field in all cases.
Static perimetry results revealed relative and absolute defects in the central
30° area, being more pronounced in the NOP than the ALP group.
To test cone and rod function loss we measured dark adaptation thresholds for
red and green targets.. All patients showed significantly elevated thresholds
for red and green stimuli, although the elevation was more pronounced in the NOP
group with a final rod threshold of −1.9 log cd/m<sup>2</sup> and a cone
threshold of 0.4 log cd/m<sup>2</sup> compared to −2.6 log cd/m<sup>2</sup> and
−0.3 log cd/m<sup>2</sup> for the rod and cone threshold in the ALP group.
Thresholds estimated for normals were −1.5 log cd/m<sup>2</sup> and −3.7 log
cd/m<sup>2</sup> for red and green stimuli, respectively. For green stimuli the
rod-cone break was normal (11.0 min for the NOP and ALP group, normal: 10.7 min)
and for red stimuli the rod-cone break tended to appear earlier (after 15.6 and
15.9 min for NOP and ALP respectively, normal: 16.9 min).
## Electrophysiology
Basic clinical investigation included the Ganzfeld ERG according to the ISCEV
standard, for which all patients showed the previously described characteristic
responses (see for typical results of a patient from the NOP and ALP group).
Most interestingly, oscillatory potentials (OPs) were almost completely absent
in the patients' ERG recordings. shows for each subject the amplitudes and
implicit times of the a- and b-waves for the scotopic response series
recordings. Typical low or undetectable response amplitudes to weak flashes were
evident, with markedly delayed implicit times of the a- and b-wave component.
There was also an abrupt increase in amplitude with increasing flash intensity
accompanied by a normalization of b-wave implicit times. While mean saturation
amplitudes *V<sub>max</sub>* of the b-wave model fit were similar for both
groups and normals (526 µV for normals, 528 and 493 µV for the NOP and ALP
groups, respectively) the intensity *K* at semi-saturation was significantly
shifted to higher intensities (−2.5, −1.7 and −1.7 log cd.s/m<sup>2</sup> for
normals, NOP and ALP, respectively). Additionally, the peak a-wave amplitude was
normal within the entire stimulus intensity range, but the peak implicit times
were prolonged for each stimulus step.
In this study the rod a-wave showed three interesting features (.): First, in
some patients the a-wave of the response to the highest intensity stimulus (4.7
log td\*s) was smaller than that to the 4.2 log td\*s stimulus. This is depicted
for one subject in. While R<sup>2</sup> of the fits in normal subjects was above
0.02 in only two eyes, it was higher in seven of the patient eyes. The
difference in goodness of the fit is seen in the normal subject in and in the
representative patient BCM5 in.
Second, the latency of the negative deflection from baseline appeared with
normal delay (average of 4.1±0.92 ms and 4.3±0.35 ms for patients and normals
respectively).
And third, while the maximum response amplitude Rm<sub>PIII</sub> estimated from
the model fit was not different from that of normals (on average 241±71 µV and
270±83 µV for patients and normals respectively), the sensitivity parameter S
was significantly lower in patients (0.73±0.38 SD) than in normals (1.14±0.36
SD).
*P<sub>II</sub>* responses were calculated by removal of the fitted
*P<sub>III</sub>* (.). The latency at which *P<sub>II</sub>* reaches 50 µV is
plotted as a function of stimulus intensity on log-log coordinates in. In the
normal retina we found a slope of −0.18±0.014 SD. In patients with *KCNV2*
mutations the mean slopes of regression lines were not significantly different
from each other or from the normal mean slope (−0.24±0.065 SD and −0.20±0.038 SD
for the NOP and ALP group, respectively), however, a similar and clear shift
either representing a response delay or a horizontal shift to higher intensities
of approx. 1 log unit was observed in both groups. Latter was consistent with
the psychophysically (dark adaptation) estimated rod threshold elevation of
approx. 1 log unit.
We additionally recorded responses to various flicker frequencies under scotopic
conditions. Magnitude and phase of the responses to increasing flicker
frequencies are demonstrated. The individual data under scotopic conditions
showed reduced magnitudes and a phase difference, which was independent from
flicker frequency thus suggesting either a rather small constant prolongation of
rod photoreceptor recovery or may result from the sensitivity reduction to the
flash strength.
Cone responses recorded under photopic conditions showed a marked suppression of
response amplitudes, which remained diminished even at the higher flash
intensities (.). Additionally, the amplitudes did not show a photopic hill
phenomenon. Implicit times were markedly prolonged for each stimulus step. The
photopic negative responses (PhNR) were almost undetectable in patients with
*KCNV2* mutations, as depicted in.
As found for scotopic conditions, the photopic responses to stimuli of
increasing frequency (.) showed a similar dependency but also lower amplitudes
and a shift in phase. Interestingly, even at the highest frequency of 45 Hz the
response waveform was still significant with an almost normal phase.
Finally, multifocal ERGs showed reduced amplitudes and delayed implicit times in
every ring (.) being more distinct in the central 2 rings. In the outer rings
more preserved responses could be obtained. The dysfunction was more sharply
limited to the central two rings in the NOP group, which was not observed that
clearly in the ALP group. This could be related to the slightly better VA of the
ALP group.
## Morphology
Fundus photographs, FAF images and OCT scans for each subject can be seen in.
There was a range of macular appearances including discrete disturbances of the
retinal pigment epithelium (RPE) and bull's eye maculopathy. Patients with a
complete absence of Kv8.2 (NOP group) showed more pronounced changes in the
macular area, the mean central retinal thickness was 103.5 µm for the NOP, and
135.1 µm for the ALP group, respectively. In FAF imaging mild RPE-alterations
were present as small areas with decreased autofluorescence. In contrast, marked
RPE-atrophies were seen as sharply demarcated areas of absent autofluorescence
surrounded by a ring of increased signal. The oldest patient (RCD307) revealed
additional RPE-defects of the posterior pole and epiretinal gliosis. The OCT
images also demonstrated the variety of morphological findings. In the milder
cases –mainly in the ALP group- OCT revealed a thinner photoreceptor layer (PRL)
in the foveal area, there was no disruption in the inner segment/outer segment
(IS/OS) border. In the more severe cases (NOP group) the PRL was missing, the
IS/OS border was diminished and an increased backscatter from the choroid was
observed due to RPE-atrophy. In one case additional granular echoes were present
due to deposits on the fundus of patient BD27.I. Based on the SD-OCT volume
scans detected from the central 30°×15° retinal area the peripheral outer
retinal structure was well preserved in every patient.
# Discussion
This study describes the genotype and phenotype of six patients with a retinal
dystrophy due to changes in the *KCNV2* gene. In three patients the mutations
resulted in a complete absence of Kv8.2 encoded by the *KCNV2* gene (NOP group).
In the other three patients heterozygous mutations of the first allele resulted
in a lack of protein product, mutations of the second allele led to mutant
subunits with presumably remaining pore function (ALP group).
On the morphological level, macular pathology varied from mild RPE disturbances
to large atrophic areas, while the periphery was normal in every case. These
findings are consistent with the results described by Robson et al. and
Sergouniotis et al., It is interesting to see that the changes are strictly
limited to the central macular area in every case, while the specific functional
changes of this retinal disorder affect rod-rich mid periphery as well. The
question of why cones degenerate and rods keep at least their morphological
integrity still remains to be unraveled, but the distribution of Müller cells in
the retina (i.e. absence in the fovea) could be one important factor, since
their regulatory and buffering effect on the extracellular K<sup>+</sup> is
missing in the cone-rich area, making cones more vulnerable.
Although there was a tendency for a more pronounced macular lesion (seen in the
OCT and FAF imaging), higher myopia and more elevated dark adaptation thresholds
in the NOP group, no clear-cut line can be drawn between the two groups.
Interestingly, the only homozygous patient (RCD307) seemed to differ in a few
aspects from the heterozygous patients: night blindness, protanomaly and
reported progression were present only in his case. There was no family history
of color disturbances in this subject and the further genetic analysis of the
L-M pigment genes showed an intact OPN1MW/OPN1LW gene cluster, indicating a
severe protanomaly rather than protanopia. Furthermore, his morphological
results revealed more distinct changes of the fovea resembling a macular hole on
the left eye. However, the additional RPE-defects of the posterior pole, the
macular hole formation and the accompanying epiretinal gliosis could be due to
age related changes, explaining the decreasing visual acuity observed by the
60-year-old patient. However, the investigated cohort is small, further large
studies are necessary to better highlight the correlations between genotype and
phenotype.
Several studies exist on the electrophysiological characteristics of KCNV2
retinopathy.. Nevertheless, there are still several aspects of this special
retinal disorder, for which an explanation is needed
The lack of Kv8.2 or the presence of mutant subunits eliminates the functional
characteristics of Kv2.1/Kv8.2 heteromers leading to a retinal disorder. Only
the intact heteromers have the essential specifics to function as a high-pass
amplifier and so regulate photoreceptor responses to light flashes. Kv2.1 can
form homomeric channels, but without the intact Kv8.2 subunits they activate
more slowly and inactivate faster, whereas the voltage dependence of the steady-
state inactivation remains unchanged.
The absence of intact Kv8.2 subunits therefore leads to a positive shift of the
steady-state membrane potential, decreasing the dark current and elevating
intracellular K<sup>+</sup> level. Kv2.1 channels alone do not produce a
permanent outward K<sup>+</sup> current, which also affects the K<sup>+</sup>
homeostasis of the photoreceptors. While restoring the integrity of the
K<sup>+</sup> levels, secondary mechanisms can also lead to a small drop of
intracellular Ca<sup>2+</sup> levels, probably due to altered/enhanced function
of the Na<sup>+</sup>/Ca2<sup>+</sup>-K<sup>+</sup> exchanger. The small drop of
cytoplasmic Ca<sup>2+</sup>, however, can result in increasing cGMP levels due
to disinhibiting the activated guanylate-cyclase and finally increases the
number of open cyclic nucleotide-gated channels. This shift to a more
depolarized state in the dark may also have consequences for recovery: for very
brief light flashes the membrane potential may remain below the critical limit
of −50 mV before the hyperpolarization-activated cyclic nucleotide-gated
channels (HCN channels) become activated, thus leading to a prolonged
hyperpolarization phase. These mechanisms are well reflected in the
electrophysiological findings observed in patients with KCNV2 retinopathy. The
responses are characteristically undetectable or markedly reduced with delayed
implicit times for dimmer stimuli and there is an abrupt rise in amplitudes and
shortening of implicit time with increasing stimulus intensity. The intensity
*K* at semi-saturation is significantly shifted to higher intensities, the
estimated difference in our cohort is around 1 log cd.s/m<sup>2</sup>. This
shift correlates with the threshold elevation during dark adaptometry (for rod
thresholds approximately 1 log unit elevation). However, most of our patients do
not suffer from nyctalopia, confirming other reports of this inconsistency
between subjective and objective light sensation.
Our results are in accordance with previous reports and also confirm that
“supernormal rod responses” in the ERG - believed to be characteristic for this
rare condition - often seem to be missing, as responses, even to high intensity
flashes, stay within normal limits in many cases. The dynamics of the b-wave
intensity-response function is a more constant feature.
In addition, our detailed electrophysiological data show other specific
features. The initial phase or leading edge of scotopic response waveforms
reflects the activity of photoreceptor cells and arises from light-evoked
closure of Na<sup>+</sup> channels along the plasma membrane of the outer
segments. Based on the model fits to our electrophysiological data we conclude
that phototransduction activation in this retinal dystrophy is normal, since the
onset of the deflection from baseline appears with normal delay. While the
maximum amplitude Rm<sub>PIII</sub> was within normal limits, the sensitivity
parameter S was significantly lower. Similar results were reported for the
patient described by Tanimoto et al.. On the contrary, Hood et al. found
essentially normal sensitivity S and a slightly lower Rm<sub>PIII</sub>, the
maximum amplitude, for the rods. However, our patients were included in the
study based on confirmed alterations of *KCNV2*, while patients in the study of
Hood et al. were chosen on a clinical-electrophysiological basis (i.e. retinal
dystrophy with supernormal rod responses). While higher Rm<sub>PIII</sub> might
be explained by an overshoot due to delayed HCN channel activation, lower
sensitivity may be related to higher cation channel sensitivity to cGMP due to
lower Ca<sup>2+</sup> levels.
There is also a delayed postreceptoral response, which seems independent from
flicker frequency. The delay of the emerging b-wave may have two different
origins. Firstly, voltage dependent transmitter release may be delayed due to a
small prolongation in reaching hyperpolarization. Secondly, a delayed HCN
channel activation and depolarization of the photoreceptor might result in an
overshoot of the response of the downstream neuron. Such overshoot would not be
associated with changes in the G-protein activation cascade of the bipolar cell,
which can be assessed with the *P<sub>II</sub>* response analysis. In our
*KCNV2* patients the kinetics of the ON-bipolar cell G-protein cascade seems to
be normal, however, the cascade is activated with a clear delay. In the study of
Robson et al. including 25 patients with the characteristic scotopic b-wave
signs, this delay was seen for the ON-responses and particularly for the OFF-
responses as well. A further interesting finding was the marked reduction or
even absence of the oscillatory potentials, either suggesting an altered
function of the inner retina (i.e. amacrine or interplexiform cells) or more
likely a significantly reduced cone function.
Moreover, we studied the temporal characteristics of the retina for the first
time in this specific retinal disorder. Repetitive stimulation is very demanding
for the metabolic process in neurons and changes in the temporal dynamics (e.g.
in channelopathies) are evident in flicker ERGs. The impairment of temporal
response characteristics can occur due to photoreceptor disturbances as well as
postsynaptic mechanisms. Kv2.1/Kv8.2 heteromeric channels contribute to the
generation of the K<sup>+</sup> current responsible for the dynamic signal
amplification of photoreceptors. The hyperpolarizing overshoot in response to
rapid onset illumination has an important role in increasing the sensitivity to
fast changes of illumination. Altered Kv2.1/Kv8.2 heteromers lose their ability
to function as a high-pass amplifier, which explains the altered temporal
characteristics observed in our patients (i.e. there is a constant prolongation
of photoreceptor recovery time). Interestingly, even at the highest frequency of
45 Hz the response waveform was still significant with an almost normal phase,
which indicates that even though we find clearly reduced amplitudes and a phase
shift due to cone dystrophy, the temporal dynamics seem to be almost normal.
There is little known about critical flicker fusion in retinal dystrophies in
humans, but it has been shown in RCS rats that with progression of the
degeneration the amplitude for higher frequency waveforms declines and the
critical flicker fusion frequency is shifted to lower frequencies. However,
Kv2.1/Kv8.2 heteromeric channels are not the only components of the outward
K<sup>+</sup> current in photoreceptors, although they are essential for their
functional properties.
These special electrophysiological features are considered to be strictly
associated with *KCNV2* mutations, but recently Thompson et al. have shown
somewhat similar changes of the dark-adapted electroretinogram in patients with
*KCNJ10* mutations. The K<sup>+</sup> channel expressed by the *KCNJ10* gene
(Kir4.1) has previously been recognized as pathogenic in man, causing a
constellation of symptoms, including epilepsy, ataxia, sensorineural deafness
and a renal tubulopathy (EAST syndrome). Kir4.1 constitutes the primary inward
rectifying potassium channel of retinal Müller cells and is responsible for the
regulation of extracellular K<sup>+</sup>. Thompson et al. nicely demonstrated
similar dynamics of scotopic intensity-response function in two of four
patients. ERGs to dimmer flash stimuli showed a delay of up to 20 ms before the
onset of the b-wave, and with increasing intensity a sudden elevation of
amplitudes and normal implicit times could be detected. These similarities of
the scotopic ERG could be explained with an altered sensitivity at the synapse
between rod and ON-bipolar cells due to mutations in *KCNJ10*. Photopic ERGs of
all patients showed reduced amplitudes of the photopic negative response (PhNR)
and showed a delay in b-wave time to peak, but the photopic hill was preserved.
However, these patients did not develop a cone dystrophy, since mutations in
*KCNJ10* primarily affect the Müller cell functions, while mutations in *KCNV2*
lead to disturbed functional integrity of the photoreceptors and probably impair
their postreceptoral signaling.
KCNV2 retinopathy is considered a very rare retinal disorder associated with
high but often normal mixed rod-cone response amplitudes, a marked prolongation
of b-wave implicit times and a delayed, almost sudden, steep amplitude-versus-
intensity relationship under scotopic conditions. Furthermore, while rod
phototransduction is intact, there is a constant delay of the responses, which
suggests changes in the synapse or in postreceptoral signaling pathway. Inner
retinal involvement is also probable, since oscillatory potentials are almost
absent. These findings are diagnostic and are exclusively linked to *KCNV2*
mutations.
[^1]: The authors have declared that no competing interests exist.
[^2]: Performed the experiments: DZ SK BW EZ HJ. Analyzed the data: DZ SK BW
EZ HJ. Contributed reagents/materials/analysis tools: DZ SK BW EZ HJ. Wrote
the paper: DZ. corrected the manuscript: SK BW EZ HJ. |
# Introduction
2-Hydroxypropyl-Beta-Cyclodextrin (HPβCD) is a commonly used excipient to
stabilize and solubilize pharmaceuticals. HPβCD reduces cholesterol and lipid
accumulation, and has emerged as a possible treatment for Niemann-Pick type C
disease, Alzheimer’s disease, and atherosclerosis. But, a negative side effect
of HPβCD-based treatments is hearing loss. Recent studies on the chronic effects
of HPβCD administered systemically or directly into cerebrospinal fluid found
graduated losses of outer hair cells (OHC) along the cochlear spiral, with more
severe losses at the cochlear base than the apex. While the origins of HPβCD
toxicity within the ear remain uncertain, its effects can modulate cochlear or
OHC electromechanics in excised cochleae. In experiments reported here we used a
variety of physiological measurements to understand the effects of acute
administration of HPβCD, and HPβCD analog methyl-β-cyclodextrin (MβCD), directly
into cochlear perilymph. Results were compared to measurements made during
treatment with salicylate, which has effects that have been well characterized,
and the effects of injecting artificial perilymph alone.
# Methods
## Animal preparation
We used NIH-strain pigmented guinea pigs of either sex (between 400–600 g).
Initially animals were anesthetized with an intraperitoneal injection of sodium
thiobutabarbital (100 mg/kg). Cutting shears were used to shave head and neck
fur. A tracheotomy was performed, and the animal was artificially ventilated
with isofluorane (\~1% in oxygen) with respiratory volume maintained (5% end-
tidal CO<sub>2</sub>). We monitored heart rate, O<sub>2</sub> saturation, and
expired CO<sub>2</sub> level with a pulseoximeter/CO<sub>2</sub> analyzer. The
right cochlea was accessed with a ventral surgical approach. Soft tissue of the
right ear canal was removed. The animal was mounted with hollow ear bars that
allowed delivery of acoustic stimuli. A cannula placed in the left jugular vein
was used to administer pancuronium bromide (0.06 mg/kg) to eliminate middle-ear
muscle contractions just before the start of making auditory measurements. Body
temperature was maintained (38°C) with a dc-powered heating blanket and rectal
thermometer system. Experimental protocols for this study were approved by the
Animal Studies Committee of Washington University (protocol numbers 20120113 and
20130069).
## Solution administration by injection into the cochlear apex
Ototoxic solutions were administered from a pipette sealed into the cochlear
apex. Previously we have shown that apical injection drives solutions toward the
cochlear aqueduct at the base of scala tympani, allowing the entire scala to be
uniformly treated. This overcomes the limitations of classic administration
techniques to the cochlear base that do not reach therapeutic levels at the
regions tuned to mid to low frequencies that code speech vowels and background
noise. Administering into the cochlear base is certainly not the ideal approach
to understand how HPβCD affects the entire cochlear length. Solutions injected
into the cochlear apex can be administered slowly if the goal of the experiment
is to sequentially affect finely spaced cochlear regions contributing to a
response. For the experiments reported here, we used a constant, relatively fast
(0.5 μL/min) injection rate.
Animals received either artificial perilymph alone (controls, n = 3 guinea pigs)
or, in artificial perilymph, 20 mM salicylate (n = 3 guinea pigs), 13 mM HPβCD
(n = 3 guinea pigs), 27 mM HPβCD (n = 3 guinea pigs), or 13 mM MβCD (n = 3
guinea pigs). The composition (in mM) of artificial perilymph was NaCl (127.5),
KCl (3.5), NaHCO<sub>3</sub> (25), CaCl<sub>2</sub> (1.3), MgCl<sub>2</sub>
(1.2), NaH2PO<sub>4</sub> (0.75), and Glucose (11). Animals treated with 13 mM
HPβCD were in the “low-dose” group and those treated with 27 mM HPβCD were in
the “high-dose” group. Salicylate alters surface cisternal system of hair cell
bodies, reduces turgor pressure, and effects OHC motility. In short, salicylate
attenuates the gain of the cochlear amplifier. Solutions were injected from a
pipette sealed into the cochlear apex in the 4<sup>th</sup> cochlear turn. The
mucosa covering the apex was removed with a damp, cotton wrapped applicator or
cellulose wipe. Cyanoacrylate glue was applied to the dry bone of the apex, then
a thin layer two-part silicone elastomer was applied. This effectively made the
surface hydrophobic. The cochlear apex was fenestrated through the adhesive
layers on the bone. The fenestration was made with a 30º, 1/3mm oval window pick
by resting the pick on the surface at one location and then lifting the pick off
the surface. The fenestra diameter was made to fit a 20–30 μm diameter tip
pipette that was pulled from 100 mm x 0.58 mm inner-diameter glass tubing. We
made a fluid-tight seal between the glass injection pipette and the hydrophobic
surface by wicking fluid (accumulated from either condensation or cochlear fluid
accumulation) and applying additional cyanoacrylate glue. Solutions were driven
at 500 nL/min for 15 min for a total of 7500 nL through a 50 μL Hamilton gas-
tight syringe (1710TLL), glued to a World Precision Instruments plexiglass
coupler (MPH6S10), mounted on a computer-controlled World Precision Instruments
Ultrapump.
## Acoustic stimuli and physiologic measurements
Electrophysiologic measurements were made using procedures that we have
previously described. Measurements were made with Tucker-Davis System 3 hardware
controlled by custom-written software in Visual Basic (Microsoft) on a personal
computer. TD-RP2 modules were used for stimulus generation. Stimuli were passed
through TD-PA5 attenuators, and TD-HB7 headphone amplifiers. Acoustic stimuli
were delivered in a closed sound system: an Etymotic ER-10C coupled to the
hollow ear bar. Calibrations were completed in individual ears by tracking 70 dB
SPL tones from 125 Hz to 26 kHz in ¼ octave steps. Cochlear response
measurements were made differentially between an Ag/AgCl electrode in the round
window niche and a platinum-needle electrode in the vertex. Measurements were
made with an optically-coupled TD-HB7amplifier (1000X gain, 0.005–15 kHz
bandpass filter), routed to TD-RP2 modules for digitization (48.8 kHz) and
averaging. Animals were electrically grounded with an Ag/AgCl pellet electrode
coupled to the exposed soft tissue of the neck by a fluid bridge.
## Histological preparation
At the conclusion of the apical injection experiments, the experimental cochleae
were prepared for fixative injection. The pipette used to inject ototoxins was
removed, and the fenestration was occasionally enlarged to a small extent to
accept a 20–30 μm diameter tip pipette pulled from 100 mm x 0.58 mm inner-
diameter glass tubing used to inject fixative. The pipette for fixative was
sealed into the cochlea by wicking away any cochlear or condensation fluid
accumulation while applying cyanoacrylate glue to form a fluid-tight junction.
The round window membrane was perforated before the start of fixative injection.
We injected 2.5% glutaraldehyde and 1.5% paraformaldehyde in a 0.065 M phosphate
buffer. This solution was injected at 2000 nL / min for a total of 7500 nL.
Cochleae were extracted and placed in fixative solution for at least two days at
4°C. Cochleae were decalcified (0.1 M EDTA with 0.4% glutaradehyde) for 14 days,
osmicated (1% OsO<sub>4</sub> in dH<sub>2</sub>O) for 60 minutes, dehydrated in
ethanols and propylene oxide, embedded in Araldite resins, and sectioned
parallel to the spiral axis of the fourth cochlear turn at 40 μM with a carbide
steel knife. Sections were mounted in Permount on microscope slides and cover-
slipped. Sections were analyzed with light microscopy by an author who was
blinded to the treatment each ear received.
# Results
The time course of cochlear action potential (CAP) threshold shifts differed
across treatments and tone-burst frequencies. The ≤10 dB threshold shifts
occurring 30 minutes after the start of injecting artificial perilymph alone
(control experiments) quantify the extent to which the apical injection
procedure itself influenced neural thresholds. The direction and rate (dB / min)
of CAP threshold shifts differed between HPβCD 13 and 27 mM treatments, in that
threshold shifts essentially raised and then plateaued for 13 mM treatment or,
in contrast, steadily increased to a maximum or to the abolition of CAPs for 27
mM treatment. Quick CAP threshold shifts followed by gradual CAP recovery is
consistent with the well-known temporary effects of salicylate (e.g.. Maximal
effects of salicylate and 27 mM HPβCD on CAP thresholds are consistent with
elimination of cochlear amplifier gain, as targeted deletion of prestin in mice
raise neural threshold by 40 to 60 dB. Average threshold shifts of 20 to 40 dB
from 13 mM HPβCD are far greater than the 6 dB threshold shifts caused by
heterozygote prestin knockout mice when electromotility is halved. Total CAP
abolition during, or soon after, MβCD treatment is itself an indication of far
greater effects than simple cochlear amplifier gain attenuation (see the
histological data from these ears presented later). In normal ears CAP to low-
level tone bursts originate from the peak of the traveling wave (reviewed in).
Across all toxic treatments, CAP thresholds to 2, 4, and 8 kHz started to rise
soon after the injection start (i.e., zero minutes re. injection start), but
\~3–8 additional minutes was needed to start abolishing CAP thresholds to 16
kHz. This demonstrates that we did not simultaneously treat the length of the
cochlea. Comparing maximal effects on CAP threshold shifts in helps to
understand the extent to which cochlear amplifier gain was attenuated across
treatments.
Auditory Nerve Overlapped Waveform (ANOW) measurements show that the apical
injection technique can treat the apical half of the cochlear spiral, a region
that classic round window administration cannot. The ANOW originates from
afferent auditory nerve fibers in the apical cochlear half and can quantify low-
frequency auditory thresholds. ANOW amplitudes from supra-threshold, 50 dB SPL
sound levels were ablated by salicylate, HPβCD, and MβCD treatments, but not by
artificial perilymph alone (control;). Subtle changes to ANOW from control ears
during the time of injection suggest that perhaps some ANOW changes in ears
treated with toxic solutions resulted from mechanical disturbances from the fast
apical injection approach used for these experiments. Nevertheless, unlike CAPs,
ANOW amplitude measurements were fully abolished with all four treatments,
consistent with the ANOW being more sensitive than CAPs to cochlear
manipulations and diseased states.
Cochlear microphonic (CM) measurements to 90 dB SPL 500 Hz tone bursts were made
from inside the endolymphatic space where responses are not influenced by neural
excitation to the sound used to evoke the desired hair-cell-based response.
Slight transient changes in CM amplitude from control, 13 mM HPβCD, and
salicylate treatment are consistent with slight mechanical disruption caused by
the relatively fast injection rate that was used here. The CM remaining after
treatment with 27 mM HPβCD originates from current flow through remaining OHC
and IHC, perhaps IHCs more than OHCs (as suggested from histological data
below). Total abolition of CM measurements occurred for MβCD.
Endocochlear potential (EP) measurements were made in the third cochlear turn.
Artificial perilymph injection (control) did not markedly affect the EP.
Treatment with 13 mM HPβCD and salicylate caused transient and temporary
effects: 13 mM HPβCD caused EP to increase during injection and then returned to
pre-injection values during the time immediately after injection while, in
contrast, salicylate caused EP to decrease before gradually returning toward
pre-injection values. Treatment with 27 mM HPβCD moderately affected the EP,
causing transient increases during injection that returned to pre-injection
values during the time immediately after injection before markedly declining
further. MβCD caused a brief increase in EP then total abolition before the
injection was complete. EP changes are mirrored by changes in the magnitude of
the silent current, e.g., an EP decrease is consistent with a decrease in the
standing current through outer hair cells in silence. The time courses of
effects on EP during treatment are not identical to the time courses of effects
on CM (cf. Figs), which is consistent with the origin of these measurements
being different even though they were made with the same electrode in the
endolymphatic space of the third cochlear turn.
Cubic DPOAE amplitudes were measured at 2*f*<sub>1</sub>- *f*<sub>2</sub> with
an arbitrarily chosen *f*<sub>*2*</sub> ≈ 6 kHz. Individual-ear DPOAE amplitude
fine structure was considered by choosing the *f*<sub>*2*</sub> primary-tone
frequencies closest to 6 kHz that produced a peak in the 2*f*<sub>1</sub>-
*f*<sub>2</sub> DPOAE amplitude. Injection of artificial perilymph did not
affect DPOAE amplitudes in control ears. With 13 mM HPβCD treatment, DPOAE
amplitudes dramatically declined during the injection but gradually recovered
toward near pre-injection levels. With salicylate and 27 mM HPβCD treatments,
effects on DPOAE amplitude were longer, and did not fully recover. MβCD was the
only treatment that totally abolished DPOAE amplitudes.
Sensory cells within the organ of Corti were evaluated using light microscopy
and serial sections of plastic embedded cochleae. All sensory and non-sensory
cells were well preserved in the control and 13 mM HPβCD groups. Effects from
treatment with 27 mM HPβCD were variable, ranging from well-preserved to
severely damaged outer and inner hair cells. Ears with severe IHC and OHC damage
had greater effects at the base than apex. While some of this variability in OHC
loss may be attributable to the relatively short time-frame of our acute study,
interanimal variability is nevertheless consistent with that reported from
Crumling et al. and Cronin et al. who studied the chronic effects of HPβCD
treatment. Treatment with MβCD consistently caused severe damage directly to the
OHCs and IHCs or the regions around these sensory cells. During the time course
of these experiments with HPβCD, MβCD, or artificial perilymph (controls),
lateral wall structures, including the spiral ligament fibrocytes, stria
vascularis, spiral ganglion neurons did not appear to be altered. Specific
morphometry was not performed in these structures, but there was no evidence of
stria swelling or fluid leakage, the spiral ligament fibrocytes populated the
ligament as in control ears, and there were no signs of myelin unwrapping from
the spiral ganglion cell soma or evidence of cell swelling. Histological
analysis was not performed for treatment with salicylate as the effects are well
known or well-characterized.
# Discussion
Our apical injection procedure overcomes the limitations of classical approaches
of administrating drug treatments to the more surgically accessible cochlear
base. In particular, apically injected solutions can treat the entire length of
the cochlear spiral. We studied the acute effects of injecting HPβCD, and HPβCD
analog MβCD, directly into the cochlea. We injected \~2x the volume of guinea
pig scala tympani, most of which would have been driven out with our technique
through cochlear aqueduct during the injection duration. The experiments ended
and the animals were sacrificed 60 minutes after the start of treatment. Results
were compared to those from salicylate treatments, as well as artificial
perilymph alone (controls). Treatment with 13 and 27 mM HPβCD and salicylate
ablated the ANOW amplitude and raised CAP thresholds by approximately 40 to 60
dB, consistent with attenuation of cochlear amplifier gain. Other measurements
such as the endocochlear potential, cochlear microphonic recorded from inside
the third cochlear turn endolymphatic space, distortion product otoacoustic
emissions from the ear canal, and histological measurements generally showed
that effects from MβCD were greatest, followed in order of decreasing effect by
27 mM HPβCD, 13 mM HPβCD, salicylate, and artificial perilymph injections alone
(controls).
## HPβCD effects on an analysis of the electrical cochlear response
Here we address how treatment with 13 mM HPβCD affected harmonic distortion in
the electrical cochlear response measured with a round window electrode.
Sigmoidal, saturating, nonlinear functions are commonly used in analyses of
empirical, gross measurements to study general transfer of acoustic sound into
neural excitation (*f*<sub>TR</sub>, e.g.,). Even and odd order distortions
respectively associate with the asymmetry and saturation of *in vivo
f*<sub>TR</sub> and analyses of distortions can be used to estimate the
operating point of *f*<sub>TR</sub> that is associated with the amplitude of
distortion products (e.g.. Expressing an even order distortion product as a
function of operating point is proportional to the second derivative, and an odd
order distortion product is proportional to the third derivative, of a function
used to describe the sigmoidal, saturating, nonlinear *f*<sub>TR</sub> (e.g..
Cochlear response harmonics to 90 dB SPL 500 Hz tone bursts were measured from
animals in the control, salicylate, and 13 mM HPβCD groups. These are the
experimental groups where cochlear response was still present after solution
administration. Artificial perilymph injections (control) did not affect even
order harmonic distortion (*f*<sub>TR</sub> asymmetry, gray), but caused a small
and transient decrease in odd order harmonic amplitude (*f*<sub>TR</sub>
saturation) at \~18 min. after the injection start (black). Salicylate caused a
slight and brief increase in even order harmonic distortion amplitude
immediately after injection (olive), and a decrease in odd order harmonic
amplitude that recovered to levels greater than pre-injection levels. (i.e., and
“overshoot”;, green). HPβCD caused opposing effects in even and odd order
harmonic distortion. The amplitude of even order harmonics maximally decreased
at \~21 min after the injection start. In contrast, the amplitude of odd order
harmonics increased to a maximum at \~22 min. Both even and odd order harmonics
recovered to near pre-injection levels. We suspect that recovery of even and odd
order harmonics, as well as slight recovery of DPOAE amplitudes, may have
originated from minimal damage that precedes cell death and causes temporary
functional deficit. These effects and recoveries suggest that a function which
can describe the sigmoidal, saturating, nonlinearities involved with
transferring acoustic sound into neural excitation was morphing during our acute
experiments.
We will now use a Boltzmann analysis of electrical cochlear response
measurements from exemplar ears to estimate the operating point of
*f*<sub>TR</sub> and express harmonic distortions as a function of operating
point estimates. The cochlear response is a gross measurement of the cochlear
microphonic from inner and outer hair cells, summating potentials, changes to
the lateral wall potential from slow or sustained current through hair cells,
excitatory postsynaptic potentials, onset or phase-locked compound action
potentials from cochlear regions tuned to frequencies of the sound stimulus or
those located more toward the base, and the coalescence with spontaneous
excitation of single-auditory-nerve-fibers associated with cochlear regions that
are not excited by the sound stimulus. We are thus studying cochlear
nonlinearity in the general terms of influence on DPOAE amplitude, certainly not
mechanoelectric transduction at the apical pole of an OHC as is commonly done
with Boltzmann analyses. The Boltzmann analysis of the electrical cochlear
response measurements provide a more unique perspective that builds on our
qualitative description immediately above. Operating point estimates were
obtained by adjusting Boltzmann parameters until the modeled output matched
empirical cochlear responses. The Boltzmann function was V<sub>t</sub> =
V<sub>EP</sub> + (− V<sub>sat</sub> + 2 V<sub>sat</sub> / (1 + exp(-2
S<sub>B</sub> / V<sub>sat</sub> (P<sub>t</sub> + OP)))) where V<sub>EP</sub> was
a DC potential representing the endocochlear potential magnitude (mV),
V<sub>sat</sub> as the saturation voltage of the Boltzmann function (mV),
S<sub>B</sub> represented the slope of the Boltzmann function at its mid-point
(mV/Pa), P<sub>t</sub> represented the input pressure (Pa) as a function of
time, OP represented the operating point of the Boltzmann function (Pa).
Harmonic distortions did not markedly vary in control ears when expressed as a
function of operating point estimates. Even and odd harmonic distortion
variations with operating point estimates during salicylate injections were
consistent with results from previous experiments that utilized gel injections
into the cochlear apex to cause sustained displacement of the organ of Corti and
simulate the effects of endolymphatic hydrops. Variations to harmonic
distortions expressed as a function of operating point estimates during 13 mM
HPβCD injections were novel in that the trends deviated dramatically from the
second and third derivative of the *in vivo f*<sub>TR</sub>. HPβCD caused
unprecedented effects to physiologic measurements independent of apparent OHC
loss that are not consistent with sustained displacement of the organ of Corti,
such as what can happen if, for example, alteration to OHC bodies manipulate
stereocilia coupling to the tectorial membrane.
Above we used empirical measurements in a Boltzmann analysis to understand how
13 mM HPβCD treatment changed operating point estimates of *in vivo
f*<sub>TR</sub>. We now use the Boltzmann analysis to understand how these
operating point estimates can describe the trends in 2*f*<sub>1</sub> and
3*f*<sub>1</sub> harmonic distortion measurements we found during the apical
injection procedure. Recall that even (e.g., 2*f*<sub>1</sub>) and odd (e.g.,
3*f*<sub>1</sub>) order harmonics respectively quantify the asymmetry and
saturation of *in vivo f*<sub>TR</sub>. Using a Boltzmann analysis, we used a
sinewave input (P<sub>t</sub>) and systematically varied OP with estimates
obtained from fitting empirical data (i.e., the x-axis values of). The
amplitude of the harmonics in the simulated output (V<sub>t</sub>) were measured
and expressed as a function of OP. Results show that varying the operating point
of the Boltzmann analysis yielded simulated harmonic distortions that were
qualitatively similar to empirical harmonics (cf. Figs and, shades of purple).
These results add additional support for the interpretation that 13 mM HPβCD
caused morphing in the sigmoidal, saturating, nonlinearities involved with
transferring acoustic sound into neural excitation during our acute experiments,
without causing apparent OHC loss.
## HPβCD effects on electrical cochlear responses help to understand effects on DPOAEs
Here we discuss how the effects to the asymmetry, saturation, and sensitivity of
*f*<sub>TR</sub> related to the changes found to DPOAE amplitude measurements.
Administering 13 mM HPβCD did not cause apparent changes to CM amplitude
recorded inside the endolymphatic space but caused a large reduction of DPOAE
amplitudes (cf. Figs and purple, \~15 minutes after the start of injection).
These results suggest intact mechanoelectric transduction but marked attenuation
of cochlear amplifier gain. We investigated this further by analyzing CM
measurements made before, and 30 minutes after, the start of injection. Before
treatment, the CM (red) fit well to a typical Boltzmann function (blue) but not
to a Boltzmann function without saturation (V<sub>sat</sub> from the Boltzmann
analysis, green). These fits to pre-treatment empirical CM can be seen both in
the Boltzmann analysis and the time domain. During treatment, the CM (red) fit
well to a simple sine wave (light green). But, to achieve a fit to a Boltzmann
function, V<sub>sat</sub> had to be set to infinity (light blue). These fits to
empirical CM measurements made during treatment can be appreciated in both the
Boltzmann analysis and the time domain. We suspect that saturation of CM
amplitude would have occurred at sound pressure levels much higher than what was
used for these experiments because there was no apparent loss of OHC stereocilia
and bodies after 13 mM HPβCD treatment. The unexpected result of normal CM
amplitude measured inside scala media in the face of maximal decreases in DPOAE
amplitude during treatment of 13 mM HPβCD treatment likely originate from lack
of saturation to the *in vivo f*<sub>TR</sub>. The near normal CM amplitude
measured inside the endolymphatic space and transient enhancement of EP
measurements made during 13 mM HPβCD treatment could be explained if decreased
current flow through OHCs increases the overall resistance between endolymph and
perilymph. That is to say, decreased current flow would reduce the OHC-generated
potential, but with less shunting to perilymph the amplitude recorded from
endolymph may not be reduced to the same degree.
## HPβCD does not affect the auditory nerve or lateral wall of scala media
Here we revisit the finding that \~40 min after the start of 13 mM HPβCD
treatment DPOAEs returned to near-baseline amplitudes but CAP thresholds shifted
to an asymptotic level that is consistent with fully attenuating cochlear
amplifier gain (cf. Figs and purple). These findings naturally lead to the
question: Does HPβCD cause an auditory neuropathy?
Previous work found that intra-cochlear administration of the sodium channel
blocker tetrodotoxin (TTX) can be used to study excitatory post-synaptic
potentials (EPSPs) recorded with a round window electrode. EPSPs leading to
normal spike generation have shorter latencies than CAP latencies. But, post-
treatment latencies could decrease from broadened tuning caused by HPβCD
affecting the cochlear amplifier, or from the need to increase stimulus level
and thus probe a wider region of cochlear tuning curves to achieve a measureable
response in damaged ears. Broader filters have shorter delays than sharper
filters. TTX effectively causes an auditory neuropathy with raised neural
thresholds in the face of normal DPOAE amplitudes and endocochlear potential. It
is thus possible that the voltages recorded during the asymptotic threshold
shifts in were EPSPs masquerading as CAPs with an amplitude ≥ the typical 10 μV
criteria for thresholds while DPOAEs amplitudes approximated pre-injection
levels (cf. Figs and \~40 min after injection start). To address this
possibility we compared round-window electrode measurements made before and
after treatment with 13 mM HPβCD and 250 ng/ml TTX. We used measurements made
within the first and last 10 minutes of a 70-minute experiment that used the
same 15-minute injection procedure used in the other experiments discussed in
this report. CAP measurements before HPβCD and TTX administration are
comparable, as expected (red). After HPβCD treatment, the amplitude of the
response was reduced but waveform morphology was more like the pre-HPβCD
waveform than the post-TTX waveform, consistent with what would be expected by
simple reduction of stimulus sound pressure level, or attenuation of cochlear
amplifier gain, that drive neural responses (blue). In contrast, after TTX
administration the waveform resembles the well-known morphology of gross EPSPs
as seen from a round-window ball electrode (blue). *We conclude that HPβCD does
not have its primary effect by directly acting on auditory neurons*.
CM amplitudes and endocochlear potential measurements made after the apical
injection stopped were minimally affected by 13 mM HPβCD. The effects of HPβCD
(and salicylate) on distortion products are thus different than those from
furosemide which reduces both the DPOAE amplitudes *and* the EP. *Our results do
not suggest that HPβCD acts on the lateral wall of scala media*.
## Other theories on the origin of HPβCD ototoxicity
β-cyclodextrins can have widespread actions on cochlear function, potentially
impacting any process involving cell stiffness or membrane biophysics. In other
model systems, cyclodextrins can impact tight junctions, mechanotransduction and
synaptic function. Without implicating any one of these, or potentially other
interactions, it is clear that the effects of β-cyclodextrins can have multiple
origins. While our data suggest the origins extend beyond outer hair cells, it
remains unclear whether targets are permanently affected by β-cyclodextrins.
## Do HPβCD effects differ between species and along the length of the cochlear spiral?
Histological data from 27 mM HPβCD treatment showed that severe OHC and IHC
damage was more pervasive in the basal cochlear half than the apical half, a
finding that is consistent with that found by Crumling et al. and Cronin et al.
who studied the chronic effects of HPβCD in mice by administering systemically
and directly into cerebrospinal fluid. We suspect Cronin et al.’s drug entered
the cochleae in cerebral spinal fluid through the cochlear aqueduct in the base
of scala tympani, and was therefore at a lower concentration in the apical half
of the cochlear length (cf. studies of chronic effects on measurements from mid-
frequencies with administration to the cochlear base). In contrast, it is
unlikely that our solutions were diluted by mixing with cerebral spinal fluid
because our 500 nL / min injection rate is larger than the \~30 nL / min
sustained entry of cerebral spinal fluid through the cochlear aqueduct.
Methodological differences aside, our histological data showing severe hair cell
damage to be more pervasive at the cochlear base than at the apex is consistent
with Crumling et al. and Cronin et al.’s findings and suggests that *the graded
effects of HPβCD on histological measurements along the cochlear length do not
differ between species*.
Histological data alone cannot determine if HPβCD has varying effects on
*measurements of hearing* along the cochlear length. CAP thresholds to the
highest tone burst frequency (16 kHz) were affected less than to the lowest tone
burst frequency (2 kHz) by 13 mM HPβCD and salicylate, but were similarly
affected by 27 mM HPβCD. Our neural threshold measurements are not consistent
with those from Crumling et al. and Cronin et al. who found that auditory
brainstem response thresholds to low-frequency tone burst (4 kHz) were shifted
by \~40 dB after HPβCD treatment but those to high-frequency tone burst (16 & 32
kHz) shifted \~50–60 dB. The difference between our results and those from
Crumling et al. and Cronin et al. might be explained by their low-frequency
baseline (control) thresholds that are slightly high in the strain used, a
result attributed to greater OHC death. Or, disagreeing data may originate from
differences in our methods used to study the acute effects of HPβCD and their
methods used to study the chronic effects of HPβCD. Since our sets of
histological data agree in that HPβCD has a lesser effect in the cochlear apex,
one would ideally want to study the chronic HPβCD effects with an approach that
could ensure treatment of the entire cochlear length in many different species
and use physiologic measurements that can be obtained throughout the cochlear
length. But, as it currently stands, the presently available physiological data
suggests that *the graded effects of HPβCD on measurements of hearing from along
the cochlear length do differ between species*.
Coupling together the available, the histological and physiological findings
leads to the hypothesis that apical cells may be less susceptible to death, but
are equally susceptible to functional deficit. This hypothesis predicts a
continuum of a dose-response gradation from functional deficit to cell death.
Addressing the possibility of longitudinal gradients in cell physiology may be a
promising area of study, particularly once the perilymph concentrations of
clinically applied HPβCD are known.
# Conclusions
We studied the acute effects of HPβCD injected directly into the perilymph of
intact and sealed cochleae. We found that a low-dose of HPβCD raised CAP to an
extent that was consistent with attenuating cochlear amplifier gain, had no
apparent effect on the EP, altered general nonlinearities involved with
transferring acoustic sound into neural excitation without causing apparent OHC
loss, and the CM measured from inside the endolymphatic space. In contrast,
DPOAEs measured in the ear canal were greatly diminished. A high-dose of HPβCD
elevated CAP thresholds, markedly affected the EP, CM and DPOAEs, caused
sporadic OHC losses that were consistent with previous studies on the chronic
effects of HPβCD. Neither the low- or high-dose of HPβCD caused apparent
disruption of the scala media lateral wall or the auditory nerve. But, for the
duration of our acute studies, known intra-cochlear concentrations high-dose of
HPβCD caused variable effects on OHCs throughout the length of the cochlear
spiral.
We thank Dr. Uzma S. Wilson and Professor Isabel Varela-Nieto for productively
criticizing an early version of this manuscript.
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** JTL KH CAB RKD ANS. **Formal analysis:** JTL
ANS. **Funding acquisition:** JTL KH ANS. **Investigation:** JTL.
**Methodology:** JTL ANS. **Resources:** JTL KH ANS. **Visualization:** JTL.
**Writing – original draft:** JTL KH CAB RKD ANS. |
# Introduction
In the malignant progression of a solid tumor the gain of an invasive phenotype
is the first and a necessary step in the development of metastases. The sequelae
of metastasis account for 90% of cancer-related mortality. To date, the
biological events underlying invasion and metastasis remain poorly understood.
The development of effective anti-invasive/antimetastatic tools and drugs is
therefore an unmet clinical need.
Our laboratories have been characterizing and validating
4-fluoro-3’,4’,5’-trimethoxychalcone or C16 as a novel invasion inhibitor for
the study and potential treatment of metastatic cancer. C16 has nanomolar anti-
invasive potency in the chick heart and Matrigel *in vitro* invasion models
against several human cancer cell lines (MCF-7/6 breast cancer, BLM melanoma and
SK-OV-3 ovarian carcinoma). The molecule shows a defined structure-activity
relationship and possesses adequate *in vitro* absorption, distribution,
metabolism, and excretion (ADME) properties (*see section ‘Stability and
metabolism’ and*). Given its discovery in a phenotypic model, the biological
target of C16 is unknown. We have differentiated the molecular mechanism of
action of C16 from that of other antimetastatic agents, and are currently
characterizing its biochemical interactions in detail.
The aim of the present research was to explore the drug metabolism and
pharmacokinetics (DMPK) and toxicity profile of C16 in rodents, in preparation
of an evaluation of its efficacy as a pharmacological tool in animal models of
disease. Here, we have developed appropriate vehicles to accommodate the low
bioavailability of C16, including a formulation as a medicated gel suitable for
chronic oral dosing. Furthermore, we have analyzed the pharmacokinetic
properties and metabolism of C16, as well as its maximum tolerated dose and
repeat-dose toxicity profile. Routine pharmacokinetics (PK) and metabolite
profiling was conducted in the rat. Additional PK evaluation as well as
tolerability and toxicity testing was performed in the mouse, as this species
will be used in future proof of efficacy studies.
C16 is a chalcone, a chemical class to which various biological activities have
been attributed, albeit often at high concentrations. Despite this body of
literature, follow-up studies on the DMPK properties of these molecules are
rare, and it thus often remains unclear whether sufficiently high plasma levels
can be obtained for a sustained period in order to assess *in vivo* actions. The
present study may therefore have relevance to research on other members of the
chalcone chemical class.
# Materials and methods
Additional protocols are available in.
## Statistics
Data was processed using IBM SPSS Statistics 23 or higher. All data were tested
for normality (Shapiro-Wilk test) and homogeneity of variances (homoscedasity,
Levene's test). If both conditions were met, a one-way ANOVA with post-hoc Tukey
hsd was conducted. If the data was not normally distributed, then a non-
parametric Kruskal-Wallis H test or Mann-Whitney U test was performed. If the
data was not homoscedastic, Welch’s t-test and a post-hoc Gamess-Howell test
were conducted.
## Animal welfare
All aspects of this work related to *in vivo* experiments, including housing,
experimentation, and animal disposal were performed in general accordance with
the *Guide for the Care and Use of Laboratory Animals* (Eighth Edition).
Relevant protocols were approved by the Committee on the Ethics of Animal
Experiments of Ghent University (Permit Number: ECD 15/59). The persons who
carried out the described experiments received appropriate training in animal
care and handling. Animal numbers were kept low due to the exploratory nature of
the study. Exact numbers were based on relevant literature work using similar or
identical animal studies. Animal health was monitored as indicated in the
individual protocols, including during every animal manipulation. Throughout all
studies, the following criteria were used to remove an animal from the study and
humanely euthanize to prevent undue pain or distress: inability to eat and/or
drink, inability to thermoregulate, weight loss (\>20%), moribund condition,
prolonged bleeding, seizures, paralysis. These endpoints were not reached. No
unexpected deaths occurred in any of the described studies. Animals were
euthanized by CO<sub>2</sub>, except for those that underwent cardiac
exsanguination after Avertin anesthesia. Analgesics or anesthesia were
administered as indicated in the individual protocols.
## Chemicals
C16 was prepared as described earlier. A tribromoethanol injectable solution
(Avertin) was prepared as described in Section E in. In-house prepared Milli-Q
water was used. Cremophor EL (Sigma, Germany), corn oil (Wako, Japan), dimethyl
sulfoxide (DMSO, Sigma-Aldrich, Germany) anhydrous N,N-dimethylacetamide (DMA,
Wako, Japan), ethanol (Merck, Germany), Medigel Sucralose (2 oz cups, ClearH2O,
USA), 2-methyl-2-butanol (amylene hydrate, Sigma-Aldrich, Germany)
1,2-propanediol (propylene glycol, PG) (Wako, Japan), polyethylene glycol (PEG)
400 (Sigma, U.S.A), PEG 600 (Alfa Aesar, Great Britain or Sigma, USA), sodium
heparin (5000 units/mL injectable solution, LEO Pharma, Belgium) Solutol HS-15
(BASF, Germany) 2,2,2-tribromoethanol (Sigma-Aldrich, Germany) were used as
obtained from the indicated suppliers.
## Vehicle development
### Nephelometry
The solubility of C16 was tested at 10 and 30 mg/mL for the indicated vehicles
using laser nephelometry in 96-well plates (BMG LabTech NEPHELOstar microplate
reader, BMG LabTech, USA). Solutions were prepared using solubilizing techniques
including sonication, heating up to 37 °C and vortex. Solubility data was
categorized as soluble (result ≤ +15 of blank), slightly soluble (result between
+15 and +25 of blank) or not soluble (result \> +25 of blank).
### Preparation of a C16 solution in 20% DMSO / 10% (DMSO / Cremophor EL 1:1) / 70% H<sub>2</sub>O
For a 2 mg/mL final concentration, 240 μL of DMSO was added to 2.4 mg of C16 in
a glass vial. The mixture was vortexed resulting in a clear solution. Of this
stock solution, 210 μL was mixed with 105 μL of a 1:1 DMSO / Cremophor EL
mixture and 735 μL of water, yielding a clear solution.
### Preparation of a C16 solution in 10% Solutol HS-15 / 90% PEG 600
Solutol HS15 and PEG 600 were warmed to 37 °C. The pre-calculated volume of
Solutol HS15 was added to C16. The mixture was vortexed and then kept at 37 °C
with sonication until C16 had completely dissolved. PEG 600 was then slowly
added and the mixture was vortexed again, whereupon the solution was visually
clear. Solutions up to 30 mg/mL were prepared in this way.
### Stability test of C16 in 10% Solutol HS -15 / 90% PEG 600
Solutions of 30 mg/mL and 10 mg/mL of C16 were prepared and stored at room
temperature and 4°C for 24 h. The stability of C16 was evaluated at 0, 1, 2, 4,
8 and 24 h after formulation by high-performance liquid chromatography-mass
spectrometry/mass spectrometry (HPLC-MS/MS) (electrospray ionization, ESI+)
analysis using oxybutynin as an internal standard (IS). Analyte samples were
diluted 500x with acetonitrile (ACN)/H<sub>2</sub>O (20:80). This solution was
further diluted 100x with an oxybutynin solution (1 ng/μL in ACN/H<sub>2</sub>O
20:80), giving a final concentration of 0.6 ng/μL and 0.2 ng/μL, respectively,
for HPLC-MS/MS analysis (multiple reaction monitoring scan mode, MRM, *see*
Section A of for more details). All analyses were performed in triplicates.
### Preparation of C16-doped medicated gel
A pre-weighed amount of C16 was dissolved in 1 mL of DMSO and the resulting
solution was delivered into a cup of Medigel sucralose (pre-warmed to room
temperature) with a syringe through the foil lid. In order to facilitate even
distribution of C16 in the gel, the injection was spread over several points.
The resulting suspension of C16 in gel was dispersed evenly by vigorous shaking
for at least two minutes. Visual control confirmed a uniform and finely
dispersed suspension of C16 particles in the gel. The final DMSO concentration
in the gel was 1.64%.
### Tolerability test of vehicles
Vehicles of 10% Solutol HS-15 / 90% PEG 600 and 10% DMA / 90% PEG 600 were
evaluated for tolerability by oral gavage at 10 mL/kg using the same protocol as
for the single dose *per os* (PO) maximum tolerated dose (MTD) study (*see
below*). The tolerability of DMSO-doped medicated gel was evaluated during the
repeat-dose toxicity test.
## PK studies
### Plasma PK study (IV and PO) in male Sprague-Dawley rats
Six male Sprague-Dawley rats (200–350 g) were obtained from Hilltop Labs.
Animals were assigned randomly to two groups (IV or PO) of 3 upon arrival.
Duration of acclimation was approximately two days. Animals were healthy at the
start of the trial.
Animals subjected to IV dosing were fitted with a jugular vein cannula (JVC)
under isoflurane anesthesia (induction 4%, maintenance 2.0%, oxygen 1 L/min) and
under an external heating source. Post-operative analgesia was provided in the
form of 2.5–5 mg/kg of ketoprofen subcutaneously. Benzylpenicillin (60 mg) was
given intramuscularly to prevent infection. Sterile 0.9% NaCl was injected
subcutaneously under the back skin using a 21G needle during recovery to replace
fluid loss. Animals were placed under a heat lamp and monitored until they
regained full consciousness. Animals were returned to a sterile cage in the
animal care room only after full recovery and when exhibiting normal behavior.
Surgically modified animals were housed individually, monitored hourly for the
first 4 h and then daily over a 2-day recovery period.
Animals were maintained in a well-controlled temperature (20–24°C) and humidity
(30%-70%) environment with 12 h light/dark cycles. Conventional cages with wood
chip bedding were used. Animals were identified by a cage label. The study was
not blinded. Food was withheld from all animals for a minimum of twelve hours
prior to test article administration and returned at approximately 4 h post-
dose. Water was supplied ad libitum.
C16 was dosed in cassette with two undisclosed compounds of similar chemical
structure. Dosing solutions were prepared in 100% DMSO on the day of dosing. The
concentration of C16 was 1 and 5 mg/mL for IV and PO dosing, respectively. C16
was administered at 1 mg/kg IV via JVC or at 10 mg/kg PO.
Sampling took place pre-dose, 5, 15, 30 min, 1, 2, 4, and 8 h post-dose. Blood
samples (0.3 mL) were collected via the JVC, placed into chilled tubes
containing sodium heparin and kept on ice until centrifugation. Plasma
preparation involved centrifugation at a temperature of 2 to 8°C at 3,000 *g*
for 5 min. Plasma samples were stored frozen at -70°C until analysis. No
necropsy was performed.
### Whole-blood PK study (IV, IP and PO) in male CD-1 mice
Nine male CD-1 mice (25–35 g) were obtained from Hilltop Labs. Duration of
acclimation was at least two days. Animals were randomly assigned to the three
dose groups (IV, PO, intraperitoneal (IP), *N* = 3 per group), healthy at the
start of the trial, housed one per cage (conventional type with wood chip
bedding material), identified by a cage label and maintained in a well-
controlled temperature (20–24 C) and humidity (30%-70%) environment with 12 h
light/dark cycles. Animals intended for IV dosing were fitted with a jugular
vein cannula (JVC, procedure see rat plasma PK study above). The study was not
blinded. Food was withheld from the animals for a minimum of 12 h prior to test
article administration until 4 h post-dose. Water was offered ad libitum.
C16 was dosed in cassette together with two undisclosed compounds of similar
chemical structure. The dose of each test article was 1 mg/kg (IV, JVC, 0.5
mg/mL) or 10 mg/kg (IP and PO gavage, 2 mg/mL). Two respective dosing solutions
were prepared (protocol *see above*) containing 0.5 and 2 mg/mL of C16 (and
equal concentrations of the other two test articles) in 20% DMSO / 10% (DMSO /
Cremophor EL 1:1) / 70% H<sub>2</sub>O. Formulations were prepared one day prior
to dosing and stored at 4 °C.
Blood samples were taken pre-dose and 5 (except for PO administration), 15, 30
min, 1, 2, 4, and 8 h after dosing. The samples were collected via the tail vein
or by cardiac puncture (8 h time point). Blood (25 *μ*L) was collected and
pipetted into a tube with 25 μL heparinized water (1 *μ*L of sodium heparin
(1000 unit/ml) + 24 *μ*L water) within 30 seconds of collection. The sample was
pipetted up and down five times, immediately frozen on dry ice and stored at
-60°C to -80°C until analyzed. No necropsy was performed.
### Data processing
Pharmacokinetic parameters were estimated by a non-compartmental model using
WinNonlin (v5.2.1 or higher) software. The maximum plasma/blood concentrations
(*c*<sub>0</sub>) after IV dosing were estimated by extrapolation of the first
two time points back to t = 0. The maximum blood concentration
(*c*<sub>max</sub>) and the time to reach maximum blood drug concentration
(*t*<sub>max</sub>) after PO dosing were derived from the data. The area under
the time-concentration curve (AUC) was calculated using the linear trapezoidal
rule with calculation to the last quantifiable data point, and with
extrapolation to infinity if applicable. Plasma/blood half-life
(*t*<sub>1/2</sub>) was calculated from 0.693/slope of the terminal elimination
phase. Mean residence time (MRT) was calculated by dividing the area under the
moment curve (AUMC) by the AUC. Clearance (CL) was calculated from dose/AUC.
Steady-state volume of distribution (*V*<sub>ss</sub>) was calculated from
CL\*MRT. Bioavailability was determined by dividing the individual dose-
normalized PO AUC values by the respective average dose-normalized intravenous
(IV) AUC value. Samples that were below the limit of quantification were not
used in the calculation of averages, and were treated as zero for
pharmacokinetic data analysis.
## Stability and metabolism
For other stability and metabolite experiments, *see* Section C in.
### Whole blood stability and metabolite profiling
C16 (1 *μ*M) or vehicle (DMSO) were incubated with fresh rat blood (Sprague
Dawley, male) at 37°C at 5 time points in duplicate over a 120-min period. The
final DMSO concentration in the incubation was 0.25%. Reactions were terminated
following 0, 15, 30, 60 and 120 min by acetonitrile containing internal
standard. The sampling plate was centrifuged (3000 rpm, 45 min, 4 °C) and the
supernatants from each time point analyzed for parent compound by HPLC-MS/MS.
The percentage of parent compound remaining at each time point relative to the
0-min sample was then calculated from HPLC-MS/MS peak area ratios (compound peak
area/internal standard peak area).
Following the blood stability assay the time point at which 30–70% of parent had
degraded (15-min sample) was analyzed by HPLC-MS over a mass range of 200–1000
Da, using an extended run time. Potential metabolites were identified by
searching against expected biotransformations. Unexpected metabolites were also
searched for by comparison to a control sample (0-min time point and/or negative
control) run under the same analysis conditions. Metabolites had a ratio of at
least two compared to a corresponding peak in the control sample.
The metabolite sample was re-injected and data collected for the product ions in
the mass window of 50 Da to a point above the metabolite mass, with the
precursor ion fixed on the metabolite mass in the time window observed for the
metabolite in the original chromatogram. MS/MS spectra of the metabolites were
collected and processed into a table representing product ion fragments for the
parent and each metabolite.
## Single-dose PO and IP MTD studies
Male ICR mice (20–26 g), were obtained from BioLASCO Taiwan. Animals were kept
in conventional cages with wood chip bedding. Space allocation for 3–5 animals
was 30 x 19 x 13 cm. All animals were healthy at the start of the trial and
maintained in a well-controlled temperature (20–24°C) and humidity (30%-70%)
environment with 12 h light/dark cycles. Free access to a standard lab diet
\[MFG (Oriental Yeast Co., Ltd., Japan)\] and autoclaved tap water were granted.
A single PO and IP dose progression test was conducted in which animals were
dosed at a minimum of 72- or 24-h intervals, respectively. C16 (vehicle: 10%
Solutol HS-15 / 90% PEG 600) was administered by oral gavage at 10 mL/kg to
randomly assigned groups of 2 male and 2 female mice, or by IP injection at 5
mL/kg to randomly assigned groups of 5 male mice. Animals received an initial
dose of 10 mg/kg. If 50% of the animals survived for 72 h (PO) or 30 min (IP),
the dose for the next cohort was increased. If one or more animals died, the
dose for the next cohort was decreased. The next dose level was determined by
the decision scheme in.
The testing stopped when all animals survived at the upper bound, when four (PO)
or three (IP) dose levels had been tested or when the upper or lower bound had
been reached. At each dose level, animals were observed for the presence of
acute toxic symptoms and autonomic effects during the first 60 (PO) or 30 (IP)
min, and again at 2, 6 (IP only), 24, 48 (PO only) and 72 h (PO only). Body
weights were recorded before dosing and at the 72- (PO) or 24-h (IP) time point.
Gross necropsy was performed in all animals without tissue collection.
## 7-Day repeat-dose toxicity study
Fifteen 7-week old male CD-1 mice, weighing 27 to 34 g, were obtained from
Janvier, France. Animals were randomly allocated to one of four cohorts (*N* = 4
for the 0 mg/kg, 100 mg/kg and 300 mg/kg C16 cohorts, *N* = 3 for the water
control group). Animals were healthy at the start of the trial. Each cohort was
housed in one conventional cage with wood chip bedding and kept in a room with a
well-controlled temperature (20–24°C) and humidity with 12-h light/dark cycles.
Duration of acclimation was five days, during which all animals had ad libitum
access to feed and tap water. Feed was obtained from Carfil, Belgium (Complete
feed for rats and mice, article n° 10783915). Animals were ear-marked under
isoflurane anesthesia (induction 5%, maintenance 2.5%, oxygen 1 L/min) on day -3
of the study. The study was not blinded.
### Study design
On the morning of day -2 of the study, tap water access was removed from the 0
mg/kg, 100 mg/kg and 300 mg/kg C16 cohorts. These groups were granted access to
two non-medicated cups of Medigel sucralose (one in a tailor-made plastic holder
with magnetic fixation system to the side of the cage, one in a cardboard holder
on top of the bedding material) for an acclimatization period of three days.
Note: the magnetic fixation system is preferable, as animals tear and consume
the cardboard holders.
Medicated gels were prepared in order to provide animals with a PO C16 dose
equaling oral gavage dosing at 0, 100 and 300 mg/kg three times per day (0,
78.43 and 235.29 mg C16 per cup, respectively, assuming a consumption of 7 mL of
gel per day and an average animal weight of 30 g). On the morning of day 0 of
the study, treatment with two medicated gels was started and maintained up to
the morning of day 7 (i.e. a 7-day treatment). Fresh cups were prepared and
provided every day. Meanwhile, the water control group was continuously granted
unlimited access to tap water. Throughout the study, all animals had ad libitum
access to feed, except for the water control group, which was fasted on the
evening of day 6, i.e. twelve hours prior to the single PO test article
administration. Animal weight, feed and water/gel consumption and the presence
of toxic symptoms and autonomic effects were monitored on a daily basis for all
cohorts.
On the evening of day 6, blood was collected via the tail vein for the 0, 100
and 300 mg/kg C16 cohorts for determination of the whole blood C16 level. On the
morning of day 7, mice of the 0, 100 and 300 mg/kg C16 cohorts were anesthetized
by IP injection with 250 mg/kg of Avertin (20 mL/kg, preparation *see* Section
E). Blood for C16 determination and hematological and biochemical analysis was
collected via cardiac exsanguination (600 μL to 1 mL). Detailed analysis
protocols are described below. Afterward, animals were sacrificed by cervical
dislocation.
On the morning of day 7, the water group received a single PO administration of
300 mg/kg C16 (10 mL/kg) for high-dose PK evaluation, using a 30 mg/mL solution
of C16 in 10% Solutol HS-15 / 90% PEG 600 (prepared as described above). To that
extent, blood was collected via the tail vein at the 30 and 60 min time point,
and via cardiac exsanguination after 180 min (protocol as for other cohorts).
Samples for hematological and biochemical analysis were also prepared.
Afterward, animals were sacrificed by cervical dislocation.
All animals were necropsied and assessed for gross pathology. Liver, kidneys and
spleen weights were recorded, and fragments of these organs were fixed in a 4%
formaldehyde solution and processed for microscopic evaluation.
### Whole blood C16 determination
Blood samples (25 *μ*L) were collected via the tail vein or cardiac puncture
(last time point). Blood was pipetted into a tube with 25 *μ*L heparinized water
(1 *μ*L of sodium heparin (1000 units/mL) + 24 *μ*L water) within 30 seconds of
collection. The sample was pipetted up and down five times, immediately frozen
on dry ice, and stored at -60°C to -80°C until analysis.
### Hematology and biochemical analysis
Cardiac puncture as described above afforded 0.6–1 mL of blood. Approximately
500 *μ*L thereof was collected in a Greiner Bio 0.5 mL
ethylenediaminetetraacetic acid (EDTA) Eppendorf tube for hematological
parameter determination and kept at room temperature. The remaining volume was
collected in a regular Eppendorf tube and allowed to clot completely at room
temperature over 45 min. The tubes were centrifuged at 1,900 x *g* (3000 rpm)
and 4 °C for 10 min using a swinging bucket rotor. The upper serum phase was
transferred to a new tube with conical bottom. Care was taken not to disturb the
intermediate buffy coat layer. The serum samples were centrifuged at 16,000 x
*g* and 4 °C for 10 min in a fixed-angle rotor. The cleared supernatant was
transferred to a new tube without disturbing the pellet, affording 200–500 *μ*L
of serum. Samples were diluted to 500 *μ*L with normal saline, flash frozen in
liquid N<sub>2</sub> and stored at -80 °C prior to analysis. Hematological
analysis was conducted on an XE-5000 Automated Hematology System (Sysmex, USA),
biochemical analysis on a cobas 8000 series system (Hitachi/Roche, USA).
# Results
## Vehicle development
### Vehicles for single-dose studies
C16 has low aqueous solubility, which limits the choice of preclinical
formulations for *in vivo* studies. In this work, thirteen vehicles were
evaluated for their applicability in single-dose studies. For commonly used
systems (entries a-e) visual determination of solubility was conducted, while
for the more complex systems solubility was assessed by laser nephelometry.
As expected, C16 proved highly soluble in DMSO (entry a). A more preferable
formulation with 25% DMSO in Cremophor and water (entry e) also proved suitable
at low concentrations (up to 2 mg/mL). These vehicles are appropriate for
single-dose studies with low amounts of C16, e.g. in PK studies.
Dimethylacetamide / 90% PEG 600 and Solutol HS-15 / PEG 600 gave clear solutions
at 10 and 30 mg/mL (entries p,q and t,u). These potential high-dose vehicles
were further evaluated for tolerability and stability. Solutions of 10 and 30
mg/mL C16 in 10% Solutol HS-15 / 90% PEG 600 proved stable at both room
temperature (20–25 °C) and 4°C over 24 h (ANOVA/Tukey HSD or Welch/Gamess-
Howell, Table A). No adverse signs were elicited by both vehicles on PO
administration (*see section* ‘*Single dose PO and IP MTD studies’*). IP dosing
of the more common of these two vehicles, Solutol HS-15 / PEG 600, at 5 mL/kg
induced mild adverse effects (decreased exploratory behavior, decreased muscle
tone) within 30 min after administration (*see section* ‘*Single dose PO and IP
MTD studies’*, 10% DMA / 90% PEG 600 not tested). 10% Solutol HS-15 / 90% PEG
600 thus is a valid vehicle for high-dose experiments, e.g. MTD studies.
### Vehicle for repeat-dose studies
For repeated oral dosing over an extended period of time, a formulation of C16
in medicated gel (Medigel Sucralose, ClearH2O) was developed as an alternative
to repeated oral gavage. Medigel Sucralose is a non-wetting sucralose-flavored,
low calorie water gel with a sweet taste. The chosen formulation as a medicated
gel is a stable suspension of C16 particles in water. The stability of this
dispersion at room temperature was confirmed over 144 h ( and Tables C-D), and
the compound proved uniformly distributed (*see* Table E). The tolerability of
Medigel Sucralose had been explored by others. Observations on the tolerability
of DMSO in the gel (this co-solvent is used as a solubilizing agent during
preparation) are mentioned in the discussion of the repeat-dose toxicity test.
## PK parameters of C16 in the rat and mouse
C16 plasma pharmacokinetics were determined in male Sprague-Dawley rats after
intravenous and oral administration at 1 and 10 mg/kg, respectively (Tables F-G
and Figs B-C). In this exploratory experiment, C16 and two other (undisclosed)
compounds were dosed in cassette in 100% DMSO. No adverse effects were observed
after the intravenous and oral administration of the materials. Following
intravenous dosing of C16 at 1 mg/kg, a *c*<sub>0</sub> of 1257 ng/mL was
reached with an average half-life of 1.93 h. The average clearance was 4.95
L/h/kg, the volume of distribution 4.77 L/kg. After oral dosing at 10 mg/kg, a
*c*<sub>max</sub> of 32.2 ng/mL and oral bioavailability of 6.91% was found. The
half-life value of 3.67 h was longer than that observed after IV administration.
Next, the whole-blood pharmacokinetics of C16 in male CD-1 mice were evaluated
after intravenous (1 mg/kg), intraperitoneal (IP, 10 mg/kg) or oral (PO, 10
mg/kg) administration in cassette dosing with two other (undisclosed) compounds
(Tables H-J and Figs D-F). In this experiment, 20% DMSO/10% (DMSO/Cremophor EL
1:1)/70% H<sub>2</sub>O was used as a more preferable vehicle with respect to
pure DMSO. No adverse reactions were observed. Following intravenous (IV) dosing
at 1 mg/kg, C16 had a half-life of 0.746 h, a clearance rate of 45.0 L/h/kg, and
a volume of distribution of 32.6 L/kg. Following IP and PO dosing at 10 mg/kg,
C16 reached a maximum blood concentration of 107 and 12.5 ng/mL, respectively,
both at 15 min post dosing. The average IP and PO bioavailability was 54.7% and
10.5%, respectively.
## Stability and metabolism
Interpretation of the above PK data required the generation of additional *in
vitro* stability and metabolism data, and revision of known data. As illustrated
above, solubility and lipophilicity are acceptable for exploratory work *in
vivo* when using suitable vehicles. C16 has a high Caco-2 classification, with a
discrepancy in recovery for the A→B and B→A-directions.
The compound is stable in simulated intestinal and gastric fluid and in PBS. A
much higher intrinsic clearance was obtained for rat liver microsomes in
comparison with earlier results for humans. C16 does not significantly inhibit
three major drug-drug interaction (DDI)-inducing human cytochromes P450 (CYPs,
1A2, 2D6 and 3A4). Despite the high protein binding of 99%, C16 has a high
blood-to-plasma ratio of 9.2. A remarkable difference in half-life in rat plasma
and whole blood was obtained. In the whole blood stability assay, comparison of
the 15-min sample against the 0-min control revealed one major metabolite (,
Tables M-P). HPLC-MS/MS profiling showed that this compound had the mass of a
C16-glutathione conjugate. Remarkably, also the *Z*-isomer of C16 was detected
in this experiment. Fast partial isomerization apparently occurred under the
conditions of this experiment, as the configuration of C16 was initially purely
*E* (confirmed by nuclear magnetic resonance (NMR) analysis). This isomerization
was also observed after prolonged dissolution in Medigel Sucralose (*see below*
and Table C). A similar isomerization was also made by Gutteridge et al. for two
other chalcones. Both the *E*- and *Z*-isomer of C16 showed a comparable short
half-life in rat whole blood. The extent to which this isomerization occurs *in
vivo* is unknown, yet it does not cause tolerability issues (*see below*).
## Single-dose PO and IP MTD study
The MTD of C16 in mice, using 10% Solutol HS-15 / 90% PEG 600 as a high-dose
vehicle, was determined for both PO and IP administration. PO administration
(oral gavage) was evaluated in male and female ICR mice (*N* = 2 per group)
using a starting dose of 10 mg/kg. The compound was tolerated at 10–300 mg/kg
though mild adverse effects such as vocalization and increase in sensitivity to
touch were observed at 100 and 300 mg/kg (*see also* Section D). All animals
survived during the 72-h observation period and no gross lesions were discovered
after necropsy. Body weight gain was not affected between cohorts of the same
sex (Gamess-Howell).
C16 was also administered intraperitoneally (IP) to a group of 5 male ICR mice.
The vehicle 10% Solutol HS-15 / 90% PEG 600 at 5 mL/kg induced adverse effects
such as decreased exploratory behavior and decreased muscle tone within 30 min
after administration ( and Section D). Additional effects such as decreased
sensitivity to touch, decreased exploratory behavior, decreased spontaneous
activity, decreased muscle tone, deep respiration, decreased palpebral size and
hunch back were elicited by C16 at the three doses levels (10, 30 and 50 mg/kg),
with more severe effects observed at higher doses. The effects were reversible,
and all animals survived without significant changes in food consumption across
groups (Kruskal-Wallis) and with moderate weight decrease during the 24-hour
experimental period. Only between the 10 and 50 mg/kg cohorts, a significant
difference in average weight decrease was noted (*p* = 0.037, Kruskal-Wallis).
In addition, no significant changes were observed during gross necropsy.
## Repeat-dose toxicity study
Finally, an assessment was conducted of the suitability of the medicated gel as
a vehicle for oral delivery, and of the toxicity of C16 on continuous dosing via
this route of administration. Animals were given the equivalent of *ter in die*
(TID, three times per day) dosing of 0, 100 and 300 mg/kg of C16 in Medigel
Sucralose (1.64% DMSO) over a 7-day period. The 300 mg/kg value was chosen as it
was the highest evaluated dose in the single-dose MTD study. As a control, a
fourth cohort received regular drinking water during the same period. Overall,
the Medigel vehicle and compound were well-tolerated (raw data is available in
Section E). All animals survived during the 7-day observation period.
As seen in the single-dose MTD study, mild behavioral changes were observed in
the C16-treated cohorts. From day 2 on, animals exhibited hyperactivity and
increased anxiety on opening of the cages. This behavior was absent in the 0
mg/kg and the water groups. The effects were mild, ranging from slightly
noticeable in the 100 mg/kg cohort to noticeable in the 300 mg/kg cohort. The
magnitude of these effects did not increase in time. No significant difference
in average weight between the four cohorts was noted during the study (Tukey HSD
or Kruskal-Wallis). A slight but non-significant decrease in weight was observed
during the acclimatization period for all three Medigel cohorts with respect to
the water cohort, indicating slight neophobia towards the non-medicated gel.
Animals recovered fast from day 0–1 on, despite the start of the treatment (no
neophobia towards C16 or DMSO). Average food intake per animal was around 6
g/day with a slight upward trend over the test period in all cohorts. Throughout
the study, no significant differences in average food consumption were observed
between study groups (Tukey HSD). Decrease of gel per day was between 8 and 16
g/animal, and did not differ significantly between the gel cohorts (Tukey HSD).
An identical, slowly increasing trend in consumption was observed. The actual
gel consumption lies a fraction lower than the reported amounts because of loss
of gel due to sanitation of the cups pre-weighing. We estimate that the actual
consumption will be close to that of the water cohort, which is commensurate
with the manufacturer’s findings.
No gross lesions were discovered on detailed necropsy. Liver and spleen weights
were identical across the Medigel-treated cohorts, while a slight but
significant decrease in kidney weight was noted for the 300 mg/kg cohort with
respect to the 100 mg/kg and to the 0 mg/kg cohort (, ANOVA with post-hoc Tukey
HSD, *p* = 0.001). No comparison was made with the water cohort, since these
animals were fasted overnight prior to necropsy. Histology of the kidney and
spleen revealed no abnormalities. A slight hepatocyte toxicity (centrilobular)
was noted in some animals of the Medigel-treated groups, but not in the water
cohort.
Clinical chemistry and hematology parameters were determined in all cohorts at
the end of the study. All measurements fell within or were close to normal
ranges (*see* Table Z). A (marginally) statistically significant difference
between cohorts was only noted for the glucose measurements of the treatment
groups versus the blank. This result was expected, since only the blank animals
were fasted overnight prior to analysis.
On the last evening (day 6) and last morning (day 7) of the study, blood samples
were taken from the Medigel-treated cohorts to determine the systemic
availability of C16 via this route of administration. At both time points, whole
blood C16 concentrations were above its projected EC<sub>50</sub>. No difference
was found in C16 levels between the 100 mg/kg and 300 mg/kg cohort, in both the
evening (ANOVA) and morning (Mann-Whitney U test) samples. As expected from
their dietary pattern, average C16 levels were higher in the morning than in the
evening. Nevertheless, this difference was not statistically significant for the
100 mg/kg cohort (*p* = 0.200, Mann-Whitney U test) and only borderline
significant for the 300 mg/kg group (*p* = 0.057, Mann-Whitney U test).
A final aspect of this 7-day study was a comparison between attainable plasma
levels of C16 on administration *via* gel and oral gavage. On the final morning
of the study, animals in the water cohort received a single PO administration of
300 mg/kg of C16 in 10% Solutol HS-15 / 90% PEG 600 via oral gavage. Blood
samples were collected after 30 min, 1 h and 3 h. Maximum attainable
concentrations via this route of administration were not higher than via the
medicated gel. Extrapolation of the time-concentration curve obtained for oral
gavage reveals the need for a dosing frequency of at least three times per day
in order to obtain sustained levels above the projected EC<sub>50</sub> of the
compound.
# Discussion
4-Fluoro-3’,4’,5’-trimethoxychalcone or C16 is a small molecule with nanomolar
anti-invasive activity, a defined structure-activity relationship and a
satisfactory ADME and toxicity profile *in vitro*. In order to examine its
potential as a pharmacological tool, prior exploration of its *in vivo*
pharmacokinetic profile, toxicity and tolerability was required. The obtained
data is also relevant to several other members of the chalcone chemical class,
which have been attributed biological activities (mostly at elevated
concentrations), but for which associated DMPK profiling is lacking.
## Vehicle development
DMSO is an excellent solvent for C16 but its applicability *in vivo* is limited.
Other formulations were therefore evaluated. For low single dose studies,
DMSO/Cremophor/water was identified as a suitable vehicle, while for high
single-dose evaluation, Solutol HS-15 / PEG 600 proved appropriate. C16 is
stable when dissolved in Solutol HS-15 / PEG 600, and the vehicle is well
tolerated in the mouse when given *via* oral gavage. It however generated
moderate adverse effects on IP administration, which prohibits its IP use in
chronic dosing studies.
The suboptimal DMPK properties of C16 (*see* below) would necessitate at least
three oral gavage administrations per day to maintain the desired plasma levels
in chronic experiments. We therefore turned to self-medication, as this reduces
animal handling and stress, and does not disrupt the diurnal rhythm of the
animals. It also reduces the burden on the investigator significantly.
Administration of C16 via regular drinking water was not possible, as the
compound crashes out of solution and moreover is unstable in an aqueous
environment upon prolonged dissolution. At the start of this study, a limited
number of reports on the use of medicated gels as a route of administration were
available. Medicated gels have advantages over mixing in the feed, as new
batches can be prepared on a flexible basis and without the need for special
equipment. Daily gel intake is comparable to that of regular drinking water, the
gel can thus serve as a complete replacement of drinking water over prolonged
periods of time. A major aim of this study was the development of a C16-doped
gel as a vehicle for chronic dosing. A suitable formulation was developed in the
form of a stable suspension of C16 in Medigel Sucralose. The compound proved
stable in this preparation over 144 h, and was shown to be uniformly spread.
Earlier reports had validated the tolerability of Medigel Sucralose in rodents.
Validation of C16-doped Medigel Sucralose for use in mice was conducted in a
repeat-dose toxicity study (described below).
## Pharmacokinetics
The IV, IP and PO pharmacokinetic profile of C16 was determined in rat plasma
and mouse whole blood. Regardless of the vehicle (DMSO or DMSO/Cremophor/water),
sampling (plasma or whole blood) and species (rat or mouse), the compound shows
low (PO) to moderate (IP) bioavailability. In both cases, slow dissolution is a
limiting factor, resulting in a similar t<sub>max</sub> for IP and PO dosing,
prolonged exposure and rather flat (rat) or multiple peak (mouse) concentration-
time profiles on PO dosing. The use of cassette dosing may have accentuated this
phenomenon, though the main cause can be found in the physicochemical profile of
C16. It should be noted that the significance of the average t<sub>max</sub> for
PO dosing in rats is limited, as a flat average concentration-time profile was
obtained. Despite its good permeability, the low A→B recovery of C16 in the
Caco-2 assay suggests cellular metabolism, which also limits oral
bioavailability. While the high extent of protein binding may also necessitate
elevated oral dosing, the high recovery levels suggest reversible binding.
Regardless of the sampling mode (plasma or whole blood), both rat and mouse
half-life on IV dosing are short. This is in line with data published for other
chalcones. Nevertheless, an important difference in initially attained IV
concentrations was noted, which can be attributed to species and sampling
factors. Clearance in both species is much higher than hepatic blood flow (rat:
3.3 L/h/kg, mouse: 5.4 L/h/kg), which points towards extra-hepatic elimination.
C16 shows high apparent blood partitioning and a large difference between plasma
and whole blood half-life. Rather than irreversible binding to erythrocytes, the
compound was found to be metabolized rapidly with glutathione conjugation as the
major mechanism. Erythrocytes are a major site of biotransformation and contain
cytosolic glutathione S-transferases. The conjugates are then actively
transported outward across the red-cell membrane. Metabolism in red blood cells
thus seems the major route of extra-hepatic elimination for C16, explaining its
much shorter half-life in blood versus plasma. This route of metabolism was not
observed for other rapidly metabolized chalcones such as cardamonin. As a
further consequence, the higher reported blood-to-plasma ratio is not accurate
because it was not based on measurements of both blood and plasma fractions.
## Tolerability, toxicity and chronic systemic availability
The maximum tolerated single dose of C16 in the mouse on administration via oral
gavage is \>300 mg/kg. The compound was well tolerated, and induced only mild
behavioral effects at 100 and 300 mg/kg (vocalization and increased sensitivity
to touch). The pre-defined humane end points (*see* *section*) were not reached.
No other treatment-induced phenomena were noted on observation or necropsy. The
animals proved much more sensitive to intraperitoneal delivery of a single dose
of the same vehicle and compound, though humane end points were again not
reached. These observations can be correlated to a low tolerability of the
vehicle itself, and the earlier observed higher C<sub>max</sub> for C16 on IP
delivery with respect to oral administration. Moderate to severe behavioral,
neurologic and autonomic effects were noted in a dose-response pattern. These
results indicate that the vehicle and C16 up to 50 mg/kg may be tolerated for
single-dose IP administration in the mouse, but not for chronic experiments.
Despite its low oral bioavailability, the high oral tolerability of C16 offers
opportunities to obtain sufficiently high systemic levels for efficacy testing.
This hypothesis was tested in a repeat-dose experiment. The 7-day continuous
dosing experiment validated Medigel sucralose as a vehicle for self-medication
of C16 in a chronic setting. Substitution of drinking water for blank gels
caused a slight but non-significant decrease in weight during the
acclimatization period due to neophobia. During the further course of the
experiment, however, treatment with solvent-doped gels (DMSO) did not generate
significant differences in weight, water and food intake, and hematological and
biochemical parameters with respect to the water cohort. No gross lesions or
differences in organ weights were noted on necropsy. A slight hepatocyte
toxicity, however, was noted for some gel-treated animals, which was absent in
the water cohort. This effect may be due to the relatively high DMSO content of
the gel. DMSO hepatotoxicity has been reported in chronic trials in laboratory
animals. In our setting the effect was not major, nor is it reflected in the
clinical chemistry panel. The DMSO dose was also well below the LD<sub>50</sub>
in the mouse (21–28 g/kg, PO). Overall, Medigel Sucralose thus proved a suitable
vehicle for chronic studies.
Oral C16 dosing via medicated gels over a 7-day period caused sustained systemic
levels above its projected EC<sub>50</sub> value (6.32 ng/mL). This is a
significant result, as a major reason for poor correlation between *in vitro*
and *in vivo* efficacy of chalcones has been their poor pharmacokinetics and
bioavailability, yielding plasma levels below the EC<sub>50</sub> of the
compounds. Gel intake was close to the anticipated amount for all cohorts,
resulting in an actual daily oral dose close to the intended. C16 showed
nonlinear uptake as systemic levels did not differ significantly between the 100
mg/kg and 300 mg/kg cohort. Given the physicochemical profile of C16, this is
likely due to limits in dissolution. Lower dosing regimens may thus still
generate sufficient systemic levels, but were not evaluated in this work.
Importantly, as expected from the low-dose PK studies, the high-dose
concentration-time profile obtained after a single administration of 300 mg/kg
C16 via oral gavage confirms the need for TID dosing in order to reach sustained
levels above the EC<sub>50</sub>. This underpins the value of the developed
medicated gel formulation in the present research and for others working on
related molecules.
The confirmed seven-day exposure to the molecule at levels above its
EC<sub>50</sub> did not engender treatment-associated differences between the 0,
100 and 300 mg/kg cohorts, except for a slightly lower kidney weight in the 300
mg/kg cohort. Nephrotoxicity was however not detected in any of the cohorts
during histological examination. Hematological parameters were all satisfactory.
Some individual measurements fall slightly outside of reported reference ranges,
but these ranges itself vary strongly upon the cited source and underlying
animal-related variables. The only valid comparison of therapy response thus is
to the age-, strain-, and sex-matched controls of the water cohort. No
differences were found, except for a lower average glucose reading in the water
cohort due to fasting prior to sampling.
Oral delivery using our medicated gel formulation thus is a suitable tool for
evaluating the efficacy of C16 in a range of invasion and metastasis models.
Depending on the models and more specifically on the interrogated phenomena
(local invasion, regional or distal metastasis), compatible dosing schedules may
include prophylactic treatment over a 7-day period before inoculation (e.g. in a
peritoneal metastasis model), intermittent or low-dose chronic treatment in
long-term experiments (months) or high-dose treatment in short-term, more
aggressive models (weeks). From the data gathered in this report, adequate
exposure and good tolerability of C16 can be expected in these settings.
# Conclusions
C16, like other members of the chalcone family, shows suboptimal
pharmacokinetics in the rat and the mouse. Its limited solubility and cellular
metabolism causes poor intestinal uptake and limited oral bioavailability. The
compound is rapidly metabolized, with a clearance rate exceeding hepatic blood
flow. Glutathione conjugation in erythrocytes was identified as a major route of
extra-hepatic elimination, explaining the much shorter half-life of C16 in blood
versus plasma. An oral dosing routine using medicated gels was developed that
solved these issues. A schedule providing amounts equivalent to oral gavage at
100 mg/kg three times per day yielded sustained whole blood levels above the
EC<sub>50</sub> in a 7-day chronic study. The compound showed good tolerability
during the latter experiment. Evaluation of C16 as a pharmacological tool in
animal models of metastatic disease, using the medicated gel formulation, is
currently ongoing. Besides, we are preparing analogs with a superior DMPK
profile in search of improved clinical relevance.
# Supporting information
The authors thank prof. Marc E. Bracke for helpful discussions regarding the
repeat-dose toxicity experiment.
ACN acetonitrile
ADME absorption, distribution, metabolism, excretion
AUC area under the time-concentration curve
AUMC area under the moment curve
C16 4-fluoro-3’,4’,5’-trimethoxychalcone
CL clearance
DMA N,N-dimethylacetamide
DMPK drug metabolism and pharmacokinetics
DMSO dimethyl sulfoxide
EC<sub>50</sub> half maximal effective concentration
EDTA ethylenediaminetetraacetic acid
HPLC-MS/MS high-performance liquid chromatography-mass spectrometry/mass
spectrometry
IP intraperitoneal
IS internal standard
IV intravenous
JVC jugular vein cannula
MRM multiple reaction monitoring
MRT mean residence time
MTD maximum tolerated dose
NMR nuclear magnetic resonance
PEG polyethylene glycol
PG propylene glycol
PK pharmacokinetics
PO *per os*
SAR structure-activity relationship
TID *ter in die*
V<sub>ss</sub> steady-state volume of distribution
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in
clinical practice. AF is a well-known risk factor for thromboembolic events,
silent cerebral infarcts, strokes, congestive heart failure and mortality in the
general population and in high stroke risk patients, and it has become a major
public health problem worldwide. Although the underlying mechanisms of AF are
not yet fully understood, autonomic dysfunction, unbalanced
inflammation/oxidative stress and renin-angiotensin system activation have all
been shown to be related to AF. However, the pulmonary vein (PV) is confirmed to
be the most important and critical trigger for AF, and PV isolation (PVI) using
catheter ablation is the cornerstone therapy for symptomatic AF refractory to
antiarrhythmic drugs. The 2016 European Society of Cardiology (ESC) AF
guidelines also recommend catheter ablation as a more effective therapy than
antiarrhythmic drugs (AADs) for restoring and maintaining sinus rhythm (SR) in
patients with symptomatic paroxysmal AF (PAF) and persistent AF (PerAF).
Radiofrequency (RF) ablation is a well-established treatment for AF that
achieves PVI by creating consecutive, transmural ‘point-to-point’ lesions with
heat energy. Recently, cryoballoon (CB) ablation, which achieves PVI by a
single-shot deployment of a CB with frozen energy, has become a substitute for
RF ablation, as CB ablation has the advantage of being an easier and faster
ablation procedure than RF ablation. Several previous studies have shown that
the efficacy and safety of CB ablation therapy are comparable to those of RF
ablation in patients with PAF. Compared with first-generation equipment, second-
generation CB (2G-CB) ablation devices have preponderant cooling capacity and
seem to reduce the procedure duration. However, 2G-CB ablation to achieve PVI
has some shortcomings, including an inability to perform ablation of roof linear
(RL) lesions, complex fractionated atrial electrograms (CFAEs) and non-PV
triggers. On the other hand, early studies of PVI involving only patients with
PerAF revealed suboptimal success rates. Therefore, the ‘PVI-plus’ ablation
strategy that combines 2G-CB ablation to achieve PVI and RF ablation and
addresses additional cardiac substrate modification and extra-PV lesions during
the same surgery might be a better strategy for treating PerAF and long-standing
PerAF. However, the effectiveness and safety of the ‘PVI-plus’ ablation strategy
have not been sufficiently discussed.
To this end, we executed a pooled analysis and meta-analysis of data from
existing studies and trials investigating the efficacy and safety of ‘PVI-plus’
ablation vs. ‘PVI-only’ ablation in patients with PerAF.
# Methods
Our systematic literature search was performed according to the Meta-Analysis of
Observational Studies in Epidemiology (MOOSE) guidelines and conducted using a
predetermined protocol by the Preferred Reporting Items for Systematic Reviews
and Meta-Analyses (PRISMA) statement.
## Literature search strategy
The search strategy was conducted in the PubMed, EMBASE and Cochrane library
databases until September 1, 2018. The search terms were as follows: (“Catheter
Ablation” OR “Cryosurgery” OR “Second-Generation Cryoballoon”) AND (“Atrial
Fibrillation” OR “Persistent Atrial Fibrillation”). No restrictions were applied
on regions or languages. We also manually searched the reference lists of all
publications and review articles to identify other relevant studies.
## Study selection
Two investigators (JS and LXS) independently scanned all the titles and
abstracts to identify studies that met the inclusion criteria and extracted data
from these studies. Discrepancies between reviewers were resolved by discussion
with a third reviewer (MJS).
The inclusion criteria were as follows: (1) involved patients with drug-
refractory symptomatic PerAF who underwent 2G-CB; (2) involved patients who were
treated with a ‘PVI-only’ strategy or a ‘PVI-plus’ strategy using 2G-CB (during
surgery, PVI and PVI-plus had to be carried out simultaneously); (3) reported
1-year clinical success rates; (4) involved follow-up (FU) periods longer than
12 months; (5) published as a full-text article; and (6) provided data regarding
efficacy and safety.
Studies were excluded for the following reasons: (1) conference abstracts, case
reports, case series, editorials, or review articles. (2) They did not report
clinical success rates. (3) The maximum FU period was shorter than 12 months.
(4) They were animal or in vitro studies.
## Data extraction and quality assessment
Two primary investigators (JS and LXS) independently evaluated and extracted
data from each study. Data on the first author, publication year, study
population, age, sex ratio, CHA2DS2-VASc score, underlying disease, medication
usage and ablation strategy were collected using a predesigned electronic form.
All studies were reviewed twice, and disagreements were discussed and resolved
by consensus in a meeting with a third investigator (MJS). We defined the
primary outcome criterion as no episode of AF or any atrial arrhythmia lasting
longer than 30 s without administration of AADs after a single ablation
procedure using 2G-CB with an FU period of at least 12 months (considering an
initial blanking period (BP) of 3 months). Group analysis was performed based on
the ablation strategy: ‘PVI-only’ strategy versus ‘PVI-plus’ strategy, the
latter of which involved PVI plus another substrate modification. The secondary
outcomes were complication rates, including the phrenic nerve (PN) palsy (PNP)
rate, the rates of cardiac and vascular assess site complications (including
hematomas, pseudoaneurysms, and fistulas), and the rates of other complications
(pericardial effusions and/ or tamponade, PV stenosis or atrioesophageal
fistula).
Whenever the data of interest were not available in the literature, the
investigators tried to contact the authors by email to obtain the data. We
described incomplete data as “not reported” in our manuscript.
We used the Newcastle-Ottawa Scale (NOS) to further evaluate the quality of the
observational studies, and a NOS score ≥ 7 was considered good quality.
## Statistical analysis
Statistical analysis was performed using the Cochrane RevMan version 5.3
software (The Cochrane Collaboration, UK). The results are reported as weighted
mean differences and relative risks (RRs) with 95% confidence intervals (CIs)
for continuous and dichotomous outcomes, respectively. Heterogeneity was
assessed using the *I*<sup>*2*</sup> test, Cochran’s Q statistic and the
*I*<sup>*2*</sup> index. *I*<sup>*2*</sup> values of 25%, 25–50%, or 50%
indicated low, moderate, or high heterogeneity, respectively. Funnel plot
analysis was used to evaluate potential publication bias. In all analyses, a *P*
value \< 0.05 was considered statistically significant.
# Results
## Study characteristics
Finally, as illustrated in, four studies involving a total of 879 patients who
underwent ablation with 2G-CB for PerAF were enrolled in this study. The safety
and efficacy of a combination of extra-PV lesions (linear ablations and/or
atrial substrate modifications) or PVI alone with 2G-CB for PerAF ablation were
identified.
The characteristics of the included studies are described in. All of the
included patients completed at least one year of FU. Five studies were analyzed.
Unfortunately, we did not find randomized controlled trials meeting the
inclusion criteria. The quality and bias of the included studies are shown in.
Four full-text studies had an NOS score of 8, and the other study had an NOS
score of 7.
The patients were predominantly male (69.5%); 70.1% had hypertension, 15.6% had
diabetes, and 13.35% had structural heart disease. In all the studies, 87.6% of
patients were treated with oral anticoagulants for PerAF. The mean CHA2DS2-VASc
score was 2.1.
The mid-term FU (≥ 12 months, considering an initial BP of 3 months) occurred at
27 months. Regarding monitoring during FU, 2 studies implemented 24-h Holter
recordings during FU, while two studies recorded data from 7-day Holter or
external cardiac event recorders; the other studies recorded data from 2-week
mobile cardiac telemetry monitoring performed at 6 weeks, 3\~6 months, 13
months, and 18 months.
## Publication bias
Regarding the proportion of patients who were free from AF recurrence during FU,
all the included studies had publication bias that was approximately symmetrical
on visual inspection of the funnel plot **.**
## Ablation strategy
In 3 studies, a bonus freeze protocol was implemented with an additional
freezing cycle applied to each PV after successful PVI. Subgroup analysis to
compare freezing protocols was not performed because the majority of patients
were treated with a bonus freeze protocol. All studies (including 543 patients)
performed PVI plus other linear ablations or substrate modifications. There was
no significant methodological heterogeneity in terms of patient characteristics
and ablation strategies (*I*<sup>*2*</sup> = 0%, *P* = 0.82).
The result of the study by Aryana et al. demonstrated the ablation of non-PV
triggers in 12.8% of all patients treated with 2G-CB without further
specifications. The mean procedure time was 131.9 ± 7.2 min in the ‘PVI-plus’
group and 124.2 ± 13.2 min in the ‘PVI-only’ group. No interaction was found
between the ablation approach and clinical success rate (*P* = 0.47). However,
the results of the study by Su et al. confirmed that application of 2G-CB could
achieve large-area atrial substrate modification mostly with left atrial (LA)
roofline and Coumadin-ridge ablation (atrial tissue between the LA appendage and
left superior PV). Akkaya et al. showed that among patients with PerAF and LA
enlargement, PVI with 2G-CB in addition to the creation of RLs may be a viable
alternative to point-by-point RF ablation, as indicated by a lower arrhythmia
recurrence rate with this strategy than with the ‘PVI-only’ strategy. Aryana et
al. demonstrated that ablation of PVI in conjunction with isolation of the
posterior LA wall is associated with improved clinical outcomes in certain
patients with AF.
## Primary outcome
### Recurrence
Data from 5 studies were included. At a mid-term FU of 27 months, the overall
success rate of 2G-CB in patients with PerAF was 66.1% ****. In the ‘PVI-plus’
group, the success rate of 2G-CB in patients with PerAF was 73.8%. In the ‘PVI-
only’ group, the success rate of 2G-CB in patients with PerAF was 53.6% \[RR:
0.52; 95% CI 0.42\~0.63, *P*\<0.00001\].
The heterogeneity among studies was not significant (*I*<sup>*2*</sup> = 0%,
*P* = 0.82). The rate of AAD use at the FU was 48.96%. The rate of AAD use at
the FU was reported in 4 studies.
However, because of the lack of correlated studies, we could not analyze
subgroups in detail. Meanwhile, the heterogeneity among studies was not
significant. Categories including PerAF (short-term PerAF, long-standing PerAF
or unspecified PerAF), FU strategy (24-h Holter monitoring vs. 7-day Holter
monitoring or event recording), and AF population (PerAF alone, mixed AF, such
as PAF and PerAF) could not be included in further subgroup analyses.
### One-year recurrence
At the one-year FU, compared with that in the ‘PVI-plus’ group, the one-year
success rate associated with 2G-CB in the ‘PVI-only’ group was 55.1% (vs. 75.1%
in the ‘PVI-plus’ group) \[RR 0.49; 95% CI 0.40\~0.61, *P*\<0.00001\] ****. The
heterogeneity among studies was not significant *(I*<sup>*2*</sup>* * = 0%, *P*
= 0.61).
## Secondary outcomes
### Overall complications
The overall complication rate was 5.2% ****. Phrenic nerve palsy (PNP)/PN injury
were the most frequent complications. Compared with that in the ‘PVI-plus’
group, the complication rate associated with 2G-CB in the ‘PVI-only’ group was
5.4% (*vs*. 5.2% in the ‘PVI-plus’ group) \[RR 0.98; 95% CI 0.57\~1.67, *P* =
0.93\].
### Phrenic nerve palsy
PNP was found in all studies. Transient PNP was defined as PNP that resolved by
discharge. Persistent PNP was defined as PNP that persisted beyond discharge.
The overall rate of PNP was 2.8% ****. Compared with that in the ‘PVI-plus’
group, the rate of PNP associated with 2G-CB in the ‘PVI-only’ group was 1.8%
(*vs*. 3.5% in the ‘PVI-plus’ group) \[RR 1.84; 95% CI 0.83\~4.10, *P* = 0.14\].
Two studies reported persistent PNP that recovered during FU (the maximal time
to PN recovery was not reported).
### Vascular access site complications
The overall rate of vascular access site complications was 1.6% ****. Compared
with that in the ‘PVI-plus’ group, the PNP complication rate associated with
2G-CB in the ‘PVI-only’ group was 2.1% (*vs*. 1.3% in the ‘PVI-plus’ group) \[RR
0.61; 95% CI 0.22\~1.65, *P* = 0.33\]. In one study, 5 patients had a groin
complication with a hematoma that required more than 2 weeks for full recovery,
and 3 patients had a pseudoaneurysm that required a thrombin injection for
patient recovery. Another study reported that 3 patients had minor groin
hematomas.
### Other complications
No death, myocardial infarction, PV stenosis or atrioesophageal fistula related
to the procedure was reported. One patient had a perforation that resulted in
cardiac tamponade requiring pericardiocentesis and immediate surgical treatment.
One patient had a stroke after ablation and was treated conservatively.
# Discussion
## Principal findings
In this detailed meta-analysis and systematic review of trials, we evaluated
different approaches to 2G-CB in patients with PerAF. The main findings are as
follows: (i) the ‘PVI-plus’ strategy had comparable clinical efficacy and safety
as the ‘PVI-only’ strategy; (ii) we detected a reduction in the proportion of
recurrent AF when either RL ablation, ablation of CFAEs or substrate
modification was executed in addition to ablation with PVI; and (iii) compared
with ‘PVI-plus’ involving 2G-CB, ‘PVI-only’ involving 2G-CB had similar rates of
severe complications.
## Efficacy
We used different approaches for 2G-CB. In the ‘PVI-plus’ group, the success
rate of 2G-CB in patients with PerAF was superior to that in the ‘only PVI’
group. Additional ablation can isolate other targets and reduce recurrence.
Previous studies have shown that the expression of circulating miRNAs changes
after ablation of AF, and these miRNAs participate in atrial electrical
remodeling and fibrosis, which might be one of the reasons why additional matrix
ablation reduces the recurrence of PerAF. However, such ablation may be
arrhythmogenic.
The STAR AF II trial reported no added advantage from a more extensive ablation
than PVI with RF for PerAF. This study showed a success rate of 68.9% at a mean
FU of 16.7 months. Compared with the results of recent studies involving cohorts
treated with RF energy, 2G-CB may be the optimal treatment strategy. In our
study, the mid-term FU was 27 months, and there was a greater than 70% success
rate at the mid-term FU. However, in other studies evaluating PVI alone for
PerAF, such as a previous meta-analysis, a success rate of only 51.9% was
observed. The improved success rates noted in our meta-analysis are likely
multifactorial and related to differences in the patient population and the use
of wide RL ablation, which incorporates some substrate and technological
changes.
## Ablation strategy
Patients with PerAF were recognized to have more advanced atrial remodeling than
those with paroxysmal AF. Until now, the main strategy for ablation of PerAF was
atrial substrate modification (in addition to PVI) to achieve acceptable success
rates. Importantly, similar to the results of our study, some meta-analyses have
demonstrated improved ablation outcomes when substrate ablation (CFAE or linear
ablation) was performed in addition to PVI in PerAF patients.
Linear ablations (the most common type of ablation) were successful in this
study when cryoenergy freezes lasting 180 s were applied along the LA roof by
caudocranial ascending activation and a conduction delay next to the ablation
line of \> 120 s when pacing at the right atrial upper septum during SR. In
another study involving AF without conversion to SR after PVI, additional LA
rooflines were created with point-by-point lesions using contact force (CF)
catheters. The acute endpoint was the elimination of local bipolar electrograms,
with 20- to 40-s lesions or with a force-time integral \> 400 s. CF ablation was
performed for documented atrial flutter prior to or during the procedure. This
study reported that when AF ablation required more than CB PVI, an ablation
catheter and technique were used on additional lesions. Catheter maneuvers
necessary to achieve electrical blockage, substrate modifications and clinical
outcomes were recorded. The 11 methods of CB catheter substrate modification
that are described in this study included all the extra-PV lesion sets applied
for 120 s\~180 s at each location. In the other study, LA linear ablations and
substrate modifications, including cavotricuspid isthmus ablation and substrate
ablation of CFAEs and other non-PV triggers, were performed. RF ablation was
performed using an externally irrigated, non-force-sensing catheter with a
3.5-mm tip. Power was limited to 40 W over the anterior walls and 30 W over the
posterior walls. At the end of the study, using a high-density mapping catheter
and the available data, between one and two 120\~180 s cryoapplications were
delivered to each PV with guidance by time to PVI. Once PVI was achieved, the
cryoballoon was used to perform PWI in patients undergoing this treatment.
PVI+PWI can be achieved safely and effectively using the cryoballoon. However,
the latter required adjunct RF ablation for completion of PWI in approximately
one-third of the patients.
Therefore, we evaluated PVI, which is the cornerstone treatment for ablation of
AF, and atrial substrate modification. This combination had a higher success
rate than other methods. We found that ‘PVI-plus’ with 2G-CB for the treatment
of PerAF was an effective ablation strategy that potentially reduced the
procedure and fluoroscopy times. The improved effect with 2G-CB is likely due to
structural improvements in the device, which optimized the refrigerant flow and
distribution, providing a larger, more uniform freezing zone at the entire
distal hemisphere of the balloon irrespective of balloon orientation and
enabling a shorter application time.
## Safety
In terms of overall outcomes, no deaths, myocardial infarctions or clinical
cerebral emboli were reported in a total of 879 patients. The overall
complication rate was 5.2% and mainly included PNPs (2.8%) and vascular access
site complications (1.6%).
Previous studies have reported similarly high rates of PNP (up to 5.4%) and
vascular complication. Casado-Arroyo et al. reported that 2G-CB was more likely
to cause PNP than other methods, possibly due to the larger cooling surface
area, ablation area being more adjacent to the PN, and deeper damage foci.
Moreover, the percentage of complications excluding PNP seemed to be lower with
2G-CB than with RF ablation. On the other hand, the incidence of pericardial
effusion and/or tamponade was very low with 2G-CB and was consistently lower
than the previously reported incidence with RF.
## Heterogeneity analysis
In this study, significant heterogeneity was observed in the incidence of total
complications and the rate of PNP. Due to the lack of correlated studies, we
could not analyze detailed subgroups. On the other hand, the enrolled
populations in the included studies may have been affected by the selection bias
associated with a single-center experience and the preferences of different
centers that refer patients for AF ablation because different centers used
different protocols and tools, which ultimately resulted in substantial
heterogeneity.
In addition, there was no significant heterogeneity in recurrence and one-year
recurrence in the overall population, and funnel plot analysis did not provide
evidence of significant publication bias. Therefore, it was believed that the
included studies had sufficient similarities. In conclusion, the outcome of the
meta-analysis was reliable.
## Previous meta-analysis
A previous and similar meta-analysis summarized data on the safety and mid-term
efficacy of PVI using 2G-CB in patients with PerAF. A total of 11 studies were
analyzed. After FU, 68.9% of patients were free from recurrence. Complications
occurred in 5.5% of patients.
Compared with the abovementioned meta-analysis, our meta-analysis used different
inclusion criteria and required ‘PVI-only’ and ‘PVI-plus’ to be performed
simultaneously. However, studies that included patients treated with other
substrate modifications were permitted as long as PVI was performed using 2G-CB
at least once during surgery. Similarly, we used the inclusion criterion that
PVI was performed using 2G-CB. Thus, only 4 studies were included in this
review.
## Limitations
The limitations of this article include the following:
1. We did not find randomized trials that were eligible for this analysis,
and the total sample size was not sufficient.
2. Heterogeneity among studies was significant in total complications and
the rate of PNP, and we could not perform a more detailed subgroup analysis.
In addition, the inclusion of only published studies may have resulted in
publication bias toward more favorable ablation outcomes from more
experienced centers with variable FU and assessment of arrhythmia
recurrence. For example, in 2/4 studies, postablation monitoring was
performed with only 24-hour monitors, and previous studies have shown that
continuous monitoring and telemonitoring to detect clinical and subclinical
AF events are more effective than 24-hour monitoring. Moreover, the use of
AADs varied between studies, and postablation monitoring was performed
with only 24-hour monitors.
<!-- -->
1. Two studies included in this meta-analysis may have involved the same
study population. We tried to contact the authors via email to evaluate the
data. Unfortunately, we did not receive any response from those authors.
2. The studies we included were not direct studies of PVI-only *vs*. PVI-
plus adjunctive ablation strategies for PerAF; rather, we extracted the data
that we were interested in. In addition, some of the data needed to be
calculated based on the results of the studies.
# Conclusions
In conclusion, this meta-analysis suggests that ‘PVI-plus’ involving 2G-CB for
the treatment of patients with PerAF seemed to have a superior success rate and
similar rates of severe complications compared with ‘PVI-only’ involving 2G-CB.
However, we recommend that large, prospective, randomized, controlled studies
should be performed in the future to validate our results.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Despite advances in vaccine development, vaccine distribution infrastructure
remains inadequate in many parts of the world, and it is estimated that up to
40–50% of all vaccine doses are wasted in certain countries. Crucial to
current vaccine transport is the idea of the cold-chain–a series of refrigerated
enclosures with tight temperature control that allows for stable transport of
vaccine from manufacturer to patient. Strict control of temperature is important
for whole pathogen vaccines, as these are particularly prone to stability
losses. In addition, pathogens with lipid membranes, such as bacteria and
certain viruses including influenza, are especially prone to osmotic stress, and
changing salt concentrations due to temperature-driven solvent evaporation can
lead to pathogen shrinkage and destruction. The development of vaccines
that can maintain viability outside of the cold chain would decrease vaccine
wastage and increase immunization rates in regions of the world with
underdeveloped vaccine distribution infrastructure.
Although recombinant, subunit protein vaccines have been proposed as a more
stable alternative to whole pathogen vaccines, issues with low immunogenicity
and appropriate adjuvant choice have slowed their development as a viable
option. Protein nanoparticles, made entirely of crosslinked protein
antigens, are a means of delivering antigen and adjuvant in the same delivery
vehicle, and are thus an excellent candidate for testing cold-chain-independent
vaccine stability\[–\].
Monomeric influenza hemagglutinin is a 63 kDa protein responsible for mediating
viral entry into host cells. We have shown previously that protein
nanoparticles made from trimerized, H7 hemagglutinin (3HA) were able to
protectively immunize mice against a 10xLD<sub>50</sub> H7 influenza
challenge. Given the immunogenicity of these nanoparticles, as well as the
*in vitro* hemagglutination assay that can provide a simple measurement of
protein conformation in nanoparticles, we have examined the viability of cold-
chain-independent storage of 3HA nanoparticles. We sought to assess whether
storing nanoparticles at room temperature (\~25°C) or 37°C for several months
resulted in a loss of hemagglutinating capability or immunogenic potential. We
found that nanoparticles stored at room temperature retained both
hemagglutinating activity and immunogenicity, while nanoparticles stored at 37°C
retained hemagglutinating activity for 2 weeks.
# Materials and methods
## 2.1 Nanoparticle synthesis and characterization
Trimerized H7 hemagglutinin (3HA) protein was produced and purified from Sf9
cell culture, and nanoparticles were synthesized and characterized exactly as
described previously. Briefly, 400 μL ethanol was added to 100 μL of a 1.6
mg/mL 3HA solution at a rate of 1 mL/min under constant stirring at 600 rpm. The
particles were collected by centrifugation, and resuspended in sterile
phosphate-buffered saline (PBS) with sonication. 800 μg soluble 3HA protein was
added at a final concentration of 1.6 mg/mL to 480 μg desolvated 3HA
nanoparticles and an amine crosslinking reaction was performed using 3 mM
3,3´-Dithiobis\[sulfosuccinimidylpropionate (DTSSP, Thermo Scientific, Waltham,
MA) for 12 hours while stirring to coat the nanoparticles. Coated nanoparticles
were collected by centrifugation, and protein concentration was measured by a
bicinchoninic acid (BCA) assay according to the manufacturer’s instructions
(Thermo Scientific) to estimate the total protein content in nanoparticles.
Dynamic light scattering (DLS) was performed in PBS with a Malvern Zetasizer
Nano ZS (Malvern Instruments, Westborough, MA) to assess nanoparticle size
distributions.
Hemagglutinating capability of 3HA nanoparticles was tested as previously
described. Briefly, 5 μg of 3HA protein or 3HA protein nanoparticles in 100
μL of PBS was serially diluted by half across 11 wells of a 96-well plate. 50 μL
of a 0.5% turkey RBC suspension in saline was then added to each well, and
incubated at room temperature for 1 hour. The hemagglutination titer was read as
the last well in the serial dilution that did not form a red button of settled
RBCs.
## 2.2 Extended storage
Extended storage of 3HA nanoparticles was performed in PBS at room temperature
(25°C) or 37°C. Nanoparticles and soluble protein were diluted to 200 μg/mL, the
concentration for vaccination, in 100 μL aliquots in 2 mL centrifuge tubes. The
tubes were sealed with parafilm and incubated for up to 1 month at 37°C, and up
to 112 days at room temperature. At each time point, one 100 μL aliquot was used
to measure hemagglutination activity of the nanoparticles, while two 300 μL
aliquots were stored at room temperature for *in vivo* immunizations.
## 2.3 Immunization and sample collection
All animal work was done in accordance with the IACUC guidelines of Georgia
State University, which specifically approved this study under IACUC approval
number A16024. Female, 6 week old Balb/c mice (Charles River, Wilmington, MA)
were intra-muscularly immunized with 10 μg aged 3HA nanoparticles, 20 μg aged
3HA nanoparticles, 10 μg freshly prepared 3HA nanoparticles, 10 μg soluble 3HA
protein, or PBS as a control. Five mice per group were immunized twice i.m. at a
3-week interval. To compare antibody responses, sera were collected 2 weeks
after each immunization by submandibular venipuncture. No anesthesia or
analgesia was used in this study. All animals were euthanized by CO<sub>2</sub>
asphyxiation according to the American Veterinary Medical Association Guidelines
for the Euthanasia of Animals.
## 2.4 Serum IgG titer
Serum IgG titer was assessed as previously described. Briefly, ELISA plates were
coated with 1 μg/mL 3HA protein in PBS and incubated overnight at room
temperature. Plates were blocked with 1% bovine serum albumin (BSA) in PBS for 1
hour. Mouse serum samples were initially diluted 1:100 in 1% BSA, and serial
half-dilutions were made in 1% BSA across the 96-well ELISA plate. Serum sample
dilutions were incubated for 1 hour, and 1 μg/mL HRP-conjugated goat-anti-mouse
IgG (Life Technologies, Grand Island, NY) in 1% BSA was used as a detection
antibody. Chromogenic quantification was performed by the oxidation of
tetramethylbenzidine by hydrogen peroxide (R&D Systems, Minneapolis, MN)
according to the manufacturer’s instructions. Two times the absorbance of naïve
group’s serum samples was considered the cutoff for measuring the endpoint
titer.
## 2.5 Hemagglutination inhibition assessment
Serum hemagglutination inhibition (HAI) activity was assessed according to a
protocol adapted from the World Health Organization. 10 μL mouse serum was
incubated with 30 μL of receptor-destroying enzyme (RDE) (Denka Seiken Co,
Tokyo, Japan) at 37°C overnight, followed by 56°C incubation for 30 minutes to
inactivate non-specific agglutinating proteins. Inactivated serum was diluted
with 60 μL room temperature PBS, and centrifuged at 500xg for 8 minutes to
collect treated serum samples. Eight, 25 μL serial half-dilutions of 1:10
treated sera were mixed in a round-bottom 96-well plate with 25 μL of 2 μg/mL
3HA protein and incubated at room temperature for 30 minutes. This concentration
of 3HA corresponded to 8 Hemagglutination Units (HAU) in 50 μL of PBS, as
prescribed by the WHO protocol. To this mixture, 50 μL of a 0.5% turkey RBC
suspension was added, and the wells were incubated for 1 hour to develop. HAI
titer was read as the inverse dilution of the last well able to prevent
hemagglutination, or in which a red button of settled RBCs was formed.
## 2.6 Statistics
IgG titers were assessed by the Kruskal-Wallis one-way analysis of variance
(ANOVA) for non-parametric samples. Hemagglutination inhibition titers were
analysed by comparing the number of wells neutralized to the standard HAI titer
of 40 (2 wells) established by the US Food and Drug Administration (FDA) as
protective, using a one-sample t test. P values less than 0.05 were considered
significant.
# Results
H7 hemagglutinin nanoparticles were synthesized and characterized as described
previously. The resulting nanoparticle size distribution was similar to that
of previously synthesized batches of nanoparticles. A pilot hemagglutination
study of three replicates showed identical hemagglutinating titers, and one
replicate per timepoint was used for further hemagglutination assays to conserve
materials. Nanoparticles stored in PBS at room temperature did not lose
agglutinating activity or appreciably change in size after 112 days, while
nanoparticles stored at 37°C retained full hemagglutinating activity for 2
weeks, and lost activity at 1 month. Soluble 3HA in PBS showed similar stability
to nanoparticles when stored at room temperature; no hemagglutinating activity
losses were observed after 56 days.
To compensate for any potential loss in hemagglutinating activity, we immunized
mice with a single dose (10 μg) or double dose (20 μg) of nanoparticles stored
for 112 days in PBS at room temperature. As controls, mice were immunized with
10 μg freshly-prepared nanoparticles or 10 μg soluble 3HA protein. Serum samples
were collected 2 weeks after priming and boosting immunizations and were
assessed for anti-3HA IgG titers. No significant differences in IgG titer were
observed between the nanoparticles stored at room temperature and freshly made
nanoparticles. The double dose of aged nanoparticles induced significantly
higher titers compared to soluble protein after the priming immunization, but
that significance disappeared after the boost immunization.
Hemagglutination inhibition (HAI) titer is a measurement of the ability of serum
antibodies to block hemagglutinin binding to sialic acid residues. This
measurement is directly correlated to the ability of an animal’s antibodies to
block influenza viruses from infecting host cells, and is a means of indirectly
predicting neutralizing antibody production. The World Health Organization has
defined a serum HAI titer of 40 or above to provide good protection against
influenza infection. Groups immunized with 10 μg stored and fresh
nanoparticles had significantly higher HAI titers than 40, while groups
immunized with soluble protein or PBS did not have significantly higher titers
than 40.
# Discussion
Nanoparticles were stored at room temperature for 112 days in PBS with no loss
in hemagglutinating activity. According to a report by the United Nations
Children’s Fund (UNICEF) and the WHO, approximately one month is needed for
cold-chain transport of vaccines from a manufacturer in the developed world to a
developing region. Storage of protein nanoparticles for almost 3 times this
length without additional excipients is especially promising, given that viral
and subunit vaccine storage typically requires the addition of polyethylene
glycol or sucrose to achieve weeks-long cold chain-independent storage.
Surprisingly, soluble 3HA was also able to retain hemagglutinating activity for
56 days. This could be due to the inherent stability of this particular protein,
and of recombinant subunit antigens generally.
Nanoparticles stored at 37°C in PBS were able to retain hemagglutinating
activity for 2 weeks, but started to lose activity at 1 month. Although we did
not immunize mice with nanoparticles stored at 37°C, we hypothesize that a
similarly high antibody titer would be achieved based on the correlation between
*in vitro* vaccine hemagglutinating activity and *in vivo* immunization
efficacy. Two weeks is a sufficient amount of time to reach rural health
clinics from a distribution center, but may not be enough time to store the
vaccine there for a prolonged period of time. Future studies should investigate
the mechanism of elevated-temperature-related activity loss and whether
stabilizing additives could mitigate any potential losses in immunogenicity.
Hemagglutination inhibition data suggested that our room temperature-stored
nanoparticles could be just as effective at inducing neutralizing antibodies as
freshly made nanoparticles. The FDA benchmark HAI titer of 40 allows us to
compare our vaccine to an accepted standard of immunity, and our data
suggests that our nanoparticles stored at room temperature can confer protective
immunity. Given the ability of freshly-made 3HA nanoparticles to protect mice
against a 10xLD<sub>50</sub> challenge of H7N9 influenza, we relied on
antibody measurements in this work. However, future challenge studies should
examine whether nanoparticles stored at warm temperatures can also confer
protective immunity.
The ability of 3HA nanoparticles to induce better HAI titers than soluble 3HA
concurs with current nanoparticle vaccine literature theorizing that multivalent
epitope presentation is a mechanism of recombinant antigen nanovaccine
adjuvancy. Our previous results demonstrating enhanced dendritic cell TNF-α and
IL-1β responses to OVA-coated OVA protein nanoparticles also support this
hypothesis.
In conclusion, we have shown that 3HA nanoparticles can be stored for up to 112
days at room temperature with no loss in *in vitro* hemagglutinating activity or
*in vivo* antibody and hemagglutination-inhibiting responses. The fact that 3HA
nanoparticles can remain immunogenic outside of cold-chain storage is a
promising sign that nanoparticle vaccines made from recombinant protein antigens
can survive both transport to and storage in the developing world. Future work
in assessing and engineering protein nanoparticle vaccine stability should aim
to (1) corroborate these results with other types of antigen nanoparticles, (2)
examine the mechanism of elevated temperature-related antigen nanoparticle
instability, and (3) extend viable storage at elevated temperatures beyond 2
weeks.
# Supporting information
The authors would like to thank Dr. Jong Rok Kim for the synthesis and
purification of the 3HA protein used for this study.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
The placenta is the key organ for successful pregnancy and fetal growth. It
performs key transport, metabolic, and secretory functions to support fetal
development. Term placental villi are covered by the multinucleated
syncytiotrophoblast that shares a basement membrane with a subjacent,
discontinuous layer of cytotrophoblast. The syncytiotrophoblast is in direct
contact with maternal blood and regulates maternal-fetal exchange.
To allow the efficient supply with oxygen and other key molecules to the
placenta and hence the fetus, the invasive cell population of the extravillous
trophoblast (EVT) invades the uterine decidua and myometrium (interstitial
trophoblast). A subgroup of the interstitial trophoblast migrates towards the
uterine spiral arteries, reaches the walls of such arteries (intramural
trophoblast) and can be found in the lumen of such vessels (endovascular
trophoblast), transforming them into large conduit vessels of low resistance.
This physiological transformation is characterized by a gradual loss of the
normal musculo-elastic structure of the arterial wall and replacement by
amorphous fibrinoid material in which trophoblast cells are embedded. The
interstitial route of trophoblast invasion through the placental bed has been
well described by Kaufmann et al.). These physiological changes are thought to
be required for a successful pregnancy.
At the same time, proliferation, migration, and invasion of extravillous
trophoblast are regulated by a large number of locally derived molecules
including members of the VEGF and the angiopoietin families to maintain a
healthy uteroplacental homeostasis. Extravillous trophoblast dysfunction has
been implicated in IUGR, one of the leading syndromes causing preterm delivery
and perinatal morbidity. This dysfunction is characterized by reduced numbers of
both, interstitial and endovascular trophoblast. Severe early onset IUGR is
often associated with preeclampsia, a leading cause of maternal death worldwide.
In preeclampsia, hypertension is associated with widespread maternal endothelial
dysfunction, leading to significant maternal morbidity.
Oxidative stress of the placenta is considered to be a key intermediate step in
the pathogenesis of preeclampsia, but the cause for this stress remains unknown.
In about 80% of all preeclampsia cases the extravillous trophoblast is not
affected, while the other 20% of preeclampsia cases also suffer from IUGR with
trophoblast malinvasion. In any case, placental hypoxia does not occur in cases
with trophoblast malfunction such as IUGR and preeclampsia. Hence, the cause for
the presence of oxidative stress with increased lipid peroxidation products, in
preeclampsia remains unknown. It is also still unknown whether in cases with
IUGR and preeclampsia, the malfunctional extravillous trophoblast also suffers
from oxidative stress.
A sensitive marker of oxidative damage and lipid peroxidation is
4-hydroxy-2-nonenal (4-HNE), a highly toxic aldehyde product of lipid
peroxidation which can be evaluated by immunohistochemical staining.
Significantly higher levels of 4-HNE have been consistently reported in
pathological placentas associated with oxidative stress.
A battery of genes encoding antioxidant enzymes is orchestrated upon exposure to
reactive oxygen species (ROS). This coordinated response is regulated via the
antioxidant response element (ARE) contained within the regulatory regions of
so-called “safeguard” genes such as glutathione peroxidase, and heme oxygenase-1
(HO-1). Activation of the nuclear factor erythroid 2-related factor-2 (Nrf2) as
a consequence of oxidative stress initiates and enhances transcription of these
safeguard genes, thus protecting cells against oxidative stress as well as a
wide range of other toxins.
Mann et al. were the first to discuss a link between Nrf2, vascular homeostasis,
and preeclampsia. Our laboratory provided the first experimental data that Nrf2
is active exclusively within villous cytotrophoblast of the preeclamptic
placenta, strongly suggesting that these cells suffer from oxidative stress.
Loset et al. reported that the Nrf2-mediated oxidative stress response was
overrepresented in the decidua of patients with preeclampsia, without indicating
the presence or absence of IUGR.
Furthermore, in the human placenta VEGF, PlGF and their two receptors are
differentially expressed throughout gestation. VEGF expression in the placenta
and placental bed declines as pregnancy advances, while PlGF and Flt-1 increase
towards term. Moreover, a recent study has shown that VEGF prevents oxidative
damage via activation of the Nrf2 pathway in the choriocarcinoma cell line BeWo.
This suggests that that decreased VEGF bioavailability during preeclampsia could
result in reduced basal defense against oxidative stress.
As an in vitro interplay was established between Nrf2 and VEGF, we hypothesized
that severe early onset IUGR and preeclampsia could be associated with
alterations in Nrf2 expression in the placental bed, particularly in the
extravillous trophoblast, since it is well known that VEGF and its receptor
VEGFR-1 (Flt-1) are expressed in these cells.
To test this hypothesis we used immunohistochemistry to examine the expression
of Nrf2, VEGF, and the oxidative stress marker 4-HNE in third trimester
placental bed tissues in cases of severe early onset IUGR/PE and control
pregnancies.
# Materials and Methods
## Tissues
All samples were collected from caesarean hysterectomy subjects received for
pathological examination at the Medical Institute, Ashgabat, Turkmenia (by M.K.)
apart from 2 control subjects collected in Aachen, Germany. These materials were
used in a former study by Kadyrov et al.. In all instances, permission was
granted for the histological studies, regarding the samples that were collected
in Turkmenia, approval was obtained from the Ethics Committee of the mother and
child medical centre in Ashgabat; protocol Nr. 047/1991 and Nr. 101/1992. For
the other samples our protocol was approved by the Ethics Committee of the
Medical Faculty of the University of Technology, Aachen, Germany (EK 512).
Written informed consent was obtained from each patient enrolled in this study.
Uterine tissues of control subjects were derived from caesarean hysterectomies
performed in five healthy normotensive women delivering term infants (38–40
weeks gestation) for reasons unrelated to placental development (fibroids,
cervical carcinoma, or uterine atony after caesarean section).
Uterine tissues of pathological subjects were collected from six women with
severe early onset IUGR and preeclampsia (29–34 weeks gestation). The selected
criterion for complicated pregnancies was severe preeclampsia with IUGR. Severe
Preeclampsia was defined following the criteria of the American College of
Obstetricians and Gynecologists, new onset hypertension (systolic blood pressure
≥160 mmHg or diastolic blood pressure ≥110 mmHg at least twice, measured six
hours apart) and proteinuria (5 g or higher per 24-hour period) after 20 weeks
of gestation. IUGR was defined as birth weight below the 10th centile of
customized birth weight for gestational age. The clinical characteristics of
patients enrolled in this study are summarized in.
## Immunohistochemistry
The placental bed was identified macroscopically and later verified
immunohistochemically by identification of extravillous trophoblast expressing
cytokeratin 7.
At least five samples measuring about 2×2×2 cm per uterus, covering the full
thickness of the uterine wall of the placental bed, were dissected, and fixed in
4% neutrally buffered formalin. The tissues were oriented during embedding in
such a way that the uterine layers were perpendicular to the plane of section.
To prevent bias each tissue block was rotated randomly around a virtual axis
from the endometrium to the perimetrium and embedded vertically in paraffin at
56°C. All the uterine layers can be seen in A) revealing the distribution of
extravillous trophoblast (positively stained) that invaded perpendicularly from
the endometrial-myometrial border into the myometrium.
Routine immunohistochemical procedures were performed on serial sections of 5 µm
thickness with antibodies against cytokeratin 7 (trophoblast marker; clone OV_TL
12/30, dilution 1∶200, DAKO, Denmark), VEGF (sc- 7269, 1∶30, Santa Cruz, USA),
Nrf2 (ab31163, 1∶50, Abcam, UK), and 4-HNE (ab46545, 1∶200, Abcam, UK). Binding
of species-specific biotinylated secondary antibodies was visualised with AEC
substrate chromogen (AEC) (Invitrogen, Germany). Sections were counterstained
with hematoxylin.
## Evaluation of Immunohistochemical Staining
Each immunostained section was analyzed semi-quantitatively using a modification
of the “quick score” method described by Detre et al.. In brief: An intensity
score was made on the basis of the average intensity of staining: 0 = negative,
1 = weak, 2 = intermediate and 3 = strong, then the percentage of positive
cells (endovascular and interstitial, mononuclear and multinuclear extravillous
trophoblast cells) for each staining was rated as: 1 = 0–25%, 2≤25–50%,
3≤50–75%, and 4≤75–100%. The whole of the section was assessed.
Two independent pathologists examined the immunohistochemical slides while
blinded to the clinical history of the patients. The intensity score and the
proportion score were then multiplied and scores summed to give a range of the
possible score of 0 to 15. For example, negative staining in 25% of the
extravillous trophoblast (0×1 = 0), weak staining in 50% (1×2 = 2) and strong
staining in 25%(3×1 = 3) would give a total score of 0+2+3 = 5.
## Statistical Analysis
Statistical analyses were performed using Student’s unpaired t test for dual
comparisons. Mean differences were considered to be significant when p\<0.05.
All statistical graphs and analyses were created with GraphPad Prism 5.0
(GraphPad Software, La Jolla, CA).
# Results
Several different cell types in the placental beds and uterine wall biopsies
were positive for Nrf2, 4-HNE and VEGF, including extravillous trophoblast B, 4
B, 5 A), decidual stromal cells A arrows), myometrial cells, leukocytes in
decidua and myometrium B arrows), and vascular endothelial cells (A, B arrows).
Since the aim of this study was to evaluate the differential immunostaining of
the aforementioned proteins in extravillous trophoblast, other cells were not
analyzed in detail. Semi quantitative analysis was confined to
endovascular/intramural and interstitial extravillous trophoblast.
In control pregnancies, extravillous trophoblast expressed Nrf2 to a similar
extend as has been described previously for villous trophoblast by Wruck et al..
However, immunostaining for Nrf2 was stronger in endovascular and interstitial
trophoblast in pregnancies complicated by early onset IUGR/PE B). Immunostaining
scores showed that immunostaining of cytoplasmic Nrf2 in extravillous
trophoblast was significantly increased in the IUGR/PE group (p\<0.0001) when
compared with the control group E).
In control pregnancies, interstitial and endovascular trophoblast, which stained
positive for cytokeratin 7 C), were virtually negative for 4-HNE A). By
contrast, these cytokeratin 7-positive cells D) showed stronger 4-HNE
immunoreactivity in IUGR/PE cases B) in comparison with the same cell type of
the control group. Immunostaining scores showed that immunostaining of 4-HNE in
extravillous trophoblast was significantly increased in the IUGR/PE group
(p\<0.005) when compared with the control group E).
VEGF was immunolocalized in both populations of the extravillous trophoblast,
interstitial and endovascular trophoblast of the placental bed of the control
group A). VEGF immunostaining was almost absent on extravillous trophoblast from
IUGR/PE cases B). Semi quantitative analysis of VEGF immunostaining revealed a
statistically significant difference between the control and IUGR/PE groups
(p\<0.05) E).
# Discussion
The feto-placental circulation in severe early onset IUGR/PE cases is
characterized by abnormal umbilical blood flow velocity waveforms, thought to be
indicative of increased placental resistance. In such cases, the uteroplacental
blood is altered as well showing a ten-fold increased blood flow velocity from
the spiral arteries into the intervillous space. The distorted blood flow
through spiral arteries that have not been transformed adequately may also
induce changes in oxygen delivery to the surrounding decidual tissues. This in
turn may result in a marked overproduction of ROS (reactive oxygen species),
generated mainly in mitochondria, and will cause oxidative stress. Increased
production of ROS will trigger a cascade of events to enhance the cellular
defense against oxidative stress, mainly by the Nrf2/ARE system. This is the
first study to examine the expression of Nrf2 in the placental bed of IUGR/PE
samples. As the transformation of spiral arteries is mediated by invasive
extravillous trophoblast, we focused on the expression of Nrf2 in these cells.
Although our data were limited because of the shorter duration of gestation in
the early onset IUGR/PE group, it is very difficult to obtain a normal control
group of the same gestational age. At the same time, a comparison between
specimens from early onset IUGR/PE and normal term controls does not necessarily
limit the significance of the data because transformation of the myometrial
spiral arteries largely occurs during the transition between the first and
second trimester. Consequently, uterine artery Doppler waveforms can identify a
high proportion of women who develop early onset IUGR/PE already at 12 weeks ,
while they show low resistance patterns in normal pregnancies at 22–24 weeks.
Therefore, trophoblast populations and spiral artery modifications are very
unlikely to change further between 30 and 40 weeks of gestation.
Endovascular and interstitial extravillous trophoblast populations show
decreased densities in early onset IUGR/PE as already described by Kadyrov et
al.. In these pathological samples the extravillous trophoblast revealed a
significant increase in staining intensity for Nrf2 in the cytoplasm of these
cells (B, E). This data suggests that the extravillous trophoblast in IUGR/PE
suffers from (oxidative) stress leading to increased Nrf2 expression.
Activation of Nrf2 has been correlated with transfer of this protein into the
nucleus which does not become obvious in our study. It seems as if Nrf2 is not
activated in these cells similar to what has been described for Nrf2 in
preeclamptic villous trophoblast These authors described lower transfer of Nrf2
into the nucleus, indicative for a lower activity of Nrf2. Hence, although the
extravillous trophoblast in IUGR/PE upregulates Nrf2 expression it seems as if
activation of Nrf2 fails under these conditions and thus a defense system to
combat oxidative stress in the extravillous trophoblast is not effective.
Vascular endothelial growth factor (VEGF)-A is expressed by extravillous
trophoblast and binds to the fms-like (Flt1) and kinase-insert domain-containing
tyrosine kinase receptors, which are expressed on extravillous and villous
trophoblast. In cases with early onset IUGR/PE, which are associated with a
failure in spiral artery invasion, there is down-regulation of VEGF in the
extravillous trophoblast and placental up-regulation of soluble Flt1. In an
earlier study we found that VEGF induces Nrf2 activation leading to prevention
of oxidative stress. Therefore, we tested whether increased Nrf2 expression is
associated with higher immunostaining for VEGF in these cells.
In contrast to Nrf2, VEGF expression was reduced in extravillous trophoblast in
the placental bed of IUGR/PE pregnancies B) confirming already published data.
This data suggests that Nrf2 activation is not a consequence of increased VEGF
expression, but may be a secondary adaptive response to ROS signaling.
Since Nrf2 upregulates the expression of antioxidative and detoxifying enzymes,
we further tested the level of oxidation-mediated changes in lipids (4-HNE) in
extravillous trophoblast in term controls and IUGR/PE by immunohistochemistry
(A, B). There was a marked increase in 4-HNE immunostaining in extravillous
trophoblast of IUGE/PE compared with the control group B).
The picture that develops from our data is as follows: In IUGR/PE extravillous
trophoblast experience oxidative stress (increased 4-HNE) and try to counteract
by increased expression of Nrf2. However, since these cells fail to upreguate
VEGF at the same time, activation of Nrf2 does not occur. In addition, several
studies have shown that IUGR/PE is associated with reduced levels of antioxidant
enzymes, which lead to further trophoblast damage. This in turn may result in
increased apoptosis of extravillous trophoblast and decreased densities of such
cells in the placental bed.
Taken together, we suggest that the extravillous trophoblast at late stages of
IUGR/PE pregnancies demonstrate an impairment of the Nrf2 signalling pathway, in
spite of the increased cytoplasmic Nrf2 expression, related to the cellular
oxidative damage occurring at earlier stages of the syndrome.
In conclusion, it can be hypothesized that decreased VEGF bioavailability during
early stages of preeclampsia results in insufficient Nrf2 activation, reduced
basal defense against oxidative stress and a higher vulnerability of trophoblast
to oxidative cell damage. This does not seem to be true for the villous
trophoblast only but may be extended to the extravillous trophoblast in cases
which combine IUGR and preeclampsia. The resulting damage causes increased
apoptosis and will further speed the vicious circle of shallow invasion in such
cases. Consequently, one would expect that these disturbances will limit
trophoblast invasion into the walls of spiral arteries of women destined to
develop early onset IUGR/PE. Specific attempts to strengthen the fetal
endogenous defence system against oxidative stress during early gestation could
prove to be a possible treatment option and may in turn reduce the risk of the
combination of IUGR and preeclampsia and associated perinatal complications.
The authors thank Dr. rer. nat Lars Ove Brandenburg and Dr. rer. nat Mersedeh
Tohidnezhad for performing the IRS scoring in this work.
We would like to thank Michaela Nicolau for her excellent technical assistance.
We thank Wolfgang Graulich for the production of the illustration figure (1). We
also gratefully thank the Department of Histology, Faculty of Medicine at
Damascus University because of the scholarship of Nisreen Kweider.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: NK BH MK. Performed the
experiments: NK RB MK. Analyzed the data: NK BH CJW WR TP MK. Contributed
reagents/materials/analysis tools: NK BH CJW RB WR TP MK. Wrote the paper:
NK BH MK. |
# Introduction
Microarray technology allows the capture of diverse aspects of genetic,
environmental, oncogenic and other factors as reflected in global mRNA
expression and opens the possibility of personalizing treatment of disease.
Multiple studies have taken a “top-down” approach to profiling gene expression
in human cancers, and this has led to the identification of tumor subtypes
unrecognized previously as well as gene signatures predicting various clinical
phenotypes. Alternatively, other studies have taken a “bottom-up” approach to
determine the change of gene expression caused by specific manipulations of
cultured cells *in vitro*. In these studies gene expression serves as a common
phenotype to recognize similar features in human cancers *in vivo* and to
provide a direct linkage between the known biological perturbation and the
clinical contexts.
Though many such studies have shown promise in using *in vitro* cell
manipulations to understand *in vivo* biology, this approach cannot fully
reflect the enormous phenotypic variation seen in human cancers. From such
studies, one can derive *signatures*. These we define to be lists of genes that
are differentially expressed along with their associated levels of differential
expression (which we call weights). However, there is nearly always a poor match
between these signatures and expression patterns of the same genes *in vivo*.
Therefore, a conceptual framework is needed to further dissect, enhance and
extend the *in vivo* utility of the *in vitro* derived signature. Here, we
present a technique for achieving this purpose. We propose deriving multiple
factors, based on human cancer gene expression studies, from an experimentally
defined signature. These derived factors will retain their relationship to the
original signature but represent distinct biological processes. Importantly, we
show that different derived factors can be combined to provide much better
predictive values for the clinical outcomes. Different factors also reflect
different biological processes and are linked to various aspects of molecular
and clinical features of human cancers.
There are a number of possible approaches to this problem. One popular approach
has been to compare the identities of the differentially expressed probes to
databases of pre-defined pathways. Descriptions of such approaches can be found
in. While these approaches are appealing for their interpretability, they rely
on the appropriately pre-defined pathways rather than the structure of the data
under study. Alternatively, one may simply define the signature activity level
for a sample as the weighted average of *in vivo* expression levels (where the
genes over which to compute the weights and the weights themselves are drawn
from the original signature). Although some studies have shown the power of this
concept, it is clear that one can not hope to capture the heterogeneity of *in
vivo* biology from the one-dimensional controlled biological response the *in
vitro* signature reflects.
The inherent heterogeneity of environment and cell type in tissue samples means
that the genes in a signature may potentially involve many additional activities
not evident *in vitro*. Further, experiments on cloned cell lines of a single
cell type grown under tightly controlled conditions for a fixed (and relatively
short) length of time may contrast starkly with clinical samples extracted from
living organisms containing multiple cell types that have been in a dynamic
environment for months or years. There is no clearly “correct” method for taking
what is learned by microarray experiment in culture and applying it to assess
pathway activity in tissue samples. Some genes may be poorer representatives of
pathway activity *in vivo* because they are more likely to be involved in other
pathways, because they react to environmental conditions that are not present
*in vitro*, or for a myriad of other reasons. It is, therefore, important to
provide a statistical and conceptual framework which can allow us to use the *in
vivo* expression data to further dissect, refine and enhance the *in
vitro*-derived gene signatures.
*Signature Factor Profiling Analysis* (SFPA), based on sparse statistical factor
models, is a framework for mapping *in vitro* signatures to a collection of *in
vivo* factors. While this sounds similar to hierarchical clustering (which has
become the default method for this type of problem), there are important
distinctions. First, while hierarchical clustering can be used to break a set of
samples into groups, within which expression patterns are similar in some way,
it does not quantify that similarity. Second, hierarchical clustering requires
that each observation (gene) be a member of just one cluster. This precludes
assigning clusters to biological pathways, because many combinations of pathway
activity are possible. Lastly, because the factors are generated within a
statistical model, it is possible to identify the levels of activity in each of
the factors on a newly measured sample without redoing the statistical analysis.
While there are techniques other than hierarchical clustering which address some
of these issues, for example soft-clustering and k-means clustering, our
algorithm addresses them all within a single coherent statistical framework.
SFPA provides:
- Robust statistical modeling of both experimental gene expression and tissue
sample expression.
- Identification and correction of assay artifacts, which are known to be a
significant issue associated with the use of microarray technologies.
- A mapping from a single signature, generated *in vitro*, to a collection of
factors that retain the pertinent characteristics of the signature while
better reflecting heterogeneity *in vivo* associated with the biological
perturbation the signature represents.
- A model for imputing the values of factors in new collections of tissue
samples even though these samples may originate from different groups and at
different times.
We explore this analysis approach in translating a collection of gene signatures
reflecting cellular response to five known tumor microenvironmental factors,
discovered *in vitro*, with particular emphasis on the signature associated with
response to lactic acidosis. We demonstrate that multiple factors arising in a
breast cancer context remain representative of the individual microenvironmental
pathway responses from which they are derived. Furthermore, these factors
differentiate key biological phenotypes in breast cancer, are able to improve
clinical predictions across multiple cancer data sets, and retain their
predictive ability even when applied to samples taken at vastly different times
or at different study centers.
# Results
## Context, Data and Analysis Strategy
We begin with five signatures defined by the transcriptional responses of
cultured human mammary breast epithelial cells to five microenvironmental
perturbations: hypoxia, lactic acidosis, hypoxia plus lactic acidosis, lactosis,
and acidosis. Each of these is seen in human cancers and carries prognostic
information with respect to clinical outcomes. The signatures represent changes
in expression of genes between a set of control observations and cells grown in
the presence of lactic acidosis (25 mM lactic acid, pH 6.7), hypoxia (2% O2),
lactic acid plus hypoxia, lactosis (25 mM sodium lactate, neutral pH), and
acidosis (pH 6.7 without lactate). Expression assays used Affymetrix U133+ 2.0
microarrays and signatures reflecting each of the microenvironmental factors
have been described. As shown in, hypoxia, lactic acidosis and acidosis have
strong prognostic significance in several studies of breast cancers. Our aim
here is to explore the various components of the original gene signatures to
evaluate the opportunity for further enhancing their prognostic values and
dissecting them into distinct biological pathway-relevant factors with clinical
relevance.
We use Bayesian Factor Regression Modeling (BFRM) to define and estimate factors
based on a given signature. This begins with a small collection of genes that
are highly responsive to the original intervention (highly differentially
expressed between control and experimental groups in cell culture) and then
iteratively refines the gene set, based on co-expression in an in vivo data set,
in the context of a statistical factor analysis. First, common patterns of
expression (factors) are discovered within the subset of genes currently under
consideration. Next, the association between these factors and the full set of
genes on the array allows us to identify additional genes to be included in a
revision of the factor analysis. The rationale for this is that, while
evaluating factors underlying the initial selected signature genes allows us to
elucidate *in vivo* variability that is not present *in vitro*, adding genes
from outside the original signature can improve the characterization of these
factors while providing linkages to other relevant pathways. Running SFPA on
each of the five signatures independently, we obtain 11 hypoxia factors, 10
lactic acidosis factors, 20 hypoxia plus lactic acidosis factors, 17 lactosis
factors and 9 acidosis factors. SFPA stops discovering factors once most of the
variability in the original gene set has been explained.
## Signature-Factor Relationships
We will focus, for now, on the ten lactic acidosis factors. Examining the genes
in each of the factors shows that all factors have representatives from the
original signature in addition to genes added during the process of fitting the
factor model. It is important to be sure that in the discovery of these ten
factors, we have not lost our original signature. We check this by regressing
the 10 sets of derived factor scores on the lactic acidosis signature scores.
(Calculation of a signature score is described in the section.) Witin a single
multivariate regression model, we find that 7 of the 10 are significant at
the.01 level, and that when we eliminate the remaining three factors from the
multivariate regression, those seven remain significant. Thus, at least seven of
the factors show a significant association to the original signature.
shows the fitted values from the regression of the lactic acidosis signature
score on the lactic acidosis factors from the analysis of the 251 tumor sample
data set from. The for this regression is high (.74), but it is possible these
ten factors might be able to explain many different signatures. In order to show
that this is not a spurious association, we test the hypothesis that this level
is independent of which genes are assigned which weights. We re-sampled the
weights 10,000 times, each time regressing the signature score vector computed
from these weights on the 10 lactic acidosis factors and computing an value. Of
the 10,000 values of so computed under the null hypothesis, the maximum was.48
ensuring that the p-value ≪10<sup>−4</sup>. If we approximate the distribution
of values by a beta distribution (calculated by method of moments) we get a very
close fit and estimate the p-value to be ≈10<sup>−13</sup>. Because only the
list of highly differentially expressed genes from the lactic acidosis
signature, and not the weights, are used in the factor discovery, and because
the weights are critical for the computation of the lactic acidosis signature
scores, the ability to recover signature scores from factors is strong evidence
of the relationship between the two.
The three factors derived from the lactic acidosis signature that were not
important in the prediction of signature scores may still represent activity
relevant to the presence of lactic acid, but they are not strongly predictive of
the original signature. They may also simply represent the activity of
biological pathways that involve very large sets of genes, and are thus
discovered from many different possible starting points. Nonetheless, they
represent significant structure in expression of the expanded signature gene set
in tumor data, and none of these factors would be detectable from studying the
signature alone as a phenotype.
Factors can reflect distinct aspects of biological activity. shows which of the
67 factors (all factors discovered from each of the five starting signatures)
have high correlation with the 10 lactic acidosis factors from the Miller breast
data analysis. Notice that no two of the lactic acidosis factors are highly
correlated, thus these factors seem to describe distinct processes. Some of the
10 factors, such as lactic acidosis factor 8 for example, are highly correlated
with multiple other factors, indicating that these factors have been identified
from multiple initial signatures. Most, however, show low levels of pairwise
correlation. Among the 67 factors, 40 principal components are required to
account for 95% of the observed variability (supplementary) implying that a
relatively high biological “dimension” underlies the 67 factors – they reflect a
diverse set of biological activities, and presumably pathways altered in the
cellular responses to lactic acidosis within human breast tumors. shows the
connections between genes and the 10 lactic acidosis factors in the analysis.
The genes include the initial selected signature genes and those added through
the iterative enrichment analysis. The SFPA-derived factors retain a high
percentage of genes that have been shown to exhibit a change in expression when
cells are exposed to the presence of lactic acid *in vitro*, showing in another
way that these factors still maintain their connection with the original
signature. The cross-talk between factors, in terms of genes defining more than
one factor, is also evident.
## Factors Predict Molecular Features
SFPA-derived factors can represent distinct aspects of biological processes
associated with clinical phenotypes. To evaluate this, we explored subset
regression models to predict a number of clinical phenotypes in the Miller data
set - the phenotypes including ER and PgR status, p53 status and survival times.
The molecular status indicators were modelled with binary probit regressions on
the factors, and survival with standard Weibull survival models. We utilized the
Shotgun Stochastic Search (SSS) method, to identify small subsets of the factors
showing predictive value with respect to each of these phenotypes. SSS is a
variable selection model which allows the use of model averaging (based on
posterior likelihood) for prediction. Model averaging has been shown to perform
better than algorithms which use the single best model for prediction (such as
AIC or BIC) because it gives a truer estimation of uncertainty. This analysis
was performed on the data set from, and then the resulting fitted/trained
regression models were used to predict phenotypes in each of five separate and
biologically diverse breast cancer data sets. All data sets are available from
the Gene Expression Omnibus (GEO).
### Factors predict ER status
The analysis indicates that highly scoring regression models for the prediction
of ER status utilize one of the factors – Acidosis 1, Hypoxia 4, Lactic Acidosis
2, or Lactosis 5. From, one can see that the correlation between any two of
these factors is high, so we will refer to them collectively as the ER factors.
demonstrates the ability of this factor to predict ER status on the training set
and 3b shows prediction on a distinct and completely unrelated test set. To
examine the gene ontology (GO) composition of the list of genes involved in the
ER factors, we applied the GATHER analysis and find that GO terms associated
with cell cycle, proliferation and and mitosis are greatly enriched in these
factors, corroborating well-known connection between cell progression and ER. It
is also expected that the presence of lactic acid or hypoxia acts to shut down
the cell cycle and the ER factor appears to directly link the two processes.
### Factors predict PgR status
Estrogen and progesterone are known to be antagonists, so it is expected that ER
factors can predict progesterone receptor status. Using SSS we find that the
highly scoring regression models for PgR status involve the ER factor in
addition to Lactic Acidosis factor 10 – we label this the PgR specific factor.
show the fitted and predictive ability of these two factors used in a binary
regression model fit to progesterone receptor status. There is no significant
correlation in tumor expression between the PgR and ER factors. Gene ontology
for the genes in the PgR specific factor bear out some of the known links
between progesterone and RNA metabolism in breast cancer.
### Factors predict p53 status
The third binary phenotype, wild type versus mutant p53 gene, is present in only
the data set from. SFPA was re-run on a randomly selected 50% of these data and
used to predict the other 50%. Highly scored models for p53 involve the ER
factor, the PgR specific factor, and one of either Hypoxia 1 or Lactic Acidosis
3. The correlation between these latter two factors is 99%, so we label them
collectively as the p53 specific factor. Gene ontology for this factor is
identical to that for the ER factor with the exceptions that “cell
proliferation” and “DNA replication initiation” are replaced by “nuclear
division” and “M phase”. For all gene ontologies listed in the top eight for
these two factors, the Bayes factors are ≥10. Because of the high degree of
similarity in the gene ontology, it is tempting to try to equate these two
factors. shows a scatterplot of the activity of the tumors in the data from on
each of the two factors. The p53 factor is significantly bimodal, and the mild
correlation one can see is due entirely to this bimodality, as tumor samples
with high ER factor activity are more likely to be in the second mode of the p53
factor. We theorize that this bimodality is associated with a particular subtype
of the p53 mutation. However, there is no evidence of multimodality in the ER
factor, and the p53 specific factor predicts ER status poorly. Because of these
differences, and because cell replication is a complex process, it is likely
that these two factors are related to distinct features of cell development.
We stress that, if we restrain ourselves to considering the original *in vitro*
lactic acidosis signature, we have no ability to fit or predict any of these
biological phenotypes. Additionally, these factors were generated entirely
without regard to the ER status, PgR status, or p53 status of the samples. This
is in contrast to a more typical design in which signatures associated with
phenotypes are defined strictly based on genes with expression profiles that
match those phenotypes (for example). This type of design is plagued with
difficulties that arise from the large number of genes, out of the tens of
thousands on an array, with expression patterns that match any arbitrary
phenotype. With SFPA, we search for genes that are expressed together without
regard to phenotype, and we are therefore much less likely to be plagued by
false discovery (as proven by our out of sample predictive accuracy).
## Factors Predict Clinical Phenotypes
SFPA offers a technique for interrogating a single independent tumor sample
against any number of biologically determined signatures, and then consequent
linking of factors to phenotypes may include clinically relevant outcomes such
as patient survival outcomes and drug response.
### Factors improve prediction of breast cancer survival
Subsets of the 67 factors were evaluated in Weibull survival regression models
using the SSS method to identify and score models predicting survival. Each
model in a resulting set of highly scoring models produces fitted survival
curves and also may be used to predict survival for new samples. Bayesian
analysis mandates averaging predictions from such a set of models, and this was
done to result in. This shows fits of survival curves for the training data set,
together with out of sample predictions in four of the other data sets for which
information regarding survival exists. Recall that these are data sets from
quite distinct and diverse studies, so we are assessing a model fitted to one
data set on four quite challenging out of sample validation data sets. Though
not described further here, the BFRM statistical model analysis used by the SFPA
also addresses issues of gene-sample-study specific effects within the analysis
and is able to correct enough of the idiosyncracies and bias inherent in
microarray assays to retain predictive accuracy. The results demonstrate that
the factorprofiles of these *in vitro* environmental signatures can improve
survival prediction significantly across several test data sets. Similar results
are obtained for the prediction of metastasis-free survival.
### Factors predict Tamoxifen response
Four of the breast cancer data sets have clinical annotation pertaining to
treatment with Tamoxifen. Though the 67 factors are in no way specifically
targeted at Tamoxifen, we do know they are associated with relevant biological
pathways. From our 67 factors, we found that Lactic Acidosis 1 is predictive of
Tamoxifen resistance. It differentiates metastasis-free survival in patients who
received the drug and shows no predictive ability in patients who did not (the
analysis underlying this followed the same approach as for survival discussed
above). Because all of the patients who received Tamoxifen were ER positive,
drug resistance associated with this factor must be independent of the
antagonistic action of the drug on estrogen receptors. Since none of these data
sets were used in the training of the factor model, the ability of these factors
to distinguish resistance to Tamoxifen is remarkable and demonstrates that they
are robust to the collection biases often seen in microarray experiments. We
again used GATHER to study the ontology of the genes included in this factor.
This connects with the known association of Tamoxifen with phosphate transport,
as well as cell adhesion. In particular, Cowell et al. report that p130Cas/BCAR1
is a cell adhesion molecule that promotes resistance to Tamoxifen via a
particular phosphorylation pathway. In addition to these connections to the
secondary effects of Tamoxifen is the well-known connection between survival of
patients on Tamoxifen and toxicity associated with blood coagulation. Further
study of the genes in this factor may lead to insight into the mechanism behind
Tamoxifen resistance in ER positive breast cancer.
### Discovery of organ-specific factors from lactic acidosis signatures
While the same biological processes may contribute to tumor phenotypes in
different cancers, the process by which this happens may be entirely different
given the particular cellular context, tissue-specific gene expression and
epigenetic influences. Since SFPA can utilize *in vivo* cancer gene expression
to dissect the *in vitro*-generated gene signature, it offers the possibility of
identifying tissue and organ-specific factors associated with the same gene
signatures. This application has the potential to distinguish sub-pathways that
are conserved across many tissue types from those that are organ-specific. To
illustrate this point, we utilize the lung cancer data set published in and the
ovarian cancer data set from. We obtained the lung cancer data from GEO and the
ovarian cancer data from the Duke Integrative Cancer Biology Program (ICBP) web
site (<http://data.cgt.duke.edu/platinum.php>). We performed SFPA analysis of
the same lactic acidosis signature as a starting point for factor discovery from
the lung and ovarian cancer data sets.
In the case of the lung cancer, the analysis discovered 20 factors associated
with lactic acidosis. When we compared the expression levels of the 10 lactic
acidosis factors in the breast cancer data with the 20 lactic acidosis factors
discovered in the lung cancer data, we found that several factors are highly
conserved, including the tamoxifen factor, the p53 specific factor, as well as
factors 7 and 8. In contrast, the ER and PgR factors are only found in breast
cancers. If we look specifically at standardized raw expression levels for the
genes in the ER factor in the breast data as compared to that for the lung data
we see that there is consistent variation in the breast data which is not
present in the lung data. In contrast, the standardized raw expression for the
conserved tamoxifen factor shows a coherent expression pattern in both breast
and the lung cancers. Additionally, within this data set, our newly discovered
factors also possess significant prognostic value, being able to distinguish
between the two types of cancers as well as distinguish between high and low
risk patients. Similar observations are also present in ovarian cancer since the
model averaged survival using the 8 lactic acidosis factors discovered in the
ovarian cancer dataset can clearly differentiate high versus low acuity
patients. Additionally, we see the same patterns of loss of the ER factor and
conservation of the tamoxifen factor. Finally, we find that the exact same three
factors, the p53 specific factor, factor 7, and factor 8, have analogous factors
in the ovarian cancer data with greater than 90% correlation (as computed on the
251 breast cancer samples).
# Discussion
It is increasingly common for investigators to use gene expression signatures
directly as phenotypes to link various biological processes and perturbations to
disease phenotypes and chemical agents. Although these signatures derived *in
vitro* offer a way to understand the *in vivo* biology, there is still
considerable limitation due to the differences between these two settings. Here,
we have exemplified a statistical approach to further improve the *in vitro*
gene signatures based on the gene expression in *in vivo* cancer samples.
Elaborating the factor profile underlying the original signatures can, as we
have seen, improve the *in vivo* relevance by more fully describing the
diversity of *in vivo* expression patterns. This may enhance prognostic value
and provide mechanistic insights into how biological processes affect clinical
phenotypes. As an example, we have found direct links between factors generated
by the use of SFPA on the lactic acidosis signature. Such links are intriguing,
and open questions regarding causation as well as questions about the biological
associations of the remaining factors. However, regardless of links to known
biology, this strategy and analysis seem to provide an advance in our ability to
obtain consistent results across many different data sets collected at different
times by different groups. This is a significant advance, as data collection
inconsistencies are one of the main roadblocks to the use of microarrays in a
clinical setting.
There are several possible explanations for the enhancement of the prognostic
values achieved with SFPA. It is possible that certain genes or pathway
components in the original gene signatures are simply noise or artifact due to
their *in vitro* origins. These genes may offer no or even negative prognostic
values for *in vivo* biology. By using SFPA to separate different components, it
is possible to enhance the prognostic value by selecting only the relevant
components or genes for predictive purposes. By so doing, it is also possible to
examine the genes comprising those factors with strong links to clinical
phenotypes which will lead to biological insights into this association.
Another opportunity this analysis raises is the ability to uncover the pathways
which would be “hidden” in the *in vitro* signature. In our current study,
factor one was not immediately recognizable as a clinically relevant list of
genes, but the ability of this factor to predict patient resistance to Tamoxifen
points to an important connection which would not have been possible to discern
otherwise. This observation will lead to efforts in investigating the biological
roles of this factor and how it is related to Tamoxifen treatment and cellular
response to lactic acidosis. For example, it is well known that tumor hypoxia
negatively impacts clinical outcomes, but the actual mechanism by which this
occurs is complex and may include radiation resistance, increased tumor
invasion, migration, increased survival and decreased drug sensitivities.
Although these hypoxia-induced effects occur in cancer patients, many of these
events cannot be replicated or modeled in any particular *in vitro* setting. It
is possible to uncover these processes via of the use of SFPA for the cancer
gene expression. In a similar fashion, it is unclear how lactic acidosis
responses are linked to good prognosis, and SFPA will allow us to explore *in
vivo* gene expression to dissect this response and develop testable biological
hypotheses. Equally importantly, the mechanisms by which hypoxia and lactic
acidosis link to different clinical outcomes may vary among different cancer
types, and the use of SFPA can specifically pinpoint the relevant biological
processes to target or intervene to modulate clinical courses of cancer
patients.
Tremendous resources continue to be expended on the discovery of biomarkers for
drug susceptibility. The ability to predict susceptibility to a given drug has
the potential to significantly increase efficacy while decreasing morbidity and
mortality in the relevant patient population. Additionally, it opens the
possibility of facilitating the process of bringing new drugs to market. We have
demonstrated the efficacy of SFPA for translating signatures discovered *in
vitro* into factors which are clearly related to specific biological processes
and which can be used to assess important clinical outcomes. The factors may be
applied to just one observation (an important consideration for use in a
clinical setting), and remain consistent across many different data sets. We do
view this as a useful step ahead in thinking about how gene expression genomics
will advance us towards the goals of personalized medicine.
# Methods
A total of five signatures were derived from two different experiments on Human
Mammary Epithelial Cells (HMEC). The details of the collection of gene
expression data from these cell lines are in. Signatures from these experiments
were derived using the Bayesian Factor Regression Modeling (BFRM) software
detailed in, and that has been used in multiple previous analyses of similar
data. The workspaces used for BFRM are available in the supplementary, and the
software is publicly available.
In designed experiments such as, BFRM provides a sparse ANOVA framework for
studying changes associated with environmental stresses. It includes
functionality for correcting systematic laboratory bias which come about due to
differences in conditions under which the microarray data is collected. These
systematic differences are reflected in the doping control genes, which are used
to construct the correction factors.
## Sparse Regression for Experiments with Known Variation
BFRM is a Bayesian modeling framework. As such, we assume that all of the
parameters of our model are random variables. In order to learn more about the
values of these parameters, we specify prior distributions, which are
subsequently updated based on the data. The result of fitting the model to data
in this way is a joint posterior distribution for all of the model parameters.
In our case, the parameters of interest are the coefficients of the regression.
The general model implemented in BFRM is as follows. Let be a matrix of
expression values where (row, column) is the expression of gene from sample
where. Denote the design matrix (describing known sources of variability) by
having elements on sample and design or regression variable. The model may be
written as a separate linear regression for each probe on the array:
Or alternatively in matrix notationwhere is a matrix of regression coefficients
having elements and is a diagonal covariance matrix with non-zero diagonal
elements. We allow the regression coefficients to vary across both genes and
design vectors,but assign them a sparsity prior. We define to be the posterior
mean for, to be the posterior probabilities on non-zero values of the, and to be
the posterior mean of. All of these parameters are computed automatically by
BFRM (along with many others).
We have used a prior distribution for the coefficients of the regression that
has a point mass at zero. This reflects our belief that, for any particular
intervention, there will be relatively few genes (of the over ten thousand that
are measured in a microarray experiment) that are affected. For the case
outlined in this paper, we argue that growing mammary epithelial cells in the
presence of mild lactic acidosis has led to changes in the expression of some of
the genes on the array, but that most remain unchanged. Thus our posterior
distribution for each will consist of a probability that the parameter is non-
zero (corresponding to a probability that the gene is differentially expressed
in the lactic acidosis experimental group versus the control group), along with
a distribution on the magnitude of that possible change.
The prior on is assumed to be a diffuse inverse gamma distribution (which is a
standard conjugate prior), and the prior on is also given a point mass mixture
prior, reflecting the belief that we must maintain significant mass around the
extremes (zero and one) even after updating with all of the probes on the chip
(50,000+). The precise values of all hyper-parameters are available in the
parameter files in the supplementary section.
We define a signature to be a list of genes and associated weights. Using the
posterior parameters from above we define the weight of gene for experimental
group (design variable) to be. Calculation of the level of activity of a known
signature within a tumor sample requires that we initially subtract mean
expression levels and laboratory biases. These are computed with BFRM exactly as
above with the exception that the design matrix contains only the intercept
vector and correction factors (no design vectors). If is a *p*-dimensional
vector of corrected expression values associated with tumor sample *m*, then the
signature score of signature in sample *m* is the weighted average.
## Analysis of Breast Cancer Data Sets
We use six cancer data sets with Affymetrix U133+ expression samples available
on the Gene Expression Omnibus (GEO) web site. Details of the collection and
measuring are contained in,. For all but Wang, Affymetrix.CEL files were
available, and we computed RMA normalized values in these cases. For the data
set from, we used MAS5 normalized data which was obtained from the authors.
Statistical factor analysis using BFRM estimates latent factors that represent
common, underlying aspects of covariation of subsets of genes, typically
representing expression gene-by-gene in terms of contributions from possibly
several factors. The iterative analysis to expand on an initial set of signature
genes that we used here then revises the gene list by adding in genes apparently
associated with estimated factors, and then refitting the model. Full details of
this algorithm are available in. To choose a collection of seed genes associated
with experimental group *j*, we identify all genes, *g*, such that. From this
list of genes, we take the 25% with the highest absolute change in expression
level between the control group and the corresponding experimental group (as
measured by the posterior). Following this procedure, we obtain between 20 and
200 “seed” genes for factor analysis.
Given a signature, we must choose a collection of tissue samples on which to
train the factor model. Because of its relatively large size, the availability
of CEL files, and the wealth of clinical and phenotypic information, we chose
the data set from for the identification of factors. We have five sets of seed
genes, obtained as described above, from experiments on HMEC's. For each of
these five sets of genes, we independently use BFRM to obtain the factors that
are represented. We limited the number of genes to recruit into factors to a
total of 500.
To fit our binary regression and survival models, statistical analysis used
Shotgun Stochastic Search (SSS) routines from. Initialization files used by SSS
for these searches are included in the supplementary,. All Kaplan-Meier curves
showing differential survival are drawn by separating samples at the median of
the score that is relevant for that figure.
## Statistical Factor Models for Tumor Expression Data
Factor models are structured as in. If represents the column vector of gene
expression measures on *p* genes as assayed in a single tumor, is regressed
linearly on a combination of an overall intercept term and assay correction
factor, plus a set of latent (i.e., to be estimated) factors. If is the column
vector of known regressors (the intercept and assay correction terms) on tumor
*i*, the model is of the formwhere is a column vector of unknown latent factor
values on tumor *i*, and *A*,*B* are coefficient matrices. In the BFRM context,
both *A* and *B* are large “tall and skinny” matrices with many more rows
(genes) than columns (the number of regressors and factors), and are described
by the same sparsity probability models introduced above for the elements of
(inducing many zeros).
Implicit in this formulation is the assumption that there is a set of vectors,
equivalent to design vectors, which describe some part of the variation observed
in the matrix of expression values. This leads to the grouping of probes in a
way that is comparable to clustering, whereby we assign genes corresponding to
non-zero values in any particular column of to the same group. This is exactly
parallel to sparsity in the coefficients associated with the design vectors in
that we are assuming that most genes are not differentially expressed with any
single latent factor.
Calculation of the activity of a set of factors, on each tumor *i*, and
estimation of the factor loadings, is then a problem of statistical estimation
of the overall model. Details of these calculations are available in. The issue
of projecting factors to a new sample, is then one of prediction that is
immediately available from the BFRM analysis framework. For completeness, we
present the formula here:
Where are approximations of the factor scores for a new observation, with gene
expression values and design variables.
# Supporting Information
[^1]: Conceived and designed the experiments: JTC. Performed the
experiments: JLYC JTC. Analyzed the data: JEL CC MW. Wrote the paper: JEL
MW.
[^2]: The authors have declared that no competing interests exist. |
# Introduction
Juvenile-onset systemic lupus erythematosus (JSLE) is a heterogeneous systemic
autoimmune disease characterized by onset before 18 years of age and multi-organ
involvement. The occurrence of JSLE was estimated at approximately 20% of all
systemic lupus erythematosus (SLE) cases. The disease course in JSLE is
relatively more severe than that in adult-onset SLE due to increased rates of
renal, neuropsychological, and hematological manifestations. Lupus nephritis
(LN) and central nervous system involvement are predictors of poor prognosis in
JSLE.
Differences in clinical phenotypes related to underlying immune mechanisms were
reported to involve innate and adaptive immune responses. T and B cell
abnormalities contribute to the process of immune dysregulation and loss of
self-tolerance in this disease. Imbalance of lymphocyte subsets has been
demonstrated in SLE patients and showed some correlations with clinical
manifestations and disease activity. Reduced CD4<sup>+</sup> T cell frequency
and elevated CD8<sup>+</sup> T cell frequency were observed in JSLE patients
with high disease activity. An elevated B cell proportion was related to
increased incidence of arthritis, in line with a previous study indicating that
musculoskeletal involvement improved after receipt of B cell-targeted therapy.
Small subsets of peripheral blood lymphocytes, such as gamma delta T cells (γδ T
cells) and natural killer (NK) cells, are notably involved in the pathogenesis
of many autoimmune diseases. γδ T cells play an essential role in the
pathogenesis of SLE by acting as antigen-presenting cells, producing pro-
inflammatory cytokines, having immunoregulatory functions together with
regulatory T cells (Tregs), and enhancing autoantibody production by B cells.
Previous studies demonstrated that SLE patients had a lower frequency of γδ T
cells in their peripheral blood compared with healthy controls (HC), which may
result from infiltration of γδ T cells into target tissues such as the skin and
kidneys or from the disease itself. Tregs have a critical function in
maintaining self-tolerance through suppression of autoreactive lymphocytes.
Previous studies reported contradictory results regarding the use of different
markers and gating strategies to identify Tregs. The frequency of
CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> cells was lower in SLE patients
compared with HC and was associated with the SLE disease activity index
(SLEDAI). Moreover, the reduction in
CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> cells was linked to kidney
damage in active SLE patients. However, other studies reported unchanged
CD4<sup>+</sup>CD25<sup>+/hi</sup> cell frequency with or without
FoxP3<sup>+</sup> cells or even increased frequency of these cells in SLE
patients compared with HC. NK cells are innate immune system cells responsible
for cytotoxic functions and act as regulatory cells in the context of
inflammation. Natural killer T (NKT) cells have shared characteristics of NK
cells and T cells (CD3<sup>+</sup>). Previous reports described decreased
frequencies of NK cells and NKT cells in SLE patients related to high disease
activity and renal activity index.
Previous studies on SLE have mostly focused on the cellular components in adult
patients, with findings that the lymphocyte subsets varied according to age,
ethnicity, and environmental factors. Therefore, the findings have limited
applicability in JSLE. In addition, the complexity of the pathogenesis for JSLE
suggests the need for research targeting young patients rather than adult
patients. In the present study, we aimed to investigate the lymphocyte subsets
(CD4<sup>+</sup> T cells, CD8<sup>+</sup> T cells, γδ T cells, Tregs,
CD19<sup>+</sup> B cells, NK cells, and NKT cells) in JSLE patients and
determine their associations with clinical phenotypes and disease activity, as
well as long-term outcomes. These findings may fill the gap in knowledge on the
biological process of the disease and identify subgroups of patients with
different phenotypes and prognoses, leading to better personalized treatment
strategies in JSLE.
# Methods
## Study design and patients
This was a prospective cohort study. A total of 60 patients aged \<18 years who
were diagnosed with JSLE according to the Systemic Lupus International
Collaborating Clinics classification criteria at the Pediatric Rheumatology and
Pediatric Nephrology clinics of our hospital between May 2015 and December 2018
were included in the study. Blood samples were obtained from all 60 JSLE
patients during active disease with or without medications, and follow-up
samples were obtained in 34 of the 60 JSLE patients during inactive disease. For
a longitudinal follow-up study, 49 of the 60 JSLE patients were followed up at
0, 3, and 6 months. The HC group comprised 42 age-matched children from our
previous study. Baseline characteristics and clinical information, including
concurrent medications, were routinely collected during the follow-up visits.
Written informed consent was obtained from the legal guardians of the study
participants before enrollment. The study was approved by the Ethics Committee
of Ramathibodi Hospital (ID 055806) and conducted in accordance with the
Declaration of Helsinki.
## Disease activity measurement
We used the SLE disease activity index 2000 (SLEDAI-2K) to measure disease
activity. Inactive disease was defined as clinical SLEDAI-2K (cSLEDAI-2K),
excluding anti-double-stranded DNA (anti-dsDNA) and complement, equal to zero
regardless of medication. Clinical remission was defined as no clinical activity
(cSLEDAI-2K = 0) for 1 year. Three levels of remission were defined as follows:
(i) remission on therapy, patients with clinical remission, physician global
assessment of disease activity \<0.5 on a 0–3 visual analog scale, daily
prednisolone dose ≤5 mg/day or ≤0.2 mg/kg/day for body weight \<25 kg, with or
without antimalarials and immunosuppressants; (ii) remission off therapy,
patients with clinical remission without any medications or with only
maintenance antimalarials; and (iii) complete remission, patients with clinical
remission and serological remission (normalization of anti-dsDNA and complement
levels), without any medications or with only maintenance antimalarials.
Laboratory parameters including complement component (C)<sub>3</sub>,
C<sub>4</sub>, erythrocyte sedimentation rate (ESR), anti-dsDNA, complete blood
count, direct Coombs test (DCT), urinalysis, and urine protein-to-creatinine
ratio were collected.
## Immunophenotyping
Peripheral blood mononuclear cells were stained with fluorochrome-conjugated
antibodies (eBioscience, San Diego, CA, USA). Briefly, 10–20 μL of EDTA-treated
peripheral blood was incubated with fluorochrome-conjugated antibodies against
cell-surface antigens for 15 minutes at room temperature in the dark. The
antibodies used in the staining process were fluorescein isothiocyanate
(FITC)-conjugated anti-human CD3, allophycocyanin (APC)-conjugated anti-human
CD4, APC-eFluor 780-conjugated anti-human CD8, phycoerythrin (PE)-conjugated
anti-human γδ TCR, APC-conjugated anti-human CD19, PE-conjugated anti-human
CD56, and PE-Cy7-conjugated anti-human CD45. Red blood cells were lysed at room
temperature using Lysing Buffer (BD Biosciences, San Jose, CA, USA) for 10
minutes before measurement in a flow cytometer (BD FACSVerse; BD Biosciences).
For Treg staining, 10–20 μL of EDTA-treated blood was stained with PE-
Cy7-conjugated anti-human CD25 and APC-conjugated anti-human CD4 (1:100
dilution) for 15 minutes at room temperature, and then incubated with
Fixation/Permeabilization Solution (eBioscience) for 15 minutes in accordance
with the manufacturer’s protocol. Finally, the stained cells were incubated with
Permeabilization Buffer (eBioscience) containing 1:50 dilution of FITC-
conjugated anti-human FoxP3 at room temperature for 45 minutes and analyzed by
flow cytometry.
The gating strategies for the lymphocyte subsets were as follows:
CD4<sup>+</sup> T cells (CD3<sup>+</sup>CD4<sup>+</sup>); CD8<sup>+</sup> T
cells (CD3<sup>+</sup>CD8<sup>+</sup>); γδ T cells
(CD3<sup>+</sup>CD4<sup>−</sup>CD8<sup>−</sup>γδ TCR<sup>+</sup>); B cells
(CD3<sup>−</sup>CD19<sup>+</sup>); NK cells (CD56<sup>+</sup>); NKT cells
(CD3<sup>+</sup>CD56<sup>+</sup>); and Tregs
(CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup>). The percentages of the T
cell subsets among the total lymphocytes were analyzed using FlowJo v.10
software (FlowJo LLC, Ashland, OR, USA).
## Statistical analysis
We used IBM SPSS statistics 25 software (IBM Corp., Armonk, N.Y., USA) and
GraphPad Prism 8.3 software (GraphPad Software Inc., La Jolla, California, USA)
for the data analyses. Demographic, clinical, and laboratory parameters were
presented as median, interquartile range (IQR), and percentage as appropriate.
Categorical data were compared using the chi-square test or Fisher exact test.
For continuous data, the Mann–Whitney U test (unpaired data) or Wilcoxon
matched-pairs signed-rank test (paired data) was used for comparisons between
two groups. For comparisons between three or more groups, the Kruskal–Wallis
test (unpaired data) or Friedman test (paired data) was used with a post-hoc
Dunn test. The probability of remission on therapy was analyzed by the
Kaplan–Meier method. Predictive factors were evaluated by Cox proportional
hazards regression analysis and presented with hazard ratio and confidence
interval (CI). Statistical significance was accepted for a two-sided p-value of
\<0.05.
# Results
## Patient characteristics
The demographic, clinical, and laboratory parameters of the JSLE patients are
shown in. The JSLE patients in the study cohort had a median age of
approximately 12 years, which was comparable to that in HC. Of the 60 JSLE
patients, 34 provided blood samples for longitudinal follow-up during both
active disease (active group) and inactive disease (inactive group). The
majority of the total JSLE patients and longitudinal follow-up JSLE patients
were female. The median SLEDAI-2K at enrollment in all JSLE patients was 12 (IQR
6.00–18.00). For the longitudinal follow-up JSLE patients, the median SLEDAI-2K
was 10 (IQR 6.00–16.25) in the active group and 0 (IQR 0.00–2.00) in the
inactive group. The median C<sub>3</sub> and C<sub>4</sub> levels were low in
all JSLE patients and the active group. The median ESR was quite similar in all
JSLE patients and in the active group, but was nearly normal to normal in the
inactive group. Low median values of white blood cells, absolute lymphocyte
count (ALC), platelet count, and hematocrit were found in all JSLE patients and
the active group. The percentages of patients with anti-dsDNA positivity and DCT
positivity were 73.33% and 40% in all JSLE patients, 64.71% and 38.24% in the
active group, and 8.82% and 11.76% in the inactive group, respectively. Skin
involvement, mucosal ulcer, and fever were the most common clinical
manifestations. Approximately 35% of JSLE patients were included before starting
treatment. Prednisolone was administered with the highest frequency among the
medications.
## Differences among lymphocyte subsets between JSLE patients and HC
We first assessed the percentages of lymphocyte subsets in JSLE patients
compared with age-matched HC. The percentages of CD4<sup>+</sup> T cells, γδ T
cells, and NK cells were significantly lower in JSLE patients compared with HC.
In contrast, the percentages of CD8<sup>+</sup> T cells, NKT cells, and
CD19<sup>+</sup> B cells were significantly higher in JSLE patients compared
with HC. The percentages of Tregs and Tregs within CD4<sup>+</sup> cells did not
differ significantly between JSLE patients and HC. The data for the percentages
of lymphocyte subpopulations are provided in.
## Differences in lymphocyte subsets among non-LN JSLE patients, LN-JSLE patients, and HC
JSLE patients with LN have different disease severity and outcomes compared with
non-LN JSLE patients. Therefore, the frequencies of lymphocyte subsets in these
two groups of JSLE patients were evaluated and compared with HC. Disease
activity measured by SLEDAI-2K differed significantly between non-LN JSLE
patients and LN JSLE patients (median 10 \[IQR 6–16\] vs. median 21 \[IQR
11–26\], p = 0.0011). The frequency of Tregs in LN JSLE patients was
significantly lower than that in non-LN JSLE patients and HC. The frequencies of
CD4<sup>+</sup> T cells and CD8<sup>+</sup> T cells differed significantly
between non-LN JSLE patients and HC but did not differ significantly between LN
JSLE patients and non-LN JSLE patients. The frequencies of γδ T cells, NK cells,
and CD19<sup>+</sup> B cells differed significantly between HC and LN JSLE
patients and between HC and non-LN JSLE patients. However, there were no
significant differences in these cell percentages between LN JSLE patients and
non-LN JSLE patients.
## Associations between lymphocyte subsets and clinical manifestations in JSLE
We further evaluated whether the differences in lymphocyte subsets were
associated with other clinical manifestations. We stratified the patients into
high and low groups by the median frequency of each subset and compared the
clinical manifestations between the sets of two groups. The γδ T cells high
group and NK cells high group were significantly related to higher frequency of
mucosal ulcer, and the CD4<sup>+</sup> T cells high group was significantly
associated with higher rate of arthritis. The NKT cells high group was
substantially linked with higher number of patients with autoimmune hemolytic
anemia (AIHA). The CD8<sup>+</sup> T cells low group was mainly related to
higher frequency of vasculitis, and the Tregs low group was significantly
associated with higher rate of LN.
## Lymphocyte subset distribution during longitudinal follow-up of JSLE patients
Thirty-four of the 60 JSLE patients had follow-up samples until inactive
disease. At enrollment, 15 patients with active disease were treatment-naïve and
19 were treated with glucocorticoid therapy. After treatment, even in the
inactive disease state, the percentages of CD4<sup>+</sup> T cells,
CD8<sup>+</sup> T cells, γδ T cells, NK cells, Tregs within CD4<sup>+</sup>
cells, and CD19<sup>+</sup> B cells did not reach similar percentages to HC,
even though the median SLEDAI-2K in the inactive group was 0 (IQR 0–2). The
percentages of Tregs within CD4<sup>+</sup> cells and CD8<sup>+</sup> T cells
were substantially increased in the inactive group compared with the active
group. Meanwhile, the percentage of CD19<sup>+</sup> B cells was significantly
lower in the inactive group compared with the active group. In contrast, the
percentages of Tregs, CD4<sup>+</sup> T cells, γδ T cells, NK cells, and NKT
cells did not differ significantly between the active and inactive groups.
## Tregs in JSLE patients during the 6-month follow-up period
For 49 of the 60 JSLE patients, follow-up blood samples for the Tregs study were
collected at 0, 3, and 6 months. The percentage of Tregs in all JSLE patients
was significantly increased at 6 months of follow-up compared with 0 months
(median 1.25 \[IQR 0.53–2.70\] vs. median 0.85 \[IQR 0.31–1.82\], p = 0.0259)
(Fig). In the subgroup analysis, LN JSLE patients had a lower frequency of Tregs
than non-LN JSLE patients at 6 months of follow-up (median 0.99 \[IQR
0.39–1.53\] vs. median 1.53 \[IQR 0.65–2.84\]) (Fig). The percentage of Tregs
between treatment-naïve patients and treated patients at enrollment is shown in
. There was a significant increase of Treg percentage at 6 months in treatment-
naïve patients with LN (Table). During the 6-month follow-up, SLEDAI-2K
decreased over time in all JSLE patients, particularly in non-LN JSLE patients.
## Long-term outcomes in JSLE patients
At the end of the study, JSLE patients had a remission on therapy rate of 70%,
remission off therapy rate of 21.67%, and complete remission rate of 18.33%. As
shown in, the median time to achieve remission on therapy was longer in the
Tregs low group compared with the Tregs high group (38.80 \[CI 20.06–57.54\]
months vs. 15.27 \[CI 14.51–16.03\] months, p = 0.008). The number of patients
in remission on therapy at the end of the study was 18 (60%) in the Tregs low
group and 24 (80%) in the Tregs high group. Moreover, we also performed the
Kaplan-Meier analysis only on the treatment-naïve patients. This result
corresponded with the finding from all JSLE patients and showed the difference
between Tregs low and high groups more distinct. In the treatment-naïve
patients, the median time to achieve remission on therapy was longer in the
Tregs low group (58.40 \[CI 20.51–96.29\] months) compared with the Tregs high
group (14.70 \[CI14.16–15.24\] months) with more statistical significance
(p\<0.001) as shown in. In addition, 50% of treatment-naïve patients in the
Tregs low group and 100% of Tregs high group achieved remission on therapy.
Furthermore, multivariate analysis was performed by selecting four significant
covariates in univariate analysis, and it showed that predictors of remission on
therapy were high Tregs, high ALC (\>1.5×10<sup>6</sup>/L), positive DCT, and
non-LN JSLE at enrollment. Other lymphocyte subsets, clinical manifestations,
disease activity, anti-dsDNA positivity, medications, age, and sex at enrollment
were not related to remission on therapy.
# Discussion
The present study showed that dynamic changes in the percentages of lymphocyte
subsets occurred during the disease course of JSLE. The frequency of Tregs in LN
JSLE patients was substantially lower than that in non-LN JSLE patients and HC,
even though no significant difference was observed between all JSLE patients and
HC. The present findings further demonstrated relationships between lymphocyte
subsets and clinical phenotypes. For the longitudinal follow-up study at 0, 3,
and 6 months, the percentage of Tregs in all JSLE patients was significantly
increased at 6 months of follow-up. In the subgroup analysis, the LN JSLE group
had a lower frequency of Tregs compared with the non-LN JSLE group at 6 months
of follow-up. Predictors of remission on therapy were high Tregs, high ALC,
positive DCT, and non-LN JSLE at enrollment.
Consistent with previous studies, the percentages of CD4<sup>+</sup> T cells, γδ
T cells, and NK cells were low in JSLE patients. However, there were some
discrepancies between the percentages of CD8<sup>+</sup> T cells, Tregs, NKT
cells, and CD19<sup>+</sup> B cells in JSLE patients and HC in both the present
study and previous studies. This variation may be explained by the wide spectrum
of disease phenotypes and disease mechanisms that vary by age, ethnicity, and
environmental factors. An imbalance of lymphocyte subsets is strongly involved
in the immune response process in SLE patients. CD4<sup>+</sup> T cell functions
and cytokine production are impaired in SLE patients. Moreover, CD8<sup>+</sup>
T cells and NK cells have impaired cytotoxic functions. NK cells contribute to
the inflammatory response through type I and type II interferon (IFN) via
interactions with plasmacytoid dendritic cells. Furthermore, activated NKT cells
produce various cytokines and chemokines that regulate T cells, B cells, NK
cells, and dendritic cells. B cells are crucial for the production of
autoantibodies, resulting in immune complex activation and organ inflammation.
Not all SLE patients respond well to targeted B cell therapy, implying that the
immune cell dysfunction in SLE is not specific for B cells alone. In a previous
study, γδ T cells exhibited a regulatory function by inhibiting activated
CD4<sup>+</sup> T cells and dendritic cells. Notably, γδ T cells were related to
disease activity and disease progression in SLE patients.
From initiation of treatment to the achievement of inactive disease, the
percentages of CD4<sup>+</sup> T cells, CD8<sup>+</sup> T cells, γδ T cells, NK
cells, Tregs within CD4<sup>+</sup> cells, and CD19<sup>+</sup> B cells in JSLE
patients did not recover to the normal levels. Persistently abnormal frequencies
of CD8<sup>+</sup> and CD4<sup>+</sup> T cells may be associated with intrinsic
defects irrespective of medications received. An environment containing high
levels of circulating immune complexes could stimulate NK cell apoptosis,
leading to a decreased number of NK cells. Regarding γδ T cells, their levels in
patients with adult-onset SLE who responded to treatment gradually returned to
the normal level within 12 weeks. It will be interesting to determine whether
the persistently abnormal percentages of lymphocyte subsets during inactive
disease reflect subclinical ongoing systemic inflammation and are related to a
flare of the disease in JSLE.
Tregs have roles in immune regulation and maintenance of self-tolerance. Defects
in these cells can result in autoimmune diseases. Previous studies showed that
the percentage of Tregs was significantly decreased in the active SLE patients
compared with the inactive SLE patients and HC. In contrast, the present study
found that the percentage of Tregs did not differ significantly among active
JSLE patients, inactive JSLE patients, and HC. Moreover, we found a
significantly lower frequency of Tregs in LN JSLE patients compared with non-LN
JSLE patients and HC. These findings indicate that the role of Tregs may be more
pronounced in LN JSLE patients. In the longitudinal follow-up study, the
percentage of Tregs increased substantially over 6 months, corresponding to the
decrease in disease activity. An imbalance between Tregs and excessive T cell
and B cell activation has been demonstrated in SLE disease development. The
possible mechanism behind this finding is that Tregs are suppressed by activated
immune cells and various inflammatory cytokines in an inflammatory environment.
This hypothesis could explain the increased Tregs after treatment due to reduced
inflammation. Moreover, immune complexes, the main pathogenesis in LN JSLE
patients, induce a high IFN-α response that leads to Treg suppression, and this
could be another mechanism for the altered numbers of Tregs in these patients.
In a previous study, SLE patients under immunosuppressive medications had
significantly increased Tregs levels compared with untreated patients. We
demonstrated percentage of Tregs between treatment-naïve and treated JSLE with
LN during the 6-month follow-up period and showed a clearer alteration pattern
of Treg percentages in treatment-naïve JSLE patients. LN patients with naïve
treatment also had a lower percentage of Tregs than treated patients at
baseline. In addition, they showed significantly increased Treg percentage at 6
months, implying that lower Treg percentage at baseline derived from the active
disease more than immunosuppressive medications. However, further study with a
larger sample size regarding the treatment affecting Tregs should be performed.
The contradictory results of Tregs in various studies are from multiple factors.
First, since there are several markers of Tregs with multiple phenotypic
features, the variable Treg markers in each study cause the difference of Tregs
results. The earlier studies found that the percentage of Tregs was
significantly lower in active SLE compared to controls when CD25<sup>+</sup> or
CD25<sup>high</sup> cells were used as Treg markers. In contrast, other studies
that used FoxP3<sup>+</sup> or CD127<sup>low</sup> staining showed a comparable
percentage of Tregs between active SLE and controls. A previous study suggested
that CD25 alone should not be classified as Tregs because many of these cells
were FoxP3 negative, and other activated T cells can express CD25.
Regarding extracellular staining
CD4<sup>+</sup>CD25<sup>+</sup>CD127<sup>-</sup> cells, a previous study
reported that CD127 is also downregulated during early activation of effector T
cells, and around one-third of CD127<sup>low</sup> cells did not express FoxP3.
Therefore, low expression of CD127 might not be a good marker that represents
the Treg population. Unlike an important regulator in Treg development, a
transcription factor FoxP3 is a more specific marker and remains the best marker
of Tregs up to this point. Second, each study had a different definition of
active SLE disease, and the studies with higher cut-off SLEDAI scores tended to
have a lower percentage of Tregs. Our study results also supported this finding
that the percentage of Tregs had increased while the disease activity had
decreased. Third, since SLE is a heterogeneous disease, it is difficult for all
studies to have the same patients’ baseline characteristics, especially the
frequency of lupus nephritis, which might have an influence on the percentage of
Tregs the most.
The present results demonstrated associations between clinical phenotypes and
alterations to lymphocyte subsets. We found that γδ T cells and NK cells were
linked with mucosal ulcer, CD8<sup>+</sup> T cells were associated with
vasculitis, CD4<sup>+</sup> T cells were associated with arthritis, NKT cells
were related to AIHA, and Tregs were associated with LN. In previous studies,
SLE patients with predominant skin damage were related to γδ T cells, while SLE
patients with LN were associated with γδ T cells, NK cells, CD8<sup>+</sup> T
cells, and Tregs and the pathology of affected tissues such as the skin and
kidneys confirmed local damage arising from cellular immunity. Abnormal
activation of B cells and other lymphocyte subsets contributes to the
pathogenesis of SLE and is related to certain features. Therefore, targeted
therapies restricted to these cells may show promise for JSLE patients. The
discrepancy of the results in the above studies could be explained by
heterogeneity in the cellular defects and differences in the disease phenotypes
among the JSLE patients. In the present study, the most frequent clinical
feature of JSLE patients at enrollment was skin disease, followed by mucosal
ulcer, fever, AIHA, and LN, which may affect the altered lymphocyte subsets.
Regarding the relationships between lymphocyte subsets and long-term outcomes,
we found that assignment to the Tregs high group at enrollment could predict
clinical remission on therapy, and this subgroup of patients required a shorter
time to achieve clinical remission on therapy. These results were supported by
the finding that the Tregs low group was associated with LN, and the fact that
LN is a known factor related to morbidity and mortality. Tregs tend to exhibit
less proliferation in LN JSLE patients compared with non-LN JSLE patients due to
the extensive inflammatory environment in LN, leading to more suppression of
their function and proliferation. There is evidence from lupus-prone mice that
adoptive transfer of Tregs effectively ameliorated glomerulonephritis and
increased survival. The present findings strengthen the role of Tregs in the
pathogenesis of JSLE and suggest that Treg-based therapies may have a benefit
for the treatment of JSLE patients, particularly JSLE patients with LN.
The limitations of the study were the small number of enrolled patients,
including both treatment-naïve and treated patients, and the single-center
design. Despite the relatively small number of patients, particularly those
under longitudinal follow-up, we observed significant changes in the lymphocyte
subsets in individual patients and revealed more aspects of the cellular
involvement in JSLE.
# Conclusions
We demonstrated that changes in percentages of lymphocyte subsets were strongly
associated with clinical phenotypes and the disease course in JSLE. Due to the
heterogeneity of JSLE, patients with different clinical phenotypes had different
dynamic changes in lymphocyte subsets. Therefore, stratification of JSLE
patients based on lymphocyte subsets could help toward the establishment of
personalized treatment strategies, leading to better outcomes. Furthermore, low
levels of Tregs could help physicians identify a subgroup of JSLE patients with
severe clinical manifestations and worse long-term outcomes who require more
intense regimens. In particular, this subgroup of patients may be candidates for
Treg-based therapies.
# Supporting information
We would like to thank the patients who participated in the study. We also thank
Chompunuch Klinmalai, MSc, Division of Infectious Disease, Department of
Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University,
Bangkok, Thailand, for performing the flow cytometry. Finally, we thank Alison
Sherwin, Ph.D., from Edanz (<https://www.edanz.com/ac>) for editing a draft of
the manuscript.
10.1371/journal.pone.0263536.r001
Decision Letter 0
Kuwana
Masataka
Academic Editor
2022
Masataka Kuwana
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
6 Dec 2021
PONE-D-21-35318Associations of lymphocyte subpopulations with clinical
phenotypes and long-term outcomes in juvenile-onset systemic lupus
erythematosusPLOS ONE
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Reviewer \#1: Lerkvaleekul B et al. evaluated the association of lymphocytes
subsets with clinical manifestations in patients with JSLE. The manuscript
sounds interesting, but I have some concerns which authors need to answer.
1\. Previous studies have demonstrated quite contradictory results regarding
with frequency of Treg cells. Some reported a reduced frequency of circulating
Foxp3+ Treg cells, but others found an increased or comparable frequency of
circulating Foxp3+ Treg cells. I suggest authors to discuss this issue in
discussion section.
2\. Since the subsets of peripheral lymphocytes were dramatically changed by the
background treatment, including glucocorticoid dose, and immunosuppressive
agents. I suggest author to add background treatment in Table 1 and 2.
3\. Authors compared the clinical manifestations between high and low frequency
of each subset in Figure 3. I wonder how did authors select the manifestations,
only mucosal ulcer, arthritis, AIHA, vasculitis and LN? Please show all
manifestations listed in BILAG and compare them between high and low frequency
of each lymphocyte subset.
4\. Before showing Table 4, additional Table needs to be created comparing
clinical characteristics depending on achievement of remission on therapy and
select covariates for multivariate analysis using items having a P-value 0.05 in
univariate analysis. There was no data supporting the items were properly
selected as covariates for multivariate analysis in Table 4.
5\. In Figure 4 and Figure 5 showed the serial change of each subset and Treg
proportion and Figure 6 showed the probability of clinical remission depending
on the frequency of Treg cells. These results were influenced by background
treatment, and I suggest authors to show these results were independent of
background treatment.
Reviewer \#2: There are no reports of lymphocyte subsets in Asian children with
SLE, and this is an important finding that correlates with the phenotype. Of
particular importance is the significantly lower proportion of regulatory T
cells (Tregs) found only in patients with lupus nephritis (LN), a major cause of
refractory disease.
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10.1371/journal.pone.0263536.r002
Author response to Decision Letter 0
19 Jan 2022
Response to reviewers
19 January 2022
Dear Editor,
Thank you for allowing us to revise this manuscript. We have responded and
revised this manuscript as reviewers' suggestions. We highlighted the revised
sentences in yellow color throughout the entire manuscript. We also revised the
format of the manuscript and added minimal data set as the journal's
recommendation (2 supplement figures and 4 supplement tables). We removed the
funding information from the manuscript and did not amend the previous funding
statement given to the journal.
With Regards,
Soamarat Vilaiyuk
Comments from reviewers
Reviewer \#1: Lerkvaleekul B et al. evaluated the association of lymphocytes
subsets with clinical manifestations in patients with JSLE. The manuscript
sounds interesting, but I have some concerns which authors need to answer.
1\. Previous studies have demonstrated quite contradictory results regarding
with frequency of Treg cells. Some reported a reduced frequency of circulating
Foxp3+ Treg cells, but others found an increased or comparable frequency of
circulating Foxp3+ Treg cells. I suggest authors to discuss this issue in
discussion section.
Response: We would like to thank the reviewer for your valuable comments on this
manuscript.
We have discussed this issue and added it in the discussion section as follows.
The contradictory results of Tregs in various studies are from multiple factors.
First, since there are several markers of Tregs with multiple phenotypic
features, the variable Treg markers in each study cause the difference of Tregs
results. The earlier studies found that the percentage of Tregs was
significantly lower in active SLE compared to controls when CD25+ or CD25high
cells were used as Treg markers. In contrast, other studies that used FoxP3+ or
CD127low staining showed a comparable percentage of Tregs between active SLE and
controls \[20,22\]. A previous study suggested that CD25 alone should not be
classified as Tregs because many of these cells were FoxP3 negative, and other
activated T cells can express CD25 \[20\].
Regarding extracellular staining CD4+CD25+CD127- cells, a previous study
reported that CD127 is also downregulated during early activation of effector T
cells, and around one-third of CD127low cells did not express FoxP3 \[24\].
Therefore, low expression of CD127 might not be a good marker that represents
the Treg population. Unlike an important regulator in Treg development, a
transcription factor FoxP3 is a more specific marker and remains the best marker
of Tregs up to this point \[20,22\]. Second, each study had a different
definition of active SLE disease, and the studies with higher cut-off SLEDAI
scores tended to have a lower percentage of Tregs \[22\]. Our study results also
supported this finding that the percentage of Tregs had increased while the
disease activity had decreased. Third, since SLE is a heterogeneous disease, it
is difficult for all studies to have the same patients' baseline
characteristics, especially the frequency of lupus nephritis, which might have
an influence on the percentage of Tregs the most.
2\. Since the subsets of peripheral lymphocytes were dramatically changed by the
background treatment, including glucocorticoid dose, and immunosuppressive
agents. I suggest author to add background treatment in Table 1 and 2.
Response: We have added more details about medications in Table 1 and Table 2 as
your suggestion. (Highlight in yellow color)
3\. Authors compared the clinical manifestations between high and low frequency
of each subset in Figure 3. I wonder how did authors select the manifestations,
only mucosal ulcer, arthritis, AIHA, vasculitis and LN? Please show all
manifestations listed in BILAG and compare them between high and low frequency
of each lymphocyte subset.
Response: We analyzed all clinical manifestations but showed only the clinical
manifestations that were significantly different as stacked bar chart. More
details about lymphocyte subsets in each clinical manifestation are in
supplement table (S1 Table). We also highlighted the yellow color in the
clinical presentations and cells that were statistically different between the
high and low frequency of each subset.
4\. Before showing Table 4, additional Table needs to be created comparing
clinical characteristics depending on achievement of remission on therapy and
select covariates for multivariate analysis using items having a P-value 0.05 in
univariate analysis. There was no data supporting the items were properly
selected as covariates for multivariate analysis in Table 4.
Response: We have added the supplement table (S4 Table) that showed the
comparison of clinical characteristics between patients who had achievement and
non-achievement of clinical remission on therapy. We found that absolute
lymphocyte count and lupus nephritis were significantly different between both
groups (yellow highlight). The presence of the direct Coombs test also tended to
have a significant difference. Therefore, we selected these three factors with
our interesting factor, high Tregs, and performed univariate analysis. All of
them showed significance in univariate analysis. Furthermore, we performed the
multivariate analysis using these four factors as covariates, which showed
significance. We also added other factors that readers might be interested in
and showed in the univariate analysis. However, those factors were not
significant.
We also added more details as your suggestion in the result section of the
manuscript, as shown below.
Furthermore, multivariate analysis was performed by selecting four significant
covariates in univariate analysis, and it showed that predictors of remission on
therapy were high Tregs, high ALC (1.5×106/L), positive DCT, and non-LN JSLE at
enrollment (Table 4).
5\. In Figure 4 and Figure 5 showed the serial change of each subset and Treg
proportion and Figure 6 showed the probability of clinical remission depending
on the frequency of Treg cells. These results were influenced by background
treatment, and I suggest authors to show these results were independent of
background treatment.
Response: We have changed the presentation of Figure 4 and Figure 5 per your
suggestion. Instead of presenting all JSLE patients, we classified patients into
treatment-naïve and treated patients at enrollment. Overall results corresponded
with the initial one (all JSLE patients), and it showed a clearer pattern,
especially a significant increase of Treg percentage at 6 months in treatment-
naïve patients with lupus nephritis. We also added more details in the result
and discussion sections below (yellow highlight).
In the result section.
The percentage of Tregs between treatment-naïve patients and treated patients at
enrollment is shown in Fig 5A. There was a significant increase of Treg
percentage at 6 months in treatment-naïve patients with LN (Table in S3 Table).
During the 6-month follow-up, SLEDAI-2K decreased over time in all JSLE
patients, particularly in non-LN JSLE patients (Fig 5B).
In the discussion section.
In a previous study, SLE patients under immunosuppressive medications had
significantly increased Tregs levels compared with untreated patients \[55\]. We
demonstrated percentage of Tregs between treatment-naïve and treated JSLE with
LN during the 6-month follow-up period and showed a clearer alteration pattern
of Treg percentages in treatment-naïve JSLE patients. LN patients with naïve
treatment also had a lower percentage of Tregs than treated patients at
baseline. In addition, they showed significantly increased Treg percentage at 6
months, implying that lower Treg percentage at baseline derived from the active
disease more than immunosuppressive medications. However, further study with a
larger sample size regarding the treatment affecting Tregs should be performed.
Regarding Fig 6, we have added another graph of Kaplan Meier analysis, which was
performed only in treatment-naïve patients (Fig 6B). The graph of treatment-
naïve patients demonstrated the same pattern with all JSLE patients (Fig 6A).
However, the graph in treatment-naïve patients showed a more significant
difference. We have added more details in the result section as below.
Moreover, we also performed the Kaplan-Meier analysis only on the treatment-
naïve patients (Fig 6B). This result corresponded with the finding from all JSLE
patients (Fig 6A) and showed the difference between Tregs low and high groups
more distinct. In the treatment-naïve patients, the median time to achieve
remission on therapy was longer in the Tregs low group (58.40 \[CI 20.51-96.29\]
months) compared with the Tregs high group (14.70 \[CI14.16-15.24\] months) with
more statistical significance (p0.001) as shown in Fig 6B. In addition, 50% of
treatment-naïve patients in the Tregs low group and 100% of Tregs high group
achieved remission on therapy.
Reviewer \#2: There are no reports of lymphocyte subsets in Asian children with
SLE, and this is an important finding that correlates with the phenotype. Of
particular importance is the significantly lower proportion of regulatory T
cells (Tregs) found only in patients with lupus nephritis (LN), a major cause of
refractory disease.
Response We would like to thank the reviewer for your valuable comments on this
manuscript.
10.1371/journal.pone.0263536.r003
Decision Letter 1
Kuwana
Masataka
Academic Editor
2022
Masataka Kuwana
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
21 Jan 2022
Associations of lymphocyte subpopulations with clinical phenotypes and long-term
outcomes in juvenile-onset systemic lupus erythematosus
PONE-D-21-35318R1
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Academic Editor
PLOS ONE
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Reviewer \#1: All comments have been addressed
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The manuscript must describe a technically sound piece of scientific research
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Reviewer \#1: Yes
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Reviewer \#1: Yes
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The [PLOS Data policy](http://www.plosone.org/static/policies.action#sharing)
requires authors to make all data underlying the findings described in their
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refer to the Data Availability Statement in the manuscript PDF file). The data
should be provided as part of the manuscript or its supporting information, or
deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
participant privacy or use of data from a third party—those must be specified.
Reviewer \#1: Yes
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5\. Is the manuscript presented in an intelligible fashion and written in
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10.1371/journal.pone.0263536.r004
Acceptance letter
Kuwana
Masataka
Academic Editor
2022
Masataka Kuwana
This is an open access article distributed under the terms of the
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, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
28 Jan 2022
PONE-D-21-35318R1
Associations of lymphocyte subpopulations with clinical phenotypes and long-term
outcomes in juvenile-onset systemic lupus erythematosus
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# Introduction
It is well documented that the prevalence of adult hypertension is different by
race in the US with Non-Hispanic Blacks having a higher prevalence compared to
Non-Hispanic Whites. Considering the tracking of blood pressure (BP) levels from
childhood to adulthood, it is critical to examine racial disparities
among children and adolescents and to better understand the associated risk
factors in order to narrow the racial gaps among adults.
Relatively few studies have investigated racial differences in pediatric
hypertension (PHT), with mixed findings. Most of the earlierstudies
characterized racial differences in hypertension among children and adolescents
as non-significant. However, Rosner and colleagues reported a significant racial
difference between Hispanic and White boys, which persisted after controlling
for body mass index (BMI). Body size and sex appeared to modify the association
between race and PHT. Significant differences were also observed in subsequent
studies by Lo et al. and Rosner et al. for Black and by Shay et al. for both
Black and Hispanic youth compared to their White counterparts. In addition, low
birth weight (LBW) has been linked with high risk of adult hypertension,
although the evidence was less consistent among children and adolescents The
role of LBW in racial disparities in PHT is unclear.
The objectives of this study were to identify racial differences in the
prevalence of PHT in the US and to explore whether such differences can be
explained by body size and birth weight using the most updated nationally
representative data from the National Health and Nutrition Examination Survey
(NHANES) 1999–2010.
# Methods
## Study Population
The study population included children and adolescents aged 8–17 years who
participated in the NHANES 1999–2010. Details of the NHANES design and
procedures are available on the Center for Disease Control and Prevention (CDC)
website. In brief, the 1999–2010 NHANES included 6 cross-sectional surveys of
the non-institutionalized US population. These surveys applied a stratified
multistage probability sampling design. Low-income populations, adolescents,
seniors, Non-Hispanic Blacks, and Hispanics were over-sampled. Each survey
consisted of a household interview and a physical examination in a fully
equipped mobile examination center. The procedures and protocols of NHANES
1999–2010 were approved by the institutional review board of the CDC/ National
Center for Health Statistics (NCHS). Informed consent was obtained from study
participants or their guardians where appropriate. This study was a secondary
analysis using the de-identified data released from the CDC, therefore, no
additional consent procedures were carried out. For the current study,
individuals who self-identified as “Other Hispanics” (6.37%) or “Other Race”
(6.73%) were excluded due to small sample size. All the data are available at
the CDC NHANES website and are open to public access
**(**<http://wwwn.cdc.gov/nchs/nhanes/search/nhanes11_12.aspx>**).**
## Measurement of BP and Definition of PHT
For children and adolescents aged 8 years or above, BP was measured in a seated
position following 5 minutes of quiet rest during the physical examination. For
each individual, up to three completed BP measurements were taken at one visit
by a physician using a mercury sphygmomanometer with an appropriate size cuff.
The measuring procedure followed the American Heart Association standard
protocol. Quality control of BP measurements was reinforced in the NHANES. The
mean of the three measurements were used to determine PHT status.
PHT was defined as a systolic blood pressure (SBP) or diastolic blood pressure
(DBP) ≥ 95th percentile of age-, sex-, and height-specific reference level for
the US population, according to the Fourth Task Force Report. A height z-score
relative to the 2000 (most recent) CDC growth charts was calculated for each
child according to the CDC Box-Cox transformation estimation procedure. The
age-, sex-, and height-specific BP percentile was then calculated using the
methods provided by the Fourth Task Force Report, applying the following steps:
1) compute the expected BP value for a given age, sex, and height z score; 2)
convert the participant’s observed BP into a Z score; and 3) convert the BP Z
score into a percentile.
Individuals who were taking antihypertensive medications were also considered as
having PHT. Antihypertensive medication use was determined by utilizing
information from the Prescription Medications questionnaire of NHANES, which was
verified by survey interviewers who asked to see the medication containers when
possible. It was defined as reported use of at least one of the following
classes of medications: angiotensin converting enzyme inhibitor, beta-adrenergic
blocking agents, calcium channel blocking agents, diuretics, angiotensin II
inhibitors, other antihypertensive agents (other antiadrenergic agents,
vasodilators, renin inhibitors, agents for hypertensive emergencies, agents for
pulmonary hypertension, and antihypertensive combinations). Antihypertensive
medications were classified using the Multum Lexicon Drug Database
**(**<http://www.multum.com/Lexicon.htm>**)**.
## Measurements of Race, Age, Body Size, and Birth Weight
Race was self-reported during the household interview. It was derived by
combining responses to questions on race and Hispanic origin and classified into
the following categories: Non-Hispanic Whites, Non-Hispanic Blacks, Mexican
Americans, Other Hispanics, and Other Race. Age was grouped into two categories:
children aged 8–12 years and adolescents aged 13–17 years.
Anthropometric measurements were conducted by a trained examiner following the
standard protocol in the mobile examination center during the physical
examination. Body weight was measured to the nearest 0.05 kg using the Toledo
digital scale while wearing underwear, a disposable gown, and foam slippers.
Standing height was measured to the nearest 0.1 cm by using a fixed stadiometer
with a vertical backboard and a moveable headboard. BMI was calculated as weight
(kilogram) divided by height (squared meters) and classified into three
categories: normal weight (\<85.0<sup>th</sup> percentile of sex-and-age-
specific BMI), overweight (85.0<sup>th</sup> -94.9<sup>th</sup> percentile of
sex-and-age-specific BMI) and obesity (≥95.0<sup>th</sup> percentile of sex-and-
age-specific BMI).
Birth weight (in pounds and ounces) was self-reported and collected during the
interview from participants aged 15 years or younger and their guardians.
Previous studies have shown that maternal- and self-reports of birth weight and
LBW status have high validity. The specificity of self-reported birth weight
below 3,000 grams was 93% among middle-aged and elderly women participating in
the Danish Nurse Cohort Study. The specificity is likely to be higher among
children and adolescents given a shorter recall period. Birth weight at or below
5.5 pounds or approximately 2,500 grams was defined as LBW.
## Statistical Analyses
Results of descriptive analysis were expressed as mean or percent, with the
corresponding standard error (SE) presented. Odds ratio (OR), 95% confidence
interval (CI) and *P* values based on multivariable logistic regressions were
conducted to evaluate the independent association between race and PHT after
controlling for BMI, LBW and their interactions. This was realized through a
stepwise modeling approach, in which race, BMI categories, LBW, and the
interaction between BMI categories and birth weight categories were added to the
regressions sequentially. Age categories were also adjusted for in all
multivariable analyses where needed.
The analyses were conducted separately for boys and girls following previous
practices. For each sex, interactions between race and age and race and BMI
categories were also examined. Results were reported for the following age-and-
sex-specific subgroups: (a) boys aged 8–12 years, (b) boys aged 13–17 years, (c)
girls aged 8–12 years, and (d) girls aged 13–17 years based on the evidence of
potential marginally significant interactions (P = 0.08) between race and age
among boys. Significant interactions between race and BMI categories were also
observed in boys aged 13–17 years and stratified analyses by BMI categories were
further performed among this subgroups. Interactions between race and LBW were
not explored considering the small sample size of participants born at a LBW
with PHT. Analyses involving birth weight were only performed among children
aged 8–15 years, the ages for which birth weight was available in NHANES.
All analyses took account of the complex survey design including stratification,
clustering, and weighting to consider the oversampling of subgroups, unit non-
response and non-coverage in the NHANES. This was realized by applying the set
of survey commands (with prefix ‘svy-’) in STATA 12 with the sampling errors
estimated by the Taylor linearization method. Statistical significance was set
at the level of P ≤ 0.05 (2 tailed).
# Results
## Background Characteristics
Of 9,250 children and adolescents aged 8–17 years who were included in this
analysis, 31.24% were Non-Hispanic Whites, 32.18% were Non-Hispanic Blacks, and
36.57% were Mexican Americans. On average, Black and Mexican American
participants had significantly higher mean BMI and BMI z scores compared to
their White peers (mean BMI: 22.58 and 22.48, vs. 21.28, respectively, both
P\<0.001; BMI z score: 0.71 and 0.75, vs. 0.45, respectively, both P\<0.001).
Black and Mexican American youth also had significantly higher prevalence of
obesity than Whites (22.88% and 24.68%, vs. 15.84%, respectively, both
P\<0.001). Although Blacks and Mexican Americans on average were born at
significantly lower birth weight than Whites, only Blacks had a significantly
higher prevalence of LBW (14.56% vs. 5.89%, P\<0.001). Overall, Blacks had a
higher SBP compared to Whites (107.80 vs. 106.32, P\<0.001), and Mexican
Americans had a lower DBP than Whites (57.90 vs. 59.25, p = 0.001).
Antihypertensive medication use was low in general.
## Prevalence of PHT among children and adolescents by race and other factors
In 1999–2010, the prevalence (SE) of hypertension in U.S. children and
adolescents aged 8 to 17 years was 6.07% (0.39) among both sexes, 5.58% (0.49)
in girls, and 6.56% (0.58) in boys. No significant difference was observed
between boys and girls (P = 0.20). Mexican Americans had the lowest PHT
prevalence at 5.36% (0.57), followed by 5.60% (0.55) in Non-Hispanic Whites and
7.10% (0.59) in Blacks. Compared to non-Hispanic White youth, the unadjusted
prevalence of PTH was significantly higher in non-Hispanic Blacks (P = 0.04),
but the difference in Mexican Americans did not approach significance (P =
0.77). When stratified by sex alone, no racial differences were observed among
boys or girls. When further stratified by age and sex, among boys aged 13–17
years Blacks had significantly higher PTH than Whites 9.59% (1.02) vs. 6.59%
(1.04) (P = 0.04).
## Racial Differences in PHT Prevalence and Their Association with Body Size and Birth Weight
It was further assessed whether observed racial differences in PTH can be
explained by body size and birth weight using multivariate logistic regressions.
After controlling for age and sex, Black youth had a 29% increased odds of
hypertension (model 1, Odds ratio (OR) = 1.29; 95% CI: 1.01–1.65; P = 0.044)
compared to White youth. The Black-White difference in hypertension rate showed
little change (model 2, OR = 1.25; P = 0.047) after additional adjustment for
birth weight, but was attenuated (model 3, OR = 1.16; P = 0.19) to the degree
that it lost statistical significance with further adjustment for current BMI.
When stratified by sex and age group combined, no significant racial differences
were identified in age adjusted models and after controlling for BMI categories,
LBW status and their interaction among boys aged 8–12 years. However, among boys
aged 13–17 years Blacks were at significantly higher odds of PHT compared to
Whites in the age adjusted model (OR: 1.51; 95% CI: 1.03–3.43; P = 0.041) and
with the addition of control for LBW (OR: 2.00; 95% CI: 1.09–3.71; P = 0.027),
and then for BMI categories (OR: 1.50; 95% CI: 1.01–2.14; P = 0.048). There was
no significant racial difference among girls for both 8–12 and 13–17 age groups.
No significant differences in PTH were observed between White and Mexican youth
in any models including those for each sex-age group.
Further analysis stratified by the BMI categories among boys aged 13–17 years
shows that significant Black-White differences were only found in boys with
normal body size (OR = 2.16; 95% CI: 1.22–3.80; P = 0.008), but not among those
who were overweight or obese. No significant differences in PTH were observed
between Whites and Mexican across all BMI categories. When the overweight and
obese categories were combined to potentially increase statistical power, the
results remain the same (OR: 1.03; 95% CI: 0.65–1.66; P = 0.91 in Black and OR:
0.71; 95% CI: 0.44–1.13; P = 0.15 in Mexicans).
# Discussion
The study investigated racial differences in the prevalence of PTH among
children and adolescents aged 8–17 years and examined their association with
body size and birth weight using the nationally representative sample of NHANES
1999–2010. The prevalence of hypertension in U.S. children and adolescents aged
8 to 17 years was 6.1%. We found that Black youths had a 29% increased odds of
hypertension compared to their White peers. When stratified by sex and age
groups, the Black-White difference was only significant in boys aged 13–17.
Compared to Whites, Black youths also had an elevated prevalence of obesity and
lower birth weight. However, the racial difference in PTH among boys aged 13–17
was not explained by their obesity and low birth weight status. No significant
differences in PTH were observed between White and Mexican s youth taken as a
whole or by sex-age group.
It is well documented that in the US Black adults have higher prevalence of
hypertension than White adults. However, it is unclear whether such racial
disparity also exists in US children and adolescents. It is also unknown at
which age the Black-White differences in BP emerge. While most previous studies
found no significant racial differences in hypertension among US youths, Rosner
and colleagues reported a significant racial difference between Hispanic and
White boys, but not between Black and White boys. For girls, both Blacks and
Hispanics were found to have higher prevalence as compared to their White peers.
In contrast, we found a Black-White difference in PTH among US youths in the
current study. When stratified by age and sex, such a racial difference was only
significant among boys aged 13–17, which persisted after controlling for birth
weight and BMI. Although Mexican American youth had a higher rate of LBW and
obesity compared to Whites, they had little difference in PTH after adjusting
for age, sex, birth weight and obesity. The differences in BP measurement and
hypertension definition might partially explain the differences in findings
between the current and Rosner’s study. The timing of when the study data were
collected may also influence the two studies' results. The Rosner study dataset
encompassed all children in the Pediatric Task Force Database which included
surveys and studies conducted between 1973 and 2000. Our study, however, applied
the most recent national representative sample collected between 1999 and 2010.
Our results are consistent with 3 recent studies in US populations and provided
additional data stratified by age and gender. Nevertheless, more studies are
warranted to shed further light on the racial differences in pediatric
hypertension prevalence in the US.
Another important finding of this study is the Black-White disparity was
observed among adolescent boys with normal weight, but not among overweight and
obese boys. Similar findings that Blacks at normal weight were more likely to
have PHT than Whites have been reported previously. However, the racial
difference was significant among female adolescents in Rosner et al., and male
children and adolescents in Rosner et al.. It is unclear why among Black
adolescents only those at normal weight, but not those overweight or obese were
at increased risk of PTH than their White peers. Although we cannot rule out the
possibility that the lack of significant results in overweight and obese groups
is due to smaller sample size, results were similar when we combined the
overweight and obese youths together.
The current study has several limitations. First, the 3 BP measurements were all
obtained in one visit during physical examinations in the NHANES. Ideally,
hypertension should be defined according to 3 repeated BP measures obtained on 3
separate occasions. Secondly, the reliability of our findings in analyses
stratified by age-sex group could be compromised by small sample size, although
each age-sex group consisted of at least 600 individuals. Thirdly, NHANES is a
cross-sectional survey by design, which is not the best design to examine the
age onset of racial disparity in PTH. Lastly, data on LBW were self-reported and
only available among participants aged 8–15 years. Misclassification in self-
reported birth weight, although likely small, could introduce bias into the
association between LBW and PHT. Further, when controlling for LBW the study
sample was limited to those aged 8–15 years. A sensitivity analysis demonstrated
that restricting all analyses to that age range yield qualitative unchanged
results. Future studies are warranted to control for a better measured LBW
status.
Despite these limitations, this study contributes to the literature by examining
the most recent nationally representative samples of the US population including
extensive relevant data with PTH defined using three measurements of BP. Other
notable advantages of this study include the relatively large sample size
available in each race group, objective measure of weight and height, and the
combination of stratified and multivariate analyses guided by both previous
studies and empirical data (test for interactions).
# Conclusions
In current analysis of the most recent nationally representative samples of US
children and adolescents aged 8–17 years, we found Non-Hispanic Black children
and adolescents to have a significantly higher prevalence of hypertension than
their non-Hispanic White counterparts. This racial difference in pediatric
hypertension was only shown in boys aged 13–17 when stratified by age and sex.
The racial disparity of PTH among boys aged 13–17 could not be explained by low
birth weight and current obesity of the Black youth. No significant differences
in PTH were observed between White and Mexican Americans in all models and in
each age-sex group. Our findings are not entirely consistent with previous
findings. Future research is needed to further investigate racial differences in
hypertension in children and adolescents.
We thank the NHANES participants and staff for their contributions to the study.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: LC LL NS. Performed the
experiments: LC LL. Analyzed the data: LC LL. Contributed
reagents/materials/analysis tools: LC LL. Wrote the paper: LC LL NS. |
# Introduction
Myotonic dystrophy type 1 (MD<sub>1</sub>), or Steinert's disease, is the most
common form of dystrophy in adolescents and adults, and the second most common
neuromuscular disease. The classic form of MD<sub>1</sub> is characterized by
weakness and atrophy in skeletal muscles, myotonia, cardiac conduction
abnormalities, cognitive impairment and myocardial changes. Additionally, the
involvement of respiratory muscles is common with reduction of respiratory
muscles strength, especially expiratory muscles, resulting in death,
specifically due to respiratory insufficiency or pneumonia,.
The respiratory impairment in MD<sub>1</sub>, is rather complex because
progressive muscle weakness is associated to different degrees of central
respiratory control abnormalities causing alveolar hypoventilation. In fact,
CO<sub>2</sub> insensitivity occur in MD<sub>1</sub> independently of lung
function impairment and respiratory muscle weakness. In addition the
participation of myotonia in DM1 respiratory restriction is not fully clarified.
Myotonia is characterized by a delayed relaxation of skeletal muscles after an
intense contraction and is one of the most important clinical features of
MD<sub>1</sub>. The presence of myotonia determines the appearance of symptoms
such as muscle stiffness and cramps. Although few studies have been published,
there are indications of its presence in the respiratory muscles. In 1954,
Benaim & Worster-Drougth showed, through electromyography, the presence of
myotonia in the intercostal muscles, but not in diaphragm. In 1959, Kilburn et
al. observed in two cases a delayed return of the diaphragm to its resting
position. Smorto et al. found high amounts of high frequency electrical
activity, considered myotonia, in MD<sub>1</sub> patients. In another case
study, Estenne et al. were able to detect abnormal muscle relaxation and
registry of myotonia of the diaphragm and abdominal muscle by performing
electromyographic and nasal inspiratory pressure test (SNIP) studies in a
patient with congenital myotonia (Thompsen disease). However, due to the
presence of abnormalities in relaxation of the diaphragm, which was confirmed by
variations in abdominal pressure, the presence of myotonia was attributed to the
abdominal muscles and not to the diaphragm. In contradiction, Fitting et al.
using similar methodology, found no changes in the abdominal muscles, but in the
diaphragm. Finally, Rimmer et al. in a study with 11 MD patients, found only two
patients with electrical activity consistent to the presence of myotonia, as
measured by surface electromyography (sEMG) of the scalene, parasternal and
external abdominal oblique muscles. Therefore, the presence of myotonia in the
inspiratory muscles of patients with MD is doubtful, since most of the studies
involve few patients with MD and, in some cases, inadequate techniques.
For some years, the maximum relaxation rate (MRR) of the inspiratory muscles has
been used as an indirect marker of fatigue and overload of the inspiratory
muscles. A decreasing MRR of skeletal muscle means muscle overload and precedes
the failure of power generation. Considering the muscle relaxation as an active
process that consumes energy that can be affected by different conditions,
including fatigue, muscle weakness and atrophy of muscle fibers, a decreasing
MRR during the sniff test could be useful for evaluating the presence of
inspiratory muscles overload, signs of myotonia, and fatigue. Therefore, the
objectives of this study were to evaluate the MRR and electrical activity of the
inspiratory muscles in patients with MD<sub>1</sub>. We hypothesized that
markers of myotonia could be found in this population by analyzing different
parameters describing the strength, the relaxation and the electrical activity
of respiratory muscles. More specifically, we have considered maximal
inspiratory pressure (PImax), maximal expiratory pressure (PEmax) and SNIP test
as indexes of strength and MRR and sEMG as indexes of muscle relaxation and
contraction, respectively.
# Methods
## Subjects and study design
This is an observational, quasi-experimental study, with control group. Subjects
were individuals diagnosed with MD<sub>1</sub> recruited from a clinical follow-
up with a neurologist at a University Hospital from January 2015 to June 2016.
All patients and healthy subjects were assessed in a single day after receiving
initial information and recommendations on the preparation for the assessments.
Inclusion criteria for the MD<sub>1</sub> group were: patients aged between 18
and 65 years, properly diagnosed and accompanied by a neurologist. Those who
were smokers (or ex-smokers) or presented obstructive respiratory disease,
musculoskeletal comorbidities or impaired intellectual ability (established by
medical diagnosis) that would prevent the assessments, who failed to perform
tests for other reasons or quit from the study were excluded. The control group
included self-reported healthy subjects matched for gender and age. Individuals
who were smokers (or ex-smokers) or presented cardiac, respiratory or
musculoskeletal comorbidities, deviated septum, rhinitis or nasal obstruction,
as well as those who failed to perform the tests or gave up from participating
of study were excluded. Additionally, healthy subjects should not use any type
of drug that could interfere with the test results, such as antihypertensive or
bronchodilator drugs. All subjects agreed to participate in the study by signing
an informed consent form, which was approved by the Ethics Committee of the
Federal University of Rio Grande do Norte/Brazil (protocol n<sup>o</sup>:
752.322) according to the Declaration of Helsinki.
## Clinical phenotype and muscle impairment MD<sub>1</sub> classification
The MD<sub>1</sub> patients were classified in relation to degree of muscle
impairment and clinical phenotypes by the neurologist according to the Muscle
Impairment Rating Scale (MIRS), and the MD<sub>1</sub>-specific form according
to age and onset and clinical symptoms. The MIRS is structure in five degrees of
impairment in relation to muscle involvement, progressing from distal to
proximal: grade 1, no muscular impairment; grade 2, minimal signs (myotonia and
digit flexors and craneal muscle wasting and weakness); grade 3, distal weakness
(no proximal weakness except isolated elbow extensor weakness); grade 4, mild to
moderate proximal weakness; grade 5, severe proximal weakness. The phenotype
classification includes four categories: 1- congenital, 2- juvenile, 3- classic
adult onset and 4- mild late-onset.
## Lung function
Spirometry was performed through a KoKo DigiDoser<sup>®</sup> spirometer (nSpire
Health, Inc. Longmont, USA) and considered complete when three acceptable curves
were produced (with variation equal to or lower than 5% of the highest value).
The technical procedure, the acceptance and reproducibility criteria as well as
the standardization of equipment followed the recommendations of the ATS/ERS.
The reference values used in this study had been previously published for the
Brazilian population.
## Respiratory muscle strength
Respiratory muscle strength was assessed from measurements of PImax, PEmax and
SNIP by a digital manometer (NEPEB-LabCare/UFMG, Belo Horizonte, Brazil),
following the procedures previously published by the Brazilian Society of
Pneumology. PImax was measured from residual volume and PEmax from total lung
capacity, using a nose clip. The highest value obtained in a maximum of five
tests (with variation equal to or lower than 10% of the highest value) was used
for each assessment. SNIP was obtained from functional residual capacity, with
the subject having one nasal cavity occluded by a plug coupled to a catheter
connected to a digital manometer, while maintaining the other nostril open.
Previously published reference values were used.
In order to identify respiratory muscle weakness, cutoff values were adopted and
calculated as the mean of the normal population, published previously, minus
1.96 multiplied by the standard deviation. The reference values of Neder et al.
were used for PImax and PEmax; and the reference values of Araujo et al. were
used for SNIP. The cutoff values established for men and women, respectively,
were: 59.1 cmH<sub>2</sub>O and 70.4 cmH<sub>2</sub>O for PImax; 58.2
cmH<sub>2</sub>O and 53.9 cmH<sub>2</sub>O for SNIP; 101.1 cmH<sub>2</sub>O and
69.2 cmH<sub>2</sub>O for PEmax.
## Assessment of maximum relaxation rate of inspiratory muscles
MRR was derived from SNIP test and calculated using LabChart Reader 8.0 software
(ADInstruments, New Zealand). The values were obtained as the first derivative
of pressure-time curve (dP/dT) over the first half of the relaxation curve by
calculating the tangent at the steepest part of the pressure curve. To exclude
the effect of pressure oscillation amplitude on MRR, dP/dT was normalized and
expressed as percentage of the pressure drop in every 10 milliseconds (dP/dt) /
Psniff\*100 (%10ms). The following criteria were established for choosing the
best SNIP value for calculation of MRR: (1) sniff maneuver performed from FRC;
(2) peak pressure maintained for less than 50 milliseconds; (3) total duration
of the maneuver less than 500 milliseconds; and (4) shape of SNIP curve showing
soft peaked curves with upward and downward movement,.
## Surface electromyography
Capture and processing of myoelectric signals were performed using TeleMyo DTS
Desk Receiver<sup>®</sup> (Noraxon, USA, Inc., Scottsdale, USA) electromyograph
and 4 wireless Clinical DTS (Noraxon, USA, Inc., USA) sensors with 16-bit
resolution and common-mode rejection ratio \> 100 dB. The sampling frequency of
the captured signals was 1500 Hz, with 500 Hz low pass filter and signals pre-
amplified 1000 times. The software used to capture and store the myoelectric
signals during PImax, PEmax and SNIP tests was MR 3.2 (Noraxon, USA, Inc., USA).
Ag/AgCl bipolar surface electrodes were placed along the direction of the muscle
fibers, all in the right side of the body to avoid contamination by the cardiac
electrical signals. The skin of the volunteers was properly prepared in order to
reduce impedance, favoring the capture of a signal with greater amplitude, with
less interference and noise. To capture electromyographic signals, all
recommended procedures were strictly followed.
Electrodes were placed over the parasternal muscle on the second intercostal
space (2<sup>nd</sup>IS), 3 cm from the sternum; the sternocleidomastoid muscle
(SCM), on the muscle belly, 5 cm from the mastoid process; the rectus abdominis
(RA), 4 cm from the umbilical scar; and the scalene muscle (SCA), on the muscle
belly, 5 cm from the sternoclavicular joint and 2 cm above that mark.
Electromyographic signals were analyzed using the RMS (root mean square) and
normalized from the average of three maximum voluntary isometric contractions
for SCM, SCA, RA; and three SNIP and PImax tests for 2<sup>nd</sup>IS.
In order to quantify possible delayed muscle relaxation, the duration of
electrical activity of each muscle studied was measured and normalized by the
duration of the different maneuvers (SNIP, PImax and PEmax). The duration of
each respiratory muscle contraction (i.e., the difference between onset and
offset time during the maneuver) was automatically calculated by analyzing the
time course of pressure curves using the MR3.2 software (MyoMuscle Module,
Noraxon, USA).
## Sample size and statistical analysis
Sample size was calculated through a pilot study with five MD<sub>1</sub>
subjects. The standard deviation of the MRR variable was used for a hypothetical
t test. Twelve subjects in each group were required to achieve 80% power with
P\<0.05. Data normality were checked using Shapiro-Wilk test. Parametric data
were compared using the Unpaired t-test, while non-parametric data were compared
using the Mann-Whitney test. Significant levels were set at a 2-sided P\<0.05.
Receiver operating characteristic curves (ROC) were calculated using the MRR,
SNIP%, PImax%, PEmax% between MD<sub>1</sub> patients and healthy subjects. ROC
curve was calculated as described previously by Hanley and McNeil.
Prism<sup>®</sup> version 5 (GraphPad Software Inc, USA) was used for data
analysis and G\*Power version 3.1.9.2 (Franz Faul—Universität Kiel, Germany) for
sample size, effect-size and power calculation.
# Results
## Sample size
Initially, 74 individuals were invited to participate in the study: 44 patients
with MD<sub>1</sub> and 30 healthy subjects. After exclusion criteria a total of
18 MD<sub>1</sub> subjects and 11 healthy were included in the study.
Considering the mean of each group and the difference between standard deviation
of MRR variable, a Cohen’s d = 3.89 (considered large) was found. The power of
the study (1-ß), considered the effect size found, was 1.0.
## Symptoms, severity and rate of progression of the disease
Demographic features of the patients are reported in. The mean age of the
patients at time of evaluation was 42.3 ± 11.3 and the mean time elapsed from
diagnosis were 6.83 ± 5.98 years. In relation to phenotype, 88.8% (n = 16) were
classified as a Classic form and 11.2% (n = 2) as a Mild type. Regarding the
classification on MIRS we found that 11,11% (n = 2) were classified as MIRS 1,
55.5% (n = 10) classified as MIRS 2, 22.22% (n = 4) classified as MIRS 3 and
11,11% (n = 2) classified as MIRS 4.
## Pulmonary function and respiratory muscle strength
The spirometric values for the MD<sub>1</sub> group showed, on average, a
moderate restrictive pattern. Regarding inspiratory muscle strength in the
control group, 25% of women were below the cutoff point, while in the
MD<sub>1</sub> group was 40%. Regarding male gender, 14.3% of control group and
54.5% of MD<sub>1</sub> group were below the cutoff point. In the SNIP test, all
subjects were above the cutoff point in the control group, while in the
MD<sub>1</sub> group 63.6% of men and 80% of women were below that point. In the
PEmax assessment, where the biggest difference was observed between the two
groups, all healthy subjects met or were above the cutoff point for muscle
weakness. In the MD<sub>1</sub> group, 90.9% of men and 80% of women did not
reach that value.
For all maneuvers previously mentioned, the differences between the two groups
were statistically significant: in absolute values SNIP (P = 0.0014), PImax (P =
0.0006), PEmax (P = 0.0002) and in percentage of predict values SNIP (P =
0.0015), PImax (P = 0.0016) and PEmax (P\<0.0001). Spirometric and respiratory
muscle strength data are shown in and.
## Electromyography of the respiratory muscles
In the MD<sub>1</sub> group, 2 subjects were excluded due to the poor quality of
the signal captured by the sEMG. Obtained signals from the surface
electromyography showed increased muscle activity in MD<sub>1</sub> subjects in
most of the maneuvers carried out, with significant differences found at rest
for the SCM (P = 0.002) and SCA (P = 0.004). It was also found significant
difference during the PEmax test for the 2<sup>nd</sup>IS (P = 0.003), and
during SNIP for the SCM (P = 0.02) and SCA (P = 0.03).
Electrical activity time of respiratory muscles in patients with DM<sub>1</sub>
was significantly increased compared to controls.
## Maximum relaxation rate of inspiratory muscles
To assess MRR, four DM<sub>1</sub> subjects were excluded from the analysis
because their SNIP test did not meet the criteria for proper data acquisition.
The MRR was significant lower in MD<sub>1</sub> group compared to control group
(P = 0.001; 5.7±1.2 vs. 7.9±1.7, respectively).
## ROC analysis
The results, considering the Area Under the Curve, showed, for all variables, a
good ability to discriminate between those individuals with the disease and
those without it. The thresholds of sensitivity/specificity, confidential
interval of area and p values are shown in.
The area under the curve (AUC) above 0.80 associated with a P\<0.05, for all the
variables (maneuvers), lead to the conclusion that these variables show a good
ability to discriminate between those individuals with the disease and those
without it.
# Discussion
The study results showed that the MRR is reduced in MD<sub>1</sub> subjects
compared to healthy. The MRR of inspiratory muscles was considered sensitive to
identify the presence of reduced MRR in MD<sub>1</sub> patients and specific to
discard it in healthy individuals. According to the results of the ROC curve
analysis, which determined a cutoff point of 5.65; the electrical activity of
the accessory muscles of respiration (SCM, SCA and RA) was increased in
MD<sub>1</sub> compared to controls (P = 0.004; P = 0.009; P = 0.045,
respectively). In addition, lung function was also altered in MD<sub>1</sub>
group as well as reduced respiratory muscle strength showing considered as
muscle weakness.
The MRR of inspiratory muscles has been studied for about 25 years. This is an
index of fatigue and overload of inspiratory muscles, developed from sniff
maneuvers obtained by measuring esophageal or transdiaphragmatic pressure, which
are invasive methods. Subsequent studies have observed a high relationship
between the MRR obtained through sniff maneuvers associated with esophageal
pressure and with nasal pressure, providing a non-invasive measurement of that
variable. Despite the technical characteristics of MRR as a measure capable of
identifying the delay in inspiratory muscle relaxation in MD<sub>1</sub> and,
therefore, suitable for assessing the presence of myotonia, we have not found
previous studies of MRR obtained through nasal inspiratory pressure in
MD<sub>1</sub>.
Fitting & Leuenberger were the first authors to study MRR in a single case in
1989. These authors evaluated the effects of procainamide, an anti-myotonic
drug, in the diaphragm through transdiaphragmatic pressure. Due to the type of
study and the technique used, comparisons are inappropriate. Another study
published by Jammes et al., aimed to analyze the electrical activity of the
diaphragm and intercostal muscle during loaded inspiratory and expiratory
breathing. The authors suggest that the absence of relaxation in diaphragm
muscle during expedition may be related to the persistence of repetitive
discharge due to a failure o neural inhibitory circuits or to myotonia. More
recently, Garcia-Rio et al were the first to assess noninvasively the MRR of
inspiratory muscles using SNIP curve in 20 patients with different neuromuscular
diseases (5 amyotrophic lateral sclerosis, 3 duchenne muscular dystrophy, 2
myotonic dystrophy, 7 myasthenia gravis, 2 congenital myopathy and brachial
plexus palsy) and 10 healthy subjects. These authors used a similar methodology
to calculate MRR and found a significantly lower MRR in the neuromuscular group
compared to healthy (7.21 ± 0.68 vs. 9.31 ± 1.22). Regarding our results, we
also found a significantly lower MRR in MD<sub>1</sub> patients however; the
study of Garcia-Rio et al. used a heterogeneous sample with several different
neuromuscular diseases.
Neuromuscular disorders, despite having some common physiopathological
characteristics, have varying degrees of severity and progression, which makes
unsuitable grouping these diseases for assessment of MRR and its subsequent
comparison to a control group. It is important to report that the MD<sub>1</sub>
is characterized by the presence of myotonia in the skeletal muscles and
possibly, in the inspiratory muscles, which could potentially affect the
relaxation of these muscles and reduce the MRR. Regarding the ROC curve, the
high sensitivity and specificity found for MRR quantifies with high statistical
value the overall capacity of the MRR to discriminate the pattern of inspiratory
muscle relaxation in MD<sub>1</sub> compared with healthy subjects. Regarding
pulmonary function, our study confirmed previous results as it was found the
presence of moderate restrictive disorder in MD<sub>1</sub>. In relation to
respiratory muscle strength, Gillam et al presented, in a series of 10 patients,
values considered below the cutoff point for muscle weakness of the expiratory
muscles in 50% of subjects, with values less than 80 cmH<sub>2</sub>O for men
and 60 cmH<sub>2</sub>O for women. Recently, similar results have been reported
in the literature, in which approximately 50% of patients presented expiratory
muscle strength with values below the cutoff point for expiratory muscle
weakness. These same authors also showed PEmax, PImax and SNIP percentage of
predicted values of about 64%, 70% and 80%, respectively. Additionally, in a
study analyzing respiratory muscle strength involving different neuromuscular
diseases, patients with MD<sub>1</sub> have shown a reduction in respiratory
muscle strength and PEmax/PImax ratio below normal. In the present study,
changes in respiratory muscles strength were reinforced by the changes observed
in the sEMG.
A major limitation of our study was the small sample size due to the limited
time and financial support. However, we may consider the high power and effect
size found and the low prevalence of the disease. Additionally, the results
found in our study add new perspectives regarding the MRR assessment (through
the SNIP test) of subjects with MD<sub>1</sub>. We must interpret the results of
non-invasive MRR of inspiratory muscles in MD<sub>1</sub> with caution,
considering that non-invasive MRR of inspiratory muscles in MD<sub>1</sub>
should be validated with golden standard methods as an invasive measurements of
MRR during assessment of SNIP.
# Conclusion
The study results may provide information that can contribute to a better
understanding of myotonic dystrophy type 1 and its impact on the respiratory
system (either in its functional or muscular aspect). These results may also
contribute to the development of new evaluation methods that may be routinely
used in clinical practice. Moreover, we have brought new approaches to the
surface electromyography, which is still underused to assess respiratory muscles
and may be a complementary method for neuromuscular diseases researches.
The study was performed at the PneumoCardioVascular Lab/HUOL, Empresa Brasileira
de Serviços Hospitalares (EBSERH), Universidade Federal do Rio Grande do Norte
(UFRN), Natal, Rio Grande do Norte, Brasil and PneumoCardioVascular Lab/HUOL,
Empresa Brasileira de Serviços Hospitalares (EBSERH), Universidade Federal do
Rio Grande do Norte (UFRN), Natal, Rio Grande do Norte, Brasil.
The study received financial support of Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES), PROCAD 2013 88881.068409/2014-01, Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) processo:
400316/2012-9. Guilherme Augusto de Freitas Fregonezi is fellow from CNPq number
307353/2015-0 and Vanessa Resqueti is fellow from CNPq number 310091/2015-2.
The authors have declared that no competing interests exist.
2<sup>nd</sup>IS Parasternal muscle on the second intercostal space
MD<sub>1</sub> Myotonic dystrophy type 1
MRR Maximum relaxation rate
PEmax Maximum expiratory pressure
PImax Maximum inspiratory pressure
RA Rectus abdominis
SCA Scalene
SCM Sternocleidomastoid
sEMG Surface electromyography
SNIP Sniff nasal inspiratory pressure
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** FALD AA GAdFF. **Data curation:** MdAE METDJ.
**Formal analysis:** MdAE AS LPG VR GAdFF. **Funding acquisition:** VR
GAdFF. **Investigation:** MdAE METDJ GAdFF. **Methodology:** MdAE FALD VR AA
GAdFF. **Project administration:** VR GAdFF. **Resources:** GAdFF METDJ
FALD. **Supervision:** VR GAdFF. **Visualization:** MdAE FALD AS LPG VR AA
GAdFF GCdN. **Writing – original draft:** MdAE FALD AS LPG VR AA GAdFF.
**Writing – review & editing:** AS LPG VR AA GAdFF GCdN. |
# Introduction
As you read these words, you may be experiencing the presence of a familiar
speechlike companion. This internal speech production may accompany daily
activities such as reading (see, but see), writing (), memorising (),
future planning, problem solving or musing (for reviews see). Several studies
using experience sampling or questionnaires have shown that by deliberately
paying attention to this internal speech, one can examine its phenomenological
properties such as identity (whose voice is it?) or other high-level
characteristics (e.g., is it gendered?). Moreover, it is often possible to
examine lower-level features like the tone of the inner speech, its pitch or its
tempo. This set of basic observations leads to some important insights about the
nature of inner speech. The simple fact that we can make sensory judgements
about our inner speech tautologically reveals that inner speech is accompanied
by sensory percepts (e.g., speech sounds, kinaesthetic feelings). Some of these
introspective accounts have been examined, tested and complemented using
empirical methods from cognitive neuroscience. As summarised in, behavioural and
neuroimaging data reveal that some variants of inner speech are associated with
auditory and/or somatosensory sensations that are reflected by auditory and/or
somatosensory cortex activations. Visual representations may also be at play,
typically for inner language in the deaf population. Inner verbalising therefore
involves the reception of imaginary multisensory signals. This leads to other
fundamental questions: where do these percepts come from? Why do they sound and
feel like the ones we experience when we actually (overtly) speak?
Two main classes of explanatory theories have been offered to answer these
questions. A first class of theories, that derives from Vygotky’s views on
language and thought, and that we describe as the *abstraction view*, suggest
that inner speech is profoundly internalised, abbreviated and condensed in form.
Vygotsky suggested that inner speech evolved from so-called egocentric speech
(i.e., self-addressed overt speech or private speech), via a gradual process of
internalisation during childhood. According to him, the properties of speech are
transformed during this internalisation, and inner speech cannot merely be
described as a weakened form of overt speech (as claimed for instance by). This
has led some scholars to conceive of inner speech as predominantly pertaining to
semantics, excluding any phonological, phonetic, articulatory, or even auditory
properties. The property of abbreviation and condensation is supported by
several psycholinguistic experiments on the qualitative and quantitative
differences between overt and covert speech, as concerns rate and error biases
(e.g., but see). Such condensation implies that the auditory qualities mentioned
above would only rarely be observed during introspection and would merely be the
result of learned associations between abstract linguistic representations and
auditory percepts. A second class of theories is described under the umbrella
term of *motor simulation view*. These theories suggest that inner speech can be
conceived as a kind of action on its own, produced in the same way as overt
speech is, except that the last stage of articulatory execution is only
simulated. Most theories under this view share the postulate that the speech
motor system is involved (to some extent) during inner speech production and
that the auditory and somatosensory consequences of the simulated articulatory
movements constitute the inner speech percepts referred to in subjective
studies.
As explained in the ConDialInt model, these two views can be reconciled if
various degrees of unfolding of inner speech are considered. Fully condensed
forms of inner speech only involve semantics, and are deprived of the acoustic,
phonological and syntactic qualities of overt speech. Expanded forms of inner
speech, on the other hand, presumably engage prosodic and morpho-syntactic
formulation as well as phonological specification, articulatory simulation and
the perception of an inner voice. Between the fully condensed abstract forms and
the expanded articulation-ready form, it can be assumed that various semi-
condensed forms may exist, with morphosyntactic properties and perhaps even
phonological features, depending on the stage at which the speech production
process is truncated. Such a view was also taken by who has suggested that inner
speech varies with cognitive demands and emotional conditions on a continuum
between extremely condensed and expanded forms (see also). Therefore, the two
views of inner speech (abstraction vs. simulation) can be construed as
descriptions of two opposite poles on the condensation dimension. On the most
expanded side of the continuum, inner speech entails full phonetic specification
and articulatory simulation. It might therefore be expected that speech motor
activity could be detectable. If the motor simulation view is correct, then
motor activity could be recorded during expanded forms of inner speech. If, on
the other hand, the abstraction view applies to all forms of inner speech, then
no motor activity should be present, even in phonologically-expanded forms.
Previous research has demonstrated that it is possible to record muscle-specific
electromyographic correlates of inner speech (e.g.,). However, these
studies mostly focused on small samples of participants and sometimes used
invasive intramuscular electromyography. In contrast, more recent research
studies using surface electromyography lead to mixed results. Building upon
previous work, we describe an experimental set-up using surface electromyography
with the aim of testing the involvement of specific speech muscle groups during
the covert production of phonologically expanded speech forms.
## Inner speech as motor imagery of speech
Speech production is a complex motor action, involving the fine-grained
coordination of more than 100 muscles in the upper part of the body. In adult
humans, its covert counterpart (referred to as *inner speech* or *verbal
imagery*) has developed to support a myriad of different functions. In the same
way as visual imagery permits to mentally examine visual scenes, *verbal
imagery* can be used as an internal tool, allowing –amongst other things– to
rehearse or to prepare past or future conversations. Because speech production
results from sequences of motor commands which are assembled to reach a given
goal, it belongs to the broader category of motor actions. Therefore, a parallel
can be drawn between verbal imagery and other forms of motor imagery (e.g.,
imagined walking or imagined writing). Accordingly, studies on the nature of
inner speech might benefit from insights gained from the study of motor imagery
and the field of motor cognition.
Motor imagery can be defined as the mental process by which one rehearses a
given action, without engaging in the physical movements involved in this
particular action. One of the most influential theoretical accounts of this
phenomenon is the *motor simulation theory*. In this framework, the concept of
simulation refers to the “offline rehearsal of neural networks involved in
specific operations such as perceiving or acting”. The MST shares some
similarities with the theories of embodied and grounded cognition in that both
account for motor imagery by appealing to a simulation mechanism. However, the
concept of simulation in grounded theories is assumed to operate in order to
acquire specific conceptual knowledge, which is not the concern of the MST. In
other words, we should make a distinction between *embodiment of content*, which
concerns the semantic content of language, and *embodiment of form*, which
concerns “the vehicle of thought”, that is, proper verbal production.
A second class of explanatory models of motor imagery are concerned with the
phenomenon of *emulation* and with *internal models*. Internal model theories
share the postulate that action control uses internal models, that is, systems
that simulate the behaviour of the motor apparatus. The function of internal
models is to estimate and anticipate the outcome of a motor command. Among the
internal model theories, motor control models based on robotic principles assume
two kinds of internal models (that are supposed to be coupled and regulated): a
forward model (or simulator) that predicts the sensory consequences of motor
commands from efference copies of the issued motor commands, and an inverse
model (or controller) that calculates the feedforward motor commands from the
desired sensory states.
Emulation theories borrow from both simulation theories and internal model
theories and provide operational details of the simulation mechanism. In the
emulation model proposed by, the *emulator* is a device that implements the same
input-output function as the body (i.e., the musculoskeletal system and relevant
sensory systems). When the emulator receives a copy of the control signal (which
is also sent to the body), it produces an output signal (the emulator feedback),
identical or similar to the feedback signal produced by the body, yielding mock
sensory percepts (e.g., visual, auditory, kinaesthetic) during motor imagery.
By building upon models of speech motor control, a recent model describes wilful
(voluntary) expanded inner speech as “multimodal acts with multisensory percepts
stemming from coarse multisensory goals”. In other words, in this model the
auditory and kinaesthetic sensations perceived during inner speech are assumed
to be the predicted sensory consequences of simulated speech motor acts,
emulated by internal forward models that use the efference copies of motor
commands issued from an inverse model. In this framework, the peripheral
muscular activity recorded during inner speech production is assumed to be the
result of *partially* inhibited motor commands. It should be noted that both
simulation, emulation, and motor control frameworks can be grouped under the
*motor simulation view* and altogether predict that the motor system should be
involved to some extent during motor imagery, and by extension, during inner
speech production. We now turn to a discussion of findings related to peripheral
muscular activity during motor imagery and inner speech.
## Electromyographic correlates of covert actions
Across both simulationist and emulationist frameworks, motor imagery has
consistently been defined as the mental rehearsal of a motor action without any
overt movement. One consequence of this claim is that, in order to prevent
execution, the neural commands for muscular contractions should be blocked at
some level of the motor system by active inhibitory mechanisms. Despite these
inhibitory mechanisms, there is abundant evidence for peripheral muscular
activation during motor imagery. As suggested by, the incomplete inhibition of
the motor commands would provide a valid explanation to account for the
peripheral muscular activity observed during motor imagery. This idea has been
corroborated by studies of changes in the excitability of the motor pathways
during motor imagery tasks. For instance, measured spinal reflexes while
participants were instructed to either press a pedal with the foot or to
simulate the same action mentally. They observed that both H-reflexes and
T-reflexes increased during motor imagery, and that these increases correlated
with the force of the simulated pressure. Moreover, the pattern of results
observed during motor imagery was similar (albeit weaker in amplitude) to that
observed during execution, supporting the *motor simulation view* of motor
imagery. Using transcranial magnetic stimulation, several investigators observed
muscle-specific increases of motor evoked potentials during various motor
imagery tasks, whereas no such increase could be observed in antagonist muscles.
When considered as a form of motor imagery, inner speech production is also
expected to be accompanied with peripheral muscular activity in the speech
muscles. This idea is supported by many studies showing peripheral muscular
activation during inner speech production \[, –\], during auditory verbal
hallucinations in patients with schizophrenia, or during induced mental
rumination. Some authors also recently demonstrated that it is possible to
discriminate inner speech content based on surface electromyography (EMG)
signals with a median 92% accuracy. However, other teams failed to obtain such
results.
Many of these EMG studies concluded on the involvement of the speech motor
system based on a difference in EMG amplitude by contrasting a period of inner
speech production to a period of rest. However, as highlighted by, it is usually
not enough to show an increase of speech muscle activity during inner speech to
conclude that this activation is related to inner speech production. Indeed,
three sorts of inference can be made based on the studies of electromyographic
correlates of inner speech production, depending on the stringency of the
control procedure. The stronger sort of inference is permitted by highlighting a
discriminative pattern during covert speech production, as for instance when
demonstrating a dissociation between different speech muscles during the
production of speech sounds of different phonemic class (e.g, contrasting labial
versus non-labial words). According to, other (weaker) types of control
procedures include i) comparing the EMG activity during covert speech production
to a baseline period (without contrasting phonemic classes in covert speech
utterances), or ii) comparing the activity of speech-related and non-speech
related (e.g., forearm) muscle activity. Ideally, these controls can be combined
by recording and contrasting speech and non-speech related muscles in different
conditions (e.g., rest, covert speech, overt speech) of pronunciation of
different speech sounds classes (e.g., labial versus non-labial).
Previous research studies carried out using the preferred procedure recommended
by suggest a discriminative patterns of electromyographic correlates according
to the phonemic class of the words being covertly uttered, which would
corroborate the *motor simulation view* of inner speech. However, these studies
used limited sample sizes (often less than ten participants) and worked mostly
with children. These factors limit the generalisability of the above findings
because i) low-powered experiments provide biased estimates of effects, ii)
following the natural internalisation process, inner speech muscular correlates
are expected to weaken with age and iii) a higher sensitivity could be attained
by using modern sensors and signal processing methods.
The present study intends to bring new information to the debate between the
*motor simulation view* and the *abstraction view* of inner speech, by focusing
on an expanded form of inner speech: wilful nonword covert production. This work
can be seen as a replication and extension of previous works carried out by
McGuigan and collaborators. We aimed to demonstrate similar dissociations by
using surface electromyography recorded over the lip (*orbicularis oris
inferior*, OOI) and the *zygomaticus major* (ZYG) muscles. More precisely, given
that rounded phonemes (such as /u/) are articulated with orbicular labial
contraction, whereas spread phonemes (such as /i/) are produced with zygomaticus
contraction, if the *motor simulation view* is correct, we should observe a
higher average EMG amplitude recorded over the OOI during both the overt and
inner production of rounded nonwords in comparison to spread nonwords.
Conversely, we would expect a lower average EMG amplitude recorded over the ZYG
during both the inner and overt production of rounded nonwords in comparison to
spread nonwords. In addition, we would not expect to observe content-specific
differences in EMG amplitude concerning the non speech-related muscles (i.e.,
forehead and forearm muscles).
# Methods
In the *Methods* and *Data analysis* sections, we report how we determined our
sample size, all data exclusions, all manipulations, and all measures in the
study. A pre-registered version of our protocol can be found at:
<https://osf.io/czer4/>.
## Participants
As previous studies of the electromyographic correlates of inner speech were
mostly carried out with samples of children or young adults, used different
kinds of EMG measures (surface EMG or needle EMG), and different kinds of signal
processing methods, it was impractical to determine the effect size of interest
for the current study. Therefore, we used sequential testing as our sampling
procedure, based on the method described in and. We fixed a statistical
threshold to *BF*<sub>10</sub> = 10 and *BF*<sub>10</sub> = 1/10 (i.e.,
*BF*<sub>01</sub> = 10), testing the difference between the inner production of
labial items versus the inner production of non-labial items on the standardised
EMG amplitude of the lower lip (*orbicularis oris inferior*). In order to
prevent potential experimenter and demand biases during sequential testing, the
experimenter was blind to BFs computed on previous participants. All statistical
analyses have been automatised and a single instruction was returned to the
experimenter (i.e., “keep recruiting participants” or “stop the recruitment”).
We fixed the maximum sample size to 100 participants.
As a result of the above sampling procedure, a total of 25 French-speaking
female undergraduate students in Psychology from the Univ. Grenoble Alpes (mean
age = 19.57, SD = 1.1). took part in this experiment, in exchange for course
credits. It should be noted that this procedure did not work optimally because
we later spotted an error in the EMG signal processing workflow. Thus, the
sequential testing stopped earlier than it should have. These participants were
recruited via mailing list, online student groups, and posters. Each participant
provided a written consent and the present study was approved by the local
ethics committee (Grenoble CERNI agreement \#2016-05-31-9).
## Material
### EMG recordings
EMG activity was recorded using TrignoTM Mini sensors (Delsys Inc.) with a
sampling rate of 1926 samples/s, a band pass of 20 Hz (12 dB/oct) to 450 Hz (24
dB/oct) and were amplified by a TrignoTM 16-channel wireless EMG system (Delsys
Inc.). These sensors consist of two 5 mm long, 1 mm wide parallel bars, spaced
by 10 mm, which were attached to the skin using double-sided adhesive
interfaces. The skin was cleaned by scrubbing it with 70% isopropyl alcohol. EMG
signals were synchronised using the PowerLab 16/35 (ADInstrument, PL3516). Raw
data from the EMG sensors were then resampled at a rate of 1 kHz and stored in
digital format using Labchart 8 software (ADInstrument, MLU60/8).
EMG sensors were positioned over five muscles: the *corrugator supercilii*
(COR), the *frontalis* (FRO), the *zygomaticus major* (ZYG), the *orbicularis
oris inferior* (OOI), and the *flexor carpi radialis* (FCR). Given that the
activity of the *orbicularis oris inferior* and *orbicularis oris superior*
muscles has previously been observed to be strongly correlated and that the
activity of the OOI was more strongly affected by the experimental manipulation,
we decided to record only the activity of the OOI in this study. The two speech-
related muscles (OOI and ZYG) were chosen to show speech-specific EMG
correlates, whereas the two non-speech related facial muscles (COR and FRO) were
chosen to control for overall facial muscular activity. We also recorded the
activity of the FCR of the non-dominant forearm to control for overall (body)
muscular activity.
As reviewed in, the dominant side of the face displays larger movements than the
left side during speech production, whereas the non-dominant side is more
emotionally expressive. Therefore, we recorded the activity of control and
emotion-linked muscles (i.e., COR and FRO) that were positioned on the non-
dominant side of the face (i.e., the left side for right-handed participants),
while sensors recording the activity of the speech muscles (i.e., ZYG and OOI)
were positioned on the dominant side of the face.
The experiment was video-monitored using a Sony HDR-CX240E video camera to track
any visible facial movements. A microphone was placed 20–30 cm away from the
participant’s lips to record any faint vocal production during the inner speech
and listening conditions. Stimuli were displayed using the OpenSesame software
on a 19-inch colour monitor.
### Linguistic material
We selected ten rounded and ten spread bi-syllabic nonwords (cf.). Each class
of nonwords was specifically designed to either induce a greater activation of
the lip muscle (rounded items) or a greater activation of the zygomaticus muscle
(spread items). These stimuli were selected based on phonetic theoretical
constraints, with the aim of maximising the differences between the two classes
of non-words in their involvement of either the OOI or the ZYG muscle. More
precisely, rounded items consisted in the repetition of a syllable containing a
bilabial consonant followed by a rounded vowel, whereas spread items consisted
in the repetition of a syllable containing a lingual consonant followed by a
spread vowel.
## Procedure
Participants were seated in front of a computer screen while audio stimuli (when
applicable) were presented through speakers on both sides of the screen. A video
camera was positioned on one side of the screen to monitor facial movements. A
microphone was positioned at approximately 10 cm of the participant to record
possible speech sounds. After positioning of the EMG sensors, each participant
underwent a relaxation session aiming to minimise pre-existing inter-individual
variability on facial muscle contraction (approximate duration was 330 s). This
relaxation session was recorded by a trained professional sophrology therapist.
Baseline EMG measurements were performed during the last minute of this
relaxation session, resulting in 60 s of EMG signal at baseline. By using this
relaxation period as a baseline, we made sure that participants were all in a
comparable relaxed state. In addition, several previous EMG studies have argued
for the use of a relaxation period as a baseline, since mere resting periods may
include some inner speech production (e.g., for a review).
Subsequently, participants went through a training session, during which they
could get familiar with the main task. They trained with 8 stimuli in total (4
rounded nonwords and 4 spread nonwords, cf.). Each training stimulus appeared
in three conditions (for all participants): overt speech, inner speech and
listening. Nonwords to be produced (covertly or overtly) were visually presented
on the screen. Then, a central fixation dot appeared on the screen, indicating
to the participant that s•he should utter the nonword (either overtly or
covertly). This aimed to ensure that participants were actually producing a
nonword, not just simply visually scanning it. In the overt speech condition,
participants were asked to produce the nonword “just after the word disappeared
from the screen”, with “the most neutral intonation possible”. In the inner
speech condition, participants were asked to “innerly produce the nonword” (cf.
the for precise instructions in French), with “the most neutral intonation
possible” and while remaining as still as possible. In the listening condition,
the order of these two screens was reversed. A fixation dot was first presented
(for 1 second), followed by a blank screen (for 1 second). The audio stimulus
was presented when the blank screen appeared, while participants were asked to
remain as still as possible.
After the training, participants moved to the experimental part, that included a
novel list of 20 nonwords (cf.). Each nonword was presented 6 times in each
condition for each participant. The EMG activity was recorded during the entire
experiment. The periods of interest consisted in one-second portions, after each
stimulus presentation and during either production or listening. This resulted
in 60 observations (60 periods of 1 second) for both classes of nonword in each
test condition. The total duration of the experiment ranged between 30 min and
40 min.
## EMG signal processing
EMG signal pre-processing was carried out using Matlab r2014a (Version
8.3.0.532, [www.mathworks.fr](http://www.mathworks.fr)). We first applied a 50Hz
frequency comb filter to eliminate power noise. Then, we applied a 20 Hz—450 Hz
bandpass filter to the EMG signals, in order to focus on the 20–450 Hz frequency
band, following current recommendations for facial EMG studies.
Although participants were explicitly asked to remain still during inner speech
production or listening, small facial movements (such as swallowing movements)
sometimes occurred. Such periods were excluded from the final sample of EMG
signals. To remove these signals, we first divided the portions of signals of
interest into periods of 1 second. The baseline condition was therefore composed
of 60 trials of 1 second. The periods of interest in all the speech conditions
consisted of the 1-second interval during which the participants either produced
speech or listened to speech. It is possible that the nonword took less than 1
second to be produced, but since there was no way to track when production
started and ended in the inner speech condition, the entire 1-second period was
kept. Therefore, the overt speech condition was composed of 6 repetitions of
each nonword, that is 6x20 trials of 1 second. The “inner speech” and
“listening” conditions were similarly composed of 6x20 trials of 1 second. Then,
we visually inspected the EMG signals recorded during each trial and listened to
the audio signal simultaneously recorded. In all conditions, any time a non-
speech noise (such as coughing or yawning) was audible in a trial, the trial was
discarded (i.e., we did not include this trial in the final analysis, for any of
the recorded muscles). In the listening and overt speech conditions, if a burst
of EMG activity was present after the relevant audio speech signal, then the
trial was discarded. In the overt speech condition, if the participant started
too early or too late and only part of the nonword was recorded in the audio
signal, then the corresponding trial was discarded. In the inner speech and
listening condition, if a large EMG burst of activity was present, potentially
associated with irrelevant non-speech activity, we excluded the trial. The fact
that the artefact rejection procedures slightly differ in the various conditions
is not an issue since we do not directly compare between conditions. Instead, we
compare the EMG correlates of the two classes of nonwords within each condition.
This inspection was realised independently by two judges. Subsequently, we only
kept the trials that were not rejected by any of these two judges (i.e., we
removed a trial as soon as it was rejected by at least one judge). The agreement
rate between the two judges was of 87.82% (with a moderate Cohen’s *κ* of
approximately 0.48). The overall procedure led to an average (averaged over
participants) rejection rate of 22.96% (SD = 6.49) in the baseline condition and
18.49% (SD = 6.48) in the other conditions.
After pre-processing and artefact rejection, we computed the by-trial average
amplitude of the centered and rectified EMG signal. This provided a score for
each muscle of interest (OOI, ZYG, FRO, COR, FCR) in each condition (Baseline,
Overt Speech, Inner Speech, Listening) and for each participant. Absolute EMG
values are not meaningful as muscle activation is never null, even in resting
conditions, due in part to physiological noise. In addition, there are inter-
individual variations in the amount of EMG activity in the baseline. To
normalise and standardise for baseline activity across participants, we thus
expressed the EMG amplitude as a z-score from baseline activity (i.e., we
subtracted the mean amplitude of the centred and rectified baseline signal and
divided the result by the standard deviation of the centred and rectified
baseline signal), thereafter referred to as *δ*.
## Data analysis
Statistical analyses were conducted using `R` version 3.5.3, and are reported
with the `papaja` and `knitr` packages. To assess the effects of the condition
and the class of nonwords on the standardised EMG amplitude, we analysed these
data using *Condition* (3 modalities: speech, inner speech, and listening) and
*Class* of nonwords (2 modalities, rounded and spread, contrast-coded) as
within-subject categorical predictors, and the standardised EMG amplitude as a
dependent variable in a multivariate (i.e., with multiple outcomes) Bayesian
multilevel linear model (BMLM). An introduction to Bayesian statistics is
outside the scope of this paper. However, the interested reader is referred to
for an introduction to Bayesian multilevel modelling using the `brms` package.
In order to take into account the dependencies between repeated observations by
participant, we also included in this model a varying intercept by participant.
Contrary to what we pre-registered, we used a multivariate model (instead of
separate models by muscle). This allowed us to estimate the correlation between
each pair of muscles. Models were fitted with the `brms` package and using
weakly informative priors (see the for code details). Two Markov Chain Monte-
Carlo (MCMC) were run for each model to approximate the posterior distribution,
including each 5.000 iterations and a warmup of 2.000 iterations. Posterior
convergence was assessed examining trace plots as well as the Gelman-Rubin
statistic $\hat{R}$. Constant effect estimates were summarised via their
posterior mean and 95% credible interval (CrI), where a credible interval can be
considered as the Bayesian analogue of a classical confidence interval. When
applicable, we also report Bayes factors (BFs), computed using the Savage-Dickey
method, which consists in taking the ratio of the posterior density at the point
of interest divided by the prior density at that point. These BFs can be
interpreted as an updating factor, from prior knowledge (what we knew before
seeing the data) to posterior knowledge (what we know after seeing the data).
# Results
The *Results* section is divided into two parts. First, we present results from
confirmatory (preregistered) analyses, aiming to test whether it is possible to
dissociate the activity of the OOI and the ZYG during inner speech production,
according to the content of inner speech (here, the class of nonword). More
precisely, we expected an increased EMG activity of the OOI during the inner
production of rounded nonwords in comparison to spread nonwords. Conversely, we
expected elevated EMG activity of the ZYG during the inner production of spread
nonwords in comparison to rounded nonwords. Second, we present results from
exploratory (non-preregistered) analyses.
To foreshadow the results, we did not observe such a clear dissociation between
the EMG activity of the OOI and the ZYG muscles, neither in the inner speech
condition nor in the overt speech condition. Contrary to theoretical
expectations based on phonetics and speech production theory, the activity of
both muscles was of higher amplitude for the pronunciation of rounded nonwords
(as compared to spread nonwords) during overt speech production. Additionally,
the EMG amplitude on both muscles of interest was similar during the inner
production (or listening) of the two classes of nonwords. However, in the
exploratory analyses section, we report results from supervised machine learning
algorithms (classification using random forests), showing a reasonable accuracy
to classify EMG signals according to the class of nonwords during overt speech
production. This strategy was however unsuccessful for the inner speech and the
listening conditions.
Before moving to the statistical results, we represent the distribution of the
whole dataset, by class, by condition and by muscle for the two main muscles of
interest (OOI and ZYG) in. More precisely, the first row of this figure
represents the distribution of the standardised EMG scores in the inner speech
condition, the second row depicts the distribution of these scores in the
listening condition, whereas the third row depicts the distribution of the
standardised EMG scores in the overt speech condition. The first column depicts
the distribution of the standardised EMG scores recorded over the OOI muscle
whereas the second one represents the distribution of the standardised EMG
scores recorded over the ZYG muscle. Each individual data point is represented
as a vertical bar along the x-axis of each panel whereas the vertical coloured
line represents the class-specific median. Additionally, a vertical dashed line
is plotted at zero, which represents the baseline level. Thus, a positive value
on the x-axis represents EMG standardised scores that are higher than baseline.
In, we report the mean standardised EMG amplitude of all recorded muscles in
each condition. Given the skewness of the distribution of these scores, the mean
and the standard deviation (SD) are not the best indicators of the central
tendency and dispersion of these distributions. Therefore, we also report the
median, the median absolute deviation (MAD), and the inter-quartile range (IQR).
We also created a `shiny` application allowing for further visual exploration of
the data by muscle, by condition, and by participant, in the 3D space formed by
three (to be chosen) muscles. This application is available online (at
<https://barelysignificant.shinyapps.io/3d_plotly/>) and the associated code is
available in the OSF repository (<https://osf.io/czer4>).
## Confirmatory (preregistered) analyses
### Bayesian multivariate multilevel Gaussian model
We then compared the standardised EMG amplitude *δ* for each muscle in each
condition (Overt Speech, Inner Speech, Listening) by fitting a multivariate
multilevel Gaussian model (as detailed previously in the Methods section). We
predicted a higher increase of OOI activity during the inner production of
rounded items in comparison to spread items and conversely, a higher increase of
ZYG activity during the inner production of rounded items in comparison to
spread items. These predictions should also apply to the overt speech condition
(and to the listening condition). We should not observe any by-class differences
of FRO and COR activity in any condition.
The results of the Bayesian Gaussian multivariate model are summarised in. This
table reports the estimated average EMG amplitude in each condition and the
corresponding BF. As they are not the main focus of interest here and for the
sake of clarity, descriptive results for the other two facial muscles and for
the forearm muscle are reported in the. This analysis revealed that the EMG
amplitude of the OOI was higher than baseline (the standardised score was above
zero) in every condition whereas it was only the case in the overt speech
condition for the ZYG. Moreover, in all conditions, the EMG amplitude of the ZYG
was lower than that of the OOI. Crucially, we did not observe the hypothesised
difference according to the class of nonwords on the OOI during inner speech
production (*β* = 0.071, 95% CrI \[-0.204, 0.342\], BF<sub>01</sub> = 64.447)
nor on the ZYG (*β* = 0.005, 95% CrI \[-0.031, 0.041\], BF<sub>01</sub> =
532.811).
depicts these results by representing the distribution of the raw data (coloured
dots) along with the predictions from this model. The black dots and vertical
intervals represent the predicted mean and associated 95% credible interval for
each class of non-word, each condition and for the OOI and the ZYG. Coherently
with, this figure shows that the fitted model predicts no noticeable differences
between the two classes of non-words in any condition for the OOI muscle.
However, it predicts a higher average EMG amplitude associated with the rounded
item as compared to the spread items in the overt speech condition for the ZYG
muscle.
Before proceeding further with the interpretation of the results, it is
essential to check the quality of this first model. A useful diagnostic of the
model’s predictive abilities is known as *posterior predictive checking* (PPC)
and consists in comparing observed data to data simulated from the posterior
distribution. The idea behind PPC is that a good model should be able to
generate data that resemble the observed data. In this vein, represents the
distribution of the whole dataset (across all participants and conditions) by
muscle (the dark blue line) along with the distribution of hypothetical datasets
generated from the posterior distribution of the model (the light blue lines).
As can be seen from this figure, the distributions of the data generated from
the model differ considerably from the distribution of the observed data.
Therefore, in the next section, we turn to a more appropriate model for these
data.
### Bayesian multivariate multilevel distributional Skew-Normal model
reveals an important failure of the first model, as it fails to generate data
that look like the data we have collected. More precisely, the collected data
look right-skewed, as it usually happens with physiological measurements. To
improve on the Gaussian model, we then assumed a Skew-normal distribution for
the response variable (the standardised EMG amplitude *δ*). The Skew-normal
distribution is a generalisation of the Gaussian distribution with three
parameters *ξ* (xi), *ω* (omega), and *α* (alpha) for location, scale, and shape
(skewness), respectively (note that the Gaussian distribution can be considered
a special case of the Skew-normal distribution when *α* = 1). In addition, we
also improved the first model by turning it into a *distributional model*, that
is, a model in which we can specify predictor terms for all parameters of the
assumed response distribution. More precisely, we used this approach to predict
both the location, the scale, and the skewness of the Skew-Normal distribution
(whereas the first model only allowed predicting the mean of a Gaussian
distribution). As can been seen in, this second model seems better than the
first one at generating data that fit the observed data.
The estimates of this second model are summarised in and. According to this
model, the EMG amplitude of the OOI was higher than baseline (the estimated
standardised score was above zero) in every condition whereas, for the ZYG, it
was only the case in the overt speech condition. We did not observe the
hypothesised difference according to the class of nonwords during inner speech
production, neither on the OOI (*β* = 0.025, 95% CrI \[-0.012, 0.064\],
BF<sub>01</sub> = 64.447) nor on the ZYG (*β* = 0.004, 95% CrI \[-0.007,
0.014\], BF<sub>01</sub> = 532.811).
Predictions from this model are visually represented in. This figure differs
from (showing the predictions of the Gaussian model) in that the second model
(the Skew-normal model) predicts shifts in location for both the OOI and the ZYG
muscles according to the class of non-word in overt speech prediction. In
contrast, the first model (the Gaussian model) predicted a by-class difference
only for the ZYG muscle.
## Exploratory (non-preregistered) analyses
In the previous section, we tried to predict the average EMG amplitude by
condition on each single muscle. Although this approach was appropriate to
tackle our initial research question (i.e., can we distinguish muscle-specific
EMG correlates of inner speech production?), it is not optimal to answer more
general questions such as “can we predict the content of inner speech based on
the available EMG data?”. In, we depict the distribution of the by-word averaged
EMG scores in the 2D space formed by the OOI and the ZYG muscles. This figure
reveals that although different nonwords produced in overt speech seem difficult
to discriminate on the basis of a single muscle (cf.), it seems easier to
discriminate them in the space formed by two muscles (here OOI and ZYG). More
precisely, the two classes of nonwords seem to form two separate clusters in the
overt speech condition, but these clusters do not seem discriminable in the
inner speech or in the listening condition.
In other words, it is easier to discriminate these signals in the
multidimensional space of all speech muscles, rather than by considering each
muscle independently. Thus, we used a supervised machine learning algorithm
aiming to classify speech signals according to the class of nonwords. Broadly,
the machine learning approach seeks to find a relationship between an input *X*
(e.g., EMG recordings over the four facial muscles) and an output *Y* (e.g., the
class of nonwords). Once trained, it allows predicting a value of the output
based on some input values, whose prediction can be evaluated against new
observations.
We used a random forest algorithm, as implemented in the `caret` package. Random
forests (RFs) represent an ensemble of many decision trees (a forest), which
allow predictions to be made based on a series of decision rules (e.g., is the
score on predictor *x*<sub>1</sub> higher or lower than *z*? If yes, then …, if
not, then …). The specificity of RFs is to combine a large number of trees
(usually above 100 trees), and to base the final conclusion on the average of
these trees, thus preventing overfitting. We used three separate RFs to classify
EMG signals in each condition (Overt Speech, Inner Speech, and Listening).
To evaluate the performance of this approach, we report the raw accuracy (along
with its resampling-based 95% confidence interval), or the proportion of data
points in the test dataset for which the RF algorithm predicted the correct
class of nonwords. First, we randomly split the entire dataset into a training
(80%) and a test set (20%). The training set was used for the learning whereas
the test set was used to evaluate the predictions of the algorithm. To prevent
overfitting, we used repeated 10-fold cross-validation during the learning
phase.
### Predicting the class of nonwords during overt speech production
We first tried to predict the class of nonwords produced in overt speech, based
on the activity of the four facial muscles (OOI, ZYG, COR, FRO). Each predictor
was centred to its mean and standardised before the analysis.
This analysis revealed an overall classification accuracy of 0.847, 95% CI
\[0.814, 0.876\] (cf. confusion matrix). Examining the relative importance of
each feature (i.e., each muscle) for prediction revealed that the muscles
containing most information to discriminate the two classes of nonwords were the
ZYG and the OOI, whereas, as predicted, forehead muscles did not seem to
strongly contribute to predictive accuracy in the overt speech condition.
### Predicting the class of nonwords during inner speech production and listening
We then applied the same strategy (the same algorithm) to the signals recorded
in the inner speech and listening conditions. The results of these analyses are
reported in Tables and.
This analysis revealed an overall classification accuracy of 0.472, 95% CI
\[0.426, 0.52\] in the inner speech condition, which indicates that the RF
algorithm did not allow discriminating the two classes of nonwords better than
random guessing. As the classification accuracy in the inner speech and
listening conditions was not better than chance, we do not report the relative
importance of the predictors. Indeed, it would be difficult to interpret the
importance of predictors for a classification task at which they do not perform
better than chance.
This analysis similarly revealed an overall classification accuracy of 0.46, 95%
CI \[0.413, 0.507\] in the listening condition.
# Discussion
In the present study we aimed to replicate and extend previous findings showing
that facial electromyography can be used to discriminate expanded inner speech
content. As these studies used small samples of children, our study aimed to
examine whether such results can be reproduced using surface electromyography
and modern signal processing methods in an adult sample.
To this end, it was crucial to first show that the EMG correlates of our two
classes of nonwords were discriminable during overt speech production.
Surprisingly, the data we collected during overt speech production do not
corroborate the hypothesis according to which the average EMG amplitude of the
OOI should be higher during the production of “rounded” nonwords as compared to
“spread” nonwords. For both orofacial speech muscles (OOI and ZYG), the average
EMG amplitude was higher for rounded nonwords than for spread nonwords during
overt speech production. Moreover, whereas the average EMG amplitude recorded
over speech muscles was higher than baseline in both the inner speech and
listening conditions, we did not find differences of activation according to the
content of the material (the class of nonword). An automatic classification
approach, using the four facial muscles (OOI, ZYG, COR, FRO), revealed that
although it was possible to discriminate EMG signals related to the two classes
of nonwords with a reasonable accuracy during overt speech production, this
approach failed in discriminating these two classes during inner speech
production or during listening. We also observed a higher EMG amplitude recorded
over the facial (both orofacial and non-orofacial) muscles during inner speech
production and during the listening of speech production than during rest.
However, as pinpointed by, this observation is not sufficient to conclude that
these activations were actually related to inner speech production, because i)
both orofacial speech-related muscles and forehead non-speech related muscles
showed similar EMG amplitude changes from baseline and ii) we did not observe
different changes in EMG amplitude depending on the content of inner speech
(i.e., depending on the class of nonword to be uttered).
Before discussing the theoretical implications of these results, two main issues
are worth discussing. First, how can we explain that rounded nonwords were
associated with higher EMG amplitude during overt speech on both OOI and ZYG
muscles? Second, how can we explain the indiscriminability of inner speech
content, which seems to contradict classic as well as recent findings in the
field? We turn to each of these questions in the following.
To answer the first question, we began by comparing our results to results
obtained by another group. The authors of this study recorded surface EMG
activity from five participants while they were producing seven facial
expressions and five isolated vowel sounds (/a/, /e/, /i/, /o/, /u/), repeated
five times each. They recorded EMG activity over eight facial muscles (the
zygomaticus major (ZYG), the risorius (RIS), the orbicularis oris superior (OOS)
and inferior (OOI), the mentalis (MEN), the depressor anguli oris (DAO), the
levator labii superioris (LLS) muscles, and the digastric muscle (DIG)). We
divided these vowels in two classes to fit our own classes of nonwords. More
precisely, we have created the following two classes: a *rounded* class,
composed of the vowels /o/ and /u/, and a *spread* class, composed of the vowels
/e/ and /i/ (note that we did not include the vowel /a/ because it theoretically
does not fit in one of these two categories). We present the average EMG
amplitude recorded over the OOI and the ZYG according to the vowel class in.
We notice that have indeed observed the dissociation we initially predicted,
that is, that the EMG amplitude recorded over the OOI was higher during the
pronunciation of rounded vowels than during pronunciation of spread vowels,
whereas the reverse pattern was observed concerning the ZYG. Paired-samples
Wilcoxon signed rank tests revealed a shift in location (pseudomedian) between
rounded and spread items for the OOI (*β* = 24.12, 95% CI \[15.19, 40.77\], V =
1184, p \<.001) with rounded items being associated with a higher location than
spread items. This analysis also revealed a shift in the inverse direction
concerning the ZYG (*β* = -1.51, 95% CI \[-2.94, -0.48\], V = 275, p \<.001).
However, one crucial difference between design and ours is the complexity of the
linguistic material. Whereas used single phonemes, we chose to use bisyllabic
nonwords to increase the ecological validity of the paradigm. Although these
nonwords were specifically created to theoretically increase the engagement of
either the OOI or the ZYG (cf. the “Linguistic material” section), it is
reasonable to expect differences in the average EMG patterns between isolated
phonemes and nonwords. More precisely, we expect the *average* EMG amplitude
associated with the production of a given phoneme (e.g., /y/) to be impacted by
the production of the consonant (e.g., /b/) it is paired with, due to
coarticulation. More generally, we could hypothesise that the difference between
the *average* EMG amplitude recorded during the production of the phoneme /i/
and during the production of the phoneme /y/ could be reduced when these
phonemes are coarticulated in CV or CVCV sequences like /byby/ or /didi/ (as in
our study). In other words, we might expect an interaction effect between the
structure of the to-be produced speech sequence (either a single vowel or a
CV/CVCV sequence) and the class of the vowel. This is coherent with previous
findings showing that the muscular activity associated with vowel production is
strongly influenced by the surrounding consonants in CVC sequences. Thus,
further investigations should focus on how the average EMG amplitude is impacted
by coarticulation during the production of CVCV sequences.
With regards to inner speech, our results do not support theoretical predictions
of the *motor simulation view*, according to which it should be possible to
discriminate classes of nonwords produced in inner speech based on EMG signals.
Whereas this outcome is consistent with some recent results, it also stands in
sharp contrast with classical results in the field as well as more recent
developments. For instance, developed a wearable device composed of seven
surface EMG sensors that can attain a 92% median classification accuracy in
discriminating internally vocalised digits. There are a few crucial differences
between’s work and ours that stand as good candidates to explain the
discrepancies between our results. First, the strategy adopted to position the
sensors was radically different. Following guidelines from the field of
psychophysiology, our strategy was to position sensors precisely over the facial
muscles of interest, aligned with the direction of the muscle fibers and in
theoretically optimal positions to record activity of this muscle while reducing
cross-talk. However, precisely because of pervasive cross-talk in facial surface
EMG recordings, this strategy, whereas maximising the probability of recording
activity from a given single muscle, was also (as a result) reducing the
probability of recording activity from potentially speech-relevant neighbour
muscles. Therefore, this strategy might work sub-optimally when the goal of the
experiment is to extract the maximum amount of (relevant) EMG information to
discriminate inner speech content. However, this problem might be mitigated by
using more sensors and a more lenient sensor-positioning approach. Whereas we
recorded the EMG amplitude over only two lower facial muscles (OOI and ZIG),
analysed EMG data from seven different sensors, whose position and number was
defined iteratively in order to maximise the classification accuracy. In other
words, the parameters of the experiment were iteratively optimised to maximise a
certain outcome (classification accuracy). This strategy is radically different
from the classical approach in experimental and cognitive psychology where
experimental conditions are defined to test theoretically derived hypotheses.
Whereas the first approach is arguably more efficient at solving a particular
problem at hand, the second approach might be more efficient in tackling
theoretical questions. For instance, a recent study reported a greater EMG
amplitude of laryngeal and lip muscles during auditory verbal tasks (covert
singing) than during visual imagery tasks. By coupling EMG recording with
demographic and psychological measures, they were able to show that these
correlates were related to the level of accuracy in singing, thus shedding light
upon the nature and functions of peripheral muscular activity during covert
singing. However, adding more sensors (e.g., on the risorius), or better
optimising sensor placement, could improve the sensitivity of the present
approach.
Putting aside considerations related to methodological aspects of the present
study, these results do not corroborate the *motor simulation view* of inner
speech production. Instead, it seems to support the *abstraction view*, which
postulates that inner speech results from the activation of abstract linguistic
representations and does not engage the articulatory apparatus. However,
individual differences in discriminability highlight that the abstractness of
inner speech might be flexible, as suggested by. Indeed, although for most
participants it was not possible to decode the phonetic content of inner speech,
rounded and spread nonwords were in fact distinguishable based on OOI and ZYG
information only (by visual inspection of the 2D plot), for two of them (S_15
and S_17, cf.). This suggests either that the extent to which inner speech
production recruits the speech motor system might vary between individuals or
that it might vary within individual depending on the properties of the ongoing
task (these two suggestions are not mutually exclusive). For instance, we know
from early research on the EMG correlates of inner speech that the average
amplitude of these correlates tend to be higher when the task is more difficult.
As such, the extent to which inner speech production recruits the speech motor
system could be moderated by manipulating the difficulty of the ongoing task. In
addition, the electromyographic activity recorded during motor imagery could be
modulated by the perspective taken in motor imagery. A distinction is made
between first-person perspective or *internal imagery* (i.e., imagining an
action as we would execute it) and third-person perspective or *external
imagery* (i.e., imagining an action as an observer of this action), that may
involve different neural processes. It has been shown that a first-person
perspective may result in greater EMG activity than motor imagery in a third-
person perspective. Therefore, we hypothesise that the involvement of the speech
motor system during inner speech production may be modulated by the specific
instructions given to the participants. For instance, by instructing
participants to focus on *inner speaking* (imagining speaking), instead of
*inner hearing* (imagining hearing), and by asking them to focus on the
kinaesthetic feelings related to speech acts (rather than on auditory percepts),
we could expect to find a higher average EMG amplitude recorded over the speech
muscles. In addition, by specifically asking the participants to mentally
articulate the nonwords, as if they were dictating them to someone, rather than
just read and visually scan them, we may expect stronger articulatory
involvement.
Of course, the current study and the above discussion should be interpreted with
a few words of caution in mind. For each class of nonwords, we collected around
6 x 10 = 60 observations by condition and by participant. For 25 participants
and two classes of nonwords, this results in 25 (participants) x 120 (individual
trials) x 3 (conditions) = 9000 observations. However, after rejecting trials
with movement artefacts, we had 7285 observations in total. Although the number
of observations reported in the present study is reasonable, the sensitivity of
the experiment could be improved by increasing the number of observations and/or
by reducing two important sources of variation. More precisely, one could reduce
the variance related to the item (the specific nonword being uttered) by
selecting nonwords that are more similar to each other in the way they are
uttered, by selecting less items or simpler items. Similarly, particular
attention should be devoted to reducing inter-participant variability, which
could be done by using more guided and specific instructions, as well as a
longer training phase to familiarise the participant with the task.
In summary, we have demonstrated that whereas surface electromyography may lead
to reasonable accuracy in discriminating classes of nonwords during overt speech
production (using signals recorded over only two speech-related muscles), it did
not permit to discriminate these two classes during inner speech production
across all participants (only for two participants). These results, in
comparison with results obtained by other teams, highlight that depending on the
aim of the research, different strategies might be more or less successfully
pursued. More precisely, if the goal is to attain high classification accuracy
(problem-solving approach), then the parameters of the experiment (e.g., number
of repetitions, number of sensors, position of the sensors, parameters of the
signal processing workflow) should be optimised based on the desired outcome
(i.e., classification accuracy). However, the classical laboratory strategy used
in experimental and cognitive psychology, aiming to compare specific conditions
(or muscles) to each other in a controlled environment, is deemed to be more
appropriate when the aim of the research is to sharpen our understanding of the
psychological phenomenon under study.
# Supporting information
10.1371/journal.pone.0233282.r001
Decision Letter 0
Sulpizio
Simone
Academic Editor
2020
Simone Sulpizio
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
22 Jan 2020
PONE-D-19-27197
Can we decode phonetic features in inner speech using surface electromyography?
PLOS ONE
Dear Mr. Nalborczyk,
Thank you for submitting your manuscript to PLOS ONE. After careful
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implement with care. I only add one indication: I agree with reviewer 2
that Vygotskian theory should be cited and discussed in the paper, but I would
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Please use the space provided to explain your answers to the questions above.
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your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: Nalborczyk and colleagues present a study in which they tested the
‘motor simulation’ model of inner speech by measuring EMG to overt speech, inner
speech and listening in the OOI and ZYG muscles (and control muscles) in healthy
adult volunteers. They did not observe the expected difference in EMG activity
between the OOI and ZYG muscles to overt speech, which somewhat complicated the
comparison with the inner speech condition. They did find that it was possible
to discriminate between the phonetic content of inner speech in some
individuals, but this did not maintain at the level of the group.
Overall, I think this this paper represents a nice piece of scholarship. The
literature review was comprehensive, well written and argued, and the hypotheses
were clear and well justified. The statistics and analyses were sophisticated
and thoroughly and carefully presented. I particularly liked how the (key)
analyses were pre-registered – IMO this is a good habit and one which should be
encouraged in the field. The interpretability of the results was limited
somewhat by the study’s failure to replicate the key finding of differential EMG
activity in the OOI and ZYG muscles during the (overt) production of rounded vs.
spread nonwords. But that’s the way science goes, and it would be good to have
these data available in the literature, and I believe that the publication of
null results should be encouraged in order to provide a more accurate reflection
of the true state of the field.
I have a few minor comments that the authors could consider, primarily regarding
the experimental task itself:
\- Probably my main point is that I do not understand is the ‘artefact removal
procedure’ described on p.8. The authors state: “To remove these signals, we
first divided the EMG signals into periods of 1 second” – were these epochs
centred around something (e.g., fixation dot onset), or was the start/end
location arbitrary? How did the authors distinguish between ‘unwanted’ EMG
activity (caused by swallowing, etc.) from ‘of-interest’ EMG activity caused by
movement of the articulators? It is possible that the artefact removal procedure
removed some of the signal of interest? Could this not be problematic,
particularly if the authors only applied the artefact removal procedure to the
listening and inner speech conditions? (as seems to be implied on p.8, but I
could be wrong here – please clarify).
\- P.7. To clarify: am I right in understanding that the EMG activity in the 1
second following the presentation of the fixation dot was used as the dependent
variable? If so, were participants instructed to produce the inner/overt speech
as soon as possible after the fixation dot appeared? What was the delay (on
average) between the dot and speech production in the overt speech condition? Do
the authors assume that this delay was consistent between the inner and overt
speech conditions? If so, can they justify this assumption?
\- It appears as though the ‘baseline’ period (i.e., against which the
standardized scores were calculated) was calculated during the relaxation task
that occurred prior to the talk itself, is that correct? I don’t understand the
benefit of doing this as opposed to, say, calculating the baseline from the 1
second prior to the presentation of the fixation dot – can the authors clarify?
\- Figure 1: I think it would be appropriate to mention in the caption that the
scales between the figure panels differ markedly – these differences are
understandable (i.e., EMG activity in the overt speech condition will obviously
be higher than the inner speech / listening conditions), but still appropriate
to mention.
Reviewer \#2: The present article addresses an important but overlooked question
about the external behavioural traces of inner speech. Its reliance on
preregistered methods is really impressive, particularly in light of the
complexity of possible analyses and the risk of data ‘fishing’. We have several
main concerns. One is the theoretical framework in which the study has been
conceived, which over-emphasises a problematic distinction between simulation
and abstraction views, and pays insufficient attention to the key work in this
area.
Abstract. Second sentence should be revised; the authors do not need to describe
the steps in detail. The last sentence should also be removed as this
information should be submitted separately from the abstract (and also should be
included in the Data Availability section at the end of the manuscript).
2\. A strong claim is made about the automatic elicitation of inner speech in
reading. The picture is not quite so clear-cut; Russell Hurlburt for example has
recently published a study showing very little inner speech during reading.
9\. The discussion of the featural properties of inner speech is a little thin
and could be enhanced.
13\. The term ‘inner voice’ is problematic – does it mean ‘inner speech’? In our
view they have different connotations (see Fernyhough’s recent book on inner
speech, The Voices Within, for a discussion).
18 – 28. This section is incomplete and does not include the essential
Vygotskian theory that inner speech is internalised external speech. This is a
serious omission. See Alderson-Day and Fernyhough (2015) for a full description
of this theory. This is important not just because it is the most developed
theory of inner speech, but because it is also highly relevant to the question
of whether any behavioural traces of inner speech will be observed (see the
discussion in Jones & Fernyhough, 2007, cited here as ref 4, and also extensive
discussion in Fernyhough’s book). The fact that Vygotsky’s name is not mentioned
in this manuscript is surprising. The introduction will need to be rewritten to
represent these important views, which are significant for all of the main
hypotheses in this study and for the interpretation of the findings.
Furthermore, since Vygotsky’s is a developmental theory, it is very relevant to
the question of different methods and findings for EMG studies of inner speech
in children and adults (line 547). Giving some attention to Vygotsky’s theory
would also make a lot of sense of the authors’ surprising findings on EMG
activity during inner speech. See the discussions cited above about whether, on
an internalisation view, you would expect any motoric trace of inner speech, if
the latter is fully internalised and transformed outer speech – which also
brings in the important issue of semantic and syntactic condensation.
188\. The authors describe the sampling procedure in detail, but the
Participants section would benefit from including more demographic information
on the sample.
214\. Studies looking at EMG correlates of lip muscle activity often investigate
the orbicularis oris superior muscle alongside the orbicularis oris inferior.
The authors should discuss in more detail their justification for studying the
OOI, but not OOS.
243\. ‘during rumination’ – we think this is a mistake, and an alternative term
should be used, e.g., covert speech condition.
245\. Please give more detail here on how the stimuli were selected. Were the
stimuli piloted before being selected?
253\. More detail is needed on the instructions given in the three conditions.
Hurlburt et al. (PLOS, 2016) have recently shown a difference in brain
activations between elicited and spontaneous inner speech, questioning whether
inner speech elicited by the method apparently used here can be taken as a
reliable proxy for genuine, spontaneous inner speech. This needs some
acknowledgement and discussion, especially as it addresses many of the issues
raised in the Jones & Fernyhough (2007) paper.
More explanation is needed on the instruction given to participants during
training, e.g. how the visual cues were explained to indicate the start of the
task.
349\. Figures 6 & 7 could be moved to Supplementary Information.
583-628. The paragraph on stimuli selection should be revised. The detailed
information should be moved to the Method section, where it would have been
useful to have more information on how the stimuli were selected. More general
reflection on this process should be included in the Discussion instead.
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Reviewer \#2: No
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10.1371/journal.pone.0233282.r002
Author response to Decision Letter 0
10 Mar 2020
Response to editorial revision requests
We are very grateful to the editor and reviewers for their suggestions and
comments which substantially contributed to the improvement of our paper. We
provide a point-by-point response in the following and have included appropriate
changes in the revised version of our manuscript.
Reviewer \#1
Nalborczyk and colleagues present a study in which they tested the ‘motor
simulation’ model of inner speech by measuring EMG to overt speech, inner speech
and listening in the OOI and ZYG muscles (and control muscles) in healthy adult
volunteers. They did not observe the expected difference in EMG activity between
the OOI and ZYG muscles to overt speech, which somewhat complicated the
comparison with the inner speech condition. They did find that it was possible
to discriminate between the phonetic content of inner speech in some
individuals, but this did not maintain at the level of the group.
Overall, I think this this paper represents a nice piece of scholarship. The
literature review was comprehensive, well written and argued, and the hypotheses
were clear and well justified. The statistics and analyses were sophisticated
and thoroughly and carefully presented. I particularly liked how the (key)
analyses were pre-registered – IMO this is a good habit and one which should be
encouraged in the field. The interpretability of the results was limited
somewhat by the study’s failure to replicate the key finding of differential EMG
activity in the OOI and ZYG muscles during the (overt) production of rounded vs.
spread nonwords. But that’s the way science goes, and it would be good to have
these data available in the literature, and I believe that the publication of
null results should be encouraged in order to provide a more accurate reflection
of the true state of the field.
I have a few minor comments that the authors could consider, primarily regarding
the experimental task itself:
\- Probably my main point is that I do not understand is the ‘artefact removal
procedure’ described on p.8. The authors state: “To remove these signals, we
first divided the EMG signals into periods of 1 second” – were these epochs
centred around something (e.g., fixation dot onset), or was the start/end
location arbitrary? How did the authors distinguish between ‘unwanted’ EMG
activity (caused by swallowing, etc.) from ‘of- interest’ EMG activity caused by
movement of the articulators? It is possible that the artefact removal procedure
removed some of the signal of interest? Could this not be problematic,
particularly if the authors only applied the artefact removal procedure to the
listening and inner speech conditions? (as seems to be implied on p.8, but I
could be wrong here – please clarify).
We thank the reviewer for their comment. These elements are indeed crucial in
interpreting our data. The 1-second epochs were not chosen arbitrarily but
correspond to the periods of interest, that is, the periods during which
participants were either 1) doing nothing (that is the baseline period, composed
of 60 periods of 1-second), 2) producing overt speech (the “speech” condition,
composed of 6 repetitions of each nonword, that is overall 6\*20 periods of 1
second per participant), 3) producing inner speech (the “inner speech”
condition, composed of 6 repetitions of each nonword, that is overall 6\*20
periods of 1 second per participant), or 4) listening to overt speech (the
“listening ” condition, composed of 6 repetitions of each nonword, that is
overall 6\*20 periods of 1 second per participant).
Of course, the artefact rejection procedure could not be applied similarly in
the overt speech condition, since it is impossible to visually distinguish
speech-related activation from non- speech related activities such as
swallowing. But, in addition to the artefact rejection procedure applied to the
inner speech and listening conditions, further checks were carried out. In all
conditions, since we had recorded the audio signal, any time a non-speech noise
(such as coughing or yawning) was present, the EMG signal for that trial was
discarded. In the listening and overt speech conditions, if a burst of EMG
activity was present after the relevant audio speech signal, then the trial was
discarded. In the overt speech condition, if the participant started too early
or too late and only part of the nonword was recorded in the audio signal, then
the corresponding trial was discarded. Moreover, the fact that the artefact
rejection procedure differ in the various conditions is not an issue, since we
do not directly compare between conditions. Instead, we compare the EMG
correlates of the two classes of nonwords within each condition.
However, we agree it is a possibility that this procedure might remove some of
the signal of interest in the inner speech and listening conditions. The reason
we still applied it is because the kind of activation we can record in the inner
speech condition may be orders of magnitude lower than parasite activations such
as activation related to breathing, swallowing, facial expressions, or coughing
(amongst others). Therefore, although these “parasite activities” are expected
to be distributed uniformly across the two classes of non-words, they add a
significant amount of noise to the signal and could diminish the ability to
detect inner-speech-related EMG correlates.
To clarify this, we have made the following change in the manuscript (cf. page
9):
Although participants were explicitly asked to remain still during inner speech
production or listening, small facial movements (such as swallowing movements)
sometimes occurred. Such periods were excluded from the final sample of EMG
signals. To remove these signals, we first divided the portions of signals of
interest into periods of 1 second. The baseline condition was therefore composed
of 60 trials of 1-second. The periods of interest in all the speech conditions
consisted of the 1 second interval during which the participants either produced
speech or listened to speech. It is possible that the nonword took less than 1
second to be produced, but since there was no way to track when production
started and ended in the inner speech condition, the entire 1-second period was
kept. Therefore, the overt speech condition was composed of 6 repetitions of
each nonword, that is 6\*20 trials of 1 second. The “inner speech” and «
listening » conditions were similarly composed of 6\*20 trials of 1 second.
Then, we visually inspected the EMG signals recorded during each trial and
listened to the audio signal simultaneously recorded. In all conditions, any
time a non-speech noise (such as coughing or yawning) was audible in a trial,
the trial was discarded (i.e., we did not include this trial in the final
analysis, for any of the recorded muscles). In the listening and overt speech
conditions, if a burst of EMG activity was present after the relevant audio
speech signal, then the trial was discarded. In the overt speech condition, if
the participant started too early or too late and only part of the nonword was
recorded in the audio signal, then the corresponding trial was discarded. In the
inner speech and listening condition, if a large EMG burst of activity was
present, potentially associated with irrelevant non-speech activity, we excluded
the trial. The fact that the artefact rejection procedures slightly differ in
the various conditions is not an issue, since we do not directly compare between
conditions. Instead, we compare the EMG correlates of the two classes of
nonwords within each condition.
\- P.7. To clarify: am I right in understanding that the EMG activity in the 1
second following the presentation of the fixation dot was used as the dependent
variable? If so, were participants instructed to produce the inner/overt speech
as soon as possible after the fixation dot appeared? What was the delay (on
average) between the dot and speech production in the overt speech condition? Do
the authors assume that this delay was consistent between the inner and overt
speech conditions? If so, can they justify this assumption?
In both the “speech” and the “inner speech” conditions, the nonword was first
visually presented on the screen (for 1 second) and then a fixation dot appeared
(for 1 sec), indicating to the participants that they had to produce the nonword
that they had just seen (i.e., participants had to produce the nonword in the 1
second time window during which the fixation dot was on the screen). In the
“listening” condition however, the order of the fixation dot and the blank
screen was reversed. First, the fixation dot appeared for 1 second (so that
participants get ready for the
task, which consists, in this condition, in listening to an overtly uttered
nonword), followed by a fixation dot (for 1 second), during the presentation of
which the nonword was delivered through the speakers.
Regarding the instructions that were given to the participants, participants
were asked to produce the nonword “when the fixation dot appears” in both the
“speech” and “inner speech” conditions. Due to inattention or anticipation
effects, we might expect that some participants may indeed sometimes “miss” the
1-second time window and produce speech either too late or too soon,
respectively. We did not analyse the average “onset time” (i.e., the delay
between the apparition of the fixation dot and the beginning of the movement)
because these are not available in the inner speech condition (as the EMG signal
does not show discernible bursts of activations).
Although we generally assume that the dynamics of overt and covert speech may be
roughly similar, it is indeed possible that production in the inner speech
condition may start earlier (for instance because there is less articulatory
constraints on the production of inner speech, that is, participants do not have
to move their articulators to produce sounds). However, given that the purpose
of this study is to highlight feature-specific EMG correlates (i.e., it aims at
discriminating two classes of non-words), and because we do not have any reason
to think that the “dynamics” of (overt and covert) speech production may be
affected by this variable (i.e., the non-word class) in any way that could
affect our main measure (i.e., the average EMG amplitude), we do not think this
could be an issue in the interpretation of our data.
We have therefore clarified this section in our revised manuscript and we have
corrected a mistake in this paragraph (cf. lines 361-366). Moreover, we have
added this sentence in the description of the periods of interest:
The periods of interest in all the speech conditions consisted of the 1 second
interval during which the participants either produced speech or listened to
speech. It is possible that the nonword took less than 1 second to be produced,
but since there was no way to track when production started and ended in the
inner speech condition, the entire 1-second period was kept.
\- It appears as though the ‘baseline’ period (i.e., against which the
standardized scores were calculated) was calculated during the relaxation task
that occurred prior to the talk itself, is that correct? I don’t understand the
benefit of doing this as opposed to, say, calculating the baseline from the 1
second prior to the presentation of the fixation dot – can the authors clarify?
We indeed standardised the EMG amplitude in each experimental condition by
subtracting the baseline value to it. The main reason we chose to standardise
EMG amplitude by baseline activity (rather than pre-stimulus activity) is
because in both the “speech” and “inner speech” conditions, the second preceding
the overt/inner production of the nonwords was the second during which the
nonwords was displayed on the screen, and during which the participants were
reading the nonword. Because (some) previous studies have shown an increase in
the EMG activity of the lips during reading (e.g., Faaborg-Andersen et al.,
1958; Sokolov, 1972), standardising the EMG amplitude during inner production by
the activity during reading may obfuscate the activity that could be observed
during the task. In other words, subtracting reading (during which inner speech
may be produced) to inner speech may result in null activity. Moreover, several
previous works have argued for the use of a relaxation period as a baseline,
since mere resting periods may include some inner speech production (see e.g.,
Jacobson, 1931; Vanderwolf, 1998, for a review).
To make this more explicit, we have modified the text as follows:
Baseline EMG measurements were performed during the last minute of this
relaxation session, resulting in 60s of EMG signal at baseline. By using this
relaxation period as a baseline, we made sure that participants were all in a
comparable relaxed state. In addition, several previous EMG studies have argued
for the use of a relaxation period as a baseline, since mere resting periods may
include some inner speech production (e.g., Jacobson, 1931; Vanderwolf, 1998,
for a review).
Faaborg-Andersen, Edfeldt, K. Å. W. & Nykøbing, F. (1958). Electromyography of
Intrinsic and Extrinsic Laryngeal Muscles During Silent Speech: Correlation with
Reading Activity: Preliminary Report, Acta Oto-Laryngologica, 49(1), 478-482.
Jacobson, E. (1931). Electrical measurements of neuromuscular states during
mental activities. VII. Imagination, recollection, and abstract thinking
involving the speech musculature. American Journal of Physiology, 97, 200-209.
Sokolov, A. (1972). Inner speech and thought. New York: Springer-Verlag.
Vanderwolf, C.H. (1998). Brain, behavior, and mind: what do we know and what can
we know?
Neuroscience and Biobehavioral Review, 22,125-142.
\- Figure 1: I think it would be appropriate to mention in the caption that the
scales between the figure panels differ markedly – these differences are
understandable (i.e., EMG activity in the overt speech condition will obviously
be higher than the inner speech / listening conditions), but still appropriate
to mention.
We agree with the reviewer’s comment and we have added a word of caution about
this issue in the caption of Figure 1.
Reviewer \#2
The present article addresses an important but overlooked question about the
external behavioural traces of inner speech. Its reliance on preregistered
methods is really impressive, particularly in light of the complexity of
possible analyses and the risk of data ‘fishing’. We have several main concerns.
One is the theoretical framework in which the study has been conceived, which
over-emphasises a problematic distinction between simulation and abstraction
views, and pays insufficient attention to the key work in this area.
Abstract. Second sentence should be revised; the authors do not need to describe
the steps in detail. The last sentence should also be removed as this
information should be submitted separately from the abstract (and also should be
included in the Data Availability section at the end of the manuscript).
We thank the reviewer for their suggestion. We have removed the second and last
sentences of the abstract.
2\. A strong claim is made about the automatic elicitation of inner speech in
reading. The picture is not quite so clear-cut; Russell Hurlburt for example has
recently published a study showing very little inner speech during reading.
We thank the reviewer for this important comment. We only used “silent reading”
as a way to introduce the notion of inner speech in a practical way, as several
studies reveal that reading may be accompanied by inner speech (e.g., Yao et
al., 2011). However, we fully agree that not everyone produces inner speech
during reading (and typically not expert readers, who may well be the typical
readers of a scientific paper). We have therefore added a nuance to this
introduction, and we inserted the reference suggested by the reviewer (Brouwers
et al., 2018), as well as the discussion by Hurlburt (2018). We have also
expanded our presentation of the occurrences of inner speech. The manuscript now
reads:
As you read these words, you may be experiencing the presence of a familiar
speechlike companion. This internal speech production may accompany daily
activities such as reading (see \[1–4\], but see \[5,6\]), writing (\[7\]), 4
memorising (\[8,9\]), future planning \[8\], problem solving \[9,10\] or musing
(for reviews see \[11–14\]).
9\. The discussion of the featural properties of inner speech is a little thin
and could be enhanced.
We have provided a more thorough description of the featural properties of inner
speech and have added references to the literature on this subject. The text is
now modified to:
Several studies using experience sampling or questionnaires (e.g.,\[14,15\])
have shown that by deliberately paying attention to this internal speech, one
can examine its phenomenological properties such as identity (whose voice is
it?) or other high-level characteristics (e.g., is it gendered?). Moreover, it
is often possible to examine lower-level features like the tone of the inner
speech, its pitch or its tempo. This set of basic observations leads to some
important insights about the nature of inner speech. The simple fact that we can
make sensory judgments about our inner speech tautologically reveals that inner
speech is accompanied by sensory percepts (e.g., speech sounds, kinaesthetic
feelings). Some of these introspective accounts have been examined, tested and
complemented using empirical methods from cognitive neuroscience. As summarised
in \[17\], behavioural and neuroimaging data reveal that some variants of inner
speech are associated with auditory and/or somatosensory sensations that are
reflected by auditory and/or somatosensory cortex activations. Visual
representations may also be at play, typically for inner language in the deaf
population. Inner verbalising therefore may involve the reception of imaginary
multisensory signals. This leads to other fundamental questions: where do these
percepts come from? Why do they sound and feel like the ones we experience when
we actually (overtly) speak?
13\. The term ‘inner voice’ is problematic – does it mean ‘inner speech’? In our
view they have different connotations (see Fernyhough’s recent book on inner
speech, The Voices Within, for a discussion).
We fully agree that “inner voice” and “inner speech” should not be equated. When
we used the term of “inner voice”, we meant the audible voice signal that (may
or may not) accompany inner verbal production. When we use the terms of “inner
speech”, we mean the activity of talking silently to oneself (where “silently”
is to be understood as from the perspective of an external observer, not from
the perspective of the inner speaker, for which inner speech may (or may not) be
accompanied by sounds). To avoid confusion, we have modified the text and only
used the term voice to refer to acoustic voice quality.
18 – 28. This section is incomplete and does not include the essential
Vygotskian theory that inner speech is internalised external speech. This is a
serious omission. See Alderson-Day and Fernyhough (2015) for a full description
of this theory. This is important not just because it is the most developed
theory of inner speech, but because it is also highly relevant to the question
of whether any behavioural traces of inner speech will be observed (see the
discussion in Jones & Fernyhough, 2007, cited here as ref 4, and also extensive
discussion in Fernyhough’s book). The fact that Vygotsky’s name is not mentioned
in this manuscript is surprising. The introduction will need to be rewritten to
represent these important views, which are significant for all of the main
hypotheses in this study and for the interpretation of the findings.
Furthermore, since Vygotsky’s is a developmental theory, it is very relevant to
the question of different methods and findings for EMG studies of inner speech
in children and adults (line 547). Giving some attention to Vygotsky’s theory
would also make a lot of sense of the authors’ surprising findings on EMG
activity during inner speech. See the discussions cited above about whether, on
an internalisation view, you would expect any motoric trace of inner speech, if
the latter is fully internalised and transformed outer speech – which also
brings in the important issue of semantic and syntactic condensation.
We thank the reviewer for this thoughtful comment. We are well aware of the
Vygotskian developmental theory of inner speech and the various extensions that
have been proposed in the recent years (see for instance a more exhaustive
historical overview in the introduction of Nalborczyk, 2019). However, we do not
think the Vygotskian theory of inner speech is directly
relevant here, as it does not (contrary to the opposition between the motor
simulation view and the abstraction view) lead to testable predictions regarding
the central question of our experiment, that is, whether the two classes of non-
words will be associated with distinct orofacial EMG correlates. In other words,
it does not clearly predict whether inner speech will be associated with
phonetically-specific “observable” (using EMG) traces in our situation.
We agree, however, that the Vygotskian notion of condensation should be
mentioned, as well as the view of Charles Fernyhough and his colleagues that
inner speech can vary between two extremes, depending on cognitive demands and
emotional conditions. We are aware that condensation is, as claimed in the
ConDialInt model by Grandchamp et al. (2019), one of the important dimensions of
inner speech. We have therefore substantially modified our text, into the
following paragraph in the introduction:
Two main classes of explanatory theories have been offered to answer these
questions. A first class of theories, that derives from Vygotky’s views on
language and thought, and that we describe as the abstraction view \[18\]
suggest that inner speech is profoundly internalised, abbreviated and condensed
in form. Vygotsky suggested that inner speech evolved from so- called egocentric
speech (i.e., self-addressed overt speech or private speech), via a gradual
process of internalisation during childhood \[19\]. According to him, the
properties of speech are transformed during this internalisation, and inner
speech cannot merely be described as a weakened form of overt speech (as claimed
for instance by \[20\]). This has led some scholars to conceive of inner speech
as predominantly pertaining to semantics, excluding any phonological, phonetic,
articulatory or even auditory properties (e.g., \[19–22\]). The property of
abbreviation and condensation is supported by several psycholinguistic
experiments on the qualitative and quantitative differences between overt and
covert speech, as concerns rate and error biases (e.g., \[20,21,23,24\], but see
\[25\]). Such condensation implies that the auditory qualities mentioned above
would only rarely be observed during introspection and would merely be the
result of learned associations between abstract linguistic representations and
auditory percepts. A second class of theories is described under the umbrella
term of motor simulation view. These theories suggest that inner speech can be
conceived as a kind of action on its own \[26,27\], produced in the same way as
overt speech is, except that the last stage of articulatory execution is only
simulated. Most theories under this view share the postulate that the speech
motor system is involved (to some extent) during inner speech production and
that the auditory and somatosensory consequences of the simulated articulatory
movements constitute the inner speech percepts referred to in subjective
studies.
As explained in the ConDialInt model \[26\], these two views can be reconciled
if various degrees of unfolding of inner speech are considered. Fully condensed
forms of inner speech only involve semantics, and are deprived of the acoustic,
phonological and syntactic qualities of overt speech. Expanded forms inner
speech, on the other hand, presumably engage prosodic and morpho-syntactic
formulation as well as phonological specification, articulatory simulation and
the perception of an inner voice. Between the fully condensed abstract forms and
the expanded articulation-ready form, it can be assumed that various semi-
condensed forms may exist, with morphosyntactic properties and perhaps even
phonological features, depending on the stage at which the speech production
process is truncated. Such a view was also taken by \[29\] who has suggested
that inner speech varies with cognitive demands and emotional conditions on a
continuum between extremely condensed and expanded forms (see also \[11,28\]).
Therefore, the two views of inner speech (abstraction vs. simulation) can be
construed as descriptions of two opposite poles on the condensation dimension.
On the most expanded side of the continuum, inner speech entails full phonetic
specification and articulatory simulation. It might therefore be expected that
speech motor activity could be detectable. If the motor simulation view is
correct, then motor activity could be recorded during expanded forms of inner
speech. If, on the other hand, the abstraction view applies to all forms of
inner speech, then no motor activity should be present, even in phonologically-
expanded forms.
Nalborczyk, L. (2019). Understanding rumination as a form of inner speech. PhD
thesis. Univ. Grenoble Alpes & Ghent University. Retrieved from
<https://thesiscommons.org/p6dct/>
188\. The authors describe the sampling procedure in detail, but the
Participants section would benefit from including more demographic information
on the sample.
We have added information about the sex and age of our participants (cf. lines
254 -256).
214\. Studies looking at EMG correlates of lip muscle activity often investigate
the orbicularis oris superior muscle alongside the orbicularis oris inferior.
The authors should discuss in more detail their justification for studying the
OOI, but not OOS.
We have included more details about this choice in the Methods section (cf.
footnote n°1).
Namely, we chose to record only the OOI in this study because we previously
observed (e.g., in Nalborczyk et al., 2017; Rapin et al., 2013) that the OOS and
OOI were strongly correlated but that the OOI activity was more affected by the
experimental manipulation. Given that the information contained in these two
muscles was therefore slightly redundant and given material limitations (i.e.,
we have a limited amount of sensors), we therefore opted for recording
additional control muscles (i.e., COR and FRO) instead of the OOS.
Nalborczyk, L., Perrone-Bertolotti, M., Baeyens, C., Grandchamp, R., Polosan,
M., Spinelli,
E.... Lœvenbruck, H. (2017). Orofacial electromyographic correlates of induced
verbal rumination. Biological Psychology, 127, 53–63.
<https://doi.org/10.1016/j.biopsycho.2017.04.013>
Rapin, L., Dohen, M., Polosan, M., & Perrier, P. (2013). An EMG Study of the Lip
Muscles During Covert Auditory Verbal Hallucinations in Schizophrenia. JSLHR,
56, 1882–1893. https:// [doi.org/10.1044/1092-](http://doi.org/10.1044/1092-)
4388(2013/12- 0210)and
243\. ‘during rumination’ – we think this is a mistake, and an alternative term
should be used, e.g., covert speech condition.
We thank the reviewer for spotting this mistake, we corrected it.
245\. Please give more detail here on how the stimuli were selected. Were the
stimuli piloted before being selected?
Stimuli were mostly selected based on theoretical constraints, with the aim of
maximising the differences between the two classes of non-words (+ informal
piloting on master students). We clarified this in the manuscript (in the
“Linguistic material” section). Please note that these theoretical constraints
translate well in practice as the two classes of non-words appear to form two
clearly distinct clusters in the OOI-ZYG space in the overt speech condition
(cf. leftmost panel of Figure 6 in the updated manuscript).
253\. More detail is needed on the instructions given in the three conditions.
Hurlburt et al. (PLOS, 2016) have recently shown a difference in brain
activations between elicited and spontaneous inner speech, questioning whether
inner speech elicited by the method apparently used here can be taken as a
reliable proxy for genuine, spontaneous inner speech. This needs some
acknowledgement and discussion, especially as it addresses many of the issues
raised in the Jones & Fernyhough (2007) paper.
More explanation is needed on the instruction given to participants during
training, e.g. how the visual cues were explained to indicate the start of the
task.
We have clarified the instructions given to the participants in this section
(cf. pages 8-9). As made clearer in the introduction (see above), this study
only pertains to elicited, wilful inner speech.
349\. Figures 6 & 7 could be moved to Supplementary Information.
We agree that Figure 7 does not contain information that could not be understood
in text format and we therefore removed it. However, we do see value in having
Figure 6 in the main manuscript, as it allows the reader to immediately see how
the EMG amplitude can be clustered in the 2D space formed by the OOI and the ZYG
muscles.
583-628. The paragraph on stimuli selection should be revised. The detailed
information should be moved to the Method section, where it would have been
useful to have more information on how the stimuli were selected. More general
reflection on this process should be included in the Discussion instead.
Details concerning the selection of stimuli (of our study) are already provided
in the “Linguistic materials” section of our manuscript. It is not clear from
the reviewer’s comment what supplementary information should be added to this
section.
Please note that the details provided in the paragraph commented on by the
reviewer (i.e., lines 583-628 of the previous submission) concern additional
analyses performed on the data from Eskes et al. (2017). As this analysis is not
central to the purpose of the current study and rather subserves a general
reflection (cf. discussion in the subsequent paragraph), we feel this belongs to
the discussion section.
10.1371/journal.pone.0233282.r003
Decision Letter 1
Sulpizio
Simone
Academic Editor
2020
Simone Sulpizio
This is an open access article distributed under the terms of the
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provided the original author and source are credited.
4 May 2020
Can we decode phonetic features in inner speech using surface electromyography?
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10.1371/journal.pone.0233282.r004
Acceptance letter
Sulpizio
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Simone Sulpizio
This is an open access article distributed under the terms of the
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provided the original author and source are credited.
12 May 2020
PONE-D-19-27197R1
Can we decode phonetic features in inner speech using surface electromyography?
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# Introduction
Formation of blood vessels in the mammalian eye involves extensive tissue
reorganization including regression of embryonic vascular structures. The
developing murine eye is initially supplied with oxygen and nutrients by the
hyaloid vasculature (HV), which is later replaced by the retinal vasculature.
The HV is formed in the primitive vitreous body between embryonic days (E) 10.5
and E13.5. Concomitant with the postnatal (P) formation and maturation of the
intraretinal vasculature, the HV degenerates via apoptosis, beginning on P4 and
culminating on P7–8. On P10 most of the HV vessels have regressed and although
complete regression of the hyaloid takes a few weeks, the vitreous body is
completely avascular by P16. Vascularization of the retina is preceded by
colonization with Pax2-positive astrocyte precursors that form a network, which
becomes covered by endothelial cells. As they differentiate, these precursor
cells begin to express GFAP as well, and change their morphology.
Failure of the HV to regress, results in a congenital condition known as
Persistent Fetal Vasculature Syndrome (PFVS), or persistent hyaloid vasculature
(PHV). The consequences can be severe intraocular hemorrhage, cataract and
retinal detachment due to forces exerted on the neural retina by contractile
cells associated with the abnormal vessels in the vitreous. Although transgenic
mouse models have shed some light on possible pathways the precise molecular and
cellular mechanisms underlying the failure of the HV to regress are not yet
fully understood. Disturbance of hyaloid vessel regression was reported in mice
deficient in both Wnt7b-dependent and Wnt7b-independent Fzd4 signaling and
formation of the deeper plexus is also disrupted in these mutant mice. Wnt7b is
believed to be produced by the macrophages that play key roles in the regression
of capillaries of the HV as indicated by the finding that in heterozygous BMP4
+/−, which lack macrophages in the vitreous, the HV persists. Moreover, Arf
knockout mice and certain p53-null strains, both of which proteins are tumor
suppressors, also display persistent HV, as do Ang-2 knockout mice.
Platelet-derived growth factor (PDGF) is essential for proper development of the
retina and has been associated with proliferative retinopathies. The PDGF family
consists of four ligands, designated A, B, C and D that function as homodimers
or in the case of AB, also as a heterodimer. PDGF-AA, -AB, -BB and -CC activate
the PDGF receptor-α (PDGFRα) while PDGF-BB and –DD bind to PDGFRβ. In the normal
eye, PDGF-A is expressed by both neurons and astrocytes and, together with
PDGFRα, regulates the recruitment of astrocyte precursors to the retina and
their subsequent development at this location. In this manner, interactions
between PDGF-A and PDGFRα determine the number and distribution of astrocytes in
the retina. Maintenance of the retinal vasculature depends on signaling by
PDGF-B via the PDGFRβ. Pericytes express PDGFRβ, and their attachment to vessels
is dependent on PDGF released from endothelial cells.
Transgenic over-expression of PDGF-A in retinal ganglion cells (RGCs) results in
a dose-dependent increase in the proliferation of GFAP-immunoreactive (+) cells
in the retina, as well as inhibiting the migration and spread of these cells
across the retina, thus producing a thick carpet of GFAP+ cells close to the
exit of the optic nerve. Furthermore, over-expression of PDGF-B under control of
the rhodopsin promoter also enhances astrocyte proliferation in the retina. In
this case, folding of the retina occurs, a phenomenon also observed in MBP-
PDGF-B transgenic mice that, in addition, exhibit disorganization of capillaries
in the retina.
HV cells express PDGFRβ but not PDGFα. In the case of Arf-deficient mice with a
persistent HV, it has been proposed that inadequate repression of the PDGFRβ
promoter in mural cells stimulates their proliferation at the expense of
differentiation. This proposal implies that interactions between PDGF and its
receptors are important not only for the maintenance, but also for regression of
the HV.
To characterize the effects of over-expression of PDGF in neural progenitor
cells on the developing retina, we developed transgenic mice that express PDGF-B
under the control of a nestin enhancer element, that is active in progenitor
cells during development, but not in adult neural stem or progenitor cells.
These nes/tk-pdgfb-lacZ mice are viable and fertile but develop enlarged lateral
ventricles and mild behavioral abnormalities. In the present investigation we
report that the eyes of these mice display severe defects in retinal
development.
# Materials and Methods
## Animals and genotyping
Our experimental model, nes/tk-PdgfB-lacZ mice over-express a 730-bp PDGF-B cDNA
of human origin under control of the second intron enhancer of the human nestin
gene (nes) joined to a basic thymidine kinase promoter (tk). An internal site
for ribosomal entry (IRES) and a LacZ reporter gene were cloned downstream of
the PGDF-B gene. The nes element enhances expression in nestin-expressing cells
during embryonic development, but not in the adult cells that express this
protein. The generation and characterization of the nes/tk-PdgfB-lacZ strain
have been described elsewhere and all animals used here belongs to the \#310
line described in this earlier report.
A back-cross to C57Bl/6 mice for 5 generations was performed prior to the onset
of the experiment. All animal protocols were pre-approved by the local ethics
committee for laboratory animals. Embryonic day 0.5 (E0.5) was defined on the
basis of the presence of a copulatory plug in the morning and postnatal day 0
(P0) was defined as the morning of the day of birth. Animals were sacrificed by
CO<sub>2</sub> gas, cervical dislocation (pregnant dams and animals \>15 days of
age) or by decapitation (embryos and pups \<14 days old). The transgenic mice
were identified by PCR analysis as described elsewhere. In brief, genomic DNA
extracted from tail biopsies was subjected to PCR (95°C for 10 min, 40
cycles×\[94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min\], 72°C for 7 min)
utilizing: 5′-TGCTGCTACCTGCGTCTG as the forward primer and 5′-TTCTTCCGCACAATCTCG
as the reverse primer.
## Tissue preparation and immunohistochemical analysis
For our purposes, we chose to routinely analyze the eyes and retinas of mice at
the following stages of development in utero and post-natal life: E13.5, E15.5,
E17.5, P1, P5, P10, P15, P20, P30, P60 and P90. On occasion, other time-points
were examined as well.
To obtain cryosections, eyeballs were dissected out, fixed in 4% PFA (4°C for
15–20 minutes), washed with phosphate-buffered saline (PBS) (10 minutes at 4°C),
cryoprotected in 30% sucrose (at 4°C for 3–4 hours), embedded in OCT (Sakura,
Alphen aan den Rijn, The Netherlands) and frozen. 10 µm sections were cut,
collected onto SuperFrost Plus objective glasses (Menzel-Gläser, Braunschwieg,
Germany) and subsequently stored at −20°C or −80°C until being analyzed.
Specimens were either stained with hematoxylin and eosin (H&E) or processed for
immunostaining. For immunohistochemical staining, these sections were first re-
hydrated in PBS for 15 minutes and then blocked and permeabilized for 30 minutes
in blocking solution (PBS, 1% FCS and 0.1% Triton-X supplemented with 0.02%
thimerosal for purposes of preservation). Thereafter, the sections were
incubated with primary antibodies in blocking solution overnight at 4°C and
subsequently with secondary antibodies in the same solution for 2–4 hours at
room temperature. Following each such incubation with antibodies, the samples
were rinsed 3 times with PBS for 5 minutes each. Finally, the sections were then
mounted and coverslipped using VectaShield HardSet (with or without DAPI,
H-1400/H-1500, Immunkemi, Järfälla, Sweden).
In the case of retinal flatmounts, eyeballs were fixed in 4% PFA (at 4°C for 3
hours) prior to dissecting out the retinas, which were then washed for 10
minutes in PBS and subsequently stored in blocking solution at 4°C until being
analyzed. For staining, the retinas were incubated successively with primary and
secondary antibodies overnight at 4°C, with washing in PBS (4–6 times, 1 hr each
time after each such incubation). After the final wash, perpendicular cuts were
made at the peripheral margins of the retina to facilitate flattening. Finally,
the retinas were mounted onto SuperFrost Plus objective glasses and coverslipped
as described above.
## Antibodies and image acquisition
A list of the primary antibodies employed is presented in.
The secondary antibodies (diluted 1∶200) were obtained from Vector Laboratories,
Jackson Immunoresearch Laboratories and Molecular Probes. Samples were examined
under a Zeiss Axioplan2 microscope and images acquired using the Axiovision
software. These images have been formatted, resized, combined, enhanced and
arranged for publication in Axiovision or Adobe Photoshop.
## Cell counting in P10 retinas
For quantification of the proportions of different cell populations in the INL
and GCL, P10 retinas were co-stained for Pax6 and Isl1 and the resulting
fractions of Isl1+ only, Pax6+ only and Isl1+/Pax6 double positive cells were
manually counted in images taken using the 20× or 10× objective. Four images
from two individuals were counted and the fractions of cells single or double
positive for the markers were expressed as percentages of the total number of
stained cells per retina image. Student's t-test was performed to test for
significant differences between wild-type and transgenic mice, where p\<0.05 was
considered significant.
## Staining for X-Gal
In order to monitor the localization and temporal pattern of the activity of the
transgene via the LacZ reporter, cryosections from E13.5- P90 animals (prepared
as described above) were stained for X-gal. After re-hydration in PBS for 10
minutes and post-fixation in PBS containing 1% formaldehyde, 0.2%
glutaraldehyde, 0.02% NP40, 2 mM MgCl<sub>2</sub>, and 5 mM EGTA, pH 8.0, for 15
minutes at room temperature, these sections were washed 3×5 minutes in X-Gal
wash buffer (PBS containing 2 mM MgCl<sub>2</sub>, 0.01% Na-deoxycholate and
0.02% NP40). Visualization of X-galactosidase activity was carried out by
incubation at 37°C for 4 hours in X-Gal wash buffer supplemented with 1 mg
X-Gal/ml, 5 mM K<sub>3</sub>Fe(CN)<sub>6</sub> and 5 mM
K<sub>4</sub>Fe(CN)<sub>6</sub>. The sections were washed 2×5 minutes in X-Gal
wash buffer, then washed once in PBS and finally mounted in Entellan (VWR,
Gothenburg, Sweden).
## Determination of proliferation utilizing EdU
To examine proliferation during the earliest phases of transgene activation,
pregnant dams carrying E18.5 pups were injected intraperitoneally with 200 µg of
the nucleotide analogue 5-ethynyl-2′-deoxyuridine (EdU) and the pups analyzed 2
days later, at P1. The eyes were fixed, frozen and sectioned as described above
and the Click iT EdU imaging kit (C10083/C10337, Invitrogen, Paisley, UK)
employed in accordance with the manufacturer's protocol to visualize cells that
had incorporated EdU during S-phase.
## Measurement of intraocular pressure
The intraocular pressure (IOP) in 3–4 months-old mice was measured using the
TonoLab rodent tonometer (Icare Finland Oy, Espoo, Finland) in accordance with
the manufacturer's instructions, following sedation by i.p. injection of Rompun
(10 mg/kg, Bayer Animal Health, Copenhagen, Denmark) and Ketalar (60 mg/kg,
Pfizer, Sollentuna, Sweden). For each mouse the IOP in both eyes (with the
exception three transgenic mice in which one eye was too small to allow reliable
measurements) was determined consecutively 6 times and the means for the
transgenic (9 animals, 15 eyes measured) and wild-type mice (11 animals, 22 eyes
measured) analyzed for statistically significant differences utilizing the Mann-
Whitney test.
## Administration of STI571
STI571 was administered (100 µg/g body weight, diluted in dH<sub>2</sub>O) by
gavage once daily during three different time intervals: the first group
received this inhibitor between E17.5 and P0 or P1 (samples taken for analysis
at P1 or P2, respectively), the second group from P0–P4 (samples being taken for
analysis on P5), and the third group between P7 and P14 (samples taken on P15).
The body weights and general health of the mice was monitored daily. The eyes
were fixed, frozen and sectioned as described above.
## Statistical analyses
The data were analyzed in Excel and graphs created in GraphPad Prism (v3.02,
GraphPad Software Inc., La Jolla, USA).
# Results
## Morphological alterations in eyes of mice over-expressing PDGF-B
In the transgenic mice employed, PDGF-B is over-expressed in nestin-expressing
cells of the CNS during development. The macroscopic appearance of the nes/tk-
PdgfB-lacZ mice and their wild type littermates was generally similar, except
that the transgenic mice displayed enlarged lateral ventricles, smaller eyes and
had abnormal behavior as described previously. At the time of eye opening, the
transgenic mice displayed uni- or bilateral reduction in the size of their eyes
and/or irises and we frequently observed blood inside the eyeball in transgenic
mice. Retinal folding with frequent attachment to the lens was observed in all
the transgenic animals after birth. With increasing age of the transgenic mice,
the retinas degenerated progressively and after 2–3 months they were markedly
thinner than those of the wild-type animals (–D). Around one year of age, the
retinas had degenerated completely and consisted of only a thin sheet of cells.
Histological examination revealed a severe distortion of the transgenic retinas,
with disorganization of retinal lamination discernable already in the earliest
days of post-natal life. One prominent feature of these retinas was rosette-like
structures in the outer nuclear layer (ONL) containing the nuclei of
photoreceptors (arrow), which appeared between P5 and P10 The other nuclear
layers of transgenic retina; the inner nuclear layer (INL) and the ganglion cell
layer (GCL) were also distorted, with local variations in thickness and folding.
In general, the nuclear and plexiform layers could be distinguished from one
another although with a varying degree of distortion. Among the general laminar
disarray, local regions in some retinas appeared normal. The progressing
deterioration of the neural retina was visualized by H&E staining.
## The temporal pattern of transgene expression in the developing retina
We have previously shown that X-gal staining in this mouse strain faithfully
depicts transgenic PDGF-B expression. In order to monitor changes in the
expression of the transgene with time, the LacZ reporter gene and staining of
X-gal in sectioned tissue were therefore employed. In contrast to the brain and
spinal cord, where X-gal staining is most intense between E10.5 and E14.5
retinal expression occurred later during development. In this tissue, staining
first appeared as a faint signal distributed throughout the neuroblastic layer
(NBL) and in some cells within the putative ganglion cell layer (GCL) on E17.5.
On P1 the pattern of staining was similar, but much darker and between P5 and
P15, the robust staining persisted, albeit now restricted to a band of cells
located in the middle of the INL (note the localization of X-Gal staining in the
‘near-normal’ region shown). By P20, the intensity of the staining, although
still detectable, had attenuated significantly (arrowheads) and after P30 no
X-Gal staining could be seen ( and not shown). We conclude that transgene
expression in the retina commenced on E17.5 and attained its maximal level
between P1 and P15, before declining on P20.
## Neural development and cell death in the retinas of mice over-expressing PDGF-B
Since the onset of expression of the transgene in the nes/tk-PdgfB-lacZ retina
is concurrent with retinal neurogenesis, lamination and developmental cell death
we examined whether over-expression of PDGF-B affected these processes. First,
we analyzed the generation of the different types of retinal neurons and their
laminar positions by staining with antibodies for the five major markers: Pax6
(expressed by horizontal cells, HCs, amacrine cells, ACs, and retinal ganglion
cells, RGCs), Isl1 (bipolar cells, BPs, RGCs), calbindin (HCs and certain ACs),
neurofilament 165 kD (neurites in the plexiform layers) and the photoreceptor
(PR) markers recoverin and rhodopsin. Co-labeling with Pax6 and Isl1 revealed
that over all, the different cell types were present in the retina, and despite
the apparent laminar distortion at the correct positions relative to one
another.
To determine if there were detectable differences in cell number, we next
analyzed markers of proliferation and apoptosis. We first used an antibody
directed against cleaved caspase 3, and found that in P5 eyes the number of
labeled cells in the transgenic retinas was increased by 10% compared to
corresponding normal tissue. We also analyzed the effects of over-expression of
PDGF-B on the proliferation by using the proliferation marker Ki67 (not shown)
and incorporation of the nucleotide analogue EdU. Pregnant mice were injected
with EdU on E18.5 and analysis was performed two days later. No differences
between transgenic and wild-type mice were discernable at the ages tested.
Finally, by counting cells expressing Isl1 and/or Pax6 in P10 retinas we
estimated that here was a slight but significant decrease in the Isl1-/Pax6+
cell population.
## Altered photoreceptor development and maturation by over-expression of PDGF-B
Analysis of rhodopsin and recoverin immunoreactivity at P5 revealed that
transgenic retinas stained differently from the wild-type. Rhodopsin staining
appeared displaced relative to its normal position. In the adult, the staining
of the inner and outer segments of the photoreceptors in nes/tk-PdgfB-lacZ mice
was irregular and frequently restricted to isolated patches on the outer side of
the outer nuclear layer (ONL) or located within the lumen of the rosettes
Horizontal cells (HC) make synapses with photoreceptors within the outer
plexiform layer (OPL) of the retina. Staining for the HC markers NF165 kD and
calbindin revealed that the appearance of cells and neurites in transgenic
retinas were normal up until P5 (not shown), but exhibited abnormalities
thereafter. On P10, a time when many synapses are formed in the mouse OPL, the
neurites of transgenic HC developed ectopic extensions outside of the OPL (not
shown). These extensions reached into and across the ONL and on P30 they
surrounded photoreceptor rosettes. These ectopic HC neurites persisted, showing
little or no sign of regression even on P60.
## Glial activation in the retinas of mice over-expressing PDGF-B
Müller glia and astrocytes are the major two types of glia cells in the murine
retina. Glial fibrillary acidic protein (GFAP) is widely used as a marker for
the latter, whereas nestin is commonly employed to identify activated Müller
glia cells. Ischemia or other retinal damage triggers the expression of both
nestin and GFAP in Müller cells. Since antibodies towards Pax2 stain the
precursor cells to retinal astrocytes, a combination of antibodies directed
against nestin, GFAP and Pax2 were used to monitor glial development in the
transgenic retina. Although the expression of nestin by Müller glia cells in
transgenic retinas appeared relatively normal during early postnatal development
(not shown), on P5 GFAP-positive cells were more abundant in transgenic retinas
than in wild-type tissues. In the transgenic retinas Pax2+ and GFAP+ cells did
not spread out evenly on the nerve fiber layer between E17 and P10 as wild type
astrocytes do. Instead, on P1 and P5, astrocyte precursors staining positively
for Pax2 remained at the vitreal side, and did not populate the retina. Although
the transgenic retina contained scarce GFAP-expressing cells with normal
morphology (insert), these cells were not evenly distributed in contrast to the
wild type retina. Signs of glial activation in transgenic retinas became more
manifest with time: elevated expression of GFAP in the inner parts of the retina
and of nestin on P30 indicates the presence of either ischemia and/or mechanical
forces exerting traction on the retina. At the age of P60, the glial activation
was exacerbated.
## Over-expression of PDGF resulted in failure to vascularize the retina
During postnatal development in the mouse, retinal vascularization takes place
concomitantly with regression of the hyaloid vasculature, which initially
supplies the eye with oxygen. Upon staining cross-sections and flatmounts with
antibodies against CD31 (an endothelial cell marker) and NG2 (a pericyte marker)
on P5 virtually no CD31+ or NG2+ cells were detected in the retinas of
transgenic mice, indicating complete failure of retinal vascularization and
angiogenesis. In their wild-type littermates newly formed blood vessels had
covered more than 70% of the retina by this time (arrowhead point at the border
of CD31+ cell migration). In contrast, remnants of the embryonic vasculature was
more abundant in the transgenic mice. In addition, GFAP staining showed that the
transgenic astrocyte precursors did not spread out to populate the retina in a
normal fashion. Consequently, there was a delay in population of the retina by
vascular cells, and the network of vessels in the inner retina of transgenic
mice that finally formed was abnormal.
## Abnormal development of the retinal capillary network
At P20, the transgenic retina was vascularized, although this took place in an
irregular fashion. At P60, a notable feature of the transgenic vascular network
was the absence of large trunk vessels (compare), a finding confirmed by the
low level or absence of α-SMA immunoreactivity, characteristic of smooth muscle
cells on arteries. The NG2 and CD31 staining patterns revealed that at P60,
vessels resembling capillaries of varying diameters and thickness grew
irregularly into the retina, populating the different retinal layers in an
apparently random manner. Staining of retinal whole mounts for CD31 and NG2 at
P9 to visualize interactions between endothelial cells and mural cells show that
there was some pericyte coverage of transgenic vessels albeit irregular. Some
NG2+ cells were attached to CD31+ cells while others were found separated from
the endothelium. This demonstrated that over-expression of PDGF did not
completely abolish the interactions between endothelial and mural cells.
## The transgenic mice exhibited reduced intraocular pressure
We observed that blood and other fluid often (∼80% of the mice examined) oozed
out of nes/tk-PdgfB-lacZ eyes following puncture for retinal dissections,
indicating that the retinal blood vessels were leaky and perhaps that the
circulation of ocular fluids in these mice is abnormal. The apparent lack of
arteries also suggested that the ability to stabilize circulatory pressure in
the eyes should be reduced. When the intraocular pressure in 3–4 month-old
transgenic and wild-type mice was determined using a rodent tonometer, the
transgenic animals displayed significantly lower pressure (9.55 versus 12.4 mmHg
for the wild-type, p = 0.0006, Mann Whitney test).
## Partial restoration of astrocyte precursor migration by the tyrosine kinase inhibitor STI571
In an attempt to pinpoint the period during which over-expression of PDGF
affected the formation of various cells in the retina and possibly even reverse
the defects observed, the mice were administered the tyrosine kinase inhibitor
STI571 (Imatinib, Glivec or Gleevec) systemically by oral gavage once daily
during three different intervals: from E17.5 until P0 or P1 with samples taken
for analysis on P1 or P2, respectively, from P0–P4 (samples taken for analysis
on P5), or between P7 and 14 (samples taken on P15). STI571 blocks signaling via
PDGF receptors, and in addition, inhibits other tyrosine kinases, such as c-Kit
and Bcr-Abl. We found that E17.5 was the earliest time point at which pregnant
females could be treated with STI571 since earlier treatment resulted in the
premature birth of non-viable pups.
STI571 treatment during the late embryonic period partly restored colonization
of the transgenic retina by astrocyte precursors. In untreated transgenic P2
mice the Pax-2 positive and CD31+ cells exhibited the characteristic compact and
irregular cell mass that associates with the HYV (see also). However, in STI571
treated pups Pax2-positive astrocyte precursor cells had not been trapped by the
hyaloid to the same extent, and were more spread apart (compare 9A with 9B and
9C). Concomitantly, CD31+ cells also had an organization that was less dense
than in non-treated mice (compare 9A′ with 9B′ and 9C′). Suppression of PDGF
signaling in wild-type by STI571 during the early postnatal period (P0–P4)
inhibited colonization of the retina by vessels (–B). However, no difference
between treated and un-treated transgenic mice with respect to CD31 staining was
detected (–D).
P7–P14 was the latest time window during which phenotype rescue was attempted.
The hyaloid in un-treated nes/tk-PdgfB-lacZ mice was very often attached to both
the lens and retina, making it difficult to dissect out these structures
damaging the retina. Moreover, prominent folding of the retina often made
flatmounting challenging. In contrast, on P15, the lens of transgenic mice
treated with STI571 was easier to dissect out, and indeed, actually fell out not
being stuck in the hyaloid, indicating partial reversal of these effects. In the
case of both transgenic and wild-type mice, treatment with this inhibitor
resulted in less coverage of deep CD31+ vessels by NG2 positive cells.
# Discussion
We report here that in mice expressing elevated levels of PDGF-B in nestin
positive cells of the retina during development, astrocytes fail to populate the
retina and vascular progenitors do not colonize this tissue to produce a network
of vessels. Interestingly, with the exception of their eyes, nes/tk-PdgfB-lacZ
mice do not exhibit any profound alteration in phenotype. The transgenic eyes
were smaller than normal with an iris of a reduced diameter and with frequent
occlusions. The transgenic retinas had folds and adhered to the back of the
lens, forming a retrolental mass that made dissection difficult. This morphology
was accompanied by frequent bleeding and by the age of one year the transgenic
retina had deteriorated completely. Many of these defects in the eye are likely
to be associated with abnormal traction forces within the structures that
eventually also leads to retinal detachment. Patients with persistent fetal
vasculature syndrome (PFVS) display similar problems with a large individual
variability that was also seen among the nes/tk-PdgfB-lacZ mice. Many of the
transgenic mice developed uni-lateral microphtalmia (around 40–50% pups per
litter) and/or a cataract. These abnormalities in retinal structure of the
transgenic mice are similar to PFVS, which results from failure to degrade the
hyaloid vasculature.
The crucial role of PDGF signaling in connection with the normal formation of
retinal blood vessels is well-established. The functions of PDGFRα-signaling
include regulation of the migration and proliferation of astrocyte precursor
cells, while signaling by PDGFR-β recruits mural cells, which allows for their
proper attachment to the endothelial cells of the blood vessels. Seo and
coworkers have proposed that gain-of-function mutations in PDGF-B exert more
profound effects on retinal development than mutations in PDGF-A, due to the
ability of the former to activate both α and β receptors, present on different
types of cells. This suggestion was confirmed in studies involving
overexpression of different isoforms of PDGF in photoreceptors and further
supported by the findings of Vinores et al. In our previous study involving
transgenic expression of PDGF-B under control of the myelin basic protein
promoter a disorganized neural retina with an under-developed capillary network
was observed. The transgenic mice employed here display more severe alterations
in eye phenotype, probably because the transgene is expressed more widely, both
spatially and during the period of development examined. By binding to both the
α and β receptors the B isoform of PDGF could affect the hyaloid as well as
astrocytes and pericytes, cells that form and provide support for the
vasculature during a critical period of retinal development.
The defects in lamination discernible shortly after birth are consistent with
the onset of transgene expression on E17.5 with peak expression on P1. Since no
major effects on the representation of retinal cells, on their relative spatial
orientation or on their proliferation were seen, despite the overall disorderly
structure, we conclude that the transgene expression did not affect the general
formation, differentiation or migration of retinal progenitors.
The elevation of the number of caspase positive cells observed on P5 occurred at
a time when this tissue normally undergoes developmental cell death. Cell death
is part of the normal development of a functional nervous system and half of all
postmitotic retinal ganglion cells die during the 2 first post-natal weeks in
mice. This period of cell death is associated with trophic interactions within
the retina and with the central targets for the retinal ganglion cells, and the
extent of death is determined in part by what connections the cells can
establish. The fact that we see more apoptosis in the transgenic mice during
this period suggests that the increased cell death is associated to the
structural changes in the retina and is not a direct effect of the transgene.
Moreover, the deterioration of the retina, as seen after one year, is probably
not related to this increased apoptosis but rather related to the malfunctioned
blood supply to the eye.
The inner and outer segments of the photoreceptor layer in the transgenic
animals did not develop normally. The rhodopsin localized in patches or inside
the rosette formations and the horizontal cells, which normally form synapses
with photoreceptors, developed ectopic neurites that extended outside the outer
plexiform layer and into rosettes. This pattern persisted in the adult retina
for several months indicating that these cells may have formed synapses with the
PR, despite their anomalous location.
The up-regulation of nestin and GFAP in Müller glia cells indicated that the
transgenic retina experienced a gliotic response that is likely to be triggered
by ischemia due to the missing retinal vasculature. Mechanical traction may be
an alternative explanation since Müller glia is known to exert tractional forces
when stimulated by PDGF in vitro. It seems likely that traction also occurred in
the transgenic retinas. During normal retinal vascularization, astrocyte
precursor cells migrate from the optic nerve to populate the inner portion of
the retina, where they form a scaffold on which vascular progenitor cells can
migrate and form vessels. In the nes/tk-PdgfB-lacZ mice most of the Pax2/GFAP
positive cells remained associated with the vitreal side of the retina, and
consequently, no astrocyte network on which vessels could expand was laid down.
The lack of proper formation of new vessels was also apparent from the staining
of CD31 and NG2 in both flat-mounts and cross-sections of the nes/tk-PdgfB-lacZ
retina. This staining demonstrated defective vascularization, with few
capillary-like structures, in a seemingly random fashion and devoid of large
trunk vessels. Our results suggest that over-expression of PDGF-B delayed
regression of the hyaloid, and prevented APCs from migrating and spreading
across the retina, which in turn prevented endothelial and mural cells from
populating the retina to form a normal vascular network. Endothelial cells
recruit pericytes by secreting PDGF-B and it is well established that pericytes
require PDGF-B to remain attached to the vessel wall. The opposite situation,
i.e. an excess of PDGF-B is apparently not as detrimental, since many transgenic
pericytes remained attached to the endothelial cells. Therefore, the abnormal
vessel properties cannot mainly be related to altered coverage by mural cells.
Intraocular pressure (IOP) in mice varies between strains, underscoring the
importance of using non-transgenic littermates as controls when congenic strains
are not available. The mice examined here had been back-crossed with C57Bl/6
mice for 5 generations, but the remaining contribution of the CBA genome is
unknown. Nonetheless, the wild-type control mice, which were always siblings,
exhibited an IOP close to the reference value for C57Bl/6. The attenuated IOP in
our transgenic mice is consistent with our hypothesis that the perfusion of the
eye is severely affected in the transgenic animals. The lack of proper retinal
vessels prevented these mice from controlling the ocular circulation.
Furthermore, traction on the ciliary body can lead to acute and chronic
hypotony, followed by retinal detachment, as has been reported in patients with
PFVS.
The effect of STI571 on the retinal phenotype of the transgenic mice depended on
the time when this inhibitor was administrated. Administration prior to E17.5
caused abortion, demonstrating that PDGF signaling, and/or other tyrosine
kinases sensitive to STI571 are critical for embryo survival. From E17.5 to the
time of birth, exposure to STI571 did not affect the birth of live offspring.
Since this time period coincided with the onset and early phase of transgene
expression in the eye, the earliest effects of transgenic PDGF could be
monitored. The timing of inhibition was of importance for APC maturation and
spread. APCs were less compacted upon treatment with STI751, which apparently
allowed CD31-positive cells to follow, since these cells were also less tightly
packed in the presence of the inhibitor. However, the retinal vascularization
was not greatly improved despite the better APC colonization. The observation
that vessel distribution was not influenced at all by administration of STI571
from birth to P4 suggests that at this point it is too late to reverse the
abnormal development of the vascular network. In contrast, if STI571 was
administered to the mice during the second week of postnatal life, the hyaloid
regressed in part. Thus, even though STI571 blocks all PDGF signaling, not only
that originating from the transgene, we could discern partial rescue of the
phenotype with this inhibitor.
The present investigation reveals that timely regression of the hyaloid
vasculature and development of the adult retinal vascularization are prevented
by over-expression of PDGF-B in nestin expressing cells during development.
Failure of astrocyte precursors to form a scaffold precluded retinal vessel
formation and gave rise to a defective retina that deteriorated with time. We
also showed these mice to be useful for testing pharmacological intervention by
the ability of a small-molecular inhibitor to partially restore retinal
vascularization.
# Supporting Information
We thank the personnel at the Rudbeck Animal Facility for excellent animal care,
and Tobias Bergström for help with genotyping.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: PDE FH KFN. Performed the
experiments: PDE MN. Analyzed the data: PDE FH MVS KFN. Contributed
reagents/materials/analysis tools: KFN FH MVS. Wrote the paper: PDE FH KFN. |
# Introduction
Proximal femoral fractures are one of the most common fractures treated
surgically. The common denominator in surgical technique of fixation of these
fractures, both intracapsular and extracapsular, is the placement of a large
diameter cannulated screw or blade in the cancellous bone of the femoral head.
Their correct placement is important as misplaced implants tend to destabilize,
especially in intertrochanteric fractures, less so the in treatment of femoral
neck fractures with cannulated screws.
The placement of the implant into the femoral head is ensured by the insertion
of a guide wire under fluoroscopic guidance. Misplaced wires are corrected until
satisfactory position is achieved, and the final implant is inserted. Every
insertion of the guide wire creates a canal in the bone, often in the direct
proximity to the final screw, what raises concerns about weakening of the bone.
It has been well recognized that screw holes in cortical bone are sites of
increased fracture incidence, what has been proved biomechanically for
predominantly cortical bone, but not for cancellous bone. This study was
designed to compare the relative compressive strength of paired cancellous bone
specimens retrieved from the femoral head with and without guide wire hole. A
comparison of purely cancellous bone specimens has not been investigated before
in similar intact and drilled samples. The aim was to recreate the compression
of the cancellous bone that occurs in the femoral head between the lag screw and
the dense subchondral bone. The holes simulate additional drill holes performed
during guide wire positioning or small bone cysts around the lag screw.
Moreover, the comparison of compressive strength was made between human and
artificial cancellous bone, as synthetic bone models are commonly used to
evaluate implant stability. Our hypothesis was that guide wire drill holes
decrease the strength of cancellous bone samples. Moreover, the artificial bone
samples should have similar force at failure, stiffness as well as the decrease
of those parameters after drilling as the human samples.
# Materials and methods
## Materials
Femoral heads were retrieved from 38 consecutive patients (30 female, 8 male)
who sustained femoral neck fractures and underwent a standard bipolar hip
arthroplasty in the course of their treatment. They were operated on within 72
hours from injury. Mean age was 80 (62–96) years old (SD = 10.4). Femoral neck
fractures sustained in males above 65 years of age and in females above 50 years
of age meet the diagnostic criteria of osteoporosis, according to national
guidelines. The femoral head retrieval did not affect decision making in their
treatment and did not change the course of the surgical procedure. All patients
gave their consent to participate in the study. Femoral heads were fresh frozen
right after surgery. Patients with suspected metastatic neoplasms, arthrosis,
rheumatoid arthritis or any other condition that could affect bone quality were
excluded from the study. This study was approved by the Independent Bioethics
Committee at the Medical University of Gdansk, Poland (issued 21.05.2018,
NKBBN/228/2018).
For synthetic cancellous bone samples, Synthetic osteoporotic left femora
(LD2350.01, cortical low density/soft cancellous bone, Synbone AG, Neugutstrasse
4, 7208 Malans, Switzerland) were used.
## Sample preparation
The femoral heads were cut into cuboids using custom made templates with a
surgical oscillating saw. The cuboid was then cut in half along the sagittal
plane using templates, leaving two matching samples, an anterior and a
posterior, from the same femoral head. The size of the cuboid was described as
follows: size 1 is the size of the base of the cuboid resulting from the cut in
the sagittal plane, size 2 is the length of the base in the coronal plane, and
size 2 is the longest side of the cuboid that was directed supero-inferiorly
with regard to the long axis of the patients’ body (the sample height) to ensure
that the force applied to the sample would have the same vector as hip contact
forces for a standing patient. One of the matching samples was chosen at random
from each pair. It was drilled with an original 3.2 mm partially threaded guide
wire for cancellous screws used in femoral neck osteosynthesis (Ansis III
Threaded Guide Wire, Stryker GmbH). show the relative size, shape and direction
of the cut bone samples and the direction of the drill hole was directed as it
would have been during actual surgery.
The synthetic bone samples were prepared in a similar manner from the foam that
forms the cancellous bone in femoral head.
After sample preparation a set of 33 pairs of samples of human and 9 pairs of
synthetic bone were available for further investigation in this study. The size
of the samples as well as their weight were then recorded and the apparent
density was calculated from the volume and weight of the sample.
The mean percentage loss of cross-sectional area due to drilling was estimated
as follows:
Mean loss = (size 1 x 3.2 mm)/(size 2 x size 3) x 100%
The samples were compared statistically (Wilcoxon signed-rank test for
comparison of the samples) to ensure that the evaluated physical parameters were
similar in both sample groups.
Before testing, human bones were bathed in 37°C saline for 10 minutes after
cutting to achieve body temperature. The samples were not cleared of the bone
marrow to make sure that the bone resembles as closely as possible its
intracorporeal state.
## Mechanical testing
The uniaxial compression tests of bone blocks were carried out at room
temperature using the Zwick-Roell Z020 testing machine.
The grips separation and actual force were recorded during the test. In the
preloading protocol, 3 loading-unloading cycles of the strain range 0–0.1% were
applied. The compression loading with the strain rate of 0.5 1/s was continued
until the failure of the specimen. A small toe effect being a consequence of
adapting of the cuboid samples between testing machine grips and adaptation of
the testing machine at the beginning of the experiment, was ignored in further
analysis.
The results were not recalculated to get their representation in terms of stress
and strain relations, as the half of samples were drilled and therefore area of
their cross section was not constant along the whole specimen length. The whole
bone blocks of exactly same sizes in paired samples, and of slightly differing
sizes between pairs, were compared. Consequently, the only reliable measures
taken for further analysis were the force at failure and stiffness of the
sample.
The highest load level obtained before fracture of the specimen was defined as
the force at failure. Stiffness was calculated as the slope of the linear part
of the force-displacement curve with skipping the very initial part of the
curve, where the toe effect occurred.
The percentage difference between the mean values *X* of the obtained
characteristics for the drilled and not-drilled samples was calculated as
follows: *Difference = (X*<sub>*NOT DRILLED*</sub>*−X*<sub>*DRILLED*</sub>*) /
X*<sub>*NOT DRILLED*</sub> × 100%
Statistical analysis was performed using Statistica PL v. 13.3 software. The
Wilcoxon Signed-Rank Test was used for statistical comparison. Results were
considered statistically significant with *p* \< 0.05.
# Results
The comparison of physical parameters of bone samples are shown in Tables and.
There were no significant differences regarding size of the sample, weight,
volume and density.
The estimated mean loss of sample cross section area due to drilling was 25%
(median 24%, SD = 5.3) for human bone and 24% (median 24%, SD = 0.73) for
synthetic bone.
The strength characteristics of the drilled and not drilled human bone samples
are presented in. The results for synthetic bone are presented in. The
differences between the force at failure were statistically significant, but the
comparison of sample stiffness were not.
# Discussion
The successful treatment of proximal femoral fractures depends, among other
things, on correct positioning of the implant. In proximal femoral fractures,
optimal placement of the guide wire for the lag screw is mandatory to achieve
good results. Often, multiple trials are needed to introduce it correctly. It
seems safe to assume that multiple drill holes around the final screw or blade
of an implant may weaken the bone and lead to the more common screw cutout than
when the bone around the implant is intact.
This study has shown human cancellous bone weakening after drilling of a single
hole in a bone specimen. Drilling eliminates a portion of the cancellous bone
from the transmission of the force applied to the bone and therefore weakens it.
The mean and median force at failure are similar to the loss of cross section
area of the sample due to drilling. This suggests that the loss of strength of
cancellous bone due to drilling may be roughly proportional to the loss of cross
section area. The major drawback of this study however is that the effect of the
drill hole would be dependent on the size of the bone specimen, since the
diameter of the guide wire is constant. It is safe to presume that the smaller
the specimen, the greater the impact of the 3.2 mm hole in the bone.
In literature, the effect of weakening of the cancellous bone was not studied
before in the past the way it is done in this study. In studies of whole bone
specimens (cortico-cancellous bone) the decrease in the force to failure in
drilled specimens is between approximately 20% to 40%. The fractures occur
through the drill holes, but the tests were performed as a three-point bending
test or a torsional strength test, unlike the current study. Comparisons between
cortical and cancellous bone fracture models after drilling should be cautious.
Cortical bone is on average 20–30% stronger than cancellous bone, as well as
there are differences in elastic modulus and yield strain between those two
types of bone. Moreover, there are obvious anatomical differences between whole
bone models and a pure cancellous bone sample.
The effect of weakening of the cancellous bone in previous studies is not as
obvious as shown for cortical bone. The surprisingly high “safe” number of drill
holes (between 14 and 40) found in a recent final element analysis study
suggests that a femoral head cancellous bone can withstand multiple drilling
attempts without compromising its strength. After anterior cruciate ligament
reconstruction acute fractures usually, but not always occur through the drilled
canals. In an investigation of composite femur bones, all specimens failed
through the femoral canals of the double-bundle ACL reconstruction technique,
but in the single bundle technique the fracture patterns were similar to the
intact groups and only half occurred through the drill canal. In a similar study
of tibial fractures, it was found that load to failure was similar in all groups
(intact, single bundle and double bundle reconstructions). In a biomechanical
study of the core decompression of an equine navicular bone, which is
predominantely cancellous, it was found that the presence of unicortical bone
canals significantly decrease the strength of bone by about 20%, what is similar
to the current study. However, the size and number of drill holes did not affect
the strength of the drilled specimens.
The studies mentioned above suggest that the weakening of the bone due to
drilling may not be directly proportional to the size and number of drill holes,
and therefore to the volume of the bone stock lost because of drilling. The fact
that the loss of strength of cancellous bone that is roughly proportional to the
loss of cross-section area of the sample is a rather unexpected finding in this
study.
In the current study, no fracture occurred through the drill hole as was
expected considering other biomechanical studies. This indicates that failure
occurred rather by microfractures of the bone trabeculae of the specimens. It
must be noted, however, that in the current study a purely compressive force was
applied, and not a torsional or bending force as in others. A compressive force
would rather crush the specimen, while a torsional or bending force would
fracture the specimen through the point of least resistance, that is, through
the hole in the bone.
There are no biomechanical studies that evaluate the human cancellous bone
strength after drilling without any cortical component. The implants used in the
proximal femoral fractures are fixed in a cancellous bone, and the screw
migration occurs within the femoral head, without a typical fracture of the
femoral head. Therefore for the purpose of this study, there was a need to
examine the cancellous bone without the surrounding cortex.
This study attempts to resemble true in vivo conditions as closely as possible,
but generally the tests used to determine the effect of a hole in a bone use a
simplified protocol. In true clinical setting, the femoral head is compressed
cyclically in changing force vectors because of femoral head rotation in the
acetabulum. The site of compression in a fixed intertrochanteric fracture is
between the articular surface and the femoral head implant, which is commonly
round and threaded, or has several blades. This simplification is another major
limitation of the study. In our study, we used a compression test, as was used
in testing femoral head cancellous bone samples, as the best possible
approximation of the forces acting on the cancellous bone in this region. Care
was taken to use paired specimens of the same shape, size and origin, and the
drilling was assigned to one of the paired specimens randomly. The bone in the
anterior and posterior part of the femoral head have the same mechanical
properties. The number of paired specimens is similar to other studies. The
direction of force in this study is different than the principal compressive
region or the main anatomical trabecular direction to ensure similar force
direction as occurs in vivo between the hip contact forces of a standing patient
and the implant screw.
This study has shown statistically significant cancellous bone weakening after
drilling of a single hole in a cancellous bone specimen. The effect of weakening
cancellous bone by a guide wire hole cannot be considered yet as consistent as
the effect of screw holes in the cortical bone, but should be taken into account
in proximal femoral fracture surgery. Further studies, however, are needed to
weigh the value of ideally placed implant against weakening of the bone with
guide wire holes created by multiple attempts to insert it perfectly. This study
suggests that drill holes in the femoral head weaken the bone stock, what may
compromise the stability of the fixation. Moreover, since it has been found in
other studies that the optimal position of the screws in intracapsular femoral
neck fractures has no significant influence on the outcome, the authors of the
current study no longer attempt multiple times to place the screws in a perfect
configuration fearing that multiple drill holes around final implants may weaken
the fixation strength.
The synthetic bone model, that was similar in size and shape, shows comparable
similarities between loss of force at failure and loss of cross section area as
human bone. However, force at failure of human bone is almost twice that of the
synthetic bone, and stiffness is approximately 4 times greater in human bone.
This shows that biomechanical experiments performed on a synthetic bone cannot
be directly translated to human bone, when it comes to absolute measures of the
force to failure or stiffness, but relative loss of strength in both types of
bones that sustained some kind of damage (fracture, cyst, or implant placement)
may be comparable between human and synthetic bone. The experiments performed on
synthetic bone probably give an accurate view of the mechanical properties of
the fractures or implants compared in biomechanical studies, that is an implant
that is inferior in synthetic bone studies most likely will also be inferior in
the human bone. However, the magnitudes of forces recorded in synthetic bones
may not be accurate in human bones or in patients.
Other studies show some conflicting results regarding mechanical parameters of
human v/s synthetic cancellous bone. The screw pullout force, which is probably
the only mechanical parameter that can be attributed solely to cancellous bone,
was found to be similar to natural bone in one study, nearly 4 times greater
with standard synthetic bone, or exhibited only 40% of pullout strength of
natural bone when an osteoporotic type of artificial bone was used. A study that
compared stability of identical fracture fixations between human and standard
synthetic bone showed that the artificial samples were significantly more
stable. Those differences between types of bones and studies can be attributed
to the type of material used for comparison. The human bones may have a
different degree of osteoporosis and also different types of artificial bones
are used (standard v/s osteoporotic type, as well as different generations from
different manufacturers). In this study, we used a new type of osteoporotic
bone, and compared it to femoral heads from elderly patients who sustained
femoral neck fracture, what meets the criteria for osteoporosis. This study, as
well as previous studies suggest that a perfect replacement for human bone in
mechanical studies has not been found, but the relative differences found in
those studies between samples are most likely accurate.
In scientific research, this study supports a good scientific practice to
prepare a control group sample (for example, a perfectly reduced fracture fixed
with a standard implant, comparable with other studies), and compare differences
in force parameters of the other tested groups relative to the standard control
group. The absolute values of force to fracture (in N) should be considered with
great caution when comparing to other studies, because mechanical properties may
vary between artificial bones of different types or between bones from different
manufacturers.
The findings of this study support the hypothesis that drill holes decrease the
strength of cancellous bone, moreover, the decrease is roughly proportional to
the loss of cross section area of the sample caused by the drill hole. The
comparison between human and artificial bone shows that forces measured in
biomechanical studies on artificial bone are different than in human samples and
cannot be directly attributed to humans, but the relative differences in
mechanical properties of fractured and fixed synthetic samples may be accurate
and resemble that of human bones.
# Supporting information
The authors wish to thank prof. Andrzej Ceynowa for correcting English language.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Glutathione *S*-transferases (GSTs, EC 2.5.1.18) comprise a large superfamily of
enzymes whose soluble members primarily function as detoxification enzymes,
facilitating the conjugation of a diverse array of hydrophobic electrophilic
xenobiotics by the nucleophilic attack of glutathione. GSTs have long been known
as important components of cellular defense mechanisms in mammalian systems and
recent studies are revealing their significance in mediating allelochemical
tolerance in invertebrate-host interactions (reviewed).
The diversity of GST isoforms and their capacity to detoxify allelochemicals in
consumers has been correlated with diet breadth. For example, in a survey of GST
isoforms from five lepidopteran species, specialist herbivores expressed only
one major GST isoform, while generalists expressed multiple forms. Furthermore,
GST isoforms purified from polyphagous herbivores that regularly consume
isothiocyanate-containing cruciferous plants were able to metabolize a broader
range of isothiocyanate allelochemicals in comparison to GSTs from specialists
that did not consume crucifers and lacked the ability to conjugate
isothiocyanates. These findings imply that the evolution of generalist GST forms
favors promiscuous catalytic activity presumably needed to cope with the breadth
of dietary toxins encountered; this mirrors results for other consumer counter-
defense proteins (i.e., cytochrome P450s) whereby more catalytically flexible
detoxification enzymes may promote a greater degree of polyphagy. Moreover,
while polyphagous insects have little to no ability to increase their GST
expression upon allelochemical exposure, they often possess a higher
constitutive level of GST activity in comparison to oligophagous and monophagous
species. The difference in constitutive and inducible GST expression in
consumers may ultimately be a reflection of the non-specific role of GSTs as
antioxidant enzymes. GSTs are known to be under regulatory control by
antioxidant response elements found in their promoters. Due to the diverse range
of dietary pro-oxidants encountered in polyphagous species, sustained
transcriptional activation of GST enzymes by dietary compounds may result in the
near maximal expression of GST enzymes. Consequently, possessing constitutively
expressed GST enzymes that are catalytically versatile may confer a selective
advantage to those consumers that regularly encounter unpredictable host
chemistry.
Similar to their terrestrial counterparts, marine consumers that regularly feed
on allelochemically-rich prey may have evolved a parallel suite of biochemical
resistance mechanisms (reviewed in). The induction or high constitutive activity
of GSTs seen in several marine molluscs after allelochemical exposure has been
suggested as a protective mechanism against dietary intoxication. High cytosolic
GST activity was observed from the digestive gland of the generalist marine
gastropod *Cyphoma gibbosum*, which feeds solely on a diet of chemically-
defended gorgonians, and whose GST activity levels rival those of terrestrial
invertebrates that feed solely on allelochemically-rich prey. This gastropod
predator utilizes three families of gorgonian corals as hosts and in doing so
encounters a range of lipophilic allelochemicals that includes diterpenes,
sesquiterpenes, acetogens, highly-functionalized steroids and eicosanoids.
One gorgonian diet, *Plexaura homomalla*, has been suggested to be a favorite of
*C. gibbosum*, even though this gorgonian is known to contain impressive
quantities of the cyclopentenone prostaglandin, PGA<sub>2</sub>, which is known
to serve as a feeding deterrent against generalist reef predators. Perhaps
related to their anti-predatory properties in marine systems, the cytotoxic
nature of cyclopentenone prostaglandins occurs, in part, due to the reactive
α,β-unsaturated carbonyl group in the cyclopentenone ring, which can undergo
nucleophilic addition with electrophilic moieties, resulting in protein and DNA
adduct formation. Cyclopentenone prostaglandins of the A and J series have been
shown to be inducers of GST enzymatic activity as well as mRNA expression in
mammalian, and invertebrate cell lines. Furthermore, certain vertebrate alpha-,
mu-, and pi-class GSTs were found to enhance PGA<sub>2</sub> conjugation with
glutathione, suggesting that the overexpression of GST forms could modulate the
cytotoxic effects of cyclopentenone prostaglandins. Mammalian GSTs also have the
ability to non-catalytically bind lipophilic, amphipathic ligands, including
PGJ<sub>2</sub>, via noncovalent interactions, which effectively sequester these
ligands in the cytosol away from their nuclear targets (i.e., peroxisomal
proliferator-activated receptor, PPAR). Given that *C. gibbosum* neither avoids
*P. homomalla* nor adjusts its feeding rates to reduce toxin exposure, this
snail likely possesses effective detoxification mechanisms, possibly GST-
mediated, to contend with the high concentrations of dietary prostaglandins.
In a previous study, proteomic analysis of affinity-purified cytosolic GST
fractions from *C. gibbosum* revealed that two major GST mu-class isoforms were
responsible for the high GST activity observed in the digestive gland. Here, in
a controlled laboratory feeding study, we allowed snails to feed ad libitum for
four days on one of seven gorgonian species or a diet devoid of gorgonians, and
monitored GST activity levels and isoform expression in response to the
different suites of gorgonian allelochemicals. To investigate whether gorgonian
extracts contained possible substrates for *Cyphoma* GSTs, we used a bioassay-
guided fractionation approach, screening gorgonian crude organic extracts and
subsequent fractions of different polarities for their ability to inhibit the
1-chloro-2,4-dinitrobenzene (CDNB)-conjugating activity of GST. Selected HPLC
fractions found to inhibit GST activity were further characterized by
<sup>1</sup>NMR and LC-MS spectral analyses to identify possible bioactive
compounds. In addition, a series of commercially-available prostaglandins
representing a range of eicosanoids previously described from *P. homomalla*
were examined for their ability to inhibit *Cyphoma* GST activity.
# Results
## Gorgonian Dietary Influence on GST Activity and Subunit Expression
GST specific activity levels measured from digestive gland cytosolic
preparations ranged from 1930 to 2957 nmol min<sup>−1</sup>mg
protein<sup>−1</sup>. GST activity levels were within the range reported by
Vrolijk and Targett, but did not differ significantly between snail diets. HPLC
separation of affinity-purified GSTs identified fourteen unique peaks. HPLC peak
1 was previously identified as a theta-class GST, while HPLC peaks 2 thru 14
were identified as mu-class GST subunits. HPLC peaks 4 and 8 represented the
majority of expressed GST subunits at 25% and 68%, respectively. The relative
proportion of each GST subunit, represented by separate HPLC peaks and
calculated based on HPLC peak area, did not differ significantly as a function
of gorgonian diet when expressed either as percent of all subunits present or
when normalized to the amount of affinity-purified GST sample injected on to the
HPLC column. These results indicate that while GST activity is constitutively
expressed at high levels in *Cyphoma* digestive gland, both GST activity and
subunit abundance are unaffected by gorgonian diet.
## Inhibition of GST Activity by Gorgonian Extracts
Crude organic extracts from *B. asbestinum*, *E. mammosa*, *G. ventalina*, *P.
acerosa*, *P. americana*, *P. blanquillensis*, *P. elisabethae*, and *P.
homomalla* tested at 5% natural volumetric concentration (NC) inhibited ≥70% of
the GST activity in *Cyphoma* digestive gland cytosol compared to solvent
controls. Chloroform-soluble fractions from all gorgonian species examined
consistently showed ≥80% inhibition of GST activity compared to controls.
Aqueous fractions generally displayed minor inhibitory effects, with the
exception of fractions from *P. acerosa* and *P. homomalla*, which inhibited GST
activity by 85% and 99%, respectively. Hexane-soluble fractions exhibited
intermediate and wide-ranging inhibitory effects depending on gorgonian species.
To further investigate the source of the putative gorgonian GST
substrates/inhibitors, we used HPLC to fractionate the chloroform-soluble
extracts of each gorgonian and tested their ability to inhibit the activity of
affinity-purified *Cyphoma* GSTs. For all eight gorgonian species, HPLC fraction
1 (compounds eluting from 3 to 6 mins) exhibited the greatest inhibitory
activity, causing \>80% GST inhibition at 10% NC compared to paired solvent
controls. Inspection of HPLC chromatograms for all gorgonian species indicated
that HPLC fraction 1 consisted of a mixture of compounds. For four of these
gorgonian species (*B. asbestinum*, *E. mammosa*, *P. acerosa*, and *P.
homomalla*) affinity-purified GST activity was completely inhibited by HPLC
fraction 1 at 10% NC. Diluting HPLC fraction 1 from *B. asbestinum*, *E.
mammosa*, *P. acerosa*, and *P. homomalla* to 0.05% NC decreased the inhibitory
effect of the compound(s); however, in all cases diluted fractions still
retained the ability to inhibit \>65% of affinity-purified GST activity compared
to solvent controls.
## Identification of Prostaglandins in *P. homomalla* Extracts
Because *P. homomalla* is a favored diet of *Cyphoma*, and possesses extracts
determined to significantly inhibit the CDNB-conjugating activity of *Cyphoma*
GSTs, we focused our subsequent efforts on elucidating the compound(s)
responsible for this inhibition. LC-MS and <sup>1</sup>H-NMR spectral analysis
of HPLC fraction 1 from the chloroform-soluble extract revealed the presence of
PGA<sub>2</sub>. The <sup>1</sup>H-NMR spectrum showed a 3H triplet at 0.85 ppm,
consistent with the presence of a terminal methyl group in prostaglandins. The
mass spectrum of HPLC fraction 1 displayed a parent ion at *m/z* 333 with
fragment peaks at *m/z* 315, 271, 233, and 189, characteristic of
PGA<sub>2</sub>.
For HPLC fraction 2 from *P. homomalla*, the LC-MS signal at ∼2.5 min showed an
*m/z* 371.2, which corresponds to \[M+Na\]<sup>+</sup> of 5Z and
5*E*-prostaglandin B<sub>2</sub> methyl ester. The <sup>1</sup>H NMR spectrum of
*P. homomalla* HPLC fraction 2 showed a dominant prostaglandin-like compound
that matched the literature values (ca. \<0.25 ppm difference between literature
and experimental values) for 5*Z* and 5*E*-prostaglandin B<sub>2</sub> methyl
ester; however, this compound showed a doublet of doublets at 7.6 ppm which was
substantially further downfield than expected. The dominant compound in HPLC
fraction 2 also displayed a singlet at 2.0 ppm, suggestive of acylation. A
second peak at ∼16.6 min in the LC-MS signal for HPLC fraction 2 showed an
*m*/*z* 413.2, which corresponds to \[M+Na\]<sup>+</sup> of either 5*Z* or
5*E*-acetyl-prostaglandin B<sub>2</sub> methyl ester. <sup>1</sup>H NMR spectral
data for the dominant compound were in agreement with those from 5*Z* and
5*E*-acetyl-prostaglandin B<sub>2</sub> methyl ester, with the major exception
of the signal at 7.6 ppm, which is further downfield than expected for these
known compounds. The *m/z* of 413.2 also corresponds to 15-epi-prostaglandin
A<sub>2</sub> diester, whose <sup>1</sup>H NMR spectrum matched very closely to
the dominant compound in HPLC fraction 2, including the signal at 7.6 ppm.
However, further comparison was made difficult due to an absence of a complete
set of NMR spectral data in the literature for 15-epi-prostaglandin
A<sub>2</sub> diester. Overall, <sup>1</sup>H NMR and LC-MS spectral data
support the presence of a dominant prostaglandin-derivative in *P. homomalla*
HPLC fraction 2; however, because this fraction is a mixture of compounds, the
exact identity cannot be established.
Quantification of PGA<sub>2</sub> from *P. homomalla* HPLC fraction 1 by LC-MS
revealed an approximate whole tissue concentration of 1.6 mM (or 530 µg
PGA<sub>2</sub>/mL of wet gorgonian tissue). HPLC fraction 2 (eluting at 6–9
mins) from *P. homomalla* showed a selected ion recording (*m/z* 333.3) at the
expected retention time; however, peak intensities were below the limit of
quantification and subsequent NMR spectral analysis indicated that
PGA<sub>2</sub> was not present.
## Inhibition of GST Activity by Pure Prostaglandins
When commercially available prostaglandins representing a diversity of classes
found in *P. homomalla* were screened, those compounds containing a
cyclopentenone ring (e.g., PGA<sub>2</sub>) caused the greatest inhibition of
GST activity, whereas the methyl ester forms of PGE<sub>2</sub> and
PGF<sub>2α</sub> displayed little to no inhibitory activity in comparison to
solvent controls. The potencies of the four most inhibitory prostaglandins
(15(S)-PGA<sub>2</sub>, 15(R)-15-methyl PGA<sub>2</sub>, 15(S)-PGE<sub>2</sub>,
15(S)-PGF<sub>2α</sub>) were further evaluated at a range of concentrations
(0.2–2000 µM). All prostaglandins displayed concentration-dependent inhibition
of enzyme activities, with IC<sub>50</sub> values ranging from 75.4 µM for
15(S)-PGA<sub>2</sub> to 334.6 µM for 15(S)-PGF<sub>2α</sub>. Those
prostaglandin series with the greatest inhibitory potencies (e.g.,
PGA<sub>2</sub>) are known to be in the highest abundance in gorgonian tissues.
The K<sub>i</sub> values for 15(S)-PGA<sub>2</sub>, 15(R)-15-methyl
PGA<sub>2</sub>, 15(S)-PGE<sub>2</sub>, and 15(S)-PGF<sub>2α</sub> calculated
using the Cheng-Prusoff equation, ranged from 21.7 to 96.4 µM.
# Discussion
The exploitation of allelochemically-defended gorgonian corals by the co-evolved
predator, *Cyphoma gibbosum*, is likely to be facilitated by this predator's
ability to biotransform and/or sequester dietary allelochemicals using
detoxification enzymes, such as soluble glutathione *S*-transferases. GSTs are
integral components of the cellular xenobiotic defense system and have been
documented to mediate allelochemical tolerance in terrestrial consumers. Our
results suggest that they may also be important for marine predators that
consume chemically defended prey; *C. gibbosum*'s high, constitutive expression
of GSTs may protect this consumer from the abundance of deterrent lipophilic
compounds found in its gorgonian diet.
In a controlled feeding assay we determined that digestive gland tissues from
*C. gibbosum* constitutively express high levels of GST activity regardless of
the gorgonian diet. This finding differs from that of a previous study that
noted differences in GST activity from field-collected *C. gibbosum* from
different gorgonian hosts. The apparent differences could reflect differences in
experimental design between the two studies. Vrolijk & Targett (1992) noted
differences in GST activity among field-collected individuals for which no data
were available on the residence time of snails on their respective hosts. In
contrast, snails in the present study were subject to controlled, four-day
feeding assays. It is possible that GST enzymes could show significant levels of
induction if snails were allowed to feed on gorgonian diets longer than four
days. However, studies of GST induction in other invertebrates, suggest that
four days is sufficient for induction to occur; thus, it seems unlikely that our
design, which included those gorgonian species examined by Vrolijk & Targett
(1992), would have missed significant induction of GSTs. Snails could
conceivably extend their exposure to the same suite of allelochemicals beyond
the average 3.3 day residence time migrating to another colony of the same
species. This scenario would be favored if *B. asbestinum* and *G. ventalina*,
the two gorgonian diets eliciting increased GST activity in Vrolijk & Targett
(1992), were in higher abundance on reefs, because prey selection by *C.
gibbosum* is in proportion to gorgonian species abundance. Alternatively,
geographical or within colony differences in allelochemical content could
account for the differences between the two studies.
Although GST activity did not vary by gorgonian diet, cytosolic digestive gland
GSTs were further purified by affinity chromatography to investigate if GST
subunit composition was influenced by allelochemical exposure. *Cyphoma* GST
subunits were separated by HPLC, resulting in the identification of two major
mu-class GST subunits, which accounted for 93% of the total GST subunit
abundance. Quantification of GST subunit composition indicated that the relative
abundance of GST subunits did not differ among snails feeding on different
gorgonian diets. Interestingly, GST activity was maintained at a high level and
subunit composition did not vary in snails fed control diets devoid of
allelochemicals as compared to snails fed gorgonian diets. The presence of high
GST activity in control-fed snails could indicate that some lipophilic gorgonian
compounds and/or their metabolites may persist in snail tissues even after
feeding has ceased, causing the expression of GSTs to be maintained.
Alternatively, the constitutive expression of *Cyphoma* GSTs could be regulated
by an allelochemical-independent mechanism. In either case, having a constant
supply of ‘all-purpose’ GST enzymes may prove advantageous for predators that
consistently feed on prey containing allelochemical GST substrates.
The majority of GST substrates are hydrophobic compounds that react with the
thiol moiety of glutathione. In our bio-assay guided screening approach, we used
the ability of extracts/compounds to inhibit GST activity as an indirect measure
of their potential to act as GST substrates. The results of GST inhibition
assays indicated that the chloroform-soluble fractions from gorgonian extracts
contained the bulk of inhibitory compounds. However, in addition to containing
potential GST substrates, gorgonian extracts may also contain electrophilic
compounds that could act as potent GST inhibitors, binding to free cysteine
residues on the protein resulting in enzyme inactivation. The presence of high
affinity GST inhibitors in gorgonian tissues may represent specific counter-
adaptations of prey to thwart consumer GST-mediated metabolism of co-occurring
allelochemicals. Although our initial screening approach of gorgonian extracts
was not able to distinguish between GST substrates and inhibitors, this result
did substantiate the hypothesis that all gorgonian species contained significant
quantities of compounds capable of interacting with *Cyphoma* GSTs, which could
account for the high constitutive activity of digestive gland GSTs identified
here.
The gorgonian *Plexaura homomalla* is a favored diet of *C. gibbosum*, , despite
having high tissue concentrations of deterrent cyclopentenone prostaglandins.
Electrophilic eicosanoids, like the cyclopentenone prostaglandin
PGA<sub>2</sub>, have been recognized as high affinity substrates/inhibitors for
vertebrate GSTs,. Furthermore, NMR and LC-MS analysis showed that
PGA<sub>2</sub> was present in the *P. homomalla* HPLC fractions demonstrating
the greatest inhibitory potential. Therefore, the potential importance of *P.
homomalla*'s allelochemicals in the co-evolution of *C. gibbosum* detoxification
enzymes, coupled with the interesting ecological and biological activities of
eicosanoids, prompted us to determine if prostaglandins could serve as
substrates for *C. gibbosum* GSTs.
*P. homomalla* tissues predominately contain the fully esterified form of
PGA<sub>2</sub> (∼2% dry weight of the gorgonian), which is related to a larger
group of eicosanoids that includes the coral-derived halogenated marine
clavulones, and puniglandins, all of which display cytotoxic activities thought
to be related to the presence of a reactive α,β-unsaturated ketone. While the
exact mechanism of toxicity is unknown, the prostaglandins are transported into
the nucleus – where the electrophilic α,β-unsaturated carbonyl is free to bind
with nucleophilic sulfhydryl residues on target proteins, unless rapidly
conjugated by cytosolic GSH and transported out of the cell by glutathione-
conjugate transporters. In this study, the α,β-unsaturated carbonyl-containing
prostaglandins (15(R)-15-methyl PGA<sub>2</sub> and 15(S)-PGA<sub>2</sub>) were
the most potent inhibitors of CDNB-conjugating activity of *Cyphoma* GSTs in
both the initial screening of eight prostaglandin compounds and upon comparison
of IC<sub>50</sub> values, establishing the order of potency of prostaglandins
to be 15(S)-PGA<sub>2</sub>\>15(R)-15-methyl
PGA<sub>2</sub>≫15(S)-PGE<sub>2</sub>≈15(S)-PGF<sub>2α</sub>. The K<sub>i</sub>
values for cyclopentenone-containing prostaglandin A series were also 2.3- to
4-fold lower (greater affinities) for *Cyphoma* GSTs in comparison to those of
either PGE<sub>2</sub> or PGF<sub>2α</sub>.
If we assume PGA<sub>2</sub> is a substrate for *Cyphoma* GST(s), possibly
binding with high affinity in the active site (H-site) once occupied by CDNB, it
is reasonable to compare K<sub>i</sub> values (i.e., apparent K<sub>m</sub> for
PGA<sub>2</sub>) obtained here to K<sub>m</sub> values for PGA<sub>2</sub> cited
in other studies. The apparent K<sub>m</sub> (∼21.7 µM) for
15(S)-PGA<sub>2</sub> described here is in line with values identified for the
conversion of PGA<sub>2</sub> to its glutathione conjugate by human mu-class GST
M1a-a (26 µM), rat alpha-class GST A4-4 (12 µM) and human mu-class GST M2-2 (7.6
µM). The rank order of GST affinity for prostaglandins
(15(S)-PGA<sub>2</sub>\>15(R)-15-methyl
PGA<sub>2</sub>\>15(S)-PGE<sub>2</sub>\>15(S)-PGF<sub>2α</sub>), is also
positively correlated with the relative abundance of each prostaglandin series
in *P. homomalla* tissues. This finding may suggest that *Cyphoma* GSTs have
evolved to efficiently catalyze the conjugation of prostaglandins found in the
greatest abundance in its diet (PGA<sub>2</sub>), yet still retain a broad
enough substrate specificity to metabolize additional prostaglandin classes
(PGE<sub>2</sub>, PGF<sub>2α</sub>).
The apparent K<sub>m</sub> (Ki) values reported here indicate that dietary
prostaglandins could be high affinity substrates for *Cyphoma* GSTs *in vivo*.
However, the physiological relevance of this value would depend on the
concentration of prostaglandins occurring in digestive gland tissues of
*Cyphoma*. To obtain an estimate of these concentrations, we first calculated
the volume of *P. homomalla* tissue consumed per snail per day based on feeding
scar measurements. *Cyphoma* feeding scars on *P. homomalla* colonies averaged
12 cm in length, did not exceed 1 cm in width, and penetrated to the gorgonian
skeleton 66% of the time, a depth of 0.4 cm (K. Whalen, pers. observation).
Therefore, conservative estimates of scar volume averaged 1.44 cm<sup>3</sup>
per snail or 1.44 mL of gorgonian tissue. This tissue volume was divided by the
mean residence time of snails feeding on *P. homomalla* (2.9 d, n = 50 snails)
to yield an estimate for the volume of *P. homomalla* tissue consumed by each
snail per day (0.66 mL/snail/day). Pawlik & Fenical determined that 1 mL of wet
*P. homomalla* tissue (excluding the gorgonian axial skeleton) was equivalent to
0.86 g of dry gorgonian tissue. If 2% of the dry weight of the gorgonian is
prostaglandins, then 0.66 mL of gorgonian tissue would contain 0.011 g of
prostaglandins. Assuming the majority of prostaglandins are in the
PGA<sub>2</sub> form (FW = 348.5 g) and are completely retained within the
digestive gland upon ingestion (ave. dig. gland weight = 0.25 g, K. Whalen pers.
obs., with a density comparable to human liver ∼1 g/mL), then the upper limit of
PGA<sub>2</sub> concentrations in digestive gland would be 0.13 M. The single
day grazing rates of *P. homomalla* colonies by *C. gibbosum* of 0.17
mL/snail/day reported in were used to obtain the lower bound of PGA<sub>2</sub>
tissue concentration (0.03 M). Ciereszko and Schneider reported the fecal
pellets of *C. gibbosum* contain no appreciable amounts of recognizable
prostaglandins, suggesting that the majority of prostaglandins are being
metabolized or sequestered within the snail. If we conservatively assume that
only 1% of the ingested prostaglandins are retained within the digestive gland
during feeding (e.g., 1% concentration of PGA<sub>2</sub>∼0.3–1.3 mM
PGA<sub>2</sub>), the *in vivo* concentration of prostaglandins in this tissue
would still be 7- to 59-fold higher than the apparent K<sub>m</sub> (21.7–39.4
µM) obtained for PGA<sub>2</sub> and its methylated derivative. Even at the
lower bound of *in vivo* PGA<sub>2</sub> concentration (∼0.3 mM), *Cyphoma* GSTs
would be operating at near physiological capacity (\>93%) according to the
fractional velocity (v/V<sub>max</sub>) estimates.
Glutathione *S*-transferases are most well known for their ability to conjugate
electrophilic toxicants; however, their capacity to bind and sequester non-
substrate ligands may also be an important protective mechanism. Certain human
GST isoforms have been shown to exert their protective effects through this
ligandin-like behavior by binding with high affinity to inhibitory
prostaglandins (e.g., PGJ<sub>2</sub>), effectively sequestering them in the
cytosol away from target nuclear proteins and preventing effects on gene
regulation. A comprehensive screening of allelochemicals from host plants of the
fall armyworm *Spodoptera frugiperda* found that many act as non-competitive
inhibitors of GST activity. Overexpression of GSTs in *S. frugiperda* may serve
as a detoxification strategy by facilitating the sequestration of non-substrate
ligands and thereby preventing their interference with essential cellular
functions. The same strategy might be used by the marine chiton *Cryptochiton
stelleri*, which consumes a red algal diet rich in the feeding deterrent
lanosol, a noncompetitive inhibitor of this chiton's GST activity. Similarly,
high constitutive GST activity was observed in *Cyphoma* independent of
allelochemical diet and all of the gorgonian extracts examined contained potent
inhibitory compounds. While the type of inhibition was not quantified for
gorgonian extracts and all compounds, it is likely that gorgonian diets contain
both substrates (e.g., PGA<sub>2</sub>) and non-substrate ligands. Therefore,
the constitutive expression of GSTs may be indicative of a more *general*
biochemical resistance strategy that is capable of responding to a diversity of
compounds in the diet of a generalist consumer.
The results of this study provide the first comprehensive evaluation of the
influence of dietary allelochemicals on the expression and function of
glutathione transferases in a generalist marine consumer. Together with our
companion studies on the gorgonian diet-mediated expression of cytochrome P450
expression in *Cyphoma*, the present results add substantial knowledge regarding
the role of detoxification enzymes in determining macroevolutionary patterns of
diet preference among consumers. Controlled feeding assays showed that *Cyphoma*
digestive gland GST composition and activity did not differ with gorgonian diet.
This result in combination with evidence from *in vitro* inhibition studies with
*Cyphoma* GSTs by gorgonian extracts, suggests that the high constitutive
expression of GST enzymes in *Cyphoma* digestive gland may be necessitated by
the presence of numerous potent inhibitors/substrates in their gorgonian diets.
Furthermore, all three prostaglandin classes (A, E, F) found in the gorgonian
*P. homomalla* were able to inhibit *Cyphoma* CDNB-conjugating GST activity,
with relative potencies positively correlated with their abundance in gorgonian
tissues. Together, these findings suggest that *C. gibbosum* detoxification
enzymes may have evolved to enable the conjugation and sequestration of a broad
range of lipophilic allelochemicals resulting from this predator's close
association with chemically diverse gorgonian diets. Given the importance of
allelochemicals in shaping patterns of predation and herbivory in marine
systems, these findings suggest that co-evolved consumers have the capacity to
detoxify allelochemicals in their prey, providing these consumers with a
competitive advantage in ecosystems where allelochemically-rich prey species
abound.
# Methods
## Materials
CDNB, DTT, potassium phosphate, potassium chloride, EDTA, protease inhibitor
cocktail (4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, bestatin
hydrochloride, E-64, leupeptin, and pepstatin A), GSH-agarose (G4510) was
purchased from Sigma (St. Louis, MO). Bradford reagents were purchased from Bio-
Rad (Hercules, CA). PD-10 size exclusion columns were purchased from GE
Healthcare (Piscataway, NJ). Amicon Ultra-4 centrifugational filters were
purchased from Millipore (Billerica, MA). NanoOrange protein quantitation kit
was purchased from Molecular Probes (Eugene, OR). Prostaglandins
(15(S)-PGA<sub>2</sub>, 15(R)-15-methyl PGA<sub>2</sub>, 15(S)-PGE<sub>2</sub>,
15(R)-PGE<sub>2</sub>, 15(S)-PGE<sub>2</sub>-methyl ester,
15(S)-PGF<sub>2α</sub>, 15(R)-PGF<sub>2α</sub>, 15(S)-PGF<sub>2α</sub>-methyl
ester) were purchased from Caymen Chemical (Ann Arbor, MI). All solvents used
for extract and chemical analysis were purchased from Fisher Scientific
(Pittsburgh, PA).
## Animal Collection and Feeding Assay Design
A total of 39 adult *Cyphoma gibbosum* (ca. 2–3 cm length) were collected from
four shallow reefs (\<20m) (Big Point – 23°47.383′N, 76°8.113′W; Rainbow Gardens
– 23°47.792′N, 76°8.787′W; Shark Rock – 23°45.075′N, 76°7.475′W; Sugar Blue
Holes – 23°41.910′N, 76°0.23′W) surrounding the Perry Institute of Marine
Science (PIMS), Lee Stocking Island, Exuma Cays, Bahamas in January 2006. Snails
were immediately transported to wet laboratory facilities provided by PIMS where
a series of feeding assays were conducted with six gorgonian species (*Briareum
asbestinum*, *Eunicea mammosa*, *Gorgonia ventalina*, *Pseudopterogorgia
acerosa*, *Pseudopterogorgia americana*, *Plexaura homomalla*) observed to serve
as hosts for *C. gibbosum* in the field.
Individual snails were housed separately in 3-L polycarbonate tanks which were
placed in a 12′×20″ raceway supplied with filtered, continuous-flow seawater at
a flow rate of approximately 1L min<sup>−1</sup>. This design allowed for a
common water source to feed each tank but prevented mixing between tanks. Snails
collected from the same reefs were housed separately in the same raceways and
randomly assigned to one of seven groups – one of six gorgonian diets or a
control diet – at the start of the feeding assays. Snails were allowed to feed
ad. libum on either a control diet devoid of gorgonian compounds (i.e., alginic
acid and freeze-dried squid) (n = 12) or one of six gorgonian diets (n = 27) for
four days.
A minimum of ten colonies for each gorgonian species were collected from shallow
reefs (\<20m) surrounding PIMS and housed in a separate raceway prior to
introduction into the tanks containing *C. gibbosum*. The maximum amount of time
between gorgonian field collection and introduction into the feeding assay was
12 hours. Gorgonian colonies were cut into 2–3 inch pieces and allowed to
recover for four hours before addition to *C. gibbosum* tanks. The control diet
consisted of alginic acid and freeze-dried squid powder prepared as described in
and mirrored the average nutritional quality of gorgonian tissue. The squid-
alginate paste was pressed into sixteen 3-mm deep wells drilled into a 3″×1″
piece of Formica® resembling a domino. The domino was then placed into a 0.25 M
calcium chloride solution allowing the squid-alginate paste to harden. Both
control and gorgonian diets were replaced every 24 hours for four days and
feeding activity was monitored by the presence of feeding scars on their
gorgonian prey and empty wells on control dominos. Following the completion of
the feeding assay digestive glands were immediately dissected, weighed, frozen
in liquid nitrogen and maintained at −80°C until further processing.
## GST Purification and HPLC Subunit Analysis
Cytosolic and affinity-purified GSTs were isolated from *Cyphoma* digestive
gland samples as described in. Briefly, cytosolic GSTs were isolated by
homogenizing digestive glands (n = 39) separately in buffer (0.1 M potassium
phosphate, 1 mM EDTA, 1 mM DTT, 1.15% potassium chloride, protease inhibitor
cocktail (1×); pH 7.5), and differentially centrifuging the homogenates to
obtain the cytosolic fraction containing the soluble GST pool. Individual
cytosolic GST fractions were then applied to both a PD-10 size exclusion and
GSH-agarose affinity column in series to obtain the affinity-purified fraction
of GSTs. GST fractions were buffer exchanged to low salt concentration and
concentrated with Amicon Ultra-4 centrifugational filters (5K NMWL membrane) and
protein concentrations of Amicon concentrates were determined using the
NanoOrange protein quantitation kit.
A 30 µL aliquot of the affinity-purified GST concentrate from each of the 39
digestive gland samples was injected onto a reverse phase Vydac protein/peptide
column (model \#218 TP 52; C18 µm 250 mm×2.1 mm) and separated using a Waters
600 MultiSolvent Delivery System, with a flow rate of 0.5 mL/min. Peaks were
detected using a Waters 2487 Dual Wavelength Absorbance Detector (λ = 214 nm).
Mobile phase A consisted of 38% acetonitrile, 62% water and 0.1% trifluoroacetic
acid (TFA). Mobile phase B consisted of 80% acetonitrile, 20% water and 0.1%
TFA. The initial mobile phase consisted of 100% A. GST subunits were separated
using a linear gradient from 0 to 40% B in 22 min, and 40 to 100% B in 37 min.
The column was re-equilibrated with 100% A from 37–50 mins prior to the next
injection. Integration of HPLC peak area was achieved using the Empower 2
Chromatography Data Software package (Waters, Milford, MA) and converted to GST
subunit percent composition for each digestive gland sample.
## GST Activity Assay
Enzyme activity was measured using CDNB as a substrate by the method of
optimized for *C. gibbosum*, in a microplate format. The reaction mixture (in a
final volume of 200 µL) contained 0.1 M potassium phosphate buffer, 1.0 mM EDTA,
pH 7.5, 1 mM CDNB, 1 mM reduced GSH and 2 µg of cytosolic protein or 3.3–6.4 ng
of affinity-purified GST sample. CDNB was solubilized in ethanol and constituted
1% of the final reaction mixture volume. The reaction incubated at 25°C was
initiated by the addition of CDNB and performed in triplicate. The conjugation
of CDNB with GSH was measured as the increase in absorbance at 340 nm
(Δε<sub>340</sub> 0.00503 µM<sup>−1</sup> cm<sup>−1</sup>) using a tunable
microplate reader (Versamax, Molecular Devices, Sunnyvale, CA). Activity was
calculated using protein concentrations determined via the Bradford assay with
BSA as a standard.
## Extraction and Isolation of Gorgonian Compounds
A minimum of ten colonies for each gorgonian species were collected from shallow
reefs (\<20 m) surrounding Lee Stocking Island, Exuma Cays, Bahamas. A portion
of the gorgonian colonies, prior to their introduction into the feeding assay,
was immediately removed after field collection and immersed in seawater to
determine volumetric displacement, frozen at −80°C, and lyophilized for
subsequent chemical extraction. *Pseudopterogoria blanquillensis* was also
collected for chemical analysis; however, this gorgonian species did not
participate in the feeding assays. A 50 mL volumetric equivalent of pooled
tissue for each of the eight gorgonian species was extracted twice at room
temperature in 250 mL reagent grade acetone overnight with agitation. Resulting
crude organic extracts were vacuum-filtered through celite, dried by rotary
evaporation, and recombined into a 20 mL scintillation vial using a minimum
volume of solvent. The crude organic extracts were then completely dried using a
vacuum concentrator. Gorgonian crude organic extracts were assayed at 5% natural
concentration by volume (i.e., the extract from 0.05 mL of gorgonian was diluted
into 1 mL of assay buffer) for the ability to inhibit *Cyphoma* cytosolic GST
activity as described below. Those crude organic extracts that were able to
inhibit ≥80% of GST activity were subjected to further fractionation using a
bioassay-guided fractionation approach.
Gorgonian crude organic extracts were separated by partition between hexane and
methanol-water (9∶1) followed by partition of the methanol-water fraction
(adjusted to 6∶4) against chloroform. The chloroform-soluble, hexane-soluble,
and aqueous (i.e., methanol/water-soluble) fractions were reduced *in vacuo* and
assayed for their ability to inhibit cytosolic GST activity as described below.
Based on patterns of GST enzyme inhibition, chloroform fractions from all eight
gorgonian species were separated further using a reverse-phase semi-prep Zorbax
SB-C18 column (5 µm, 9.4 mm×2.5 cm) attached to a Waters Breeze HPLC system (515
pump) with a Waters 2487 UV detector at 215 and 254 nm. Compounds were eluted
over 33 mins at a flow rate of 3 mL/min with methanol/water (9∶1) with linear
ramping to 100% methanol. HPLC fractions were collected at three minute
intervals over 33 min, yielding ten fractions per gorgonian species. Each
chloroform fraction was assayed for GST inhibition at 10% natural volumetric
concentration. Chloroform fractions yielding 100% inhibition were further
assayed at 0.5% natural volumetric concentration.
## Inhibition Assays
GST activity measurements were performed as described above. Gorgonian crude
organic extracts and partitions were dissolved in the appropriate solvent (e.g.,
acetone, *n*-propanol, or methanol), HPLC fractions were dissolved in methanol,
and prostaglandins were dissolved in DMSO. Solvent concentrations did not exceed
5% of the experimental volume and had no effect on GST activity when compared to
non-solvent controls (data not shown). Immediately prior to the start of the
assay, inhibitor solutions were added to the buffer/GSH mixture and homogenized
to ensure equal distribution of inhibitor in all microplate wells. The data were
corrected for the non-enzymatic reaction rates and the effect of the inhibitors
on catalytic activity was measured by comparing the initial rate of reaction in
the presence and absence of the inhibitor.
Eight commercially available prostaglandins representing a diversity of forms
present in gorgonian tissue (15(S)-PGA<sub>2</sub>, 15(R)-15-methyl
PGA<sub>2</sub>, 15(S)-PGE<sub>2</sub>, 15(R)-PGE<sub>2</sub>,
15(S)-PGE<sub>2</sub>-methyl ester, 15(S)-PGF<sub>2α</sub>,
15(R)-PGF<sub>2α</sub>, 15(S)-PGF<sub>2α</sub>-methyl ester), including both
enantiomers (R and S) forms when possible, were screened at 600 µM for their
ability to inhibit crude cytosolic GST activity. From this initial screening,
only those prostaglandin compounds that demonstrated an ability to reduce GST
activity by 50% or greater were further evaluated at a range of concentrations
(0.2–2000 µM) in order to estimate the concentration producing 50% inhibition of
enzyme activity (IC<sub>50</sub>). Prostaglandin IC<sub>50</sub> values were
calculated and 95% confidence intervals were estimated using Prism 5.0 software
(GraphPad) by fitting the log transformation of the response variable by
nonlinear regression to the variable slope equation (1) and constraining the
bottom to zero but allowing the Hill Slope to vary. The variable slope equation
is:where Top is the maximum percent GST activity remaining, Bottom was
constrained to zero, IC<sub>50</sub> is the concentration of inhibitor that
produces inhibition half-way between the Top and Bottom, and \[I\] is the
logarithmic concentration of the inhibitor.
## Determining the Dissociation Constant for Inhibitor Binding (K<sub>i</sub>)
Because the IC<sub>50</sub> depends on the substrate concentration used in the
experiment, this value is only useful for comparing inhibitors within
experiments and not between laboratories unless identical assay conditions were
used. However, calculated K<sub>i</sub> (the dissociation constant of the
enzyme-inhibitor complex) values can be used to directly compare inhibitor
affinity for the enzyme between studies. K<sub>i</sub> estimates were calculated
using the IC<sub>50</sub> values obtained for 15(S)-PGA<sub>2</sub>,
15(R)-15-methyl PGA<sub>2</sub>, 15(S)-PGE<sub>2</sub>, 15(S)-PGF<sub>2α</sub>
with the Cheng-Prusoff equation (Eq. 2), where K<sub>m</sub> is the Michaelis-
Menten constant for CDNB (see below), \[S\] is the substrate concentration (1 mM
CDNB), K<sub>i</sub> is the equilibrium dissociation constant for the inhibitor,
and IC<sub>50</sub> is as defined above.To obtain an estimate of K<sub>m</sub>
for CDNB, initial-rate measurements using CDNB as the concentration-variable
substrate were performed. GST activity was measured at five concentrations of
CDNB ranging from 0.5 to 3 mM in the absence of inhibitors with 6 ng of
affinity-purified GST protein at 25°C in 0.1 M potassium phosphate buffer, 1.0
mM EDTA, pH 7.5, containing 1 mM GSH and 4% (v/v) DMSO. An affinity-purified GST
preparation from a single digestive gland was used as the protein source with a
specific activity (mean±SE) of 561±25 µmol min<sup>−1</sup> mg
protein<sup>−1</sup>. The reaction was initiated by the addition of 2 µL of CDNB
and performed in duplicate. The data from two independent experiments were
corrected for the non-enzymatic reaction rates and globally fitted to the
Michaelis-Menten equation to yield an estimate of K<sub>m</sub> = 0.41±0.14 mM
CDNB (mean±SD).
## Chemical Analysis of HPLC Fractions
Proton NMR spectra for *P. homomalla* HPLC fractions 1 and 2 were recorded in
deuterated DMSO (Cambridge Isotope Laboratories, Andover, MA) with a Bruker
DRX-500 instrument using a 5 mm inverse detection probe, and referenced to
residual DMSO (δ 2.49 ppm). Spectra collected for chromatographic fractions were
compared with the <sup>1</sup>H NMR spectrum obtained for authentic
15(S)-PGA<sub>2</sub> (Cayman Chemical, Ann Arbor, MI).
LC-MS analyses of *P. homomalla* HPLC fractions were completed using a Waters
2695 HPLC with a Waters 2996 photodiode array UV detector and Micromass ZQ 2000
mass spectrometer with electrospray ionization in both positive and negative
modes. Optima grade solvents were used in all LC-MS experiments. LC-MS
separations were achieved with an Alltech Altima C18 column (2.1×100 mm, 3 µm)
applying a gradient mobile phase of 40∶60 to 95∶5 acetonitrile∶water with 0.01%
acetic acid throughout. PGA<sub>2</sub> was detected in fractions by matching
chromatographic retention times and MS fragmentation patterns with those
obtained for pure synthetic PGA<sub>2</sub>. For fractions in which
PGA<sub>2</sub> was detected, the negative-mode ESI-MS selected ion recording
for *m/z* 333.3, corresponding to \[M-H\]<sup>−</sup> of PGA<sub>2</sub>, was
integrated and compared to a standard curve for PGA<sub>2</sub> at six
concentrations (r<sup>2</sup> = 0.94). The concentration of PGA<sub>2</sub> in
each HPLC fraction was determined by interpolation of this standard curve data.
# Supporting Information
We thank the staff of the Perry Institute for Marine Science; Carly Gaebe, Terry
Rioux and Ann Tarrant for their assistance with animal collection. We also thank
Dexter Morin for his assistance with GST separations.
[^1]: Conceived and designed the experiments: KW JK MH. Performed the
experiments: KW ALL. Analyzed the data: KW ALL MH. Contributed
reagents/materials/analysis tools: KW JK MH. Wrote the paper: KW.
[^2]: Current address: Centre for Marine BioInnovation, University of New
South Wales, Sydney, New South Wales, Australia
[^3]: Current address: Scripps Institution of Oceanography, University of
California San Diego, La Jolla, California, United States of America
[^4]: The authors have declared that no competing interests exist. |
# Introduction
Sulfotransferase (SULT) enzymes catalyze the sulfate conjugation of a broad
range of substrates and play an important role in metabolism of endogenous and
exogenous compounds including thyroid and steroid hormones, neurotransmitters,
drugs and procarcinogens. There are many isoforms of the *SULT*s supergene
family, each with different amino acid sequence identity and substrate
specificity. SULT1A1 is an important member of the sulfotransferase family
involving in the pathogenic process of various cancers.
The *SULT1A1* gene is located on chromosome 16p12.1–p11.2. Previous study
indicated that exon 7 of the *SULT1A1* gene contained a G to A transition at
codon 213 (rs9282861) that causes an Arg to His amino acid substitution. Some
studies have shown that this genetic polymorphism leads to a decrease in
enzymatic activity of SULT1A1 and the sulfonation efficiency thus associating
with susceptibility to several cancers. Although the specific role of *SULT1A1*
Arg213His polymorphism in carcinogenesis has been investigated in numerous case-
control studies, the results have been inconclusive, even conflictive. In order
to give a comprehensive and precise result, we performed this meta-analysis
study to analyze the association between this polymorphism and cancer risk.
# Materials and Methods
## Identification of eligible studies
The meta-analysis was conducted following the criteria of Preferred Reporting
Items for Systematic Reviews and Meta-Analyses (PRISMA). In this study, we did
an exhaustive literature search on studies that examined the association of the
*SULT1A1* gene polymorphisms with cancer risks. All eligible studies were
identified by searching the following databases: PubMed, Web of Knowledge and
China National Knowledge Infrastructure (CNKI, <http://www.cnki.net/>). The
following terms were utilized: “sulfotransferase, *SULT* or *SULT1A1*”,
“polymorphism, variation, variant or mutation” and “cancer or carcinoma”. In the
CNKI database, we searched with these corresponding key words in Chinese
characters. Included studies should meet the following criteria: (1) evaluating
the association between *SULT1A1* Arg213His polymorphism and cancer risk; (2)
study designed as case-control; (3) sufficient data available to estimate an odd
ratio (OR) with its 95% confidence interval (95% CI).
## Data extraction
Two investigators extracted data independently and reached consensus on the
following characteristics of the selected studies: first author's name, the year
of publication, ethnicity of the study population, matching criteria, number of
participants, genotype distribution and control source.
## Statistical analysis
Hardy-Weinberg equilibrium was assessed by Chi-square test. Crude odd ratio (OR)
and 95% confidence interval (CI) were used to estimate the association between
*SULT1A1* polymorphism and cancer susceptibility under the dominant model
(Arg/His+His/His vs. Arg/Arg), recessive model (His/His vs.
Arg/Arg<sub>+</sub>Arg/His), homozygous model (His/His vs. Arg/Arg),
heterozygous model (His/Arg vs. Arg/Arg) and allelic model (His vs. Arg). The
heterogeneity among the studies was evaluated by Q-test and *I<sup>2</sup>*
value ranging from 0% to 100% to describe the percentage of between-study
variation caused by heterogeneity. P value for the Q-test less than 0.10
indicates existing heterogeneity among studies. And then the pooled OR was
measured by a random effect model (the DerSimonian-Laird method). Otherwise, a
fixed effect model (the Mantel-Haenszel method) was chosen.
Subgroup analyses were performed according to cancer type (breast cancer,
colorectal cancer, urothelial cancer, prostate cancer, lung cancer, upper aero
digestive tract (UADT) cancer, ovarian cancer and gastric cancer), ethnicity
(Caucasian, East Asian, Indian and African) and source of controls (hospital
based and population based). When heterogeneity was detected, a multivariable
meta-regression analysis including cancer type, ethnicity, control source and
year of publication to explore potential source of heterogeneity and sensitivity
analysis were performed.
The potential publication bias was estimated using Egger's linear regression
test by visual inspection of the funnel plot. P<sub>\<</sub>0.05 was considered
statistically significant, and all P values were two-sided. Analyses were
performed using the software Review Manager 5.3 (Cochrane Collaboration), R
software ([www.r-project.org](http://www.r-project.org)) and STATA 12.0 software
(StataCrop).
# Results
## Characteristics of eligible studies
The flow diagram of literature search was given in. A total of 91 studies
focusing the association between the *SULT1A1* Arg213His polymorphism and cancer
risks were identified. 25 of them were ruled out because of unavailable data or
repeated data. Thus, the allele and genotype frequencies of the *SULT1A1*
Arg213His polymorphism were extracted from 66 articles. However, 18 articles
didn't meet with Hardy-Weinberg equilibrium and were abandoned. As a result, 53
studies of 48 articles, involving 16733 cases and 23334 controls were included
in the pooled analyses.
The characteristics of studies included in the current meta-analysis are shown
in. Among these studies, 13 were conducted for breast cancer, 10 for colorectal
cancer, 7 for urothelial cancer, 5 for prostate cancer, 5 for lung cancer, 5 for
UADT (upper aero digestive tract) cancer, 3 for ovarian cancer, 2 for gastric
cancer and 1 for myeloid leukemia, multiple myeloma, and endometrial cancer,
respectively. By ethnics, there were 27 studies of Caucasians, 11 studies of
East Asians, 4 studies of Indians, 2 studies of Africans and 9 studies of mixed
ethnics. By source of controls, 16 studies were population-based, 17 studies
were hospital-based and 20 studies were not clear.
## Overall Analysis
showed the results of overall analysis and the subgroup analysis. The analyses
on the full data set indicated a significant association of the *SULT1A1*
Arg213His polymorphism with cancer risk: heterozygous (OR = 1.09, 95%
CI = 1.01–1.19, P = 0.035), homozygous (OR = 1.20, 95% CI = 1.04–1.39,
P = 0.014), dominant (OR = 1.12, 95% CI = 1.03–1.22, P = 0.008), recessive
(OR = 1.16, 95% CI = 1.02–1.32, P = 0.027) and allelic model (OR = 1.11, 95%
CI = 1.04–1.20, P = 0.003), with high heterogeneity among studies
(*I<sup>2</sup>* = 63.1%, 62.6%, 68.5%, 58.3% and 73.7%, respectively, all
P\<0.001).
## Subgroup Analyses
We analyzed the association in cancer type subgroup. *SULT1A1* Arg213His
polymorphism can increase cancer risks in the following cancer types: breast
cancer (homozygous model: OR = 1.37, 95% CI = 1.01–1.87, P = 0.045; dominant
model: OR = 1.18, 95% CI = 1.00–1.40, P = 0.050 and allelic model: OR = 1.15,
95% CI = 1.00–1.32, P = 0.044); UADT cancer (heterozygous model: OR = 1.62, 95%
CI = 1.11–2.35, P = 0.012; dominant model: OR = 1.63, 95% CI = 1.13–2.35,
P = 0.009 and allelic model: OR = 1.52, 95% CI = 1.10–2.11, P = 0.012). Forest
plots of breast cancer risk and UADT cancer risk were shown in and separately.
Analyzed by ethnicity, a moderately increased risk was observed in Caucasians
(homozygous model: OR = 1.20, 95% CI = 1.01–1.43, P = 0.035 and allelic model:
OR = 1.10, 95% CI = 1.01–1.19, P = 0.019) and Indians (recessive model:
OR = 1.93, 95% CI = 1.22–3.07, P = 0.005). No significant association was found
in other ethnicities in any model.
By control source, significant association was observed in hospital based study,
but not the population based study.
## Meta-regression analysis
To find potential source of heterogeneity, multivariable meta-regression
analyses were conducted in total group and subgroups including cancer type,
ethnicity, control source and publication year. In the breast cancer subgroup,
ethnicity (heterozygous model, P = 0.027; recessive model, P = 0.020) and
publication year (heterozygous model, P = 0.019; recessive model, P = 0.012) are
significant sources of heterogeneity. Other variables don't affect
heterogeneity.
## Sensitivity analysis
The sensitivity analysis was constructed by repeating the meta-analysis
sequentially removing each study. In the recessive model, two studies, were
found to affect the pooled OR and the heterogeneity when removed. The study
conducted by Khvostova was focused on breast cancer and Sun's study was focused
on colorectal cancer among Caucasians, so further sensitivity analyses were
conducted in total data set and breast cancer, colorectal cancer and Caucasian
subgroups after removing the two studies. In total group, the heterogeneity was
significantly decreased (*I<sup>2</sup>* = 58.2, 42.2, 63.5, 33.1 and 66.4,
respectively). In the subgroup sensitivity analyses, removing the two studies
can significantly decrease the heterogeneity among studies, most *I<sup>2</sup>*
values less than 50%. And this polymorphism didn't show any obvious correlation
with breast cancer risk. At last, we conducted the sensitivity analyses on the
remaining studies and the result was stable.
## Publication bias
Funnel plots and Egger's test were carried out to assess publication bias. The
shapes of funnel plots indicated no obvious asymmetry. Egger's test found no
publication bias in the heterozygous (P = 0.074); homozygous (P = 0.146);
dominant (P = 0.076); recessive (P = 0.282) and allelic model (P = 0.081).
# Discussion
SULT1A1 enzyme encoded by *SULT1A1* gene plays an important role in xenobiotic
metabolism. The Arg213His polymorphism, the most widely studied polymorphism
within *SULT1A1* gene, can reduce enzyme activity and thermostability, and
consequently results in an individual's susceptibility to cancer.
There have been a few meta-analyses focusing on this mutation and cancer risk.
However, most of these analyses were conducted before the year 2012 and a new
meta-analysis is needed to give a comprehensive conclusion due to the increasing
data of case-control studies.
This present meta-analysis, including 16733 cases and 23334 controls from 53
case-control studies, explored the association between the *SULT1A1* Arg213His
polymorphism and cancer risk. This is the largest scale meta-analysis so far.
Our results suggested that the *SULT1A1* Arg213His was associated with UADT
cancer risk. As the upper aero digestive tract is exposed to numerous potential
carcinogens such as phenolic xenobiotics, polycyclic aromatic hydrocarbons and
heterocyclic aromatic amines contained in cigarette smoking, environmental
pollutants and some food, this result manifests that the mutation within
*SULT1A1* causes the low SULT1A1 activity and is associated with high
susceptibility to cancers related with environment.
In the sensitivity analyses, the study conducted by Khvostova influences the
pooled estimates and the heterogeneity most in breast cancer subgroup. And after
removing this study, the significant association between *SULT1A1* Arg213His and
breast cancer risk became null. We further checked data from Khvostova and
observed the percentage of wild homozygous genotype in Khvostova's study was
obviously lower than that in other studies thus causing great heterogeneity. At
last a robust result was achieved and failed to reveal significant association
in breast cancer subgroup. This result is similar to Wang, Lee and Jiang, but
they found a positive association of this polymorphism with breast cancer
susceptibility among Asians. While in our meta-analysis, we only recruited one
paper focused on breast cancer among Asians because other papers on Asians
deviate from *HWE* and were excluded. This is a limitation of this meta-analysis
and more independent case-control studies conducted on Asians are needed to
conclude a more comprehensive result.
In the ethnic subgroup analysis, we found that the genotype distributions of the
SNP site are different in ethnic groups. When calculating the percentage of
alleles in every ethnic, we found that His allele in Asians (9.58%) is
significantly less than in Caucasians (35.2%). Different ethnicities may have
different genetic backgrounds, thus causing different genotype frequencies in
Asian and other ethnic groups which may influence cancer susceptibility.
Li and Kotnis have conducted meta-analyses focused on environment-related
cancers, such as tobacco-related cancers and found cancer risk could be
modulated by interaction between genetic variants and environmental factors. As
exposed environmental factors are different according to cancer types, for
example smoking leads to lung cancer, while the intake of meat influences breast
cancer and colorectal cancer, and our analysis took many kinds of cancer into
account, we decided not to include environmental factors. Moreover, the
definitions of exposed environmental factors were not consistent in the studies,
which could cause great heterogeneity. Our estimates were based on crude OR
values, not adjusted OR values, which may yield inaccurate calculation.
There were several sources bringing in heterogeneity, such as study design, age
and sex distribution, and ethnicity. Meta-regression analysis was conducted to
find source of heterogeneity. In the breast cancer subgroup, publication year
could cause great heterogeneity and further attention was paid to years. We
found all the recruited studies were carried out before 2005 or after 2010, and
there were no studies between 2006 and 2009. The His allele was 29.6% in the
studies before 2005 and 33.0% after 2010, which was significantly different
(P = 0.02). This may be caused by the different study population, and needs more
case-control studies to illustrate.
In conclusion, our meta-analysis suggests that the *SULT1A1* Arg213His
polymorphism may contribute UADT cancer risk. As the result was calculated
through sampling statics and statistical difference is not the same as clinical
difference, the result can be used for clinical reference, not for clinical
diagnosis of cancer. Further detailed investigation with larger number of
worldwide participants is needed to clarify the role of this polymorphism in
cancer risk.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: XLY MHW WPW. Performed the
experiments: JJX YBZ YHZ PZ. Analyzed the data: YBZ LXF. Contributed
reagents/materials/analysis tools: JJX JGW FYS. Wrote the paper: JJX YBZ
VKK. |
# Introduction
**T**elomeres consist of long tandem arrays of TTAGGG repeats, bound by
proteins, placed at the end of linear chromosomes, which are involved in several
essential biological functions., These non-coding telomeric repeats represent a
buffer zone preventing the adjacent coding region of the genome from erosion. In
normal human cells, telomeres decrease by some 5–20 repeats with every cell
division. Therefore telomere shortening limits the number of times a cell can
divide. Hence, they can regulate the onset of replicative senescence in the
somatic cells.–
In human cells, several pathways regulating telomeres length have been
identified. The most important is regulated by telomerase, that catalyzes
extension of 5′-ends of the lagging DNA strand by adding TTAGGG repeats onto the
telomeres using its intrinsic RNA as template for reverse transcription. Two
major subunits of the human telomerase core complex have been identified, namely
h-TERC and h-TERT. The former serves as a template for telomeres elongation;
instead, the latter subunit (h-TERT) contains a reverse transcriptase domain
that catalyzes this reaction.
The length and structure of telomeres are also controlled by a variety of
proteins. Collectively, these telomeric proteins protect telomere integrity and
function, connect DNA damage/repair network with the controls of cellular
senescence, monitor telomere homeostasis and modify the access of telomerase to
telomeres. The two major proteins are the duplex TTAGGG repeat-binding factors 1
and 2 (TRF1 and TRF 2) that are localized at telomeres. These proteins play a
key role in the maintenance of telomere function and structure modifying
telomerase activity.– Recent evidence shows that TRF1 interacts with other
telomere-binding molecules. TRF1 accepts adenosine diphosphate
(ADP)-ribosylation catalyzed by the tankyrase-poli-ADP-ribose polymerase (TANKs-
PARP) complex. The ADP-ribosylation of TRF1 reduces its ability to bind
telomeric DNA, allowing telomerase to elongate telomeres and extending the
cellular life span.–
The alteration of telomere length homeostasis affects telomere structure and
leads to genomic instability by generating chromosome end-to end fusion and
chromosomal abnormalities. It has been demonstrated that telomeres shortening
could initiate successive events, such as aberrant fusion or recombination of
the end of chromosomes, genomic instability, loss of cell growth control, and
finally cancer development.,
The phenomenon of telomeres alteration during carcinogenesis and cancer
progression is well known and established at the molecular level.– Nevertheless,
studies focused on the analysis of telomere dysfunction in astroglial brain
tumors are missing. The present study was designed to investigate the expression
levels of a panel of genes controlling the length and structure of telomeres in
human astroglial brain tumors with different grade of malignancy (WHO Grade
2–4). We analyzed telomeres length, telomerase activity and the expression
levels of TRF1, TRF2, h-TERT and TANKS-PARP complex in tumor samples obtained
*in vivo*, investigating the presence of a specific genes expression profile
during the different stages of tumor progression, from low grade astrocytomas to
glioblastoma.
# Materials and Methods
## Ethics Statement
The manuscript has been submitted to the Ethics Committee on February 21, 2011.
The above mentioned Committee issued a formal written waiver for the need of
ethics approval. All patients signed a written informed consent for the purpose
of publication of clinical data, according to the internal regulation. Results
were analyzed anonymously.
## Patient population
Tumors samples, histologically verified as grade 2–4 astrocytomas, were obtained
in adult patients who underwent craniotomy for microsurgical tumor resection.
All tumors were located in the supratentorial compartment. Only patients who had
undergone gross total resection (more than 95% of the tumor volume) were
eligible for the study. Summary of demographic and clinical data are reported
in. Samples obtained from single or multiple stereotactic biopsies were not
included in the present study. We carefully excluded tumors containing
components that were suspicious of oligodendroglioma. No case of recurrent
tumors and no patient who underwent radio and/or chemotherapy before surgery
were employed in the present study.
## Tissue Samples
All tumor tissue samples were obtained from resection specimens, within 15
minutes from surgical tissue removal. Specimens were taken from viable areas of
tumor, avoiding areas of gross necrosis. Three to seven anatomically separate
areas of tumor tissue were sampled from each resection specimen, according to
the volume of resected tissue available. Tumor samples were placed in cryovials
and immediately flash-frozen in liquid nitrogen in the operating room and stored
at −70°C. Tissue samples adjacent to the frozen tissue, as well as additional
tissue submitted *in toto* from the resection specimens, were both used for
histological typing and grading according to WHO criteria. Three samples of
normal brain tissue were used as controls. Non-neoplastic brain tissue samples
were derived from the temporal lobes of patients surgically treated for temporal
lobe epilepsy, histologically verified as normal cortex and white matter.
## Telomere length analysis
Terminal restriction fragment (TRF) length measurements in tumor specimens and
in normal samples were obtained by using the *Telo* TTAGGG telomere length assay
kit (Roche Diagnostics, Milan, Italy), according to the manufacturer's
recommendations. The intensity of the hybridization was evaluated by
densitometric analysis with Quantity One software (Bio-Rad Laboratories,
Hercules, CA) and mean TRF length of a sample was estimated according to the
formula as described by Harley CB at al.
## Telomerase activity Assay
Telomerase activity was measured by a telomere repeat amplification protocol
(TRAP) assay using TeloTAGGG Telomerase PCR ELISA plus® (Roche, Mannheim,
Germany) according to the manufacturer's recommendations. The relative
telomerase activity (RTA) within different samples was calculated using the
following formula: RTA = (*A*<sub>sample</sub>/*A*<sub>sample,IS</sub>)/(*A*<sub
>TS8</sub>/*A*<sub>TS8,IS</sub>)×100%, where *A*<sub>sample</sub> = absorbance
of sample; *A*<sub>sample,IS</sub> = absorbance of internal standard of
sample; *A*<sub>TS8</sub> = absorbance of control template; and
*A*<sub>TS8,IS</sub> = absorbance of internal standard of control template.
## Extraction of total RNA and Real-Time Quantitative PCR
Total RNA was extracted from each specimen using TRIizol reagent and purified
with RNA Purification kit (Rneasy Mini Kit clumns- Qiagen). The quality and
quantity were checked respectively on agarose gel and spectrophotomety. Three µg
of total RNA from each sample was reverse-transcribed by Archive kit (Applied
Biosystems Milan, Italy). Generated cDNA was used as template for real time
quantitative PCR analysis using gene expression products according to the
manufacturers recommendations (Applied Biosystems). All reactions were performed
with a 7300 Sequence Detection System apparatus (Applied Biosystems) to measure
and compare the mRNA level of TERF-1, TERF-2, TNKS, PARP1, h-TERT, and β-actin
(as an endogenous control).
Relative quantification (RQ) for these genes was expressed as fold variation
over control, and calculated by the ΔΔCt method, using normal brain tissue
(control) as calibrators.
## Proteins extraction, electrophoresis (SDS-PAGE) and immunoblotting
Frozen tumor tissues (∼50 mg) was harvested by homogenization with a Potter
homogenizer in a 15 volumes ice-cold triple detergent lysis buffer.
Immunoblots were probed with goat polyclonal antibody anti TRF1 and TANKS, mouse
monoclonal antibody TRF2, PARP1 and β-actin (Santa Cruz Biotechnology Inc.,
Santa Cruz, California, USA). Following incubation with primary antibody (1∶200)
at RT for 2 hours, blots were incubated with a secondary antibody: mouse- anti-
goat and rabbit – anti-mouse IgG, (1∶1000; DAKO) conjugated to peroxidase at RT
for 1 hour. Enhanced chemiluminescence reagents were used to visualize
immunolabeling on Kodak Biomax ML chemiluminescent film. (ECL, Amersham
biosciences, Little Chalfont, Buckingamshire, UK).
## Quantification of telomeric proteins
Semi-quantitative evaluation of protein levels detected by immunblotting was
performed by computer-assisted densitometric scanning (AlphaImager 4.2 Digital
Imaging System, Italy). Different time of exposure were used for each blot
(15–25 seconds), and longer exposures were performed in an attempt to detect
very low levels of proteins in normal brain tissue. Data were acquired as
integrated densitometric values and expressed as percentages of the
densitometric levels obtained on scans from normal brain tissue used as control
visualized on the same blot (ADU - Arbitrary Densitometric Unit). We used two
different negative controls for each blot.
## Statistical Data Analysis
Statistical analysis was accomplished using the unpaired Student t-test to
compare the expression levels of mRNA of TRF1, TRF2, h-TERT, Tankyrase, PARP1 as
quantified on real time RT-PCR and telomere length on southern blotting. The
Spearman nonparametric correlation test was used to assess the correlation among
mRNA expression levels and the nominal variables (WHO grade). Computer software
programs (INSTAT \[version 3.0\] and PRISM \[version 4.0\]; GRAPHPAD, San Diego,
CA and MedCalc \[version 7.2.1.0\]) were used to perform the data analysis. A
probability value less than 0.05 was considered statistically significant. All
values where expressed as mean ± standard deviation. All tests are two-tailed;
values are expressed as mean ± standard deviation.
# Results
## Telomere length in normal brain and astroglial brain tumors
Telomere length varied in astroglial brain tumors with different grade of
malignancy. Telomere length was 7.4±0.23 Kbs in LGGs; 6.9±0.14 Kbs in AAs;
9.2±0.5 Kbs in GBMs; 10.93±2.35 Kbs in NBT. Elongated telomere was frequently
found in GBMs as compared with both AAs and LGGs (p\<.001). No statistical
differences in telomere length between AAs and LGGs were observed.
Interestingly, telomeres in tumor specimens were always shorter as compared with
NBT.
## Telomerase activity and h-TERT expression
Telomerase activity (TA) in NBT was weak or undetectable using the TeloTAGGG
Telomerase PCR ELISA plus. Telomerase activity was 3.9-fold in LGAs, 15.75-fold
in AAs, and 51-fold in GBMs as compared to normal control values. Telomerase
activity significantly correlates with h-TERT mRNA expression and WHO grade.
h-TERT mRNA expression was 1.71±0.90 in LGAs, 8.45±8.49 in AAs, 14.8±12.46 in
GBMs. The expression levels in GBMs resulted statistically higher as compared
with those in LGAs (P = .009). The expressions in AAs differed significantly as
compared with that in LGAs (P = .04). A correlation was found between h-TERT and
WHO grading (P\<.001). Conversely, no statistical differences in telomere length
between h-TERT and Telomere length were observed. A correlation was also found
between expressions of hTERT mRNA and PARP-1 mRNA (P = .008), and Tankyrase mRNA
(P = .058).
## Telomere-associated proteins
### Down-regulation of TRF1 and 2 and TANKS-PARP up-regulation occurs along with malignant progression in astroglial brain tumours
Summary of biomolecular data are reported in. TRF1 mRNA expression was 3.54±3.65
in LGAs, 0.30±0.34 in AAs; 0.19±0.22 in GBMs. The expression levels in LGA were
statistically different as compared with those in GBMs (P = .007). The
expression in LGAs was significantly higher than that in AAs (P = .009) ( A). An
inverse correlation was found between expressions of TRF1 and WHO grade
(P = .006).
The TRF2 mRNA expression was 0.51±0.22 in LGAs, 0.38±0.43 in AAs; 0.16±0.19 in
GBMs. The expression levels in LGAs resulted statistically higher as compared
with those in GBMs (P = .008) ( B). An inverse correlation was found between
expressions of TRF2 and WHO grade (P = .008).
The Tankyrase mRNA expression was 1.28±0.58 in LGAs, 4.46±6.23 in AAs, 1.08±1.35
in GBMs. The expression levels in AAs resulted statistically higher as compared
with those in LGAs (P = .009) and GBM (P\<.001) ( C). A correlation was found
between expressions of Tankyrase mRNA and that of both hTERT mRNA (P = .03), and
TRF-2 mRNA (P = .03).
The PARP-1 mRNA expression was 0.97±0.59 in LGAs, 17.4±5.69 in AAs, 11.5±15.5 in
GBMs. The expression levels in AAs resulted statistically higher as compared
with those in LGAs (P\<.001). The expression in GBMs showed a tendency toward
the higher expression as compared with LGAs (P = .07) ( D).
Western blot analysis of proteins confirmed previous results.
### Possible clinical implications of telomere length, Telomerase activity and Telomere-associated proteins
A statistically significant correlation between telomere length and WHO grade
was observed (P\<.001). Conversely, no statistical differences between telomere
length and both age and KPS, were found. A correlation was found between h-TERT
and WHO grading (P\<.001). No statistical differences between h-TERT and
Telomere length were observed. An inverse correlation was found between
expression of TRF1 and WHO grade (P = .006), and between expression of TRF2 and
WHO grade (P = .008).
# Discussion
Our results suggest that factors controlling telomere length are expressed at
variable level in astroglial brain tumors with different grade of malignancy
when compared with normal brain tissue. They also suggest the presence of a
specific expression profile for different tumor grade, with telomere length
depending on the balance of expression levels of different genes involved in the
control of telomere maintenance. In details, up-regulation of TRF1 and 2, and
shorter telomere featured LGGs, suggesting a pivotal role of these telomeric
proteins in the early stage of cell immortalization. A down-regulation of TRF1
and 2, and up-regulation of both telomerase and TANK-PARP1 mainly observed in
AAs and GBMs, may play a role in malignant progression of astroglial tumors
toward higher malignancy levels.
In order to better understand the role of telomere dysfunction, including
elongation or attrition, in carcinogenesis and tumor progression, we measured
telomere length in astroglial brain tumor with different grade of malignancy
(WHO Grade 2–4). Terminal restriction fragment measurement showed that telomere
length was reduced in astroglial tumors as compared with NBT. In GBM however,
telomeres were often longer than AAs and LGGs.
Telomere length is generally reduced in human tumors.– The relevance that this
shortening plays in carcinogenesis has been extensively studied using knockout
mice., – In the absence of genome checkpoint functions, telomere dysfunction
caused by telomere shortening accelerates genomic instability, facilitating
cancer initiation and progression.
On the other hand, the telomere length tended to increase along with malignancy
of tumors.– Gertler et al., showed that in colorectal tumors increased TRF
correlated with higher tumor stage, decreased overall survival, and in a
multivariate analysis, TRF was an independent prognostic factor.
Our findings support the hypothesis that hypervariability of telomere length,
already described in other human cancers, probably depends on the different
stages of carcinogenesis and tumor progression. Hence, to ascertain this
hypothesis, we evaluated, for the first time in gliomas, telomerase activity and
the expression levels of the main genes involved in telomere maintenances
including h-TERT, TRF1 and TRF2, and TANKs-PARP, in tumor tissues obtained
during surgery and their paired normal tissue. In cancer cells telomere length
depends on the balance between the loss of telomeric repeats during DNA
replication and the elongation of telomeric repeats mediated by telomerase. In
most normal human somatic cells, telomerase activity is weak or undetectable. As
in almost all tumors, malignant brain tumors are associated to higher telomerase
activity than benign tumors, such as neurinomas, meningiomas or normal brain
tissue. Increased telomerase expression has been also associated with higher
proliferative index, tumor grading, age, vascular and endothelial proliferation,
poor outcome.– In our investigation, both telomerase activity and h-TERT mRNA
levels varied in astroglial brain tumors and both were significantly correlated
with tumor grading. On the other hand, no statistical correlation between h-TERT
and telomere length was observed. Similar results have also been reported in
other studies, suggesting that a telomerase-independent mechanism for the
regulation of telomere lengthening possibly exist in these telomerase-negative
tumors. Recent studies indicate that telomere-associated proteins can regulate
telomerase accessibility in either positive or negative ways, suggesting a role
in telomere maintenance. The two major telomere- binding proteins are TRF1 and
TRF2. Both may function individually or by interacting with other binding
proteins, such as tankyrase, TIN2, hRap1, Mre11/Rad50/Nbs1 DNA repair complex,
Ku86., Recent in vitro studies indicate that, overexpression of TRF1 in
tetracycline-responsive human fibrosarcoma cell line HTCC75 resulted in a
gradual decline in telomere length and it has been reported that the forced
tethering of a large number of TRF1 molecules to a single telomere induce a
significant shortening of telomere length. These findings suggest that in human
cancer cells an upregulation of TRF1 may results in a progressive telomere
shortening. Our results documented a differential telomere-associated proteins
expression in astroglial brain tumors of different grade compared with normal
brain tissue. An inverse correlation between TRF1 and 2, and WHO grade was also
found. Several studies, dealing with correlation of TRFs expression and telomere
length in human cancers, also confirm our results. Oh et al. documented that up
regulation of telomere-binding proteins, TRF1, TRF2, and TIN2 is related to
telomere shortening during human multistep hepatocarcinogenesis. These findings
support the hypothesis that an up regulation of the telomeric binding proteins,
inhibiting telomerase, results in a progressive telomere shortening and may play
a role in immortalization of cancer cells. Our findings reveled a weak TA In low
grade tumors, TRF1 and TRF2 mRNA is up-regulated, while TA was weak and h-TERT,
tankyrase, and PARP resulted absent or minimally expressed. These data, also,
confirm our previous observation that glioma cells express higher levels of TRF1
as compared with normal brain tissue., As expected, telomere length in such
tumors was shorter as compared with normal brain tissue. In high grade tumors
h-TERT, PARP and tankyrase are over-expressed with an increased telomerase
activity. These findings, are consistent with the evidence of longer telomeres
in those tumors.
# Conclusions
Our findings, are consistent with the hypothesis that in astroglial brain
tumors, up-regulation of TRF1 and TRF2 may occur in the early stages of
carcinogenesis when telomerase has not yet been activated or is down-regulated
by TRF1. This stage is characterized by short telomeres, genomic instability,
low proliferative rate and prolonged life span, that are typical biological
behaviour of LGGs. Later, telomere dysfunction caused by telomere shortening
accelerates genomic instability, facilitating cancer initiation and progression.
At this stage, an up-regulation of PARP-Tankyrase complex and telomerase
activation may occurs. The ADP-ribosylation of TRFs, mediated by PARP1, reduces
its ability to bind to telomeric DNA, allowing telomerase to elongate
progressively telomeres and extending cellular life span. Down-regulation of
TRF1 and TRF2, increasing telomerase activity, persistent over-expression of
PARP-TANKs and elongated telomere are typical features of GBMs. Elucidation of
additional telomerase components and associated proteins will certainly
contribute to further investigations of the effect of telomerase in telomeres
elongation, telomere length maintenance, oncogenesis, and new unidentified
cellular functions. As far as we know, no other studies regarding these issues
in astroglial brain tumors are available in the literature, so further studies
should be performed to better understand the pathways involved in the telomeres
length maintenance and, consequently, in the process of carcinogenesis and
malignant progression of human astroglial brain tumors.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: DLT. Performed the
experiments: DLT AC MA SR MGDP CT. Analyzed the data: DLT AC FFA SC CA SR.
Contributed reagents/materials/analysis tools: MA SR MGDP CT CA. Wrote the
paper: DLT AG AC. |
# Introduction
Accurate assessment of respiratory muscle strength is clinically important in
patients with neuromuscular disorders (NMD) or unexplained dyspnea. Measurement
of maximum inspiratory pressure (MIP) at the mouth is the most commonly employed
test to evaluate inspiratory muscle function, as it is non-invasive and
relatively convenient. However, particularly in patients with NMD, MIP suffers
from the possible occurrence of falsely low values due to difficulties in
maintaining an effective mouth seal or sustaining a maximal inspiratory effort.
For these reasons, sniff nasal inspiratory pressure (SNIP) has been used as an
alternative non-invasive test of inspiratory muscle function which is easier to
perform for many NMD patients. It can be employed to monitor inspiratory muscle
strength over time in NMD, and a normal SNIP can also effectively rule out
inspiratory weakness in individuals with spuriously low MIP values. In
amyotrophic lateral sclerosis (ALS), the SNIP has been reported as the best
prognostic indicator.
The SNIP measurement entails brief maximal sniff efforts by the patient during
simultaneous intranasal pressure recordings within a nostril that is sealed by a
snugly fitting plug containing the pressure-sensing probe. The sniff maneuver
has long been used in the assessment of diaphragm function. It was initially
described for radiological assessment of unilateral diaphragm weakness.
Subsequently, sniffs were found to be a representative approximation of phrenic
stimulation in studies of diaphragm contraction. The sniff maneuver is now
commonly used when diaphragm strength is being assessed by measuring
transdiaphragmatic pressure (Pdi). The SNIP, in turn, has been devised as a less
invasive alternative.
In the original description of SNIP measurements, the contralateral nostril
remained unobstructed or open (henceforth referred to as SNIP<sub>OP</sub>).
Under these conditions, a reliable SNIP<sub>OP</sub> value presumably requires
inspiratory collapse of the contralateral nasal valve in order to allow for
quasi-equilibration of intrathoracic and nasal cavity pressures. The SNIP can
also be measured as a static maneuver with the contralateral nostril closed
(SNIP<sub>CL</sub>), as reported in a much smaller number of studies.
Although they appear to be used and reported interchangeably in the literature,
it is unclear if SNIP<sub>OP</sub> and SNIP<sub>CL</sub> in fact produce the
same results. Theoretically, they could be very similar in healthy subjects, but
individuals with inspiratory muscle weakness may be unable to generate
sufficient negative inspiratory pressure to collapse the nasal valve when
performing SNIP<sub>OP</sub>. Therefore, SNIP<sub>OP</sub> might poorly reflect
the actual negative intrathoracic pressure values and thus provide inaccurate
information about the true level of inspiratory muscle strength in some NMD
patients.
In the present study, we sought to determine whether there are any systematic or
clinically significant differences between values of SNIP<sub>OP</sub> and
SNIP<sub>CL</sub> in patients with known NMD as well as in patients without
clinical evidence of respiratory dysfunction. Our primary hypothesis was that
values of SNIP<sub>CL</sub> would be significantly higher than SNIP<sub>OP</sub>
in NMD patients. The secondary hypothesis was that in patients with a reduced
MIP value, SNIP<sub>CL</sub> would give results within a normal range
(suggesting an absence of respiratory muscle weakness) more often than
SNIP<sub>OP</sub>.
# Materials & methods
## Study subjects
The two groups of study subjects consisted of: 1) NMD patients recruited from a
home non-invasive ventilation program, and 2) a control group comprised of
individuals with obstructive sleep apnea (OSA) and no known NMD or significant
lung disease who were also participants in a Pompe disease screening study. The
NMD diagnosis was obtained from the medical record, as made by a clinical
neurologist. Two patients in the initial control group were identified as having
a NMD and thus transferred into the NMD group. In addition, several control
group patients (n = 8 with asthma or chronic obstructive pulmonary disease
(COPD), n = 5 with morbid obesity, n = 7 with other) had abnormal spirometry
(FEV1 or FVC \< 80% of predicted, or FEV1/FVC ratio \<70%) and were thus
excluded.
Subjects were recruited between September 2013 and November 2014, and provided
their written informed consent. Participants, none of whom were hospitalized at
the time of testing, took their usual medications without modification. All
study subjects underwent spirometry and respiratory muscle strength measurements
as outlined below, all in the sitting position, and all within a single testing
session between 10 am and 2 pm. The study was approved by the institutional
Research Ethics Board of the McGill University Health Center (13-379-BMB).
## SNIP measurements
Both SNIP<sub>OP</sub> and SNIP<sub>CL</sub> were performed using a commercially
available device (MicroRPM, VIASYS Healthcare, Hochberg, Germany) with
disposable nasal probes. Factory-set calibration of the device was verified
using a manometer. The nostril that appeared most patent clinically was chosen
for insertion of the nasal probe and the appropriate nasal probe size was
verified by ensuring the absence of air leak during sniffs. Without a prior
training period, the patient was asked to perform short, sharp sniffs of maximal
intensity from functional residual capacity (FRC) in the sitting position with
the mouth closed. Normal breathing was allowed between trials. At least 10
trials were done in total: five sniffs with contralateral nostril open
(SNIP<sub>OP</sub>), and five with the contralateral nostril closed
(SNIP<sub>CL</sub>). Half of the participants performed SNIP<sub>OP</sub> first,
whereas the reverse order was used in the other half, to account for any
potential learning or order effect. The highest value for each SNIP method is
reported for each individual. A single research assistant performed all testing.
## Standard PFT measurements
Spirometry was performed (Jaeger FlowScreen V2.6.0, Carefusion Corp, San Diego,
CA) to determine forced expiratory volume in one second (FEV1), forced vital
capacity (FVC), and peak expiratory flow (PEF) according to ATS guidelines and
established reference values. Supine spirometry was subsequently similarly
performed. Wheelchair-bound participants who could not easily transfer did not
have supine measurements, unless their wheelchair tilted to at least a 30 degree
recline. MIP was measured through a flanged mouthpiece from residual volume
(RV). The highest of at least three consistent values was recorded as
recommended. Reference values were taken from Vincken et al.. Individuals with
values reaching the upper saturation limit of the MIP manometer (≥150
cmH<sub>2</sub>O) were excluded from the analysis (n = 5 from the control group)
to avoid a ceiling effect which could introduce error into the analyses.
## Statistical analysis
Unpaired t-tests were used for comparisons of baseline characteristics between
NMD and control groups. Descriptive statistics are presented as mean and
standard deviation (SD), unless specified otherwise. Normality of outcomes data
was tested using the Shapiro-Wilk test. Control data were normally distributed
but not NMD data. Therefore, we used ANOVA to compare the SNIP<sub>OP</sub>,
SNIP<sub>CL</sub> and MIP measurements in controls, and (nonparametric) Friedman
ANOVA for the NMD group. Intraclass correlation coefficients (ICC) were
performed for combinations of SNIP<sub>OP</sub>, SNIP<sub>CL</sub> and MIP
within groups. Scatter plots and Bland-Altman plots were generated, and bias was
defined as the mean of the differences between two measurement values. Limits of
agreement were calculated as bias +/- (1.96 x SD for the difference). The Fisher
exact test was used to compare counts. Statistical significance is defined as
p\<0.05. Analyses were done using SAS software, version 9.3.
### Power calculation
Our sample size was based on a detectable difference between SNIP<sub>CL</sub>
and SNIP<sub>OP</sub> of 10 cmH<sub>2</sub>O, which we considered the minimum
that would be relevant, and assumed a normal distribution. For a sample size of
50 patients (in each group separately), using a paired t-test and conservative
estimate for the standard deviation of the difference of 20 cmH<sub>2</sub>O, we
would have a power of 93% to detect a difference of 10 cmH<sub>2</sub>O with
type I error of 0.05.
# Results
shows demographic and PFT data for the 52 NMD patients and 52 control subjects
included in the study. The two groups did not differ with respect to age,
although the control group tended to include more females and had a higher
average body mass index. NMD patients demonstrated mild to moderate reductions
in spirometric values, which were significantly lower than the control group
(73.1% vs. 98.6% of predicted for FVC, p\<0.001).
shows MIP, SNIP<sub>OP</sub> and SNIP<sub>CL</sub> values in the control and NMD
groups. As expected, all values were significantly lower in NMD compared with
control subjects (p\<0.001). Neither age nor sex correlated with
SNIP<sub>CL</sub> values in the control and NMD groups, whereas age was weakly
correlated with SNIP<sub>OP</sub> (r = 0.295, p = 0.03) in the NMD group only.
The mean SNIP<sub>OP</sub> value was significantly lower than SNIP<sub>CL</sub>
and MIP in both groups. Results were identical irrespective of the order in
which SNIP<sub>OP</sub> and SNIP<sub>CL</sub> were performed (results not
shown). Scatter plots demonstrating the relationships between these parameters
in individual patients are shown in.
To assess agreement between measurements, ICC was calculated between
SNIP<sub>OP,</sub> SNIP<sub>CL</sub> and MIP. Agreement was poorer in the
control subjects than in the NMD group for all combinations of measures. In both
groups the highest agreement was for SNIP<sub>OP</sub> vs. SNIP<sub>CL</sub>,
and SNIP<sub>CL</sub> was in better agreement with MIP than SNIP<sub>OP</sub>.
Agreement and bias were further assessed using Bland-Altman plots. These plots
indicate that SNIP<sub>CL</sub> is greater than SNIP<sub>OP</sub> for the
majority of subjects, with a mean bias of -15.04 in NMD and -19.9 in controls.
Moreover, the bias between SNIP<sub>CL</sub> and MIP was substantially lower
(consistent with better agreement) than between SNIP<sub>OP</sub> and MIP. This
was true for both NMD and control groups, although the limits of agreement are
narrower (less scatter) for NMD compared with control subjects. Visual
inspection of the plots also suggests less scatter at lower values, particularly
in NMD patients.
To assess whether SNIP<sub>CL</sub> might be more useful clinically than
SNIP<sub>OP</sub> to rule out inspiratory muscle weakness in subjects with
reduced MIP, we determined how often SNIP<sub>OP</sub> or SNIP<sub>CL</sub> were
higher than MIP in individuals with a low MIP value, as determined using three
different thresholds for MIP. For MIP \< 80% of predicted, SNIP<sub>CL</sub> was
higher than MIP more frequently than SNIP<sub>OP</sub> in NMD patients (40% vs.
14%, p = 0.03). For MIP \< 60% of predicted, SNIP<sub>CL</sub> was also more
often higher than MIP as compared to SNIP<sub>OP</sub> in NMD patients (48% vs.
10%, p = 0.02). In control subjects, MIP was \< 80% of predicted in 4 subjects
and \< 60% of predicted in 1 subject. The latter subject had both
SNIP<sub>OP</sub> and SNIP<sub>CL</sub> higher than MIP, while another control
subject had a SNIP<sub>CL</sub> (but not SNIP<sub>OP</sub>) higher than MIP.
Finally, we assessed subjects with MIP \<80 cmH<sub>2</sub>O, selected because
this represents a threshold value above which clinically significant inspiratory
muscle weakness is considered to be highly unlikely. SNIP<sub>CL</sub> and
SNIP<sub>OP</sub> were higher than MIP in 44% and 14% of 36 NMD patients with
MIP falling below this threshold, respectively (p = 0.003). In controls, this
occurred in 69% vs. 31% of 13 subjects, respectively (p = 0.12).
Lastly, we evaluated how often SNIP<sub>OP</sub> or SNIP<sub>CL</sub> values
fell within the normal range in subjects with reduced MIP. The recommended lower
limit of normal (LLN) for SNIP is 70 cmH<sub>2</sub>O for males, and 60
cmH<sub>2</sub>O for females. In NMD patients with MIP\< 80 cmH<sub>2</sub>O, 1
subject had a SNIP<sub>CL</sub> value \> LLN, whereas this did not occur for
SNIP<sub>OP</sub> in any NMD patient. In controls with MIP \< 80
cmH<sub>2</sub>O, 3 subjects had both SNIP<sub>OP</sub> and SNIP<sub>CL</sub>
values \> LLN, while 4 had only SNIP<sub>CL</sub> \> LLN and none had only
SNIP<sub>OP</sub> \> LLN.
# Discussion
Although MIP is the most widely used test of inspiratory muscle strength in
standard clinical practice, it is clear from previous work that the use of a
single test such as MIP tends to overdiagnose weakness. SNIP has thus been
recommended as a complementary test to help address this issue, particularly in
NMD patients. However, SNIP<sub>OP</sub> and SNIP<sub>CL</sub> have been
utilized in a seemingly interchangeable fashion by different investigators to
assess inspiratory muscle strength. Moreover, very few studies in the literature
have actually reported on the use of SNIP<sub>CL</sub> to evaluate inspiratory
muscle function. To our knowledge, this is the first study to formally compare
the two methods of SNIP measurement in NMD patients as well as control subjects
with normal inspiratory muscle strength. Our main findings are that
SNIP<sub>CL</sub> values are systematically greater than SNIP<sub>OP</sub> in
both NMD and controls, and that the level of agreement with MIP is also superior
for SNIP<sub>CL</sub> in comparison to SNIP<sub>OP</sub>. Therefore, in patients
with a low MIP value, SNIP<sub>CL</sub> appears to be a more useful test than
SNIP<sub>OP</sub> for excluding inspiratory muscle weakness.
Both MIP and SNIP have a learning effect and are operator-dependent, but
several aspects of SNIP may be more advantageous in NMD patients. The SNIP
requires only a short burst of maximal inspiratory muscle contraction, whereas
the MIP involves sustaining a maximal inspiratory effort for at least 1 second.
This more prolonged effort required for MIP may be difficult for some patients,
resulting in falsely low values. Furthermore, in principle SNIP can be performed
in individuals who are unable to maintain a tight lip seal around a mouthpiece,
which is frequently the case in NMD. The maneuver required for SNIP is also
generally regarded as more natural and easier to explain to patients. In keeping
with the above, SNIP was reported to be more predictive of outcomes than MIP in
ALS and Guillain-Barré syndrome.
SNIP<sub>OP</sub> has been reported to be higher than MIP in some studies, and
this appears to be more prevalent in those individuals with the least amount of
weakness. Conversely, Hart et al. found, in a group of NMD patients, that MIP
was greater than SNIP<sub>OP</sub> (4.8 cmH2O bias with both tests performed
from FRC), and SNIP<sub>OP</sub> was lower as a proportion of MIP in those
patients with the most severe impairment. We speculate that the above findings
are at least partly explained by an inability of very weak patients to generate
a sufficiently negative inspiratory pressure to collapse the nasal valve within
the open nostril during SNIP<sub>OP</sub> measurements. In contrast, by
occluding the nostril during SNIP<sub>CL</sub>, the measurement becomes a static
one such that pressures are more readily equilibrated throughout the airways.
It is interesting to note that SNIP<sub>OP</sub> (but not SNIP<sub>CL</sub>) was
also lower than MIP in the control subjects of our study, suggesting that
additional factors other than weakness are involved in the better equilibration
of pressures achieved with SNIP<sub>CL.</sub> One potential factor could be the
presence of airflow obstruction at the lower airway level, although this appears
unlikely since we excluded individuals with abnormal spirometry in our control
group. However, obstruction could occur at the upper airway (e.g., nasal) level,
which we did not assess. A possibility also exists that the pattern and/or level
of inspiratory muscle recruitment differs between SNIP<sub>CL</sub> and
SNIP<sub>OP</sub>. This might also help to explain a closer correlation between
SNIP<sub>CL</sub> and MIP, since the two are similar in being "static" in nature
compared to the more "dynamic" SNIP<sub>OP</sub>. The specific maneuver itself,
i.e., sniff vs. Mueller, is also an important element in determining inspiratory
muscle recruitment, with brief sniffs generally producing higher values of
diaphragm activation and transdiaphragmatic pressure than the inspiratory
maneuver employed for MIP. However, SNIP<sub>OP</sub> may conversely generate
lower pressures than static maneuvers due to shortening of inspiratory muscles
and the attendant pressure-velocity relationship. In a clinical context, these
sources of variability are difficult to ascertain, but the tests are not
interchangeable and should be viewed as complementary.
## Critique of methods
It should be noted that our study design contains several elements which
accurately reflect the routine clinical evaluation of NMD patients but may also
introduce increased variability in the measurements. For example, NMD patients
were comprised of a heterogeneous group of diagnoses with different levels of
weakness. In this regard, Terzi et al. previously reported much wider limits of
agreement between SNIP<sub>OP</sub> and MIP in myotonic dystrophy than in
Duchenne muscular dystrophy. In addition, our control group was a clinical one
rather than being composed of entirely healthy volunteers, although it should be
emphasized that all control group subjects had normal spirometry. The MIP was
initiated from RV as per standard clinical practice and American Thoracic
Society recommendations, whereas SNIP was measured at FRC according to the
original description of the technique. These lung volume differences would be
expected to result in a small (less than 10 cmH<sub>2</sub>O) change in
inspiratory force generation, which is quite consistent with the average
magnitude of MIP minus SNIP<sub>CL</sub> differences found in our study. Given
that the study subjects did not undergo any prior training period, one possible
limitation of the current study might be insufficient learning of the procedure.
However, this appears unlikely since results were similar regardless of which
test was performed first. Finally, as noted above we did not objectively measure
nasal resistance, which can also affect SNIP reliability.
It is important to emphasize that although SNIP test result variability may have
been increased by one or more of the above factors, our findings are likely more
generalizable to real world clinical practice for the very same reasons. In
addition, since technical measurement errors often underestimate but are very
unlikely to overestimate muscle strength, respiratory muscle pressure generation
is primarily used as a “rule out” test for muscle weakness. Accordingly, our
study suggests that the use of SNIP<sub>CL</sub> in this manner may help to
prevent clinical misclassification of certain patients who might otherwise be
considered as having significant inspiratory muscle weakness based on low values
for either MIP or SNIP<sub>OP</sub>.
# Conclusions
The SNIP<sub>CL</sub> maneuver produces values which are systematically higher
than SNIP<sub>OP</sub> and therefore likely represents a more useful test for
ruling out inspiratory muscle weakness. Accordingly, we propose that whenever
MIP is low or cannot be performed, SNIP<sub>CL</sub> should be used to obtain
further information on inspiratory muscle strength. Clearly, the use of
different tests of respiratory muscle strength should be considered
complementary in nature as previously suggested by others.
# Supporting information
We would like to thank Pei Zhi Li for her work on data analysis and figures.
[^1]: Basil Petrof has received an investigator-initiated grant and speaker
fees from Sanofi Genzyme Inc. This does not alter our adherence to PLoS One
policies on sharing of data and materials.
[^2]: **Conceptualization:** BP MK. **Data curation:** FN MK. **Formal
analysis:** MK FN. **Funding acquisition:** BP. **Investigation:** BP.
**Methodology:** BP MK. **Project administration:** BP MK. **Resources:** MK
BP. **Supervision:** MK BP. **Validation:** MK FN. **Visualization:** MK BP.
**Writing – original draft:** MK. **Writing – review & editing:** BP MK. |
# Introduction
Plant communities are known to be shaped by competition for resources such as
light, space, nutrients, and water. Differences in species' abilities to capture
or alter these resources across space and time can drive competitive
hierarchies, successional dynamics, community diversity, and invasions. One of
the best known attempts to describe these complex interactions with a simple set
of rules is R\* resource competition theory. In this theory, Tilman posits that
plant species differ in their ability to deplete a limiting resource, and the
species that maintains a positive growth rate at the lowest level of this
resource will emerge as the competitive dominant. Following from Tilman, R\* is
the equilibrium concentration of resources necessary for the consumer (plant)
species to maintain a stable population. Although seemingly simplistic, this
theory has successfully predicted the outcome of plant competition in various
ecosystems, including California grasslands, alpine meadows, and Midwestern
prairies.
Part of R\* theory's success stems from providing clear predictions and testable
hypotheses for ecologists. For example, it is relatively simple to quantify
differences in resource depletion among species by planting monocultures and
measuring resource concentrations after biomass stabilizes (R\*). Determining
whether R\* values predict competitive hierarchies and structure communities can
be accomplished by comparing these monoculture R\* values with the relative
abundance of those species in mixtures (i.e. after competition). A negative
relationship between species' R\* and their relative biomass in mixture supports
R\* theory, since the species depleting resources to the lowest levels are the
competitive dominants.
An open question is, what drives species-specific R\* values in the first place?
Differences in species R\* levels for a soil nutrient may be due to differences
in plant uptake, differences in how plants affect microbial nutrient cycling and
sequestration, or both. However, the relative contribution of plants and
microbes to the observed resource depletion remains largely untested. R\* theory
in terrestrial ecosystems has largely been tested in perennial communities or
perennial communities subjected to annual invaders,. In such systems it is
difficult to disentangle over multi-year timescales the relative contributions
to R\* of plant uptake, microbial nutrient cycling, and the indirect effects of
plant traits such as litter quality and other plant-soil feedbacks.
Plants are often assumed to be the major drivers of R\*. For example, in systems
limited by nitrogen (N), depletion has largely been assumed to be due to plant N
uptake and sequestration in plant tissues coupled with litter-mediated
feedbacks. Plant species can also alter microbial community composition, size,
and activity, and thus alter available soil N concentrations through differences
in plant traits such as quality of root exudates or root and leaf litter. These
effects of plant species on microbial N cycling rates can occur over short time
scales. Carbon-rich root exudates from grasses, for example, have been shown to
increase microbial respiration and N turnover in rhizosphere soils within 24
hours, resulting in higher N uptake by grasses.
However, microbes are also strong competitors for N in soils, given their high
surface area. Microbes are integral in the cycling of N in soils by breaking
down large organic N molecules into smaller compounds (e.g., breaking proteins
into amino acids and ammonium), and converting ammonium into nitrate. While
microbial biomass is typically not large, it turns over rapidly. As microbial
biomass turns over, N from microbial “necromass” can become less available to
plants and microbes as it is incorporated into soil organic matter and is
protected from decomposition by a range of chemical, physical, and biological
mechanisms.
To test the extent to which plant or microbial controls on N availability
underlie the R\* theory of plant competition, we established an experiment in a
California grassland ecosystem. We planted annual grass and forb species in
monoculture and mixture plots and followed them over one growing season. Our
focus on an establishing annual system is very different than the majority of
work examining R\* theory, most of which has been done over multiple years in
perennial systems and does not explicitly examine the extent to which plants or
microbes are driving the patterns. Although it has been previously established
that plant-microbial feedbacks mediated by litter are associated with species'
R\* after several years in perennial systems, this has not been studied
mechanistically (i.e., by explicitly examining the plant and microbial N pools
and fluxes). Moreover, in the first year of community establishment in an annual
system, such feedbacks are unlikely to be important drivers. Thus, examining an
annual system over a single growing season allows for a detailed examination of
short-term impacts of plants on microbial activity, and microbial activity on
plant growth and nutrient acquisition, all in the relative absence of litter
mediated feedbacks. As such, this study represents a first step in describing
the mechanisms that drive patterns of resource competition in R\* theory.
We have previously reported that the available soil N levels in monocultures in
this experiment were correlated with the competitive hierarchy among plant
species, and thus, that R\* for N (and therefore, competition for nitrogen)
strongly influences community dynamics in this system. The importance of
competition for N is not surprising, given that grasslands in this locality have
been shown previously to be N-limited. Here we examine the degree to which plant
and microbial controls drive N levels in soils, and directly or indirectly
influence plant-plant competitive dynamics.
A range of different relationships could emerge between plants, microbes, and
R\*. If differences in R\* are driven primarily by plant N uptake, we would
expect to see a strong negative relationship between plant N uptake and
dissolved inorganic N (DIN), our metric of R\*. Furthermore, we would expect no
relationship between microbial N cycling and DIN. However, if differences in R\*
are driven primarily by changes in microbial N cycling rates (*e.g.*, net N
mineralization), we would expect that microbial N cycling would be positively
correlated to DIN, while plant N uptake would have no relationship with DIN. If
differences are driven by both differences in plant uptake and in microbial N
cycling, we would expect a pattern somewhere in between the plant dominated and
microbe dominated patterns with a slight positive correlation between microbial
N cycling and DIN, and a slight negative relationship between plant N uptake and
DIN.
While we expected that both direct plant uptake of DIN and microbial N cycling
would contribute to R\*, we were interested in where on the continuum of plant
to microbial control this annual ecosystem would fall. Across the different
monoculture and bare plots, we measured: 1) pool sizes of total N in plants; 2)
DIN at the end of the growing season (our measure of R\*) and over time; 3)
microbial pools and process rates (microbial N, microbial biomass, net N
mineralization, gross NO<sub>3</sub><sup>−</sup> production, and nitrification
potential). To tease out drivers we then examined relationships in bare and
monoculture plots between DIN and: plant biomass; microbial biomass and biomass
N; and microbial process rates.
# Materials and Methods
## Site and Experimental Design
Experimental plots were established in a pasture/grassland with permission from
the owner, the Midland School, in Los Olivos, California. The climate is
Mediterranean, with hot, dry summers and cool, wet winters and an average annual
rainfall of 550 mm. The land had not been plowed or tilled since 1940, but these
soils were not undisturbed; gopher activity causes extensive physical turnover
of surface soils in California grasslands, and has been estimated to give
complete turnover of surface soils every 3–5 years. Plots have also been grazed
by cattle annually, presumably also causing soil disturbance. The vegetation is
a mixture of annual and perennial herbaceous forbs and grasses growing with
occasional oak trees. Soils are typic Argixerolls, with gravelly fine sandy loam
texture.
Study species included three native annual forbs (*Amsinkia menziesii,
Calandrinia ciliata, Clarkia purpurea*), three native annual grasses
(*Muhlenbergia microsperma, Vulpia microstachys,* and *Vulpia octoflora*), and
six exotic annual grasses (*Avena barbata, Bromus hordeaceous, Hordeum murinum,
Lamarkia aurea, Polypogon monspielensis* and *Vulpia myorus*). All species were
present in nearby grasslands (Stan Harpole, personal communication) and thus our
study plants reflect a realistic representation of current California annual
species. All data were collected in winter, spring, and summer of 2006.
Five ‘blocks’ were established within the grassland and were separated by 50 to
500 m. Vegetation was cleared by spraying with Roundup in fall 2005 and roto-
tilling with a tractor two weeks after plants had died. Within each block,
fourteen 0.64 m<sup>2</sup> plots were established separated by 1 m buffers, and
plots were randomly assigned treatments (species monocultures, mixtures, or bare
plots). There was one full replicate of the experimental design within each
block, consisting of one bare, one mixture (with all 12 species), and 12
monoculture plots, giving a total of five replicate plots for each plot type
between the five blocks. Each block was fenced to exclude cattle grazing (see
for further details). Data from mixture plots was used to establish the R\*
relationship in HilleRisLambers et al., by relating relative biomass in mixture
to DIN in monoculture plots. Because we were trying to determine what drives DIN
concentrations in the monoculture plots used to test R\* theory, we focused on
monoculture plots only for this study.
To establish plots, we collected seeds locally (*A. barbata, B. hordeaceous, H.
murinum, L. aurea*, and *V. myorus*) or used seed from local seed companies (*A.
menziesii, C. ciliata, C. purpurea, M. microsperma, P. monspielensis, V.
microstachys*, and *V. octoflora*). We added a total of 15 grams of
seed/m<sup>2</sup> to each plot, with that weight being divided equally among
the species in mixture plots. Seeds were added at the beginning of the growing
season (late November 2005) and plots were watered with a volume equivalent to 3
inches of rainfall to encourage germination and establishment. We weeded plots
twice to remove nontarget species, once soon after germination and once midway
through the growing season. Seeding amounts in monoculture and mixture plots
were sufficiently high to generate plots with little bare ground visible for all
but two species (JHRL, personal observation), including *M. microsperma*, which
failed to germinate. *M. microsperma* thus served as a type of bare plot, and
may be considered as a more appropriate control because it had an initial N
input from seed, similar to other planted plots. We refer to it as bare (seeded)
in the rest of the manuscript and figures.
## Soil N Pools and Dynamics
We collected all soils immediately after plant biomass harvest. We sampled soil
by extracting two 5 cm diameter, 10 cm depth soil cores from each plot. From
each of the five replicate plots for each species, duplicate cores were sieved
(2 mm mesh size) prior to soil and microbial analyses (see *Microbial N Pools
and Process rates*, below). Dissolved inorganic N (DIN hereafter) was quantified
by extracting ammonium and nitrate (NH<sub>4</sub><sup>+</sup> and
NO<sub>3</sub><sup>−</sup>) with 2M KCl which were then analyzed on a Lachat
flow injection autoanalyzer (Lachat Instruments; Loveland, USA), while soil
moisture was determined gravimetrically after six days drying at 60°C. DIN was
our measure of R\*. Total soil N pool (which includes DIN, as well as organic N)
was measured on dried soils after they were ground in a ball mill (Wig-L-Bug
amalgamator, Crescent Dental, Lyons IL), after which Carbon (C) and N content
were measured on a Carlo Erba NA 1500 CHN analyzer (Fisons Instruments, Beverly,
MA).
Soil bulk density was measured in June by collecting five soil cores randomly
placed within plots in each block (4 cm diameter, 10 cm deep). These were
returned to the lab, dried for 48 h at 60°C, rocks were removed, and remaining
soil weighed. Block-specific bulk densities were used to convert all soil
measures to g m<sup>−2</sup> unit values to enable comparison of N budgets
between soils, plants, and microbes. Outcomes of our statistical analyses on
soil N pools did not differ given different N measurement units (i.e., g N
m<sup>−2</sup> versus g N kg<sup>−1</sup> soil).
To determine whether our R\* measure (DIN pools in May) was representative of N
dynamics over the growing season, we used resin stakes (PRS Probes; Western Ag
Innovations, Saskatoon, Canada) to track inorganic N levels in soils throughout
the experiment. Plastic stakes with ion exchange resin surfaces were placed in
0–5 cm soils in mid-month February 2006, and switched each month thereafter
until mid-month June 2006. Thus the May sample date represents resin-available N
sampled between mid-April and mid-May. Excess soil was washed off of probes with
DI water. Probes were then extracted in 0.5 M HCl, and extracts were analyzed
for NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>−</sup> as described
above. Regenerated stakes were placed into the same slots in the soil as
previous stakes.
## Plant N Pools
Plant biomass was harvested in mid-May for all species; this timing was chosen
based on close monitoring of the diverse phenologies of the species in the
experiment. At this time, all species had reached peak biomass and set seed, but
had not senesced. Aboveground plant biomass was sub-sampled in each plot by
clipping all vegetation within one 10×50 cm quadrat. Roots were collected from
duplicate soil cores collected within the clip plots by sieving soils (see above
in *Soil N Pools and Dynamics*) in each of the five replicate plots for each
vegetation type. All plant material was dried at 60°C for six days and weighed.
Plant material was ground with a Wiley mill (Thomas Scientific, Philadelphia,
PA) followed by a ball mill. Plant samples were then analyzed for C and N on the
CHN analyzer. Total plant N (g N/m<sup>2</sup>) in monoculture plots was then
quantified as (aboveground biomass/m<sup>2</sup> × shoot % N/100) + (belowground
biomass/m<sup>2</sup> × root % N/100).
## Microbial N Pools and Process Rates
Prior to examining microbial biomass and N cycling, sieved soils were adjusted
to 35% water holding capacity (WHC) to eliminate the confounding effects of
variable water content on process rates. Soils were allowed to equilibrate for 7
days to allow the effects of sieving and drying/rewetting to pass, after which
soils were weighed out for all analyses. Lab replicates were not included given
that soils were sieved, there were five field replicates per treatment, and past
work using these same techniques in soils from this area showed tight agreement
among lab replicates.
To examine how microbial biomass N relates to R\*, we used a chloroform slurry
extraction to examine the flush of N released from microbial cell lysis and
extraction. Briefly, 25 mL of 0.5 M K<sub>2</sub>SO<sub>4</sub> was added to
each glass tube containing a 4 g soil sample. To this, 0.5 mL of EtOH-free
chloroform was added. Tubes were sealed with PTFE lined caps, and shaken for 4 h
at 150 rev min<sup>−1</sup> on an orbital shaker. Tubes were allowed to settle
for 10 minutes allowing the bulk of the chloroform and soil slurry to separate.
The top 10 mL of extract was filtered through a Pall A/E glass fiber filter
(Pall Corporation, Port Washington, NY, USA), then bubbled with air for 20–30
minutes to remove any residual chloroform. Chloroform slurries were compared to
slurries extracted without chloroform.
To test the influence of microbial abundance on R\* we used substrate induced
respiration (SIR), an index of microbial biomass. Briefly, 10 mL of a 12 g
L<sup>−1</sup> Difco Yeast Extract (Becton, Dickinson and Company, Franklin
Lakes, NJ, USA) was added to 4 g soil. Soils were capped and shaken for four
hours with 1 mL headspace CO<sub>2</sub> subsamples withdrawn at 20 minutes, two
hours, and four hours. Gas samples were analyzed on a LI-6262 infrared gas
analyzer (LI-COR, Lincoln, NE, USA). The SIR biomass was then calculated by
applying a linear fit to the change in CO<sub>2</sub> over time (average
r<sup>2</sup> = 0.99).
Because microbial biomass turnover rates are faster than those of annual plants,
microbes may have driven inorganic N availability in soils via process rates
that are not reflected in microbial biomass pools. Thus, we also quantified
potential net N mineralization, gross NO<sub>3</sub><sup>−</sup> production, and
nitrification potential. Net N mineralization is the net accumulation of
extractable NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>−</sup> in soil,
and represents the balance between microbial production and consumption. Gross
nitrification separates microbial production and consumption of
NO<sub>3</sub><sup>−</sup>, and offers insight into nitrifier abundance and
ambient substrate (NH<sub>4</sub><sup>+</sup>) availability. In contrast to
gross rates, nitrification potential assays add NH<sub>4</sub><sup>+</sup> in
excess and thus rates are driven by—and serve as an index of—the abundance and
potential activity of ammonia oxidizers. Net accumulation of inorganic N is
associated with conditions where heterotrophic microbes are not limited by N
availability, while high gross rates of nitrification and high nitrification
potential are associated with high availability of NH<sub>4</sub><sup>+</sup>
and NO<sub>2</sub><sup>−</sup> to autotrophic nitrifiers.
For net N mineralization, two sets of 4 g samples were used, with one set
extracted on the first day of incubation for one hour with 25 ml of 0.5 M
K<sub>2</sub>SO<sub>4</sub>, while the other set was incubated for 60 days at
20°C prior to extraction. Net N mineralization was then calculated as the
difference in extractable NH<sub>4</sub><sup>+</sup> and
NO<sub>3</sub><sup>−</sup> in soils between the initial and final time points.
These rates can be seen as potential rates because they were held at optimal
moisture and temperature for microbial activity. Our method has the advantage of
controlling for microclimate variability that may affect mineralization rates,
but is not expected to perfectly match field rates.
To quantify gross nitrification, we took duplicate 4 g samples of soil at 35%
WHC and added 0.25 ml of 25.5 mg N/L (98% enriched
<sup>15</sup>N-NO<sub>3</sub><sup>−</sup>) stirring with the pipette tip after
addition. Samples were then extracted for one hour with 25 ml of 0.5 M
K<sub>2</sub>SO<sub>4</sub>, with one sample extracted at 15 min, and the other
sample at 24 h. After NO<sub>3</sub><sup>−</sup> was measured, extracts were
prepared for isotope ratio mass spectrometry. First, NH<sub>4</sub><sup>+</sup>
was converted to NH<sub>3</sub> with MgO and driven out of solution. Then,
NO<sub>3</sub><sup>−</sup> was converted to NH<sub>3</sub> by Devarda's Alloy in
the presence of MgO, and the NH<sub>3</sub> was then captured on acidified
filter disks in teflon packets using the methods of Sørensen and Jensen. Filter
packs were removed and placed in a desiccator with DrieRite and concentrated
H<sub>2</sub>SO<sub>4</sub> to dry filters and trap any free NH<sub>3</sub>.
Filters were folded into tin capsules, and <sup>15</sup>N enrichment was
measured at UCSB's Marine Science Institute Analytical Lab using a Thermo-
Finnigan MAT Delta+ Advantage (Thermo Fisher GmBH, Dreieich, Germany). Gross
nitrification rates were calculated following Hart et al.. We aimed to measure
gross N mineralization in addition to gross nitrification, however
NH<sub>4</sub><sup>+</sup> pools in soils were too low, and consumption too
high, such that it precluded quantifying gross N mineralization.
Nitrification potential was measured by adding 35 ml of a stock solution
containing 8 mL 0.2 M K<sub>2</sub>HPO<sub>4</sub>, 1 ml 0.2 M
KH<sub>2</sub>PO<sub>4</sub>, 5 ml 0.2 M
(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, and 20 ml of 1 M NaClO<sub>3</sub>
(added to block the activity of nitrite oxidizers;). The rate of appearance of
nitrite was measured at three time points over 4 h using a modified Griess-
Illosvay reagent, with 1 ml of color reagent added to 4 ml of sample.
## Statistical Analyses
We asked what the patterns in DIN were over the course of the growing season
under our different monoculture types (i.e. plant community composition, time)
by fitting the resin N data with five linear generalized mixed effects models
(with block as random effect in all), including: a null model; a model with only
species identity as explanatory variable; a model with only time as the
explanatory variable; a model with both time and species identity as explanatory
variables; and a model with the main effects of time and species identity as
well as their interaction. Specifying block as a random effect with time as an
explanatory variable is analogous to a repeated measures ANOVA. The log of resin
N values was used as our response variable to ensure normality. We used AIC's to
determine the best-fitting of the five models, and the appropriate likelihood
ratio tests to determine the significance of explanatory variables retained in
the best fitting model. We also used resin-N data to test whether our one time
measure of soil DIN represented soil dynamics across the season, by correlating
species-specific cumulative resin N values for each species (over the entire
growing season) to species-specific DIN values. We used Kendall's tau rather
than Pearson's r for this correlation because both DIN values were non-normally
distributed.
We also asked whether DIN values were greater in plots without any vegetation,
as we would expect if plants influence available soil N. We did this using a
generalized mixed effects model with DIN (log transformed) as the response
variable and the categorical explanatory variable ‘vegetated’ vs. ‘bare’.
We then asked how species-specific DIN related to N pools and fluxes. First we
used 1- way ANOVAs with species as a fixed effect and block as a random effect
to ask whether plant and microbial parameters were different between species. We
then quantified the correlation between DIN and either: total plant N; Microbial
N; Microbial biomass (substrate induced respiration, SIR); Net N mineralization;
Nitrification potential; and Gross nitrification (we use the term “microbial
biomass/process rates” to encompass these measurements of biomass or process
rates hereafter). Kendall's tau was used to assess the relationship between
these variables since many were non-normally distributed. If plant uptake was
driving N dynamics, we expected to find a negative relationship between DIN and
plant N, and no clear relationship between DIN and other N pools and fluxes. By
contrast, if microbial processes were driving R\*, then we would expect to find
positive correlations between microbial biomass/process rates and DIN (our proxy
for R\*), but no clear relationship between plant biomass N and DIN. If a
combination of microbial cycling and plant uptake were driving DIN, we would
expect a modest positive correlation between microbial biomass/process rates and
DIN, and a modest negative correlation between plant biomass N and DIN.
While examining the longer term effects of litter mediated plant-soil feedbacks
is beyond the scope of this study, we used linear regression to examine the
relationship between plant C:N and plant biomass to provide some insight into
potential relationships. A positive correlation between biomass and litter C:N
would reinforce the short-term R\* patterns, with plants that reach high biomass
and draw N levels to the lowest levels further depleting soil N with C rich and
N poor litter. A negative correlation could suggest that litter-feedbacks might
drive the system in the opposite direction, with plants that achieve lower
biomass and have a higher R\* potentially slowing decomposition over longer time
scales with high C:N litter.
Finally, we asked how plant productivity (as measured by total plant biomass per
m<sup>2</sup>) related to total available N pools (summed over soils, microbes
and plant biomass). We used Kendall's tau for these correlations. All analyses
were performed using R version 2.13.1.
# Results
## N Pools and Dynamics
In examining N pools in this annual grassland, total soil N accounted for 154±4
g N/m<sup>2</sup>. Microbial biomass N and DIN pools
(NO<sub>3</sub><sup>−</sup>+NH<sub>4</sub><sup>+</sup>) comprised a small
fraction of soil N pools, with each accounting for about 0.3% of the total soil
N pool. In contrast to microbes, plants were a much larger N pool, about ten-
fold higher than microbial biomass N.
Our indicator of growing season N dynamics, resin N, varied by species over time
for all monoculture plots. A model including species identity, time, and their
interaction gave the best fit, as indicated by AIC values (data not shown).
Resin available N increased in May in all plots, with a pronounced increase in
both seeded bare plots as well as plots with low plant biomass (*Vulpia
octoflora*, Vo; *Hordeum murinum*, Hm; and *Calandrinia ciliata*, Cc). In
contrast, this response was tempered in monoculture plots with high plant
biomass (e.g., *Clarkia purpurea*, Cp).
The five-month sum of resin-available N was significantly correlated with DIN
pools measured in May. This shows that our R\* measure (May DIN pools) was
correlated with the soil N dynamics through the growing season. The temporal
trends in the resin N data showed that plots with high plant biomass maintained
lower DIN. Consistent with these data, we found that vegetated plots had lower
DIN (R\*) values than bare plots.
## Plant N Uptake
Monoculture plant biomass N was significantly and negatively correlated with
soil DIN, and gave a similar trend with resin-available N. Monoculture plots
with high plant N (e.g., *Clarkia purpurea*) consistently exhibited lower levels
of resin-available N than bare plots. When comparing N pools of plants and
microbes within monoculture plots, even for those plants that took up very
little N (*e.g., C. ciliata*), plant biomass was still a larger N pool than
microbial biomass. This difference became greater with increasing aboveground
plant biomass.
We found that plant species differed in their uptake of N into plant biomass.
Differences in plant biomass N were related to differences in total plant
biomass yielding a strong positive correlation between the two
(r<sup>2</sup> = 0.81, p\<0.001, n = 54), whereas plant % N showed a slight
negative correlation with N plant biomass N (r<sup>2</sup> = 0.10, p = 0.02,
n = 54). Plant species that obtained higher biomass in monoculture plots also
had higher C:N ratios (C:N aboveground biomass versus biomass in monoculture:
r<sup>2</sup> = 0.67, p = 0.007, data not shown).
## Microbial N Uptake and Process Rates
Microbial biomass N and SIR biomass did not differ in soils across plant
monoculture plots, and had no relationship with DIN. Similarly, there was no
relationship between DIN and our indicator of microbial N cycling rates, net N
mineralization. Our two metrics of the conversion of NH<sub>4</sub><sup>+</sup>
to NO<sub>3</sub><sup>−</sup>, nitrification potential and gross nitrate
production, did show significant positive correlations with DIN.
# Discussion
While a number of studies have shown that a plant species' depletion of soil
available N can be predictive of its competitive ability in mixture, the extent
to which plants or microbes drive the N depletion has remained largely
unexplored. Set in the context of an N-limited annual grassland ecosystem, our
study suggests that plant N uptake was more important than species-specific
effects on microbial processes in driving plant species' differences in their
R\*, and thus uptake determined their competitive dominance in the system. Three
lines of evidence support this interpretation: 1) plant N and soil DIN were
negatively correlated across the monocultures of different plant species ; 2)
microbial N was not correlated with soil DIN; and 3) net N mineralization was
not correlated with soil DIN.
## Evidence that Plants Wear the Pants
The negative correlation between plant N and soil DIN suggests that plant uptake
was largely responsible for the observed species level differences in R\* at the
end of the growing season. Additionally, throughout the growing season most of
the monocultures—especially those species that achieved high biomass—maintained
lower levels of resin-available N than bare plots, where N levels were primarily
a function of microbial processes.
We found that neither microbial biomass N nor net N mineralization was
correlated with soil DIN at the end of the growing season This suggests that
microbes were not driving differences in soil DIN via direct uptake and
immobilization of available N into microbial biomass, and that microbial
processing of organic N was not driving differences in DIN levels across plant
species. It has been suggested in the literature that, while microbes do not
drive available N via direct uptake, they do regulate N via processing and
plants take up the “leftovers”. If this were the case in the present study net N
mineralization would be expected to positively correlate with plant N uptake,
which is a relationship we did not find in our system (net N mineralization
versus plant N uptake: r<sup>2</sup> = 0.05, p = 0.47).
In fact, nitrification potential and gross nitrification were the only measured
microbial parameters that were correlated with DIN. Nitrification is a
dissimilatory process and thus only alters N form, not DIN pool size. Since
nitrifier abundance (nitrification potential) and process rates (gross
nitrification) correlated to substrate pools (DIN), we hypothesize that they
were limited in their activity by the size of their substrate pools of
NH<sub>4</sub><sup>+</sup> and NO<sub>2</sub><sup>−</sup>. This is consistent
with the current understanding of these chemoautotrophic organisms, which are
generally considered poorer competitors for soil N than heterotrophic microbes
or plant roots.
In contrast, the heterotrophic microbes which drive N mineralization and
immobilization were likely not limited by N availability. Rather, they
functioned similarly across monoculture plots regardless of N uptake by plants
and DIN concentrations. This is consistent with low soil C:N values, which
averaged 7.3 (±0.1), indicating that heterotrophic microbes were more limited by
C than N. C-limitation in turn may explain why heterotrophic microbes did not
seem to drive final DIN levels.
## Evidence of plant-microbial interactions
Our data suggest that plant N uptake drove DIN pool sizes in this annual system,
and thus drove R\*. This is consistent with <sup>15</sup>N tracer experiments
which postulate that plants immobilize a greater amount of N into plant biomass
over an entire growing season, and thus drive the longer-term patterns of N fate
in ecosystems. However, these same studies make it clear that microbes can
outcompete plants for available soil N in the short term, with some of the best
evidence for this coming from California grasslands.
Our goal was to test the mechanisms underlying R\* by determining the role of
plant uptake as compared to changes in microbial biomass and microbially
mediated N cycling rates. Understanding such mechanisms is particularly
important in such ecosystems where competition outcomes among annuals in the
first year of community assembly are likely to be important for long-term
community trajectories. By constraining our study to one year in an annual
system, we explicitly removed the confounding effect of plant-microbe feedbacks
as mediated by litter, which are more likely to develop over several years.
Short-term competition stands to be particularly important in these California
grasslands, where disturbance plays a large role in exotic annual grass
invasion, and the dominant competitors are annual.
While our data suggest that plant N uptake drove soil DIN pools, another story
emerges when the focus is shifted from looking at comparisons of individual pool
sizes and fluxes to looking at the total N in plants, microbes, and DIN for each
set of monoculture plots. Examining the sum of N in plants, microbes, and DIN
reveals that there were larger pools of biological and/or bioavailable N in
plots with higher plant biomass. This suggests that microbes released more plant
available N over the course of the growing season in those plots with higher
plant biomass and lower R\*, even though our measure of microbial N turnover,
net N mineralization, did not differ between plots. Comparing planted vs. bare
plots, there was more N in these pools (plant N, microbial N, and DIN) in
planted (5.4±0.3 g N/m<sup>2</sup>) than bare plots (1.3±0.7 g N/m<sup>2</sup>;
F<sub>1,62</sub> = 26.7, p\<0.001) and total N in these pools increased with
increasing plant biomass and decreasing R\*. Thus, while plant N uptake was
working in a manner consistent with our conceptual models, microbial N cycling
may have actually been responding in the opposite direction of our predictions.
In other words, microbial N cycling in the field may have been negatively,
rather than uncorrelated or positively correlated with DIN; in plots with lower
R\* and higher plant N uptake, microbes may have generated more plant available
N than in plots with higher R\* and lower plant N uptake.
It is possible that this pattern—where the sum of plant N, microbial N, and DIN
was higher in plots with lower R\*—could be explained by large losses of
available N from bare plots due to denitrification or leaching. Several lines of
evidence suggest that these explanations are unlikely. Denitrification depends
on labile C, which would have led to higher potential for N losses due to
denitrification in planted plots with roots exuding labile C as opposed to those
that were bare or had sparse cover. Thus denitrification would be expected to
yield more loss of N with increasing biomass, which would lead to a pattern
opposite to that observed in the total N pools. While leaching losses would have
been higher in plots with lower biomass due to decreased evapotranspiration, we
suggest it is unlikely to have been the main driver of the observed patterns.
Over 94% of the annual rainfall had fallen by mid-April, prior to peak plant
growth and strong differences in monoculture soil resin available N. The two
remaining rainfall events were small enough that they would not have caused
significant leaching.
While we cannot rule out denitrification and leaching, we hypothesize that plant
‘priming’ of microbes led to differences in microbial N cycling in the field,
which in turn altered plant uptake of DIN, and thus the total distribution of N
in different ecosystem compartments. Priming is the stimulation of microbial
activity via labile C-rich root exudates. The low C:N ratio of these soils makes
it likely that microorganisms were limited more by C than by N. Plant roots
releasing labile C into rhizosphere soil may have led to localized increases in
microbial activity leading to higher N availability in rhizosphere soil.
Because we did not separate rhizosphere from bulk soil, localized increases in
microbial activity, such as net N mineralization, were less likely to be
observed. Priming has been shown to increase plant N uptake in various
ecosystems, – including California grasslands, where it was shown to increase
gross N mineralization rates by ten-fold. Thus plant-microbe interactions may
have increased N cycling in this current experiment in plots with lower R\*, but
our measures of N cycling were not sensitive to those differences given several
factors, including: the localized nature of this interaction; the removal of
roots and fresh C inputs which would have been the drivers of this interaction;
and the short preincubation following sieving and adjustment of water content.
While our results were consistent with plants driving patterns in R\* through
uptake of N, and showed no evidence of microbial processes or biomass driving
R\*, when ecosystem N pools are taken as a whole those plots that accumulated
more N in microbial, plant, and DIN pools likely had increased cycling and
availability of N to plants. This suggests that even over the course of one
growing season, plant-microbial interactions (*e.g.*, priming) may be important
to ecosystem N cycling and ultimately, may influence plant competition.
It remains an open question as to whether these patterns would persist over
multiple growing seasons, and what kind of impact litter mediated feedbacks
would have in this annual system. In our experiment, plant species that obtained
higher biomass in monoculture plots also had lower quality biomass (higher C:N
in aboveground biomass), suggesting that the patterns observed in this study
would be reinforced over multiple years by litter mediated feedbacks. It is
likely that monoculture plots of annual species with high N use efficiency and
low N litter would depress N mineralization, helping drive low R\* and the
magnitude of this effect could increase with time as feedbacks become better
established. Thus for annuals growing in continuous monoculture, we would expect
to see similar patterns to perennial systems, where the persistence of plants
allows for strong litter feedbacks that help determine plant species' R\* for
nitrogen.
We thank the Midland School (Los Olivos, CA) for letting us perform this
experiment on their property. Ben Munger and Rick Skillin provided invaluable
help in setting up the experiment, and Peter Adler, Anna Oliver, Lilly Hayden
and Rebecca Harris assisted in data collection. We also thank Carla D'Antonio,
Steve Perakis, Josh Schimel, Alexia Kelley, Bonnie McGill, Marissa Lee, Erin
Mordecai, Louie Yang, and three anonymous reviewers.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: SGY BPC JHRL JML. Performed
the experiments: BPC SGY JHRL. Analyzed the data: JHRL SGY BPC. Contributed
reagents/materials/analysis tools: SGY BPC JHRL JML. Wrote the paper: SGY
BPC.
[^3]: Current address: Pacific Island Ecosystem Research Center, United
States Geological Survey, Kilauea Field Station, Hawaii National Park,
Hawaii, United States of America
[^4]: Current address: Department of Biology, Duke University, Durham, North
Carolina, United States of America
[^5]: Current address: Institute of Integrative Biology, Eidgenössische
Technische Hochschule Zurich, Zurich, Switzerland
[^6]: Current address: Biology Department, University of Washington,
Seattle, Washington, United States of America |
# Introduction
Delirium is a frequently observed complication in patients in an intensive care
unit (ICU), that has been associated with long-term cognitive impairment,
prolonged length of ICU stay and with increased health care costs. The
pathophysiology of delirium is complex and heterogeneous. Metabolic disorders
such as hypo- and hyperglycemia have been identified as risk factors for
delirium onset, but extensive research is lacking. To improve patient related
outcomes, identification of modifiable factors in delirium need to be further
explored.
Tight glucose control has been implemented as regular care in critically ill
patients to reduce extreme glucose deviations as hypo- and hyperglycemia,
glucose variability and to decrease the mean glucose concentration with
decreased mortality risk as result. However, the optimal blood glucose range in
tight glucose control is controversial. Intensive glucose control (glucose
target between 4.5–6.0 mmol/l (81.0–108.1 mg/dL)) has been shown to increase
mortality rates compared to conventional glucose control (glucose target ≤ 10.0
mmol/l (180.1 mg/dL)). The occurrence of hypoglycemia during intensive glucose
control may be responsible for this increased risk of death. Furthermore, it has
been reported that the mortality rate after hyperglycemia is higher in non-
diabetic patients compared to diabetic patients, due to adaptive mechanisms to
chronic hyperglycemia in patients with diabetes.
Glucose variability has been associated with higher mortality risk in critically
ill patients. A gold standard for measurement of blood glucose fluctuations is
lacking. Glucose fluctuations were frequently reported as glucose variability
and refer for example to mean glucose concentration, mean absolute glucose (MAG)
change, standard deviation (SD) or hypo- and hyperglycemia.
Delirium and glucose variability have both been associated with negative
outcomes, but their mutual relation has been poorly studied. Higher glucose
values have been reported in critically ill patients with hyperactive delirium
compared to critically ill patients with non-hyperactive delirium. Given that
delirium results from acute illness, it is plausible that this acute illness may
increase activity of hypothalamic-pituitary-adrenal axis leading to increased
cortisol release and subsequent decreased peripheral insulin sensitivity,
thereby contributing to hyperglycemia. It is unclear whether glucose variability
is higher during delirium within the window of glucose control during ICU
admission.
The aim of this study was to determine whether estimates of glucose variability
are altered during delirium in critically ill patients with and without diabetes
in the ICU.
# Materials and methods
## Setting, study design and population
Data were used from a prospective cohort study conducted in the 32-bed mixed ICU
of the University Medical Centre Utrecht (UMCU), the Netherlands. All patients
hospitalized for longer than 24 hours on the ICU in the period from January 2011
to June 2013 were included in this study, except in the case of neurological
illness, if delirium assessment was impossible or patients were unable to speak
Dutch or English. The local Institutional Review Board waived the need for
informed consent in this non-interventional investigation (IRB 010/056/c and
12/421/c) and approved further research with the anonymous data.
The mental status of all ICU patients was daily classified by the research team
as ‘delirious’, ‘awake and non-delirious’ or ‘comatose’ using a 5-step validated
algorithm (interobserver agreement, 0.94–0.97; sensitivity, 0.75; and
specificity, 0.85). This multistep algorithm incorporates a review by a research
nurse of all Confusion Assessment Method for the ICU (CAM-ICU) assessments
conducted by the bedside nurses, whether delirium treatment was initiated and a
meticulous chart review for the presence of documented terms clinically
associated with delirium. When delirium could not be ruled in or out using this
procedure, the research nurse conducted an additional CAM-ICU assessment.
Delirium episodes were recorded and delirium subtype was classified using the 3
hourly registered RASS scores (10 point scale ranging -5 (comatose) to +4
(heavily agitated)). A delirium episode ended if a patient had a classification
of ‘awake and non-delirious’ or a classification of ‘comatose’ for at least two
days.
For this study, patients with delirious and non-delirious observation days were
selected from the study cohort. In case of one delirious episode during ICU stay
all observation days were included until ICU discharge. In case of more than one
delirious episode, observation days until the start day of the second delirious
episode were included for that patient. Patients were excluded if there was no
glucose value available during a delirious episode or during a non-delirious
episode. Observation days were excluded from the study if there were no glucose
values available or if the observation day was classified as ‘comatose’.
## Data collection
Trained, assigned physicians collected data (baseline and per day) from all ICU
patients including demographic data, (chronic) co-morbidities and medication
use, ICU admission characteristics, daily physiological measurements and vital
signs, and therapeutic interventions. Diabetes was marked present if noted in
the medical record or if patients used insulin and/ or oral antidiabetic drugs
at ICU admission. Current alcohol intake was marked as positive if patients used
more than three units of alcohol per day, as documented in the medical records
or history. Current smoking was marked as positive if smoking was written in the
medical records or history. Planned admissions were those admissions which could
be postponed for at least 12 hours without adverse consequences. The Acute
Physiology and Chronic Health Evaluation (APACHE) IV classification was used to
determine the admission diagnosis, severity of disease, and infection at ICU
admission. The extent of chronic comorbidities were measured with Charlson
Comorbidity Index (CCI). The Sequential Organ Failure Assessment (SOFA) score
without central nervous system component was used daily to classify severity of
disease. The presence of severe sepsis or septic shock was classified using
international sepsis definitions at the time of study.
During the study period, a glucose regulation protocol was used to maintain the
target glucose concentration during ICU admission between 5.0 and 8.0 mmol/l
(90.1–144.1 mg/dL), except in those ICU-patients with a low risk of prolonged
hyperglycemia such as per- and postoperative patients with one bolus injection
of dexamethasone. Continuous insulin infusion was initiated in patients with
diabetes and in ICU-patients with a (drug-induced) glucose concentration \> 8.0
mmol/l. Glucose levels were measured on fixed time points between 0.5–4 hours
after the last glucose measurement (details are described in the glucose
regulation protocol) from blood samples obtained from an arterial catheter
using BeckmanCoulter AU5800 (Beckman Coulter Inc., Brea CA, USA) or if arterial
catheter was absent by finger stick using Precision Xceed Pro (Abbott, Abbott
Park, USA). Glucose levels were automatically stored in the electronic patient
data management system (EPDMS, MetaVision, version 5.45, iMDsoft).
Medication use (drug, dose, route and time of administration including total
parenteral nutrition) and glucose measurements (concentration and time of
measurement) were retrieved from the EPDMS and added to the prospectively
collected data. Continuous infusions, such as insulin, were recorded in the
EPDMS, including end date and time of administration. A change in infusion rate
resulted in a new medication record. If a continuous infusion covered more than
one day, the dose per day was calculated using the ratio between infusion times
of both days. Energy intake was defined as the sum of daily caloric intake from
continuous infusion of glucose, total parenteral or enteral nutrition, and high
caloric medication, such as propofol.
## Outcome
The primary outcome was the within-patient difference in glucose variability
during delirious and non-delirious observation days. Glucose variability was
measured each observation day, expressed by the following five measures:
1. mean glucose concentration (mmol/l)
2. SD of all glucose levels (mmol/l)
3. MAG change, defined as the mean absolute glucose change per hour
(mmol/l/hour). To calculate the MAG, all absolute changes in blood glucose
levels were added up and were divided by the time between first and last
glucose levels (in hours).
4. Daily delta, defined as the difference of daily maximum and daily
minimum glucose concentration (mmol/l)
5. Hypo- and hyperglycemia. Hypoglycemia was defined as a glucose
concentration \<3.5 mmol/l (63.1 mg/dL) and severe hypoglycemia was defined
as a glucose concentration \<2.2 mmol/l (39.6 mg/dL). Hyperglycemia was
defined as glucose concentration \> 8.0 mmol/l (144.1 mg/dL) and severe
hyperglycemia as glucose concentration \> 11.0 mmol/l (198.2 mg/dL).
## Data analyses
Patient and observation day characteristics were reported as numbers with
percentages in the case of nominal data and means with SD or median with
interquartile range (IQR) in the case of continuous data. Continuous data were
compared using Student independent sample t tests when the data was normally
distributed; otherwise the Mann-Whitney U test was used. Chi-square tests were
used to compare nominal data.
Characteristics of delirious and non-delirious days in non-diabetic and diabetic
patients were compared in a multilevel technique using linear mixed-effects
models for continuous characteristics and generalized mixed-effects models with
logit link function for dichotomous characteristics. Statistical significance
was considered at p-value \<0.05, when appropriate 95% bootstrap percentile
confidence intervals (CIs) were expressed. Two-stage bootstrap resampling
procedure with ‘patient’ as cluster variable was used for obtaining CI’s and
p-values from 1000 replications.
In the case of one glucose concentration per day the mean glucose concentration,
SD and the difference of daily maximum and minimum could not be calculated. The
MAG change was calculated if there were more than two glucose levels per day
available. Hyperglycemia and hypoglycemia were described as dichotomous outcome
per observation day, but glucose values were analysed individually. Linear
mixed-effects models and generalized mixed-effects models with logit link
function were used as multilevel techniques to test whether delirium was
associated with increased glucose variability. The effects were expressed as
regression coefficients or odds ratios, both with bootstrap 95% CIs. Covariates
were included in the model as fixed effects, when possible as time dependent
covariate. The use of medication was classified dichotomous per day. All models
included random effects for ‘patient’.
The degree of glucose variability depends on diabetic status, therefore separate
models were developed for patients without and with diabetes. The adjusted
models always included the following covariates; age, gender, total dose of
insulin (bolus injection and continuous infusion) in the 30 minutes before
glucose measurement or total dose of insulin per day and energy infusion in the
30 minutes before glucose measurement or energy infusion per day. Confounders
were selected based on p-values (\< 0.05) and effect sizes. The following
variables were tested as potential confounders: age, gender, body mass index
(BMI), current alcohol intake, current smoking, admission type, planned
admission, confirmed infection, APACHE IV-score, CCI, SOFA-scores, support of
mechanical ventilation, presence of severe sepsis or septic shock, number of
observation day, length of stay (LOS) at ICU, the use of antipsychotic drugs,
norepinephrine, corticosteroids, clonidine, ACE-inhibitors, cyclosporine or
tacrolimus, beta-blockers and beta-agonists. All statistical analyses were
carried out with R version 3.2.3 with package ‘lme4’ (R Foundation for
Statistical Computing, Vienna, Austria).
# Results
During the study period, 2669 patients were admitted to the ICU and of whom 1557
patients were excluded. Delirium was diagnosed in 535 patients. Of those
patients, 125 patients were excluded: 88 (16.4%) patients because they had only
delirious or comatose observation days during their ICU admission and 37 (6.9%)
patients because of the absence of glucose values during delirious or non-
delirious observation days. Therefore, the final population consisted of 410
patients with 1233 delirious and 1775 non-delirious observation days.
Patient characteristics are shown in. Diabetic patients were on average older,
had a higher BMI and had a higher APACHE-IV score compared to non-diabetic
patients. The number of delirious days was higher in diabetic patients compared
to non-diabetic patients. Diabetic patients had a higher maximum glucose
concentration in the first twenty-four hours of ICU-stay than patients without
diabetes.
shows the characteristics of delirious and non-delirious days in non-diabetic
and diabetic patients. During delirious days, diabetic and non-diabetic patients
had more often insulin infusions, had more insulin rate adjustments, and had a
higher average of numbers of glucose measurements in comparison with non-
delirious days.
In total 19,962 glucose levels were collected. Estimates of glucose variability
are presented per observation day in Tables and. In the unadjusted models,
delirium was associated with a higher MAG change (β:0.038; 95% CI:0.017–0.061; p
= 0.001) and increased daily delta (β:0.325; 95% CI:0.134–0.494; p = 0.001) in
patients without diabetes. After adjustments for potential confounders, the
association was not maintained in non diabetic patients using the same
definitions for glucose variability (MAG change; β adj.:0.021; 95%
CI:-0.004–0.043; p = 0.076 and daily delta β adj.:0.100; 95% CI:-0.096–0.282 p =
0.287). Delirium was positively associated with hypoglycemia in diabetic
patients (OR adj.: 2.78; 95% CI: 1.71–6.32, p = 0.005), but not in non-diabetic
patients (OR adj.: 1.16; 95% CI: 0.58–2.28, p = 0.689). Generalized mixed-
effects models with logit link function were not performed for the association
between delirium and severe hypoglycemia as the number of glucose levels below
2.2 mmol/l was insufficient.
We found similar results for glucose variability when all delirious and non-
delirious days during ICU stay were analysed compared to the observation days of
the first episode, or when consecutive episodes (delirious and non-delirious
episodes) were analysed (data not shown).
# Discussion
In this cohort of ICU patients, mean and SD of glucose concentrations, MAG
change, daily delta and the risk of hyperglycemia were unaltered during
delirious days compared to non-delirious days in non-diabetic and diabetic
patients. Furthermore, we demonstrate that in diabetic patients delirium was
associated with hypoglycemia. The association was even stronger after adjustment
for several confounding factors. This association was not found for non-diabetic
patients.
Little is published about the mutual relationship between glucose levels and
delirium. It has been reported that mean glucose levels did not differ between
patients with delirium and without delirium within non-critically ill older
patients. Although we conducted our study in an ICU cohort with critically ill
patients, our results are in concordance with their study. In the ICU setting,
one study has been conducted reporting higher mean glucose levels in patients
with hyperactive delirium compared to patients with non-hyperactive delirium. In
our study, we were not able to identify any hyperactive delirium. This may be
related to the use of sedatives. Additionally, our study was designed to compare
mean glucose concentrations during delirious and non-delirious days per
individual. In concordance with our results, tight glucose control has been
linked to increased hypoglycaemia rates and increased delirium rates. (Insulin-
induced) hypoglycaemia affect brain function. One of the strengths of our study
is that we were able to conduct our study in one of the largest high quality
cohorts with ICU patients with different delirium episodes. In addition, we had
extensive information on glucose measurements, allowing us to assess subtle and
detailed changes in glucose levels over time, both in diabetic and non-diabetic
patients. In particular, this detailed information enabled us to look at various
definitions of glucose variability. Furthermore, we were able to look at within-
patient patterns (comparing delirious and non-delirious days in each
individual), which minimizes the risk of confounding. Finally, we were able to
control for various potential confounders in a time dependent manner, such as
glucose-influencing drugs including insulin, norepinephrine, corticosteroids and
energy infusion.
However, this study has some limitations. The generalizability is possibly
limited as this study was performed as monocenter study at a university
hospital. Selection bias could have occurred because patients and observation
days without glucose measurements were excluded. Despite our rich information on
glucose levels, a potential limitation is the possibility that peaks and nadirs
in blood glucose levels have been missed as glucose levels were not measured
continuously. We considered this misclassification as non-differential as this
misclassification occurred at random during delirious and non-delirious days.
Due to the multiple testing, it remains a possibility that the association
between delirium and hypoglycemia was based on a type I error, despite the
stronger positive association after adjustment for confounders. Unmeasured
confounding may have occurred as there could have been unmeasured confounding
covariates.
The measures of glucose variability could depend on the number of glucose
determinations. Especially, the MAG-change is sensitive for higher frequency of
measurement. We consider this as less important because observation days were
compared, but not whole ICU stays. Furthermore, we adjusted for disease severity
and insulin infusion which indirectly correct for the frequency of measurement.
For the number of glucose measurements has not been adjusted because this
indices can been seen as glucose variability measure.
Hypoglycemia at the ICU has been associated with increased mortality independent
of diabetic status. For this reason, our findings suggest that in clinical
practice blood glucose levels should be monitored more often during delirium in
critically ill patients with diabetes to avoid hypoglycemia. More research is
needed to explore the impact of our findings concerning diabetic patients on ICU
outcome and determine whether any causality consists between delirium and
glucose variability.
# Conclusions
Mean glucose concentration, its SD, MAG change, daily delta and the risk of
hyperglycemia were not significantly altered during delirium in non-diabetic and
diabetic ICU patients. Delirium in critically ill patients with diabetes was
associated with hypoglycemia. This association was not found for non-diabetic
ICU patients. Our findings suggest that glucose levels should be monitored more
closely in diabetic patients during delirium at the ICU to prevent hypoglycemia.
# Supporting information
The authors thank W. Plasma, DVM., Department of Intensive care Medicine,
University Medical Centre Utrecht, Utrecht, the Netherlands for his assistance
in data acquisition.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Blood transfusion is a crucial life saving therapy to many who have experienced
road accidents, maternal hemorrhage, anemia, different surgical procedures and a
number of other medical and surgical conditions. Blood comes from blood donors
defined as “persons who donates either whole blood or blood products for
transfusion” who provide a global estimate of 112.5 million blood donations
yearly. About half of all donations come from developing countries where more
than 80% of world’s population lives. World Health Organization (WHO) further
provide estimations of nearly nine times greater average blood donations rates
in developed countries when compared to developing countries, equivalent to 4.6
donations per 1000 people in developing countries compared to 33.1 donations
recorded in developed countries. This brings greater restrain to blood donation
needs among the under five year old, who are majority users of blood in
developing countries when compared to the needs in developed countries where
elderly are the majority users.
Persons donating blood may be voluntary non-remunerated blood donors or
replacement donors as required by a member of their own family or community. WHO
recommends voluntary non-remunerated blood donors over replacement donors due to
the degree of blood safety from the two groups. Although a person can
voluntarily decide to donate blood, they may be disqualified from donating blood
due to reasons pertaining to the donors’ safety and/or recipient safety, which
is simply referred to as donor deferral. Deferral may be temporal postponement
or permanent exclusion from donating blood due to being suspected or confirmed
of having an infectious disease, hematological disease, or any other medical
condition that will either influence the safety of blood or affect donors’ own
health. However, the prevalence of blood donor deferrals varies widely, and
examples that follow substantiate the variations. In Asia, blood donor deferrals
differ from one locality to another and different studies report the prevalence
that ranges from 4.6 to 30%. Observation in different countries within Europe
shows that the prevalence of blood donor deferrals is slightly lower that from
Asia. In Africa, the prevalence of blood donor deferrals seems to be comparable
to that of middle income countries. For example, the prevalence is 10.8% in
Ivory Coast, and 7% in Zimbabwe. These studies confirm that blood donor deferral
is an issue in all countries including Tanzania however, the prevalence is not
known. Regardless of the prevalence of deferral, we may generally note that it
is an issue of concern to most blood transfusion centers in the world; it
affects both low income and high income countries and leads to inadequate blood
for transfusion due to lack of eligible blood donors. Blood donor deferral is a
sad experience to the donor and the blood bank as whole.
Analysis of the causes of blood deferrals show that the causes do differ from
one country to the other, calling for a need of setting specific analysis of
causes based on the donor selection criteria. However, low haemoglobin has
remained to be the main cause of temporal deferral in many places. For example,
in Turkey the leading cause of donor deferral is low haemoglobin 20.7% followed
by common cold and/or sore throat 17.7%, high risk sexual activities 6.7%,
hypertension 5.6% and polycythaemia 2.8%. Similar findings on the role of
hemoglobin of deferral has been reported in Netherlands and Asian countries.
However, risk factors related to infections of Human Immunodeficiency Virus
(HIV) and Hepatitis B have been reported to be the leading cause of permanent
blood deferrals in other studies. Understanding causes of blood donor deferrals
are important in instituting appropriate preventive strategies towards
conditions identified, including appropriate referral systems for clinical care.
According to WHO annual blood estimates formula, Tanzania needs 508,000 units of
blood annually, but the amount of blood collected in 2016 was less than two-
fifths of actual needs (196,735 units). About 71% of the collected blood is used
during child birth and by children under five years of age. To make sure that
there is constant supply of blood for transfusion in Tanzania, the Tanzania
national blood transfusion services (NBTS) was established in 2004. This was
followed by establishment of centralized system through the establishment of
seven zonal centers. As the part of NBTS policy, guidelines to ensure high
standards of blood safety, screening and testing for transfusion transmissible
infections (TTIs) is done and include infections such as Human Immunodeficiency
Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C virus (HCV) and Syphilis.
Screening and deferring clients with medical conditions like fever, low
hemoglobin, unstable blood pressure and social reasons like risk behaviors such
as sex workers and drug users is done. Although there has been a system to
screen and document causes of deferral in Tanzania, no studies have presented a
comprehensive analysis on the causes of blood donor deferrals in the country.
Information on deferral causes for blood transfusion is important when
considering future interventions that aim at increasing blood donation and in
the prevention of diseases and conditions associated with blood. Increasing
demand of blood for transfusion calls for a need to maximize enrollment to blood
donation program and increase the number of those who ultimately donate blood.
This study was therefore aimed at estimating the prevalence of blood donor
deferrals and to identify the causes of deferrals in the northern zone blood
centre in Tanzania.
# Materials and methods
## Study design and setting
This was a cross-sectional study where information from all participants who
presented for blood donation at the northern zone blood bank from January
1<sup>st</sup> to December 30<sup>th</sup> 2016 were retrospectively analyzed
between March and June 2017. The northern zone blood bank center is one of the
six centers in Tanzania assigned to collect blood from all donors in four
regions of Kilimanjaro, Arusha, Tanga and Manyara. The center was established in
2005 and is located in Moshi municipality about four (4) kilometers from Moshi
town, just proximal but within the compound of KCMC Hospital–a consultant
hospital in the zone. This center deals with blood donor recruitment, blood
collection, screening and distribution of blood to all health facilities in the
area. The goal of northern zone blood transfusion center was to collect 15,846
blood units in 2016, however it managed to collect 7,163 (45%) blood units. The
center serves a total population of about 6,949, 880 people from the four
regions, which is 16% of the Tanzanian population. To achieve the goal the
center had to collect blood from voluntary non-remunerated blood donors as well
as from replacement donors within the zone.
The procedures for donor intake is as follows: The clients presents for blood
donation at the center where they are received at reception and registered. This
is followed by counseling provided by trained donor counselors. This counseling
process goes in hand with checking on the eligibility for blood donation using a
standardized donor questionnaire. The questionnaire inquires about the socio-
demographic characteristics of participants such as age, marital status,
occupation and address; the type of donor–either voluntary or replacement; and
general health check of the donor in terms of diseases and risks for acquiring
transmissible infections such as HIV and HBV. Voluntary non-remunerated blood
donor was defined as a person who gives blood on his or her own free will and
receives no payment, either in the form of cash or in kind. After counseling,
prospective blood donors then receive measurements for weight, blood pressure
and hemoglobin estimation. The facility then does testing on blood samples for
HIV, Hepatitis B, Hepatitis C and Syphilis. Clients who do not fulfill the
eligibility criteria for donation are then deferred either temporarily or
permanently depending on reason for the deferral. Blood donor deferral in this
study was therefore defined as the temporally postponement or permanent
exclusion from donating blood by a person suspected of having an infection,
disease or any other medical condition that will either influence the safety of
blood or affect donors’ own health. Blood donation is done after the client has
signed informed consent.
Regarding testing for TTIs, the Tanzanian NBTS tests four TTI markers which are
HIV, Hepatitis B, hepatitis C and Syphilis. These tests are done using ELISA
antigen–Antibody combination with test kits shown in. For each marker,
appropriate screening test (Screening Test \# 1) is done with three possible
results; either negative, positive or grey zone. Those with negative test
results are recorded as negative and can be recalled for next donation. The
blood unit with positive or grey zone test results for the initial screening
test is discarded and subjected to a duplicate repeat (Screening test \# 1 in
Duplicate Repeat) and then supplemental test (Supplemental test \# 2). The donor
with the blood unit testing positive after this duplicate repeat are deferred
permanently.
## Study population and data collection
We were provided by the NBTS with information from all clients who presented for
blood transfusion at the northern zone blood bank center from January to
December 2016. This study extracted data from all registered clients with
complete information on donor type, decision to donate or defer and clearly
indicated reason for deferral among deferred clients. Participants excluded from
this analysis were those who decided not to proceed with the assessment for
qualifying to donate blood before being administered with donor questionnaire.
Information extracted from the database included those from donor questionnaire
and from laboratory where the results for TTIs and other estimations such as of
hemoglobin are documented. Information also included the final verdict of donate
or defer.
## Statistical analysis
Statistical Package for Social Studies (SPSS) program version 20.0 was used to
analyze the data. Descriptive statistics was used to summarize data where
frequency tables and cross tabulation were made while describing the data in
numbers and percentages. The association between donor types; voluntary and
replacement; and between deferrals and accepted donors, are made using Pearson
Chi-square test for categorical variables. Results of the difference were
considered statistically significant when p-value was less than 0.05.
## Ethical consideration
The ethical approval for carrying out this study was obtained from the
Kilimanjaro Christian Medical University College Research and Ethical Committee
(CREC) and the National Blood Transfusion Services Ethical Committee. The letter
to request permission to do the research and to access the database for the
registered blood donors was granted by the National Blood Transfusion Services.
The head of laboratory records, head of blood donors and head of records were
informed about the study. The information obtained was used for research purpose
only. Confidentiality of participant information was observed during the entire
period of the study.
# Results
Out of all clients 16537 presented for blood donation in northern blood
transfusion centre, 14377 (87.0%) met inclusion criteria. Reasons for exclusion
were incomplete data for the status of deferral, donation and reasons for
deferral of participants.
## Demographic characteristics of participants
Demographic characteristics of all participants are presented in below. The
majority of participant were males, who formed more than three-quarters 11377
~~(~~79.1%) of all participants. More than two third of all participants 9719
(67.6%) were aged between15 and 30 years followed by 3377 (23.5%) who were aged
31 to 45 years. Almost half of the participants 6671 (46.4%) came from
Kilimanjaro region where the center for blood transfusion in the northern zone
is located and nearly a quarter 3408 (23.7%) came from a nearby region of
Arusha. Almost nine out of every ten participants 12775 (88.9%) who presented
for blood donation were voluntary non remunerated blood donors. Family
replacement donors were significantly more likely to be females, with increasing
age above 31 years and come from nearby regions of Arusha and Kilimanjaro (P
value \<0.01).
## Prevalence of blood donor deferrals
Out of 14377 participants who presented for blood donation in 2016, 1829 (12.7%)
were deferred. About 15.6% of all men were deferred and 12.0% of all female were
deferred. Blood donor deferrals were also categorized according to age where
participants aged 46–65 years had higher deferral rate (27.6%) when compared to
those who were in the younger age groups of 18 to 30 years (12.1%) and between
31 and 45 years (17.6%). Replacement donors had higher deferral rate 15.8% as
compared to voluntary non-remunerated blood donors who had 12.3%. Deferral was
significantly more likely among females, increasing age, came from Tanga or
surrounding region of Kilimanjaro and/or a family replacement donor (P value
\<0.01).
## Causes of blood donor deferrals
Detailed analysis of permanent and temporary causes of blood donor deferrals is
shown in below. The general picture show that infections contribute to 62% of
all deferral causes in this study. Looking at temporary causes, slightly more
than half of all blood donor deferrals 927 (50.7%) were due to temporary causes.
Of all temporary causes, low haemoglobin was the leading cause; contributed one-
fifth (21.1%) of all causes of blood donor deferrals and the majority of
participants with low haemoglobin were females 224 (58.0%). Syphilis was the
second leading cause of temporary blood donor deferral, carrying about one-tenth
171 (9.3%) of the remaining causes. Majority of those who were deferred because
of syphilis were males 149 (87.1%). However, the prevalence of syphilis in the
study population was 1.2%. Of all permanent deferrals causes, hepatitis B was
the leading cause. It contributed more than a quarter (29.6%) of all deferrals.
About 90% of those deferred due to hepatitis B were males. HIV was the second
leading cause of permanent deferrals accounting for 13.3% of all deferrals. Of
all participants who were deferred because of HIV, 81.1% were males. The
prevalence of HBV, HIV and HCV in this study was 3.8, 1.7 and 0.1% respectively.
# Discussion
This study aimed to determine the prevalence and causes of blood donor deferrals
among clients presenting for blood donation in northern zone Tanzania. The
prevalence of blood donor deferrals in our study was found to be 12.7%. Analysis
of deferral causes indicated that slightly more than half of all deferred
clients were due to temporary causes. Infections were the leading cause of
deferral in this study by 62%; with Hepatitis B being the leading single cause
followed by low haemoglobin and HIV. The majority of those who were deferred
were replacement donor when compared to voluntary non-remunerated donors.
Exploring on the background characteristics of the study subjects, the majority
of participants were males, with a male to female ratio of 3.8:1. Other studies
have indicated very high male to female ratio. Having very high male to female
ratio, as compared to our study has implication to the study results as some of
deferral causes are more prevalent in either males or females. For example,
having more males in the study decreased the deferral rates in some studies
since females are more deferred than males due to low haemoglobin. The present
study also indicated that voluntary non-remunerated blood donors were nearly 90%
of all the blood donors with remaining proportion being replacement donors.
Other studies on blood donor deferrals have found a reverse composition where
replacement donors are more than voluntary non-remunerated blood donor.
Voluntary non remunerated blood donors are safer than replacement donor.
However, the reported deferral rates by these studies are within similar range
with what has been reported in the present study. The characteristics of
participants enrolled in these studies therefore influence the deferral rate,
calling for further exploration on other factors. This might be due to
differences in geographical location where these studies have been done,
difference in levels of infections and/or awareness towards blood donor
eligibility among populations where these studies were conducted.
In the present study the prevalence of blood donor deferrals was 12.7%. To the
best of our knowledge there is no similar study that have been done in Tanzania
thus no other study in the country to be compared with the present study. The
deferral rate reported in our study is comparable with what has been reported in
other studies done elsewhere. While some studies have shown congruent prevalence
results with the present study, some have reported slightlyr lower or higher
prevalence rates of deferral. The studies which reported congruent prevalence
results used the same study design and tools, and were from low income
countries. Most of these studies used donor screening questionnaire together
with TTIs result from laboratory and had employed retrospective study design. In
that regard, all the studies presenting comparable results with the present
study assessed the pre-donation and post-donation deferrals. Of the studies that
reported lower rates of deferral, two studies used information on TTI’s similar
to the methodology of the present study. Similarly, two of the studies which
reported higher rates of deferral than what is reported in the present study
included results of TTIs in their analysis. It can therefore be concluded that
the variation in deferral rate presented in these studies when compared to the
present study might be explained by the difference in donor eligibility
criteria, level of awareness of the population from where donors come on blood
donor eligibility criteria and social economic status due to difference in
geographical location. In middle and high income countries the level of
awareness on blood donor criteria is high, have good nutrition status and low
prevalence of infectious diseases which results in the prevalence of deferrals
to be low. More strict blood donor eligibility criteria and the level of
keenness of blood donor counselors on exploring risk behaviors to clients may
lead to the increased prevalence of deferrals. The differences in social-
economic status between the countries also contribute to differences in the
prevalence of deferrals. For example, with high prevalence of low hemoglobin in
low income countries when compared to high income countries, low income
countries tend to have high prevalence rates of deferrals.
The common cause of blood donor deferrals in our study was infections, which are
measured by the positivity of TTIs when screening, accounted for two third of
all deferrals. TTIs that are measured in northern zone blood transfusion center
are HBV, HCV, HIV, and Syphilis. These results correspond to the nature of low
income countries where there is a double burden of infectious diseases and non
communicable diseases. Prevalence of infectious and non communicable disease
plus malnutrition is higher when compared to the high income countries. The
present study further found that HBV was the leading cause of permanent
deferrals followed by HIV. Other studies elsewhere have indicates similar trend.
Our study has found that HBV is more prevalent among blood donors (3.8%) than
HIV (1.7). Even in the general population of Tanzanians, the prevalence of HBV
(6.0%) is higher than that of HIV(4.7%). It is important noting here that
clients testing positive for HIV and HBV will never donate blood, reducing the
pool of potential donors in the general population. However there is low level
of awareness on HBV, its mode of transmission, its causative agent and its
consequences among the general population when compared to HIV in the country.
This might be the reason as to why the prevalence of HBV is high among the
clients who presented for blood donation in northern zone blood transfusion
center. If those positive on HBV and HIV are refereed to appropriate services
then Blood Transfusion centers will have identified the diseases and referred to
appropriate care, improving the integration with other health care services. The
Health Sector Strategic Plan for the health sector and the national policy on
health emphasizes the need for continuous care and highly recommends referrals
to appropriate care. However, there is a need to balance between the risks for
test seeking behavior among blood donors which may attract high risk persons to
donate blood and increase the likelihood of window period donations. The best
practice is improving community based initiatives for screening of infections
coupled with preventive messages, while linking those tested positive for
treatment in nearby centers.
The present study has shown that low hemoglobin was the leading temporary cause
of blood donor deferrals and the second leading cause of all deferrals followed
by Syphilis. This study implies that low hemoglobin is prevalent in northern
zone. Similar finding as in our study was reported in Turkey, Netherlands, Asian
countries and in other parts of the world. The low Hb is caused by parasitic
infections like hookworm and poor nutrition status like low consumption of iron
containing diet, Vitamin B12 and folic acid. It seems that most of the
participants don’t know about their health status as Hb estimation is a simple
and cheap measure of which at least many could have known before presenting at
the blood transfusion center. Some of those with low Hb could have known their
status before they presented themselves for blood donation. This may mean that
the level of understanding on blood donation eligibility criteria is very low
among population in the northern zone. From public health perspective, low
hemoglobin is amenable to address in the short term and depending on the root
cause, re-entry programs to donation are important among clients deferred
because of low Hb levels.
The rate of blood donor deferrals in the present study was higher among
replacemenr donors than in voluntary non remunerated blood donors. Similar
findings have been documented by Stokx et al and by Meinia and Sawhney where
replacement donors were deferred more than voluntary non remunerated blood
donors. This imply that voluntary non remunerated blood donors are relatively
safer than replacemnt donors, inline with the WHO recommendation. Regarding the
proportion of type of donors, there has been ethusiasm among blood transfusion
centers in order to comply with WHO recommendation which requre 100% of all
blood to be donated from voluntary non remunerated blood donors. Some of blood
donation settings have been reported to falsify the donor status from
replacement donors to voluntary donors. We may not know if this may have
happened in our setting where 90% of the participant were voluntary non
remunerated blood donors.
When interpreting the result of this study, it is important to cosider some of
the study limitations. A considerable number of participants had incomplete
information and were excluded from this study. Due to having incomplete
information, we were not able to compare with those we included in the study.
Also the study could not capture the information from self defferals blood
donors (who included below 18 years and underweight 50kg), who were not
registered into blood donor database at transfusion centre. The potential donors
who declined interview and were not recorded may constitute special risk factors
different from those who accepted interview. We may therefore not know their
influence to the results presented in study. Regardless of these limitation, the
present study had several strengths. The study has used a very large sample
size. The study also present the combined information from blood donor screening
questionnaire and TTIs results thus studying both pre-donation and postdonation
deferral causes at the same time.
# Conclusion
A siginificant proportion of blood donor deferrals has been reported in the
present study, that accounted for the inadequate supply of blood for
transfusion. Transfusion transmissible infections are the leading cause of
permanent blood doonor deferrals where HBV and HIV forms the highest proportion
of infections. Low Hb is a leading cause for temporary deferal. There is a need
to improve blood donor recruitment plans by increasing awareness of the people
on blood donation and the causes for deferrals. Mass education on HBV is
important so as to increase awareness of the population and ultimate prevention
of HBV.
The authors would like to acknowledge staff from the northern zone blood
transfussion center for their cooperation and support during conducting this
study. Special appreciations are registered to Dr Efesper Nkya, the program
manager of National Blood Transfusion Services, for providing permission to
conduct this study. We are also grateful to Dr Sia Msuya, the Director of
Institute of Public Health and KCMUCollege for her support throughout the
process of developing this work.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Global cerebral ischemia/reperfusion (GCIR), a syndrome characterized by the
rapid interruption of cerebral blood flow, occurs in patients who suffer from
cardiac arrest, shock or complex cardiac surgery and is usually accompanied by a
broad range of neuronal death in the brain\[–\]. Many of these patients suffer
from various degrees of memory loss and learning dysfunction, suggesting an
impairment in the hippocampus, which is the primary region of the brain that
controls the formation of memories and learned behaviors. Currently,
counteracting ischemia-induced cognitive impairment is challenging, and
effective strategies that can attenuate the negative effects of ischemia are
insufficient. The increased neurogenesis, especially in the hippocampus of
adults after GCIR, has been reported to be a compensatory adaptive response to
brain injury that could counteract the negative effects of cell death and
cognitive dysfunction.
Sleep is necessary for health and survival. After decades of research into the
function the sleep-wake cycle, a large body of evidence strongly indicates that
sleep deprivation (SD) has complex detrimental consequences on rodents and
humans, though the mechanisms underlying these negative effects remain largely
unclear\[–\]. However, growing evidence also suggests that short-term sleep
deprivation (SD) with a duration of less than 48 h produces positive effects.
For example, previous studies suggest that 6–12 h of short-term SD prior to
cerebral ischemia produces neuroprotective effects by attenuating inflammatory
responses and glial reactions in the rat hippocampus\[–\]. Moreover, a recent
study showed that 24 h of short-term SD immediately following traumatic brain
injury (TBI) reduces morphological damage and enhances recovery in rats.
Hence, we suggest that the effect of sleep deprivation may largely depend on the
time window and duration of SD. More specially, emerging studies have also
reported that 12 h of short-term SD can promote neurogenesis in the hippocampus
of normal rats. Based on these findings, we hypothesized that short-term
SD may improve cognitive function in an ischemic model through neurogenesis-
induced neuronal regeneration. Similarly, the ideal durations and time window of
SD is significant.
Brain-derived neurotrophic factor (BDNF) has recently been shown to be a
homeostatic regulator of sleep. BDNF is also known to increase neurogenesis,
neurite sprouting and other processes related to the general enhancement of
hippocampal function in normal and ischemic rodents. Some reports have
demonstrated that short-term SD increases BDNF expression in the hippocampus in
normal rats. We speculate that the BDNF pathway mediates the neurogenesis
induced by short-term SD, thereby ameliorating cognitive function in global
ischemia.
In light of this, the present study aimed to determine whether different
durations of short-term SD could stimulate hippocampal neurogenesis and
ameliorate the impaired cognitive functions induced via GCIR in rats in an
appropriate time window and whether this self-repair is related to the
hippocampal BDNF pathway.
# Materials and Methods
## Animal groups
One hundred male Sprague—Dawley rats (weighing 230±20 g), were purchased from
the Experimental Animal Center of Chongqing Medical University. They were housed
in groups of five in polycarbonate cages under a 12:12 h light:dark cycle
(lights on from 9:00 to 21:00) with unlimited access to food and water at an
ambient temperature of 21±1°C and a relative humidity of 40–50%. All experiments
were approved by the Chongqing Medical University Institutional Lab Animal Care
and Use Committee and were in accordance with the National Institutes of Health
guidelines.
The rats were maintained in the institutional animal facilities for at least 2
weeks and then randomly assigned to 3 groups: the Sham group (n = 20), the GCIR
group (n = 20), and the short-term SD group. Based on the different durations of
SD, the short-term SD group was randomly divided into three subgroups: the
GCIR+6hSD\*3d-treated group (n = 20) rats were subjected to GCIR followed by
sleep deprivation for 6 h/d continuously for 3 d; the GCIR+12hSD-treated group
(n = 20), rats were subjected to GCIR followed by sleep deprivation for 12 h;
and the GCIR+12hSD\*3d-treated group (n = 20) rats were subjected to GCIR
followed by sleep deprivation treatment for 12 h/d continuously for 3 d. The
rats were sleep-deprived starting at 48 h following GCIR.
## Global ischemia reperfusion model establishment
Global cerebral ischemia/reperfusion (GCIR) modeling was performed in a manner
similar to a previous method with slight modifications. Briefly, GCIR was
induced via ligation of the bilateral common carotid artery combined with
hemorrhagic hypotension in rats. The rats underwent fasting for 12 hours prior
to surgery. After anesthesia was induced (3.5% chloral hydrate, 1 ml/100 g,
i.p.), the bilateral common carotid arteries were slightly isolated, and the
right jugular vein was cannulated with tubing connected to a heparinized
syringe. Blood (2.5 ml/100 g) was slowly withdrawn from the right jugular vein
until the volume reached standard requirements, at which time the bilateral
common carotid arteries were temporarily occluded using artery clamps for 20
min; the extracted blood was then slowly reinfused, and the catheters were
withdrawn. The rats in the sham group were subjected to the same operation
described above, with the exception of the bilateral carotid artery occlusion
and hemospasia from the right jugular vein. We discussed the success and
exclusion criteria of the GCIR model in our previous report.
## Sleep deprivation procedures
Sleep deprivation was conducted 48 h following GCIR using the modified multiple
platform method, as described previously, which does not involve immobility
stress or forced activity, resulting in less interference from other factors\[,
\]. Sleep deprivation was initiated at 09:00AM during the rest circadian phase
of the rats (light phase: 09:00AM-9:00PM). Briefly, groups of 8 rats were placed
in water tanks (75×34×17 cm) containing 8 small circular platforms (6.5 cm in
diameter). The surfaces of all platforms were 1 cm above the water level. The
rats fall into the water if they lose muscle tonus, forcing them to climb back
onto the platform, thus being awakened. Additionally, the rats in the sham and
GCIR groups were placed in an identical apparatus that was equipped with a
larger platform (18 cm in diameter) to permit sleep, serving as a control for
the small platform to filter out the effects of nonspecific stressors. All of
the rats were placed on the platform for 10 min twice per day beginning one week
prior to the experiment to adapt to the homemade device. Moreover, food and
water were provided ad libitum throughout the study, and the water in the tanks
was changed daily.
## BrdU Incorporation
BrdU (B5002, Sigma Aldrich, Munich, Germany) was dissolved at a concentration of
10 mg/ml in fresh isotonic sterile saline prepared just prior to use and the
solution was injected in a volume of 5 ml/kg/d of the body weight. And BrdU was
injected four times with a 2 h interval in the 13th day and 27th day after GCIR.
Rats were sacrificed 24h after the last BrdU injection.
## Morris Water Maze Test
The Morris water maze (MWM) procedure was employed to assess spatial learning
and memory function of the rats 7 days after the beginning of SD procedure and
included 5 days of spatial acquisition and 1 day of a probe trial. The MWM
(Institute of Materia Medica, Chinese Academy of Medica Sciences, Beijing,
China) was equipped with a diameter of 150 cm, height of 50 cm, water depth of
40 cm, and temperature of 24±1°C. A platform (10 cm in diameter) was submerged 1
cm below the surface of the water and placed in the middle of the same quadrant
throughout the training phase. During the learning process (1–5 d), the rats (n
= 6 per group) were subjected to four consecutive trials per day with intervals
of 5 min. In each trial, an individual rat was placed into the pool and
permitted to search for the submerged platform for 90 s. If a rat failed to
locate the platform within 90 s, it would be gently guided to the platform, and
the escape latency was recorded as 90 s. The mean escape latency of 4 trials was
noted as the daily result of learning ability for the animal. On the 6th day of
the test, each rat was placed into the pool after the platform had been
previously removed and then allowed to explore the pool for 90 s. The frequency
with which each rat passed the hidden platform and the resident time that each
rat spent in the target quadrant were noted as the result of the spatial memory
function.
## Preparation of paraffin-embedded tissue
Four rats were selected in each group for histopathological observation
respectively at the 14 d and 28 d timepoints after GCIR. Rats were anesthetized
with 3.5% chloral hydrate (1 ml/100 g, i.p.) and rapidly perfused with ice-cold
saline (approximately 200 ml. Then, rats were perfused with 250 ml of 4%
paraformaldehyde until the liver became pale and the limbs and neck became
straight and stiff. Finally, paraffin sections of coronal slices in the
hippocampal dentate gyrus (DG) were cut with a slice thickness of 4μm.
## Preparation of fresh tissue
On the 7th day after GCIR, the rats (n = 6) were decapitated, and their
hippocampi were rapidly removed on ice, quickly frozen in liquid nitrogen and
stored at -80°C in a refrigerator until ELISA.
## Immunohistochemistry and immunofluorescence analysis
Briefly, brains embedded in paraffin were used to examine BrdU incorporation 14
d after GCIR to assess the proliferation of newly generated cells. The sections
were immersed in 3% H2O2 for 30 min at 37°C to block endogenous peroxidase
activity. After being washed in PBS, the sections were blocked with 5% BSA for
30 min, incubated with a primary mouse monoclonal antibody against BrdU
(1:50,B2631,Sigma)overnight at 4°C, and then incubated with biotinylated goat
anti-mouse IgG(1:100,Zhongshan Inc) for 60 min. Immunoreactivity was detected
with 0.05% diaminobenzidine (DAB) containing 0.03% H2O2 for 5 min.
To further determine the cell lineage of the proliferating cells, we examined
the expression of BrdU and the neuronal marker neuron-specific enolase (NSE) 28
d after GCIR. After pretreatment with 2 N HCl, the sections prepared as
described above were incubated overnight at 4°C in anti-BrdU (1:50, B2631,
Sigma) and rabbit anti-NSE antibodies (1:300; Cell Signaling Technology,
Danvers, MA) and then incubated for 1 h at room temperature in Alexa Fluor 488
goat anti-mouse IgG(1:500, Invitrogen, Carlsbad, CA) and Alexa Fluor 594 goat
anti-rabbit IgG (1:500, Invitrogen, Carlsbad, CA) for 1 h at room temperature.
The sections were washed with PBS-T 3×10 min followed by 2× 5 min washes with
PBS and 2×1 min washes with water; they were then mounted with water-based
mounting medium containing anti-fading agents (Biomeda, Fischer Scientific,
Pittsburgh, PA).
## Image Analysis
The sections were examined by light microscopy (BX51, Olympus, Tokyo, Japan).
Cells were counted, in a blinded manner, within defined regions of interest in
the inner edge of the granule cell layer (GCL) of the DG. Cell counting in
hippocampal DG was performed in the ipsilateral DG using a series of coronal
sections between 10.0 mm and 10.6 mm from the front of the brain. In total, six
4-μm coronal sections were examined per animal (n = 4 per group), spaced 100μm
apart, under high-power (40×objective) using Image-Pro Plus 6.0 for Windows
(Media Cybernetics, MD, USA). In each section, measurements were made in ten
regions of interest. The planar area enclosed by each region was 50μm×50μm. The
average ratio of BrdU-positive cells in the proportion of the total number of
cells and the average ratio of BrdU/NSE-positive cells in the proportion of
BrdU-positive cells in the DG per section were respectively calculated.
## BDNF ELISA
The hippocampal tissue was placed in Eppendorf tubes and immersed in isopentane
cooled by dry ice. This process did not take longer than 3 minutes. These
samples were homogenized with a Dounce homogenizer (Kontes-7 ml, Vineland, NJ,
USA) in ice-cold homogenate buffer solution. The ELISA procedure was performed
according to the manufacturer’s instructions (BOSTER, China) using a microplate
reader (Bio-Rad, Richmond, CA, USA).
## Statistical analysis
The results were expressed as the means ± standard deviation of the means. Two-
factor multi-level analysis of repeated measures was used for the comparison of
the water maze escape latencies. Statistical comparisons of differences between
groups for different interventions were performed using one-way ANOVA. All
statistical analyses were performed with the Statistical Package for the Social
Sciences (SPSS 10.0) software, and p\<0.05 was considered statistically
significant.
# Results
## Effect of short-term sleep deprivation on spatial learning and memory ability in rats with GCIR
The Morris water maze results showed that rats in all the five groups exhibited
a rapid reduction in their escape latencies to find the platform over the five
training days. Compared with the S group, the rats subjected to GCIR showed a
prolonged escape latency, which implies that global ischemia significantly
impaired spatial learning ability, and this impairment occurred from training
day 1 onward (p\<0.05). Surprisingly, there was no apparent difference between
the escape latency of rats in the GCIR+12hSD group and the GCIR group. And the
rats in the GCIR+6hSD\*3d group just spent less escape latency than the rats in
the GCIR group on day 3 (p\<0.05). However, the escape latency in the
GCIR+12hSD\*3d group was significantly shorter than which in the GCIR group, and
the decrease trend substantially occurred in training day 1 onward (p\<0.05),
which indicates that the 12hSD\*3d-treated method can improve the spatial
learning ability of rats with global cerebral ischemia/reperfusion-induced
injury.
During the probe test, the platform was removed, then the resident time and
frequency with which the rats crossed the target quadrant was recorded. Relative
to the S group, the rats with GCIR showed less crossing times and resident time
in the test (p\<0.01). However, the rats in all short-term SD groups (including
GCIR+6hSD\*3d, GCIR+12hSD, and GCIR+12hSD\*3d) spent significant longer and made
substantial more frequent cross-platform movements in the original platform
quadrant than the rats in the GCIR group (respectively p\<0.05, p\<0.01,
p\<0.01). This indicates that the rats in the S and all short-term SD groups
exhibited better spatial memory regarding the original platform location.
## Effect of short-term sleep deprivation on cell proliferation in the dentate gyrus (DG) of the hippocampus in rats 14 d after GCIR
BrdU-positive cells were showed brown or black irregular granular nuclear
markers distributed along the hippocampal dentate gyrus (DG). The results showed
that a small number of BrdU-positive cells were observed in the hippocampal DG
in the S group. Relative to the S group, the number of BrdU-positive cells was
substantially increased in the GCIR group (P\<0.01). While the number of BrdU-
positive cells had no significant difference in the GCIR+6hSD\*3d group and
GCIR+12h SD group compared with GCIR group. Furthermore, the number of BrdU-
positive cells in GCIR+12hSD\*3d group was largely higher than that in the GCIR
group(P\<0.05).
## Effect of short-term sleep deprivation on cell differentiation in the hippocampal dentate gyrus in rats 28 d after GCIR
Four weeks after GCIR, BrdU/NSE double fluorescence-positive cells were rarely
found in the S group. However, the number of double-positive cells was
significantly increased in the GCIR group compared to the S group (P\<0.01). The
BrdU/NSE double-positive number in the GCIR+6hSD\*3d group had no difference
compared to the GCIR group. Moreover, a higher number of BrdU/NSE-positive cells
was observed in short-term SD groups (including the GCIR+12hSD group and
GCIR+12hSD\*3d group) compared with the GCIR group (P\<0.05).
## The expression of hippocampal BDNF in rats 7 d after GCIR
The expression of hippocampal BDNF in all short-term SD groups (including
GCIR+6hSD\*3d, GCIR+12hSD, and GCIR+12hSD\*3d) was significantly increased
relative to the GCIR group (P\<0.05), and the most substantial increase was
observed in the GCIR+12hSD\*3d group.
# Discussion
In this study, we examined the beneficial effects of different durations of
short-term SD on learning, memory function and neurogenesis in the hippocampus
of rats exposed to GCIR. We found that after GCIR, (1) short-term SD could
ameliorate the impairments in learning and memory, (2) short-term SD could
promote the proliferation and differentiation of newly generated cells in the
hippocampal DG, (3) the BDNF signaling pathway participated in the stimulation
of neurogenesis, and (4) the GCIR+12hSD\*3d-treated method may be the most
appropriate way to implementation of sleep intervention in the rat with GCIR.
As we know the brain is highly sensitive to ischemia, and reperfusion
exacerbates ischemic damage. The hippocampal neurons, particularly the pyramidal
neurons in the CA1 region, are known to be the most sensitive to the deleterious
effects of cerebral ischemia/reperfusion. And as the hippocampus is an
important region for learning and memory in mammals, the majority of
ischemia survivors suffer from various cognitive dysfunctions. Using the
current model, our study showed that global ischemia significantly compromised
spatial learning and memory function.
In the present study, the rats in the GCIR+12hSD\*3d group showed less escape
latency in the five training days relative to the GCIR group, suggesting an
improvement in spatial learning. And all groups of rats treated with short-term
SD in our experiment spent significant longer and made substantial more frequent
cross-platform movements in the original platform quadrant than the rats
underwent GCIR alone in the probe test, suggesting an attenuation of memory
dysfunction. In total, the group of rats subjected to 12 h SD for 3 d
consistently showed the greatest recovery of cognitive function. Similar results
have also been reported in the literature; for instance, Moldovan et al. found
that rats with occlusion of the middle cerebral artery (MCAO) that were
subjected to pretreatment with 6 h of SD showed better learning and memory
performance during the first week of recovery; this was also consistent with
reduced signs of morphological damage. Additionally, Martinez-Vargas showed
that 24 h of SD after a TBI had a neuroprotective effect by reducing
morphological damage and enhancing recovery in rats. The mechanism of how
short-term SD promotes neurological recovery from brain injury is largely
unclear. Some previous reports suggested that sleep deprivation prior to
transient GCIR could attenuate hippocampal damage by attenuating inflammatory
responses or glial scar formation. In the present study, we induced sleep
deprivation intervention 48 h following GCIR, and focused on observing the
neurogenesis effects of different durations of short-term SD.
A considerable degree of neurogenesis has been reported to occur following
cerebral ischemia in the adult mammalian brain. The regional correlation of
neurogenesis in the hippocampal DG has also been studied in various disorders
that cause memory impairment. Global ischemia stimulates cell proliferation in
the hippocampal DG, which peaks at 1–2 weeks after ischemia. Many newly
generated cells die in the weeks following ischemia, but the minority of cells
that survive will differentiate into neurons approximately 4 weeks later. In
our study, compared with the GCIR group, a larger number of BrdU-positive cells
was observed 14 days after GCIR in the hippocampal DG of the rats treated with
short-term SD.NSE, a neuronal marker, can further determine the cell lineage of
the newly generated cells. We also observed an increased number of BrdU/NSE-
positive cells in the short-term SD groups, especially in the GCIR+12hSD group
and GCIR+12hSD\*3d group, 28 days after GCIR compared with the GCIR group. These
results indicated that short-term SD not only promoted the proliferation of
newly generated cells but also stimulated the proliferating cells to
differentiate into neurons in the hippocampal DG. However, the most effective
proliferation and differentiation occurred in the GCIR rats with 12 h SD for 3
successive days.
Our results are in agreement with previous studies indicating that sleep
deprivation for relatively short periods(\<48 h), such as 12 h, significantly
stimulates neurogenesis in the hippocampus of normal rats by enhancing cell
proliferation and the survival of newly generated cells. Based on these
data, it may be tentatively concluded that delayed short-term SD (after 48 h)
facilitates cell repair in the hippocampus through the stimulation of
neurogenesis via endogenous progenitor cells to improve behavioral recovery and
the most appropriate way is 12 h SD for 3 successive days.
In our experiment, the ELISA results showed that the BDNF level of hippocampal
tissues was significantly increased after different periods of short-term SD 7 d
following GCIR rats, which is in line with earlier reports suggesting that 12 h
of short-term SD could increase the expression of hippocampal BDNF.
Numerous evidences have also indicated that BDNF is required for basal
neurogenesis in the hippocampal DG. The knockdown of BDNF by RNA interference
and lentiviral-mediated gene silencing in the DG reduces hippocampal
neurogenesis. Therefore, BDNF signaling is one possible mechanism for short-
term SD-induced neurogenesis, and further research is needed to clarify the
down-stream signaling mechanisms.
# Conclusion
The current study clearly demonstrates that 48 h delayed short-term SD,
especially the GCIR+12hSD\*3d-treated method, is effective in promoting
neurogenesis in the hippocampal DG and facilitating cognitive recovery following
GCIR in the adult rat. Additionally, the underlying mechanism of short-term SD
in neurogenesis may be mediated by the BDNF signaling pathway. These results
suggest that short-term SD in an appropriate way may be a potential strategy for
the clinical treatment of ischemic injury in brain tissues.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: OC RL JY. Performed the
experiments: OC RL LZ LY BY JW. Analyzed the data: OC RL LZ. Contributed
reagents/materials/analysis tools: OC RL LZ LY BY JW BC. Wrote the paper: OC
RL JY.
[^3]: ‡ These authors are co-first authors on this work. |
# Introduction
The giant clam *Tridacna crocea*, inhabiting many Indo-Pacific coral reef
communities, is remarkable for its fantastic mantle and unique ability to bore
fully into coral rock. Similar to other tridacnids, there is a symbiotic
relationship between *T*. *crocea* and symbiotic dinoflagellates, which are
commonly called zooxanthellae. Symbionts of clams are intercellular, and the
zooxanthellae live within a branched, tubular structure permeating the mantle;,
[the](http://bbs.pigai.org/t45156-1-1.html) symbionts produce nutrients via
photosynthesis to aid in the host’s autotrophy, while they gain essential
nutrients for growth and metabolism from the host. Because of the
characteristics of giant clams, they are effective ecosystem engineers playing
multiple roles in coral reefs. Nevertheless, because of environmental and
anthropogenic disturbances, such as over-harvesting and habitat destruction,
giant clam populations have been depleted, and their densities have been
insufficient for self-replenishing and maintaining their populations, which
might eventually lead to their extinction.
In recent years, the breeding of giant clams has attracted increasing attention
worldwide. However, diseases caused by bacterial pathogens have been reported to
result in losses in aquaculture populations of these commercially important
shellfish. Pathogenic vibrios can affect larval stages of cultured bivalves and
are also involved in diseases of juveniles and adults. *Vibrio coralliilyticus*
was originally known as a temperature-dependent etiological agent involved in
coral bleaching; it targets the mucus of the coral host through the use of
dimethylsulfoniopropionate as a chemotaxic and chemokinetic agent, and its use
of extracellular proteases results in coral tissue lysis and symbiont density
decrease. Lately, it was demonstrated to cause mortalities in larval oysters.
Since *V*. *coralliilyticus* is phylogenetically related to *Vibrio tubiashii*,
many marine isolates of *V*. *coralliilyticus* from shellfish were misidentified
as *V*. *tubiashii*. Therefore, previous reports of *V*. *tubiashii* in bivalve
shellfish aquaculture on the west coast of North America were possibly caused by
*V*. *coralliilyticus*. It has been reported that gross pathological changes to
the velum and cilia occurred in diseased *Crassostrea gigas* larvae. Moreover,
*V*. *coralliilyticus* has also been associated with outbreaks of vibriosis in
several other bivalve species, including Eastern oysters (*Crassostrea
virginica*), the European flat oyster (*Ostrea edulis*), the great scallop
(*Pecten maximus*), the Atlantic bay scallop (*Argopecten irradians*) and New
Zealand green-lipped mussels (*Perna canaliculus*). Consequently, *V*.
*coralliilyticus* is a significant pathogen for bivalves, contributing to
dramatic losses in mollusks worldwide.
Bivalves mostly rely on an innate immune system composed of cellular and humoral
components. Hemocytes are thought to be responsible for the main activities by
which bivalves respond to infectious agents, and those are phagocytosis,
encapsulation and nacrezation. The first action of the bivalve’s immune system
when challenged by microbial pathogens is the recognition of these foreign
organisms. This is achieved by expressing various pattern recognition receptors
(PRRs), which sense diverse pathogen-associated molecular patterns (PAMPs).
Recognition of PAMPs by PRRs activates intracellular signaling pathways that
culminate in the induction of inflammatory cytokines, chemokines, interferons
(IFNs) and the upregulation of costimulatory molecules. Toll-like receptors
(TLRs) are particularly essential members of PRRs, and they are responsible for
the recognition of PAMPs and the activation of downstream signaling adaptors.
Among these adaptors, myeloid differentiation factor 88 (MyD88), which mediates
the activation of the TLRs, except for TLR3, was originally identified as a
curial and conserved signaling proteins. MyD88 interacts with IL-1 receptor-
associated kinase (IRAK), and IRAK associates with TNF receptor-associated
factor 6 (TRAF6), which subsequently activates the NF-кB pathway. In addition,
the TLR pathway not only activates inflammation and phagocytosis but also
regulates the induction of apoptosis. Apoptosis is important in the immune
system and plays significant roles in the control of the immune response, the
removal of immune cells recognizing self-antigens, and cytotoxic killing.
Pathogens can be eliminated by the ability of hemocytes to recognize foreign
targets and induce apoptosis. Similarly, *V*. *coralliilyticus* exposure can
induce significant changes in the host TLR pathway and apoptotic systems. Since
the first TLR was identified in *Drosophila melanogaster*, large members of TLR
family have been recently investigated in marine bivalves, such as *Chlamys
farreri*, *Chlamys nobilis*, *Mizuhopecten yessoensis*, *C*. *gigas*, *C*.
*virginica*, *Mytilus edulis* and *Hyriopsis cumingii*. Among them, the innate
immune regulation of TLR genes in bivalves has been reported in *C*. *farrer*i
(*Cf*Toll-1) and *C*. *nobilis* (*Cn*TLR-1), respectively, both of which might
be involved in the immune response against pathogen invasion. In addition, 23
TLRs were identified and arranged in 4 clusters according to extra-cellular LRR
domain content in *Mytilus galloprovincialis*. Furthermore, there were 83 TLR
genes in the genome of *C*. *gigas*, 19 of which had different responses to
*Vibrio* infection. Despite the fact that *T*. *crocea* has high ecological
value and is under the stress of populations, there is limited information
available about its immune molecule response mechanisms.
In the present study, we injected adult *T*. *crocea* with *V*.
*coralliilyticus* to investigate the individual and cellular responses of giant
clams. Furthermore, high-throughput sequencing was applied to analyze the
differentially expressed genes in *T*. *crocea* hemocytes at 0 h, 6 h, 12 h, and
24 h after *V*. *coralliilyticus* challenge. Afterwards, using KEGG pathway
enrichment analysis, some molecular mechanisms of response and candidate genes
involved in *V*. *coralliilyticus* infection were identified. Therefore, we
explored the possible sensing patterns for *V*. *coralliilyticus* in the innate
immune system of *T*. *crocea*. The results provide insights for a better
understanding of *V*. *coralliilyticus* pathogenicity and the future development
of disease prevention strategies.
# Materials and methods
## Ethics statement
All *T*. *crocea* used in the present study were bred in our laboratory (Hainan
Tropical Marine Biology Research Station), Chinese Academy of Sciences, Sanya,
China. No specific permits were required for clams sample collection or
described sampling. The location was not privately-owned or protected, and the
field studies did not involve any endangered or protected species.
## Animals, tissue, embryonic development collection and challenge experiment
Adult *T*. *crocea* (average 6.7–10.8 cm shell length) were obtained from Sanya,
Hainan Province, China and maintained in tanks filled with natural seawater
(temperature: 28±1°C; salinity: 33). Cultures were illuminated with metal halide
bulbs from 6:00 am to 7:00 pm every day for two weeks prior to use.
To analyze the tissue distribution of *Tc*MyD88, the following tissues were
collected from six healthy adult *T*. *crocea*: siphonal mantle, pedal mantle,
inner mantle, byssus gland, pedis, heart, gill and hemocytes. For analysis of
the developmental expression patterns of *Tc*MyD88, samples of 0, 0.5, 1, 4, 12,
16, 24, 48, 72, 96, and 120 h-developing embryos were also obtained.
For the bacterial challenge, an in vivo infection experiment was performed.
Healthy giant clams were injected into the adductor muscle either 100 μL of
2×PBS or 100 μL of live *V*. *coralliilyticus* (1.0 ×10<sup>8</sup> CFU/mL)
suspended in 2×PBS. Specifically, purified bacterial inoculum were centrifuged
for 10 min to collect bacterial pullets and were washed 3 times with PBS and re-
suspended in 2×PBS to a concentration of OD600nm = 1.0. *V*. *coralliilyticus*
(CAIM616) was purchased from the Marine Culture Collection of China and was
cultured in Zobell marine broth 2216 (Difco Laboratories) at 28 °C overnight.
After injection, the giant clams were returned to tanks full of seawater at 28
°C for subsequent treatment or sampling. If the valves of a giant clam were not
closed and the mantle did not react after stimulation, it was considered dead.
Mortality in each tank was assessed visually and counted every 6 h after
injection. Hemocytes were collected at scheduled intervals (0, 3, 6, 12, 24, and
36 h post injection) from both the challenged and control groups. Among them,
hemocytes taken at 0, 6, 12, and 24 h post challenge were stored in liquid
nitrogen for transcriptome analysis. Five individuals were randomly sampled from
each group at every time point after injection to obtain biological replicates.
## Flow cytometric analysis of apoptosis
Hemocytes were harvested 6 h after *V*. *coralliilyticus* injection and were
then resuspended in 100 μL of binding buffer containing 5 μL of Annexin V-FITC
and 5 μL of propidium iodide, provided in an apoptosis detection kit (Vazyme,
A211); the mix was incubated at room temperature for 10 min in the dark.
Finally, another 400 μL of binding buffer was added to the solution and applied
to a Guava<sup>®</sup> easyCyte<sup>™</sup> (Millipore). At least 10,000 cells
were obtained to analyze the population by Flow Jo v10.0 software.
## De novo assembly and gene annotation
Based on the sequencing by synthesis (SBS) technique, cDNA libraries were
sequenced using an Illumina Hiseq high-throughput metering platform to obtain
raw data. The raw data were processed to discard the dirty reads and low-quality
sequences. After filtering, the remaining reads were called “Clean Reads”. The
unigenes were de novo assembled using Trinity. For gene annotation analysis, the
assembled transcripts were scanned against NR (NCBI nonredundant protein
sequences), Swiss-Prot databases and KOG (euKaryotic Ortholog Groups) using
diamond with E-values at 1.0×10<sup>−5</sup> (E-values of less than
1.0×10<sup>−5</sup> were considered significant). NT (NCBI nucleotide sequences)
and PFAM (protein family) analyses were performed using the NCBI BLAST + v2.2.28
and HMMER 3.0 programs, respectively. In addition, the unigenes were also
classified according to the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of
Genes and Genomes) databases.
## Differential expression analysis
Read counts were estimated by mapping clean reads to unigenes using Bowtie2, and
they were calculated according to the comparison result with RSEM. The
expression abundance of the corresponding Unigene was expressed by the FPKM
(expected number of fragments per kilobase of transcript sequence per millions
of base pairs sequenced) value. We used the adjusted p-value to detect
differentially expressed genes (DEGs). When the adjusted p-value was less than
0.05 and had a greater than two-fold change (absolute value of log2 ratio≥1),
the gene was considered differentially expressed in a given library.
Significantly enriched terms were obtained by mapping DEGs to the corresponding
KEGG pathways.
## RNA extraction and cDNA synthesis
Total RNA (50mg) was isolated with TRIzol Reagent (Invitrogen, 15596–026)
according to the manufacturer’s protocol. RNA was dissolved in DEPC-treated
water, and the integrity of the RNA was assessed by electrophoresis with a 1.0%
agarose gel, and then the concentration and purity were examined at 260/230 and
260/280 absorbance ratios. Purified RNA was diluted to 1 mg/ml to synthesize
first-strand cDNA using a Primer Script<sup>™</sup> First Strand cDNA Synthesis
kit (TAKARA Bio Inc. Japan). The cDNA was used as the template for amplifying
gene sequences and analyzing their expression. All primers used in this study
were designed with Primer Premier 5.0 and are shown in.
## Cloning the full-length cDNA of *Tc*MyD88
A search of the transcriptome data of *T*. *crocea* revealed a TIR contig
homologous to the MyD88 gene of *T*. *crocea*. The intermediate fragment
sequences of *Tc*MyD88 were obtained by polymerase chain reaction (PCR). Then,
gene-specific primers were designed to amplify the unknown 5’ and 3’ ends of
*Tc*MyD88 cDNA using rapid amplification of cDNA ends (RACE). For *Tc*MyD88 3’
sequencing, the primer pairs Takara3P/*Tc*MyD88-F1 and Takara3NP/*Tc*MyD88-F2
were employed for primary PCR and nested PCR, respectively. Similarly, the 5’
end of the *Tc*MyD88 gene was obtained by nested PCR using Takara5P/*Tc*MyD88-R1
and Takara5NP/*Tc*MyD88-R2. Full-length cDNA sequences were obtained by
combining intermediate fragment sequences and 3’ and 5’ end sequences.
## Sequence and phylogenetic analyses
The cDNA sequences and deduced amino acid sequences of *Tc*MyD88 were analyzed
using the BLAST program (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>) and the
Expert Protein Analysis System (<http://expasy.org/>). The nucleotide and
protein sequences were analyzed using BLASTN and BLASTX, respectively. The
molecular weights and the theoretical isoelectric points were calculated using
the Compute pI/Mw tool (<http://web.expasy.org/compute_pi/>). A structural
analysis of proteins was performed using the Simple Modular Architecture
Research Tool (SMART) program (<http://smart.embl-heidelberg.de/>). Multiple
protein sequence alignments were performed using Bio-edit software via the
Clustal W method. A phylogenetic tree was constructed using the MEGA5.0 software
based on the alignment of the complete amino acid sequences with the neighbor-
joining method and 1000 bootstrap replicates.
## Plasmid construction, cell culture and transfection
The *Tc*MyD88 ORF was amplified from *T*. *crocea* cDNA by PCR using specific
primers designed by CE Design V1.03. The target PCR products were subcloned into
eukaryotic expression vectors, pcDNA3.1-HIS (Promega, USA) and pEGFP-N1
(Promega, USA), by homologous recombination using a Vazyme<sup>™</sup> one step
cloning kit. The constructed recombinant plasmids were digested with enzymes,
and the inserted fragments of each clone were sequenced. All of the plasmids
used for transfection were extracted from overnight bacterial cultures using a
HiPure Plasmid EF Micro kit (Magen) according to the manufacturer’s protocol.
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco)
supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics
(100 mg/L streptomycin and 105 U/L penicillin, Gibco) at 37 °C with 5%
CO<sub>2</sub>, and they were subcultured every three days. Transfections were
performed using ViaFect reagent (Promega, USA) according to the manufacturer’s
instructions.
## Subcellular localization and dual-luciferase reporter assays
For subcellular localization analysis, HEK293T cells were seeded into a 6-well
cell plate, cultured for 24 h and then transfected with a pEGFP-N1-*Tc*MyD88
mix. Forty-eight hours after transfection, cells were washed once with 1×PBS (pH
7.4) and then were fixed with 4% paraformaldehyde for 10 min, which was followed
by staining with 6-diamidino-2-phenyl-indole (DAPI) (1 mg/ml) for 5 min.
Finally, the cells transfected with fluorescent vectors were directly observed
by fluorescence microscopy.
For dual-luciferase reporter assays, cells were plated in a 48-well cell plate,
cultured until they reached 40–50% confluency and then were transfected with
pRL-TK (20 ng/well), NF-κB reporter (200 ng/well) and a target plasmid (0, 100,
200 or 400 ng/well). The pRL-TK vector and NF-κB vector (Promega, USA) were used
as internal controls. After 48 h of transfection, the luciferase activity of
total cell lysates was measured using a luciferase reporter assay system
(Promega, USA). Renilla luciferase activity was expressed as the fold
stimulation relative to that of the empty vector transfected cells. The values
are expressed as the mean relative stimulation for a representative experiment
from four separate experiments, and each experiment was performed in duplicate.
## Quantitative real-time PCR analysis
qRT-PCR reactions were conducted using 2 × Real Star Green Power Mixture
(GenStar, A311) and a LightCycler<sup>®</sup> 480 II (Roche, Switzerland)
according to the manufacturer’s protocol. All the template cDNA were diluted to
200ng/μl. PCR conditions were as follows: 95 °C for 10 min, followed by 40
cycles of amplification at 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. At
the end of each qPCR, a melting curve analysis was performed to confirm the
specificity of the PCR products. The data from each experiment are expressed
relative to the expression levels of the β-actin gene to normalize expression
levels between the samples. All of the samples were analyzed in triplicate, and
the expression values were calculated with the 2<sup>-ΔΔCT</sup> method.
## Statistical analysis
Data processing and statistical analyses were performed using GraphPad Prism
v5.0.1. All data are presented as the means ± S.D. Comparisons between two
groups of samples were performed with Student’s t-tests, and comparisons among
more than two groups were performed with one-way ANOVA followed by Turkey’s or
Dunn’s post hoc test. Values \*p \< 0.05, \*\*p \< 0.01 and \*\*\*p \< 0.001
were considered significant.
# Results
## The mortality and apoptosis after vibrio challenge
To investigate the possible effects of a *V*. *coralliilyticus* challenge on
*T*. *crocea*, challenge experiment was performed. After injection with *V*.
*coralliilyticus*, the siphonal mantle of *T*. *crocea* contracted inward and
collapsed. Bacterial challenge directly affected adult survival; dead
individuals appeared at 6 h post infection, and 45.56% and 56.78% mortality
rates were observed in the two challenged groups, while no death was observed in
the control group after 36 h. Flow cytometry was performed to detect the
apoptosis of hemocytes. The results showed that the apoptosis rate of the
hemocytes dramatically increased 1.8-fold (including early and late apoptotic
cells) in the *V*. *coralliilyticus*-injected *T*. *crocea* compared with that
of the control group, which clearly implied that *V*. *coralliilyticus* could
cause apoptosis in *T*. *crocea* hemocytes.
## Transcriptome sequencing and assembly of transcripts
The transcripts of *T*. *crocea* injected with *V*. *coralliilyticus* were
assembled to analyze the molecular response mechanism of *T*. *crocea* during
*V*. *coralliilyticus* infection. After filtering dirty reads from the raw
reads, 46525916, 44448554, 49647448 and 53853546 clean reads were obtained from
hemocytes in the *V*. *coralliilyticus* challenged group at 0 h, 6 h, 12 h, and
24 h, respectively. Sequencing reads were found to be of high quality, with Q30%
values of 92.97%, 92.33%, 91.89% and 93.03%, respectively. The GC content of
nucleotides was approximately 38%. For all four sequencing libraries, the
percentages of reads that could be matched with assembled reference sequences
were higher than 64.80%. All sequencing reads were stored into the Short Read
Archive (SRA) database and available with the accession PRJNA596951.
## Annotation and function analysis of unigenes
To achieve protein identification and gene annotation, transcripts were compared
with data from the NR, NT, KO, Swiss-Prot, PFAM, GO and KOG databases. A total
of 195651 unigenes (ranging from 301 to 25533 bp) were generated with an N50
size of 810 bp. All sequences from 75468 unigenes were annotated in at least one
database, with 28385 annotated unigenes (14.50%) having significant matches with
sequences in the NR. We also assembled the species distribution of the unigenes
by aligning sequences against the NR database to learn the sequence similarity
of *T*. *crocea* with other species. A total of 62.5% of the unigenes matched
with sequences from five top-hit species, *M*. *yessoensis* (25.4%), *C*.
*gigas* (14.3%), *C*. *virginica* (14.0%), *Lottia gigantea* (5.2%) and *Lingula
anatina* (3.6%), all of which are mollusks.
GO classification is a unified gene functional classification system. The
analysis of annotated transcripts demonstrated that 36361 matched unigenes
(18.58%) were divided into 26, 20, 10 entries for three categories, biological
processes (BP), cellular component (CC) and molecular function (MF),
respectively. The unigenes in “binding,” “catalytic activity,” “signaling,”
“response to stimulus,” “membrane,” which were potentially related to immune
existed in higher percentages compared to counterparts at sub-categories at the
whole transcriptome reference. They played important roles during the defense
process after challenge. The results provided a comprehensive view for screening
candidate genes related to immune and defense mechanism.
In addition, we also used KEGG to identify the probable functional status of
assembled transcripts. A total of 12912 unigenes (6.59%) were assigned to five
main categories containing 232 KEGG pathways. Remarkably, immune system (558)
was the well-represented term in the KEGG organismal systems category. Signal
transduction (1455) was the most enriched terms in both environmental
information processing and the whole transcriptome reference, suggesting the
dominant position of various signal transduction in processing the stimulation
of environment. In the metabolism category, abundant unigenes were found in the
amino acid metabolism (510) sub-category. These results indicated that these
unigenes may play a role in mediating *V*. *coralliilyticus* exposure and
associated impacts.
## Identification of differentially expressed genes
To identify the DEGs involved in *V*. *coralliilyticus* infection, a comparison
of the relative transcript abundance for each unigene was performed. We compared
the expression levels of each unigene for 6 h, 12 h, and 24 h with 0 h,
obtaining 1941, 2172 and 1116 DEGs, respectively. By taking the union of three
sets of DEGs, we ended up with 3446 DEGs. Subsequently, KEGG pathway enrichment
analysis was used to classify the DEGs and highlight biological associations. As
expected, the results emphasized the immune system, where the Toll-like receptor
signaling pathway and the NF-кB signaling pathway were enriched in DEGs.
Furthermore, some DEGs were associated with apoptotic signaling, such as the TNF
signaling pathway. Meanwhile, we selected some of the DEGs related to innate
immunity and constructed a heat map in which 39 genes were upregulated and 16
genes were downregulated. The upregulated genes were mainly TLR pathway-related
genes, immune effector molecules (which included inflammatory cytokines
interleukin-1 (IL-1), IL-17 and tumor necrosis factor (TNF)), antimicrobial
peptides (β defensin and phage lysozyme 2), and apoptosis-related genes (such as
inhibitor of apoptosis proteins 1 (IAP1) and the anti-apoptotic genes of the
Bcl-2 family). For the TLR pathway, one TLR homolog, the adaptor molecule MyD88,
TRAF6, kinase IRAK4 and two transcription factors IкB-α and NF-кB were all
significantly upregulated after 6 h and 12 h of infection. The downregulated
genes included members of the lectin family and the complement pathway,
including perlucin, galectin, and C-type lectin. The results revealed that the
MyD88-dependent pathway was activated after *V*. *coralliilyticus* challenge,
which promoted the release of immune effector molecules such as IL-1 and TNF,
and indirectly affected the expression of some apoptosis-related genes.
## Cloning and sequence analyses of *Tc*MyD88
*Tc*MyD88 was dramatically enriched in multiple KEGG pathways and occupied an
essential position in the TLR pathway. The full-length sequence of *Tc*MyD88
(MN829819) is 1853 bp, containing a 105 bp 5’-untranslated region (UTR), a 140
bp 3’-UTR and an open reading frame (ORF) of 1608 bp. The ORF encodes a putative
protein of 535 amino acids, with an estimated molecular mass of 60.430 kDa and a
theoretical isoelectric point (PI) of 5.04.
The protein structure of *Tc*MyD88 was predicted and analyzed using the SMART
program. Typical Myd88 domains comprising a DEATH domain (residues 24–123) and a
TIR domain (residues 179–316) were identified in the *Tc*MyD88 protein.
To clarify the evolutionary relationship among variable MyD88 proteins, multiple
sequence alignments were performed with the deduced amino acid sequences of
MyD88. Compared with other species, *Tc*MyD88 displayed a high degree of
homology and conservation, especially in the TIR domain. Moreover, a
phylogenetic tree was also constructed using MEGA5.0 software using the
neighbor-joining method. The deduced phylogeny of MyD88 revealed two major
clusters, one for mollusks and the other for fish and mammals. *Tc*MyD88
initially clustered with bivalves, such as *Ruditapes philippinarum*, *H*.
*cumingii*, *C*. *virginica*, and *C*. *giga*, to form a single branch. In
conclusion, the evolution of *Tc*MyD88 is relatively well conserved in bivalves,
and it is distantly related to that of vertebrates and other invertebrates.
## Expression pattern variation of *Tc*MyD88 in tridacna
The tissue distributions of *Tc*MyD88 mRNA were detected by qRT-PCR. According
to the results, *Tc*MyD88 was widely expressed in all examined tissues,
including the siphonal mantle, pedal mantle, inner mantle, byssus gland, pedis,
heart, gill and hemocytes. With the change in the position of mantles, the
expression level of *Tc*MyD88 varied, while it was relatively highly expressed
in the siphonal mantle. Furthermore, it was expressed predominantly in the
gills, followed by the siphonal mantle and hemocytes, whereas the pedis, heart
and byssus gland contained low levels. The expression level of *Tc*MyD88 was
approximately 33.7-fold higher in gills than it was in hearts.
The mRNA expression pattern of *Tc*MyD88 was also observed throughout
developmental stages. As shown in the figure, the expression of *Tc*MyD88
maintained a stable, low level from 0 h to 72 h. At 72 h, there was a sudden
increase, and the level continuously increased after 72 h.
## Subcellular localization of *Tc*MyD88
The subcellular localization of *Tc*MyD88 was examined by transient transfection
of the *Tc*MyD88-GFP plasmid into HEK293T cells. Imaging of the GFP-tagged
*Tc*MyD88 revealed that *Tc*MyD88 was distributed mainly in the cytoplasm,
whereas the GFP protein was found in both the cytoplasm and the nucleus.
## Dual-luciferase reporter assays
To determine whether *Tc*MyD88 could modulate NF-кB transcriptional activity,
dual-luciferase reporter assays were performed in HEK293T cells. As shown in,
*Tc*MyD88 can activate the NF-κB responsive reporter, and the overexpression
significantly increased the activation in a dose-dependent manner from 100 ng to
400 ng. The most marked increase was approximately 10.9-fold (P \< 0.01) over
what was observed in cells transfected with pCDNA3.1 alone. These results
implied that *Tc*MyD88 could potentially be involved in the NF-кB signaling
pathway.
## The responses of TLR pathway-related genes in *T*. *crocea* to *V*. *coralliilyticus* challenge
On account of the DEG analysis showing that the TLR pathway was activated, we
proceeded to analyze pathway-related genes by qRT-PCR. For the TLR pathway
genes, significant differences between control and infected *T*. *crocea*
individuals were observed for TLR, MyD88, IRAK4, TRAF6 and IкB-α. Upon *V*.
*coralliilyticus* challenge, the level of TLR transcripts was significantly
upregulated at 6 h (22.9-fold; p \< 0.01) and then returned to the basal level
at 36 h. With *V*. *coralliilyticus* stimulation, the expression of *Tc*MyD88
initially significantly increased at 3 h (4.8-fold; p \< 0.01) and reached the
highest expression levels at 6 h (9.6-fold; p \< 0.01), and then it declined at
12 h. During the *V*. *coralliilyticus* challenge, the expression level of IRAK4
was upregulated at 3 h and maintained a high level throughout the whole stage,
with the highest value at 6 h (4.0-fold; p \< 0.01); then it returned to the
basal level at 36 h. With exposure to *V*. *coralliilyticus*, TRAF6 expression
was significantly upregulated by 3.6-fold (p \< 0.01) at 6 h, and then it peaked
with a 2.8-fold increase at 12 h (p \< 0.001). After *V*. *coralliilyticus*
infection, the expression of IкB-α rapidly increased at 6 h (3.9-fold; p \<
0.001) and then dropped to 1.5-fold at 12 h (p \< 0.05) before nearing the
control level at 36 h. The expression of cytokine-related genes was also
affected by infection. In response to *V*. *coralliilyticus* challenge, IL-17
exhibited an increase at 3 h and 6 h by 6.3-fold (p \< 0.001) and 10.5-fold (p
\< 0.001), respectively. There was also a significant change in apoptosis-
related gene IAP1. During *V*. *coralliilyticus* infection, IAP1 mRNA was
increased at 3 h (2.0-fold; p \< 0.01), and it dramatically increased at 6 h
with a 3.0-fold change (p \< 0.001). In summary, the qRT-PCR analyses greatly
confirmed the direction of changes determined by the transcriptome analysis.
Preliminary indications are that the expression of TLR pathway-related genes
changed after *V*. *coralliilyticus* challenge, and the downstream genes were
activated, which promoted the release of inflammatory and led to the activation
of a series of signals.
# Discussion
In a global warming scenario, an increase in the seawater temperature could
promote the potential disease outbreaks associated with *V*. *coralliilyticus*
in mollusks. In the present study, we demonstrated that *V*. *coralliilyticus*
could cause acute mortalities of adult *T*. *crocea* when previous work was
mainly focused on mortalities of shellfish larvae caused by *V*.
*coralliilyticus*. After injection with *V*. *coralliilyticus*, the siphonal
mantle of *T*. *crocea* contracted inward and collapsed, while the siphonal
mantle of healthy *T*. *crocea* extended outward. In previous studies, *V*.
*coralliilyticus* infection resulted in the loss of *C*. *gigas* larval motility
due to the gross pathological changes to the velum and cilia. Histopathology
indicated that the route of infection by *V*. *coralliilyticus* was the
digestive system in Greenshell<sup>™</sup> mussel larvae. Furthermore,
vacuolation of the tissues of the digestive tract similar to oyster *C*.
*virginica* larvae and necrotic tissue were observed. Moreover, the results of
this study suggest that *V*. *coralliilyticus* could trigger apoptosis in *T*.
*crocea* hemocytes. Diverse metalloprotease and effector genes like the pore
forming toxin hlyA were identified in the genome of *V*. *coralliilyticus* and
expressed proteases were also detected in the secretome, which caused mortality
in *Drosophila* and *Artemia* and may be involved in the infection of *T*.
*crocea*. It has been reported that hemolysins play a role in inducing
apoptosis. In addition, *C*. *gigas* larvae infected by *V*. *coralliilyticus*
showed higher activities of catalase and superoxide dismutase, two key enzymes
implicated in antioxidant defense, indicating their activation after pathogen
stimulation; further, PO activity was significantly increased in challenged
mussel larvae. While sequencing the genomes of *V*. *coralliilyticus* isolates
has indicated a varying repertoire of potential virulence factors that may
function independently or in concert to induce pathogenicity, hemolysin and
extracellular protease activities are thought to play important roles during
pathogenesis in oysters. It was found that lytic enzymes produced by aquaculture
pathogens include haemolysins and proteases. Extracellular metalloproteases
facilitate bacterial invasion and the infection process, acting to enhance
tissue permeability and leading to necrotic tissue damage and cytotoxicity in
the host. The discovery in our study filled the research blank of rapid
lethality caused by *V*. *coralliilyticus* in adult bivalves. Furthermore, we
dissected this phenomenon from a mechanistic molecular level.
To shed light on the molecular response mechanism of *T*. *crocea* during *V*.
*coralliilyticus* infection, we used transcriptome sequencing analysis and other
relevant techniques to analyze the *T*. *crocea* hemocytes at 0 h, 6 h, 12 h and
24 h after *V*. *coralliilyticus* challenge. *T*. *crocea* employs the innate
immune response as the sole defense mechanism against pathogen infection, such
as pathogen recognition and apoptosis systems. Our results revealed that the
total number of unigenes and DEGs was 195651 and 3446, respectively. More
details were uncovered by KEGG pathway enrichment analysis, where DEGs were
found to be significantly enriched in immune-related signaling pathways, such as
the TLR signaling pathway and the NF-κB pathway, and some were associated with
apoptotic pathways, such as the TNF signaling pathway. These results were
consistent with the quantitative validation, where it was preliminarily
demonstrated that the expression of TLR pathway-related genes changed after *V*.
*coralliilyticus* challenge, and downstream genes were activated, which promoted
the release of inflammatory factors such as IL-17 and led to the activation of a
series of signals. Taken together, these results coincided with previous work in
Pacific oyster larvae and adult *Nematostella vectensis* in which the TLR-to-NF-
κB pathway was activated during pathogenic conditions. However, this activation
reaction is not absolute, as shown here; some downregulated genes, such as the
partially lectin family, are components of the complement system. The complement
system enables one of the major innate immune mechanisms, which has the ability
to remove microbes and attack pathogens, and the existence of a potential
multicomponent complement system has been identified in shellfish. When *C*.
*gigas* was infected with *V*. *splendidus*, suppressed genes may have helped
the bacteria escape the giant clam response, and afterward, the bacteria were
able to establish the pathogenic intracellular and intravesicular forms mediated
by localization that enhanced bacterial protection from an innate immune attack.
Furthermore, *Tc*MyD88 of the TLR pathway was identified by KEGG pathway
enrichment analysis. MyD88 has been demonstrated to be a key adapter protein in
TLR signal transduction that triggers downstream cascades involved in innate
immunity in organisms ranging from mammals to mollusks, that includes human,
fish, Drosophila, and *C*. *gigas*. The MyD88-dependent pathway leads to the
activation of NF-κB and the expression of proinflammatory genes, such as TNF and
IL-1. *Tc*MyD88 was found to have two conserved domains, the death domain and
the TIR domain. Furthermore, the expression profile of *Tc*MyD88 and the
activation of NF-кB both revealed that *Tc*MyD88 plays a crucial role in the
regulation of the *T*. *crocea* immune system. These results verified the
existence and significance of a MyD88-dependent signaling pathway in *T*.
*crocea*. Acting as an intermediate receptor for signal transduction, MyD88
participates in the transmission of multiple signaling pathways. TNF expression
and apoptosis have been reported to share the same signal transduction molecule,
MyD88, in human myelomonocytic cells. Meanwhile, the TLR pathway and other
signaling pathways may increase the activation of MyD88. Therefore, *Tc*MyD88
may serve as a link among different immune regulatory mechanisms against *V*.
*coralliilyticus* infection.
In conclusion, we discovered that *V*. *coralliilyticus* could cause acute
mortality of adult *T*. *crocea* at 28°C, which is the first evidence of the
rapid lethality of *V*. *coralliilyticus* in adult bivalves at natural and
agricultural temperatures, since previous work was mainly focused on mortality
of shellfish larvae. Moreover, transcriptomic analyses of the differences in
molecular mechanisms between healthy and infected giant clams were obtained, and
abundant differentially expressed immune-related genes and signaling pathways
were identified, which drew our attention to the TLR pathway. Following
quantitative validation and functional analysis, the results suggested that
adult *T*. *crocea* could initiate the innate immune response through the TLR
pathway against Vibrio infection, where changes in TLR pathway-related gene
expression promoted the release of inflammatory factors such as IL-17, leading
to the activation of a series of signals driving activities such as apoptosis.
Despite the fact that *V*. *coralliilyticus* appears to be a global bivalve
pathogen, limited information about its pathogenicity, infection mechanism and
disease mitigation is available. Further studies are needed on the immune
defense mechanisms of adult giant clams. These studies will be conducive to the
development of health management in aquaculture.
# Supporting information
We would like to acknowledge the contributions of several other people to this
work. We would like to thank Chuanjie Fu, Kunna Liu and Yunqing Li for their
helps with samples collection. We appreciate the sequencing services of Novogene
(Beijing, China).
10.1371/journal.pone.0231399.r001
Decision Letter 0
Fugmann
Sebastian D.
Academic Editor
2020
Sebastian D. Fugmann
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
22 Jan 2020
PONE-D-19-35528
Mechanistic molecular responses of the giant clam Tridacna crocea to Vibrio
coralliilyticus challenge
PLOS ONE
Dear Dr zhiming,
Thank you for submitting your manuscript to PLOS ONE. After careful
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1\) Reviewer \#1 raised numerous concerns about experimental details. This
should be addressed by making appropriate changes to the Material & Methods
section and to the text of the Results / Figure legends where appropriate.
2\) The concerns about the apoptosis assay in Fig. 1 need to be addressed.
While the percentages of cells in the quadrants change upon infection, there is
no actual "cell population" coming up which in annexin V positive (and hence
early apoptotic) - it appears like a general no specific increase in
fluorescence signal. Also the is a considerable number of PI+ cells at t=0 h
raising concerns about a large number of cells dying during the cell extraction
and purification procedure. Also these cells should likely be PI+ but Annexin V
negative; thus there is an apparent issue with the compensation between the
fluorescence channels in this experiment ( take a look a the sample plots of the
vendor of the kit
used: <http://www.vazymebiotech.com/products_detail/productId=84.html>). One
important positive control in this context would be a treatment of the cell with
an apoptosis inducing agent (e.g. etoposide).
3\) The assembled transcriptome dataset should also be made publicly available
such that a consistent gene annotation for this organism can be established for
future references.
4\) The labeling in heat map shown in Figure 3 is not really helpful - instead
of only referring to Cluster XXXX YYYYY, common names should be utilized that
give a clear hint on the function of the genes being differentially expressed.
5\) The description of the results regarding the GO/ KEGG classification (lines
311-322) is currently meaningless, e.g. it is quite obvious (even without
knowing anything about the data) that the majority of genes fall in to classes
like "cellular processes", "metabolic processes", "cell", and "cell part". What
else should they fall in? I strongly urge the author to use their biological
"common sense" to omit this non-useful information (that likely was just copid
as the highest ranking scores from a bioinformatics software produces) and
rather include information that actually provides some biological insight into
the immune system of their model organism.
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deposited to a public repository. For example, in addition to summary
statistics, the data points behind means, medians and variance measures should
be available. If there are restrictions on publicly sharing data—e.g.
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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted
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grammatical errors should be corrected at revision, so please note any specific
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5\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: The manuscript presented here is of interest, well-written and
presents new data.
My main concern is about the functional demonstration of apoptosis induction by
V. coralliilyticus.
First, I am not confident in the chosen flow cytometry areas. Can I see your
dead and apoptosis-induced controls? For me, PI+ cells should be separated at
102. With such area, I am quite afraid about your control hemocyte mortality
(more than 10%?)
Secondly, to my opinion, one unique technic (on one sampling point) is not
enough to demonstrate apoptosis. Can’t you perform TUNEL assay, caspase dosage,
TEM, …?
In introduction section, I would appreciate to find more information on the huge
diversity of TLR in marine bivalves.
Material and method sections lack information
\- How many individuals were challenges?
\- Did you check bacterial suspension purity and concentration?
\- Did you analyze moribund animals to ensure V. coralliilyticus ‘imputability’
in mortality?
\- What is the difference in the hemolymph sampling explained L138-139 and
141-142 ?
\- If hemocytes sampled at 0, 3, 6, 12, 24 and 36h, can we see flow cytometry
results on all sampling points?
\- Were the RT-QPCR analyses (L440) and RNA-seq analyses performed on the same
biological samples
\- L150. Triplicate = technical replicates and not biological replicates if I
correctly understand what you mean
\- Can you precise the volume/weight of tissue for RNA extraction L177
\- For RNA, 230 nm is also informative (L180)
\- For qPCR, were the cDNA used diluted or pure? Can you precise it L241?
In discussion section, I would recommend to authors to be more careful on the
potential bacterial virulence factors that could induce the immune response
measured (L485-500). There is a diversity of hemolysins for instance. Even if
some hemolysins were described in other models as playing a role in apoptosis,
you could not say that ‘some hemolysins \[…\] in the secretome of V. cora \[…\]
may be responsible of this phenomena’ (L487). Are they expressed in vivo ? and
which hemolysin are we talking about? Except by performing dual-RNAseq, you
should be more moderate in this part of your discussion.
\*\*\*\*\*\*\*\*\*\*
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10.1371/journal.pone.0231399.r002
Author response to Decision Letter 0
3 Mar 2020
PONE-D-19-35528
Reviewer \#1
1\) Reviewer \#1 raised numerous concerns about experimental details. This
should be addressed by making appropriate changes to the Material & Methods
section and to the text of the Results / Figure legends where appropriate.
Response: We have appropriately changed the Material & Methods section as the
reviewer suggested. We totally used 330 individuals to complete the experiment.
Each experiment had biological repeats. Some imprecise statements have been
corrected in the revised MS (please see line151-152, 287, 193). And the
insufficient information has been improved and supplemented in the revised MS
(please see line142-144, 190, 256). Through the reculture of bacteria from the
moribund animals hemocytes and no deaths occurring after PBS and Vibrio
alginolyticus injection, we can ensure V. coralliilyticus ‘imputability’ in
mortality. Meanwhile, based on previous studies and our data, we chose the time
point, 6 h to perform flow cytometry in consideration of the scarcity of
experimental samples.
2\) The concerns about the apoptosis assay in Fig. 1 need to be addressed. While
the percentages of cells in the quadrants change upon infection, there is no
actual "cell population" coming up which in annexin V positive (and hence early
apoptotic) - it appears like a general no specific increase in fluorescence
signal. Also the is a considerable number of PI+ cells at t=0 h raising concerns
about a large number of cells dying during the cell extraction and purification
procedure. Also these cells should likely be PI+ but Annexin V negative; thus
there is an apparent issue with the compensation between the fluorescence
channels in this experiment ( take a look a the sample plots of the vendor of
the kit used: <http://www.vazymebiotech.com/products_detail/productId=84.html>).
One important positive control in this context would be a treatment of the cell
with an apoptosis inducing agent (e.g. etoposide).
Response: We have repeated the experiment of apoptosis as the reviewer suggested
and submitted a new version of Fig 1, and we have compensated for the
fluorescence channels appropriately according to the manufacturer’s instruction
for both this time and before. Specific experimental operations and data
processing were referred to Lin’s and Qin’s articles, where the early apoptosis
of hemocytes in marine invertebrates may not be accompanied by the new “cell
population” coming up \[1-3\]. Moreover, due to the specificity of species and
the difficulty of experimental operation, some cells died during the previous
experimental treatment. However, the control group and the experimental group
were performed simultaneously, with the same protocol and statistical methods,
which making the FACS results comparable and statistically significant
differences credible. The Annexin-positive cells in the experimental group was
significantly higher (P\<0.05) than that in the control group, although there
was no new “cell population” coming up, which indicates V. coralliilyticus
infection could induce hemocytes apoptosis (please see the details below).
3\) The assembled transcriptome dataset should also be made publicly available
such that a consistent gene annotation for this organism can be established for
future references.
Response: We will release the assembled transcriptome dataset, as soon as the
article is accepted.
4\) The labeling in heat map shown in Figure 3 is not really helpful - instead
of only referring to Cluster XXXX YYYYY, common names should be utilized that
give a clear hint on the function of the genes being differentially expressed.
Response: Thank you for reviewer’s helpful comments. We have revised the
labeling in the heat map and submitted the new version of Fig 3.
5\) The description of the results regarding the GO/ KEGG classification (lines
311-322) is currently meaningless, e.g. it is quite obvious (even without
knowing anything about the data) that the majority of genes fall in to classes
like "cellular processes", "metabolic processes", "cell", and "cell part". What
else should they fall in? I strongly urge the author to use their biological
"common sense" to omit this non-useful information (that likely was just copid
as the highest ranking scores from a bioinformatics software produces) and
rather include information that actually provides some biological insight into
the immune system of their model organism.
Response: Thank you for reviewer’s valuable comments and we changed the
description of the results as reviewer suggested (please check the MS line
320-326, 336-342 and below).
Revised: GO classification is a unified gene functional classification system.
The analysis of annotated transcripts demonstrated that 36361 matched unigenes
(18.58%) were divided into 26, 20, 10 entries for three categories, biological
processes (BP), cellular component (CC) and molecular function (MF),
respectively. The unigenes in “binding,” “catalytic activity,” “signaling,”
“response to stimulus,” “membrane,” which were potentially related to immune
existed in higher percentages compared to counterparts at sub-categories at the
whole transcriptome reference. They played important roles during the defense
process after challenge. The results provided a comprehensive view for screening
candidate genes related to immune and defense mechanism (S2 Fig).
In addition, we also used KEGG to identify the probable functional status of
assembled transcripts. A total of 12912 unigenes (6.59%) were assigned to five
main categories containing 232 KEGG pathways. Remarkably, immune system (558)
was the well-represented term in the KEGG organismal systems category. Signal
transduction (1455) was the most enriched terms in both environmental
information processing and the whole transcriptome reference, suggesting the
dominant position of various signal transduction in processing the stimulation
of environment. In the metabolism category, abundant unigenes were found in the
amino acid metabolism (510) sub-category. These results indicated that these
unigenes may play a role in mediating V. coralliilyticus exposure and associated
impacts (S3 Fig).
Reviewer \#2
Reviewer \#1: The manuscript presented here is of interest, well-written and
presents new data.
1\) My main concern is about the functional demonstration of apoptosis induction
by V. coralliilyticus.
First, I am not confident in the chosen flow cytometry areas. Can I see your
dead and apoptosis-induced controls? For me, PI+ cells should be separated at
102. With such area, I am quite afraid about your control hemocyte mortality
(more than 10%?)
Secondly, to my opinion, one unique technic (on one sampling point) is not
enough to demonstrate apoptosis. Can’t you perform TUNEL assay, caspase dosage,
TEM, …?
Response: Firstly, we have repeated the experiment of apoptosis as reviewer
suggested and submitted the new version of Fig 1, where the control hemocyte
(PBS injection group) mortality were all less than 10% (please see the details
below).
Secondly, RNA-seq and RT-QPCR have confirmed the change of apoptosis-related
genes expression. Moreover, it was at the same time point as flow cytometry,
which further proves the occurrence of apoptosis.
2\) In introduction section, I would appreciate to find more information on the
huge diversity of TLR in marine bivalves.
Response: Thank you for reviewer’s valuable comments and we supplemented more
information on the huge diversity of TLR in marine bivalves in the introduction
(please check the MS line 108-117 and below).
Revised: Since the first TLR was identified in Drosophila melanogaster, large
members of TLR family have been recently investigated in marine bivalves, such
as Chlamys farreri, Chlamys nobilis, Mizuhopecten yessoensis, C. gigas, C.
virginica, Mytilus edulis and Hyriopsis cumingii \[30-36\]. Among them, the
innate immune regulation of TLR genes in bivalves has been reported in C.
farreri (CfToll-1) and C. nobilis (CnTLR-1), respectively, both of which might
be involved in the immune response against pathogen invasion \[31, 37\]. In
addition, 23 TLRs were identified and arranged in 4 clusters according to extra-
cellular LRR domain content in Mytilus galloprovincialis \[38\]. Furthermore,
there were 83 TLR genes in the genome of C. gigas, 19 of which had different
responses to Vibrio infection \[39\].
3\) Material and method sections lack information
\- How many individuals were challenges?
Response: 330 individuals were challenged. 40 individuals were performed for
flow cytometry considering individual differences and death. 180 were used to
investigate mortality where the experiment was repeated three times, with 20
individuals in each group tested every time. Another 110 individuals were for
RNA-seq and RT-QPCR, where 5 individuals were randomly sampled from each group
at every time point and 50 of them were used to make up for the loss of death.
\- Did you check bacterial suspension purity and concentration?
Response: We have supplemented the information in the revised MS (please see
line 142-144). Specifically, for bacterial challenge, purified bacterial
inoculum were centrifuged for 10 min to collect bacterial pullets and were
washed 3 times with PBS and re-suspended in 2×PBS to a concentration of OD600nm
= 1.0.
\- Did you analyze moribund animals to ensure V. coralliilyticus ‘imputability’
in mortality?
Response: Hemocytes were collected from moribund animals and then homogenized
and diluted in PBS and spread on Thiosulfate Citrate Bile Salts Sucrose (TCBS)
Agar plates, followed by overnight culture. Through the successful reculture of
bacteria, we can ensure V. coralliilyticus ‘imputability’ in mortality (please
see the details below).
Meanwhile, when giant clams were injected with an equal amount of PBS and Vibrio
alginolyticus under the same conditions, there was no observable death, which
also demonstrated that V. coralliilyticus should be the main cause of mortality
in T. crocea.
\- What is the difference in the hemolymph sampling explained L138-139 and
141-142 ?
Response: We have corrected the misunderstanding statement in the revised MS
(please see line151-152). There is no difference in the hemolymph sampling
explained L138-139 and 141-142.
Revised: Hemocytes were collected at scheduled intervals (0, 3, 6, 12, 24, and
36 h post injection) from both the challenged and control groups. Among them,
hemocytes taken at 0, 6, 12, and 24 h post challenge were stored in liquid
nitrogen for transcriptome analysis.
\- If hemocytes sampled at 0, 3, 6, 12, 24 and 36h, can we see flow cytometry
results on all sampling points?
Response: It has been reported that after 6 h post-injection of V.
coralliilyticus, haemocyte cell concentrations in haemolymph of infected and
non-infected mussels were similar. However, the proportion of viable haemocytes
in haemolymph of infected mussels was substantially lower than that in non-
infected mussels \[4\]. Hence, we speculated that 6 h might be a critical time
point. Meanwhile, in terms of phenotype, dead individuals appeared at 6 h post
infection. Furthermore, in terms of mechanism, the expression of apoptosis-
related genes like IAP1 was dramatically increased at 6 h, which both RNA-seq
and RT-QPCR confirmed. Based on these, it is reliable that flow cytometry was
performed at 6 h post infection. On the other hand, giant clams are rare and
difficult to collect. Taking into account the loss of death, this experiment
needs a large number of samples, which should be considered very carefully and
ecological friendly.
\- Were the RT-QPCR analyses (L440) and RNA-seq analyses performed on the same
biological samples
Response: The RT-QPCR analyses and RNA-seq analyses were performed on the same
biological samples. Each sample was divided into two parts, one for RNA-seq, the
other for RT-QPCR.
\- L150. Triplicate = technical replicates and not biological replicates if I
correctly understand what you mean
Response: We have corrected the misunderstanding statement in the revised MS
(please see line287). We performed biological replicates and n=3.
\- Can you precise the volume/weight of tissue for RNA extraction L177
Response: We have supplemented the information in the revised MS (please see
line190). We sampled 50mg of each tissue for RNA extraction.
\- For RNA, 230 nm is also informative (L180)
Response: We have corrected the unspecific statement in the revised MS (please
see line193).
Revised: The concentration and purity were examined at 260/230 and 260/280
absorbance ratios.
\- For qPCR, were the cDNA used diluted or pure? Can you precise it L241?
Response: We have supplemented the information in the revised MS (please see
line256). For qPCR, all the template cDNA were diluted to 200ng/μl.
4\) In discussion section, I would recommend to authors to be more careful on
the potential bacterial virulence factors that could induce the immune response
measured (L485-500). There is a diversity of hemolysins for instance. Even if
some hemolysins were described in other models as playing a role in apoptosis,
you could not say that ‘some hemolysins \[…\] in the secretome of V. cora \[…\]
may be responsible of this phenomena’ (L487). Are they expressed in vivo ? and
which hemolysin are we talking about? Except by performing dual-RNAseq, you
should be more moderate in this part of your discussion.
Response: Thank you for reviewer’s valuable comments and we have corrected the
related description in the discussion (please check the MS line 510-514 and
below).
Revised: Diverse metalloprotease and effector genes like the pore forming toxin
hlyA were identified in the genome of V. coralliilyticus and expressed proteases
were also detected in the secretome, which caused mortality in Drosophila and
Artemia and may be involved in the infection of T. crocea.
References
1\. Lin Y, Mao F, Zhang X, Xu D, He Z, Li J, et al. TRAF6 suppresses the
apoptosis of hemocytes by activating pellino in Crassostrea hongkongensis. Dev
Comp Immunol. 2020;103:103501. doi: 10.1016/j.dci.2019.103501. PubMed PMID:
31634519.
2\. Qin Y, Zhang Y, Li X, Noor Z, Li J, Zhou Z, et al. Characterization and
functional analysis of a caspase 3 gene: Evidence that ChCas 3 participates in
the regulation of apoptosis in Crassostrea hongkongensis. Fish Shellfish
Immunol. 2020;98:122-9. doi: 10.1016/j.fsi.2020.01.007. PubMed PMID: 31917320.
3\. Wang X, Wang M, Xu J, Jia Z, Liu Z, Wang L, et al. Soluble adenylyl cyclase
mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster
Crassostrea gigas exposed to elevated CO2. Fish & Shellfish Immunology.
2017;66:140-7. doi: 10.1016/j.fsi.2017.05.002.
4\. Nguyen TV, Alfaro AC, Young T, Ravi S, Merien F. Metabolomics Study of
Immune Responses of New Zealand Greenshell™ Mussels (Perna canaliculus) Infected
with Pathogenic Vibrio sp. Marine Biotechnology. 2018;20(3):396-409. doi:
10.1007/s10126-018-9804-x.
10.1371/journal.pone.0231399.r003
Decision Letter 1
Fugmann
Sebastian D.
Academic Editor
2020
Sebastian D. Fugmann
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
24 Mar 2020
Mechanistic molecular responses of the giant clam Tridacna crocea to Vibrio
coralliilyticus challenge
PONE-D-19-35528R1
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10.1371/journal.pone.0231399.r004
Acceptance letter
Fugmann
Sebastian D.
Academic Editor
2020
Sebastian D. Fugmann
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
26 Mar 2020
PONE-D-19-35528R1
Mechanistic molecular responses of the giant clam *Tridacna crocea* to *Vibrio
coralliilyticus* challenge
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[^1]: The authors have declared that no competing interests exist. |
# Introduction
Family planning is the a technique for either limiting number of children, or
want to delay their next birth \[Spacing\] after having unprotected sexual
intercourse. Informed choice of family planning is defined as when a woman
chooses a family planning method after receiving information about the possible
side effects, what to do in case of side effects happen, and possible
alternative methods by healthcare providers. If there are no contraindications,
the choice of family planning (FP) methods is ultimately at the decision of the
user.
Clients are entitled to make voluntary, informed decisions about family planning
services based on options, knowledge, and comprehension. Ethiopian national
family planning guideline appreciate health care professionals to provide
informed decision making as the best strategy to quality family planning
services. In sub-Saharan Africa countries, the level of informed choice of
family planning is not satisfactory. Potential African countries are providing
family planning services without informing possible side effects. The level of
informed choice of family planning is very low in Burundi \[35%\], Niger
\[38%\], Gambia \[42%\], Benin \[47%\] and Mali (48%). In Ethiopia, the level of
informed decision of family planning is poor (25%) compared to other African
countries. Recent research has confirmed that the extent of informed choice for
family planning methods is inconsistent with the place of residency in Ethiopia.
Women residing in urban areas were 1.4 times more likely to be informed about
family planning methods than women living in rural slums. However, the factors
responsible for the disparity of uninformed choice between the two groups (urban
and rural) are not examined. Education about the side effects of family planning
and other alternative methods [plays a significant
role](https://www.classicthesaurus.com/play_an_essential_role/synonyms) in the
uptake of long-acting methods. The low use of long-acting family planning
methods may be related to uninformed family planning choices.
To our knowledge, and after a thorough review of literatures, previous studies
in Ethiopia have investigated the magnitude and socio-demographic factors
associated with uninformed family planning choices. However, none of these
studies examined the extent of urban-rural inequalities. Moreover, none of these
studies identified the factors contributing to the urban-rural gap in the
informed family planning decision. In addition to that, this study determined
the spatial distribution of uninformed choices of family planning in Ethiopia
and identified areas exhibiting elevated aggregation of uninformed choices
(popularly known as hotspots\], which facilitate the formulation of
interventions tailored to specific locations.
Therefore, the objective of this study is to identify and quantify the factors
that contribute to the observed urban-rural gap in informed family planning
decisions. Besides, information obtained from Georeferenced datasets could help
program developers and local decision-makers develop location-specific
strategies to address the problem of uninformed family planning choices, rather
than indiscriminately providing services to the entire part of the country.
# Methods and materials
## Study design and period
This study was conducted using information from the fourth cross-sectional
demographic health survey of Ethiopian (EDHS) communities, which was carried out
between January 18 and June 27, 2016.
## Study area
Ethiopia is the second-most populous country in Africa. The country’s overall
population was expected to reach roughly 103,000,000 by 2021, according to the
Population and Housing Census (PHC) projection from 2007. According to the 2007
Census, the majority of people lived in rural regions (83.6%), there were 4.7
people per family on average, and 47% of all women were between the ages of 15
and 49.
## Population
### Source population
All reproductive age women in Ethiopia.
### Study population
Women aged 15–49 years who were modern family planning users during the data
collection period.
### Dependent variable
The outcome variable for this study is informed choice of family planning for
the decomposition analysis and uninformed family planning choice for the spatial
analysis.
### Independent variables
According to our extensive literature reviews, maternal age, maternal education
(none, primary, secondary, higher), marital status, occupation, wealth status
(poorest, poor, middle, rich, and richest), sources of contraception (private,
non-governmental organizations (NGO), pharmacy, other), marital status
**(**never married/ever married), family planning message on mobile phone (got
messages/ not receive messages), Working status (working/ no working), visit a
health facility in the last 12 months, frequency of reading newspapers or
magazines (not at all, less than once a week, at least once a week), frequency
of listening to the radio (not at all, less than once a week, at least once a
week), and Frequency of watching television (Not at all, Less than once a week,
At least once a week) were considered as independent variables for this study.
### Operational definitions
*Informed choice of family planning*. women of reproductive age who used
contraceptive methods but were not informed of side effects, what to do if they
experienced the side effects or problems, and who were not informed of other
contraceptive methods that could be used, were labeled as “uninformed choice”
with code 1, otherwise as” informed choice” with code 0.
*Media exposure*. created by combining whether a respondent reads the newspaper,
listens to the radio, and watches television. If the respondent was exposed to
at least one of the three media this is labeled “exposed” and coded” 1”,
otherwise “not exposed” is coded “0”.
### Data source
We used the Kids’ record (KR) dataset of the 2016 EDHS to further analyze.
### Data collection tools and procedures
The website of the DHS measure
([http://www.dhsprogram.com](http://www.dhsprogram.com/)) was employed to
effectuate registration for access to the 2016 Ethiopian DHS Datasets and Global
Positioning System (GPS) data, with requisite permission having been duly
procured in order to gain access to the requested tools. Accordingly, all
required data were downloaded from the Demographic and Health Surveys Program
website. A global positioning system was used to collect the geographic
coordinates of each cluster.
### Data quality control
The data collectors of this survey documented that questionnaires were pre-
tested in all three local languages (Amharic, Afaan Oromo, and Tigrigna) to
ensure that questions were clear and understandable to respondents.
### Sample size and sampling procedures
A two-stage, stratified, clustered sampling procedure was implemented across all
11 (now expanded to 13) geographic administrative areas comprising 9 regions and
2 city administrations. The first strata included a total of 645 enumeration
areas (EAs) that were selected proportionally to the EAs size of the nine
geographical regions and two administrative cities. In the second strata, every
eleven administrative divisions were further sub-grouped into urban and rural
residents, yielding a total of 21 sampling strata. From each cluster, 28
households were chosen using an equal probability technique. This study
ultimately includes a weighted sample of 3511 participants.
### Data management
Data were processed, reviewed, sorted, and recoded using STATA/SE version 16.0.
To account for the effects of the survey’s complex sampling design or the
hierarchical nature of the EDHS dataset, to restore survey representativeness,
and to obtain reliable statistical estimates, the data were weighted via
applying the STATA command "svyset." This command was prepended to each analysis
in this study.
Arc GIS version 10.8 software was used to visualize the spatial distribution and
locate hotspot areas (clusters).To measure the deviation of the spatial
arrangement of the uninformed choice from randomness, the global spatial
autocorrelation (Global Moran’s I) was calculated. A positive value of Moran’s I
represent positive spatial autocorrelation (cluster together), whereas a
negative value indicates dispersed arrangement.
### Logit-based multivariate decomposition analysis
To find factors that contributed to the gap of informed choice of FP between
urban and rural residents, multivariate decomposition analysis was calibrated.
This analysis utilizes the output from the logistic regression model to assign
the observed change in informed choice of FP rate between urban and rural
through subdividing into components (i.e., due to endowment and coefficients).
For logistic regression, the Logit or log-odd of difference between urban and
rural is taken as:
Logit (rural)-Logit (urban) = F (X urban\*rural)–(F Xurban\*βrural)
= \[(F(X urban\*β2rural)-F (Xurban\*βrural)/E)\] + \[(F(X urban\*βrural)-F
(Xurban\*βrural)/C)\].
X indicates the outcome variable i.e., informed choice of FP Beta (β) indicates
that, regression coefficient of each selected independent variables
The E component refers to change in informed choice of FP imputable to
differences in endowments or characteristics. The C component refers to a change
in informed choice of FP imputable to differences in coefficients or effects.
During decomposition analysis the categories urban and rural were recorded as
“0” and”1” respectively. Percent contributions with a 95% confidence interval
(CI) of coefficients and a p-value \<0.05 were reported. Candidate variables for
multivariable decomposition regression were selected according to the p-value
from bivariable decomposition regression. Those variables with p-value less than
0.2 in the bivariable regression were moved to multivariable decomposition
regression analysis.
### Spatial scan statistical analysis
Bernoulli model and purely spatial Kulldorff’s scan statistical analysis was
deployed to detect clusters (areas with concentrated numbers of women making
uninformed choices FP). If the spatial pattern of uninformed choice of FP is
randomly distributed across space, it is not doubtful that the development of
site-specific strategies will be effective in curbing the high prevalence of
uninformed choice.
Only areas with a high rate of uninformed choices of FP were applied to
determine the geographical location of statistically significant clusters using
SaTScanTM v10.0.1 software. We used the Bernoulli model because the data were
binary (uninformed or informed choice). Women who were uninformed about the
choice of FP were considered cases (1), whereas those who were informed were
considered non-cases (0).
The case file (1), non-case file (0), and coordinate files (latitude and
longitude) were imported into SaTScan <sup>TM</sup> software to determine the
location of significant clusters. The maximum size of the scan window was scaled
according to the percentage of the total population at risk. The maximum radius
of the circle was adjusted to be less than 100 km. This is done to facilitate
the development of intervention strategies, where clusters with optimal radius
are easier to manage than large clusters.
To avoid overlooking very small and very large clusters, the maximum geographic
cluster size was adjusted to \< 50% of the population at risk as an upper limit.
The most likely (primary), cluster was determined using p-value and likelihood
ratio tests. The cluster with the highest likelihood ratio represented the most
likely cluster, and the remaining clusters with statistically significant log-
likelihood ratios (LLR) were designed as possible secondary possible clusters.
The relative risk (RR) parameter of the uninformed choice of FP in each cluster
was calculated to estimate the risk of uninformed choice within the cluster
areas.
## Ethics approval and consent to participate
As the authors of this manuscript did not involve collection of data from the
participants, consent to participate and ethical approval is not required. But
the authors have granted to access the dataset of 2016 EDHS through registering
to [www.dhsprogram.com](http://www.dhsprogram.com/) website. The data granted
from this website have no personal identifying information (anonymous) and the
participants’ confidentiality and privacy issue is not an inconsequential in
this scenario. All the methods were performed in accordance with the relevant
guidelines and regulations of DHS measures.
# Results
A total of 3511 (weighted) modern family planning users were included in this
study. The median age of the study participants was 28 years (IQR = from 23 to
34 years). As shown in the Figure (below), only 40.7% \[95% confidence interval:
39.12% to 42.4%\] of current contraceptive users were informed about FP choices.
Additionally, the magnitude of informed choice was 12 percent higher among urban
residents than rural residents.
The greater number of study participants were from rural slums (76.5%). More
than 60% of the study participants were from Oromia and Amhara regions. Of all
participants, 1838 (52.3%) were orthodox Christian followers, accompanied by
Protestants (25%).
## The spatial arrangement of the uninformed choice of FP
The spatial arrangement of the uninformed choice of FP in Ethiopia was not
evenly distributed. The null hypothesis of global spatial autocorrelation
assumes that the uninformed choice of FP was randomly distributed across the
study area. The positive z-score, positive Moran’s index, and statistically
significant p-value from prove that the spatial arrangement of uninformed choice
was clustered. The corresponding p-value of less than 0.01 on the right side of
the figure can be interpreted to mean that the probability of randomness of the
observed spatial pattern (i.e., clustering) of uninformed choice is less than
1%.
shows hotspot areas (areas with an overwhelming number of women who have
uninformed FP choices) among contraceptive users in Ethiopia. the primary
cluster (shaded in red) (LLR = 34.8, p-value\<0.001) focused primarily on the
southern part of the Amhara region, which included East & West Gojjam, South
Gondar, and south wollo zones and was centred at the latitude of 710.984556°N,
and longitude of 38.044450°E with a radius of 97 km.
## Geographic clustering of uninformed choice
.
## Decomposition analysis
Variables with a p-value of less than 0.2 from the bivariable decomposition
analysis were picked as the contender variable for the multivariable
decomposition analysis. The composition factors of region and religion were not
included in the multivariable composition analysis because of having a p-value
greater than 0.2 at bi-variable decomposition analysis. The multivariable
decomposition regression models revealed that 74% of the changes in informed
choice of FP could be attributed to changes in population characteristics
(population dynamics). This can be interpreted to mean that if household
characteristics related to the explanatory variables had been equalized between
urban and rural residents, the Urban-rural gap of informed choice of FP would
have declined by 74%. However, the change due to the coefficients is not
statistically significant and the detailed result of the decomposition of each
variable is not included in the table.
**Non-linear decomposition of informed choice of FP.** From the multivariable
non-linear decomposition analysis, the age group between 35 and 49 years,
receiving the last FP at private health facilities, and being visited health
facilities in the last year were the gap tightening variables (negative
percentage contribution). Conversely, the age group between 25 and 34 years,
lack of access to FP-related mobile short message service (SMS) and listening to
the radio were variables that increased the difference in informed choice of FP
between these two groups.
As can be seen from the table (above), the difference between urban and rural
areas is mainly explained by the location of FP supply (i.e., private
facilities), followed by listening to radio programs. In addition, the age of
current users of FP also had a strong influence on the urban-rural gap in
informed choice. To illustrate, if the composition (characteristics) of where FP
is offered (in private facilities) were identical in urban and rural areas, the
difference in informed choice of FP in Ethiopia would have increased by 16
percent.
Similarly, if the composition (characteristics) of women aged 35–49 had remained
the same for urban and rural participants, the difference in the extent of
informed choice would have increased by 9.3%. Oppositely, the gap in informed
choice of FP between rural and urban residents would have been narrowed by 9%,
if the composition of women aged between 25 and 34 years was the same between
rural and urban in 2016. Besides, the gap would have been narrowed by 11
percent, if the composition of radio listeners stayed the same between the two
groups.
# Discussion
This study found that the spatial arrangement of uninformed choice of FP is
clustered. Moreover, variables like woman’s age, place of FP offer, media
exposure, access to mobile SMS, and visit to health facilities are among the
contributing compositional variables to the gap of informed FP choice between
urban and rural residents. The current study concentrated on the quantitative
analysis of the informed choice of FP data from a spatial vantage point using
cluster analysis. Using the local spatial statistics (SaTScan), the spatial
arrangement of uninformed choice was concentrated in east & west Gojjam, south
Gondar and wollo zones of the Amhara region. These provinces were also
identified with the poor practice of health communication on family planning.
Moreover, earlier research also documented that informed choice of FP varied
across regions and places of residency. A possible explanation for that
inequality might be, women residing in urban areas are highly likely to get in
touch with well trained and experienced health professionals who can nourish
theme information related to the choice of FP. Tailoring plans and programs
based on geographic locations would be more effective and resource-saving
instead of random health service provisions to all areas of the country.
Moreover, women living in rural provinces were laggards compared to their urban
counterparts in having informed choices of FP. Those results are consistent with
another similar prior study in the context of India. These results are likely to
be related to rural women who might have less opportunity to access well trained
health professionals who give adequate information about methods of choice.
Rural health facilities in Ethiopia has less knowledgeable and less autonomous
in their choice of health care services. It would be interesting to focus on
rural residing women in offering information related to the choice of family
planning.
Interestingly, getting family planning services at private health facilities is
found to narrow the gap in informed choice between rural and urban residents.
This finding was in agreement with other studies. The reason for this could be
the client flow at private health facilities are lower than at public health
facilities so that informed choice of family planning can be practically
achieved in private health facilities. Moreover, since private health facilities
are commercial, they could have a good client handling approach including
delivery of detailed information about the service and treatment they are
providing to their clients. It is better to expand private health facilities to
rural areas as equal as the public health facilities to have informed choice
services of FP. Compared to the age group of 25 to 34 years being age group
between 35 and 49 years is found decrease the gap in informed choice of family
planning between rural and urban residents. Other studies also reported similar
findings. The possible reason behind this could be elder mothers from rural
areas may also seek more information about the family planning method that could
be delivered to them because they might experience side effects and discomforts
in their prior utilization of family planning methods.
Unsurprisingly, visit to health facilities in the last 12 months were found to
narrow the urban to rural gap in the informed choice of family planning, and it
was consistent with another study done in Ethiopia. This could be due to the
fact that rural resident women might remember their informed choice of family
planning if they visit health facilities recently but rural resident women who
had not visited health facilities recently may forget about whether they were
offered informed choice of family planning services. On the hand, urban women
are more likely to remember the informed choice of family planning services as
they are more educated and exposed to media than their rural counterparts.
Additionally, information flow and access are increasing over time which could
make family planning providers in rural health facilities to be more aware of
the right of being informed during the choice of their family planning method.
This study showed that Access to FP related mobile SMS significantly widened the
urban-rural gap in the informed choice of family planning. This finding was
supported by other studies. The possible justification behind this could be
related to the fact that the availability and utilization of mobile phones were
limited in rural areas. Besides, rural resident women are less educated and
could not read and understand messages sent to them via SMS. This study
demonstrated that the frequency of listening to the radio was a significant
variable that widen the urban-rural gap in the informed choice of family
planning methods. The possible reason might be there is better access and
coverage of mass media including Radio in urban areas because of improved energy
access. Additionally, urban women are more literate to understand and
internalize the information streamed through radio programs than their rural
counterparts.
## Strengths and limitations
In this study, nationally representative data were used, allowing for better
generalizability. The design effect due to the hierarchical nature of the
samples was accounted for by using the STATA command "svyset" for each
descriptive and analytical analysis. But, as this article is analyzed using
secondary data, important behavioral and other socio-demographic variables might
have declined model performance. The EDHS survey relies on respondents’ self-
reports, which may be subject to recall bias Furthermore, some women may be
embarrassed to disclose all the details (self-report) concerning FP because it
is a sensitive subject. Conclusion
In this study, visit to private health facilities, women’s age group between 25
to 39 and visit to health facilities in the last 12 months are factors that
significantly widen urban -rural gap of family planning. On the other hand,
factors such as Access to FP related mobile SMS and frequency of listening to
radio were found to narrow the urban -rural gap of informed choice of family
planning. These findings suggest that to narrow residential inequalities of
informed choice, tailoring interventional plans based on age, health seeking
behavior and family planning promotion via media are effective strategies.
The spatial irregularity of not informed choice of family planning identified in
this research needs further research to identify the factors behind these
geographic inequalities using geographic weighted regression.
The authors acknowledge MEASURE DHS for permitting us to access and download the
Ethiopian 2016 DHS datasets.
EDHS Ethiopian Demographic health survey
LLR Log-Likelihood Ratio
FP Family Planning
NGO Non-Governmental Organization
GPS Global Positioning System
RR Relative Risk
IQR Inter-Quartile Range
EA *enumeration areas*
SNNP Southern Nations, Nationalities, and People and
SMS Short Message Service
10.1371/journal.pone.0289099.r001
Decision Letter 0
Simegn
Wudneh
Academic Editor
2023
Wudneh Simegn
This is an open access article distributed under the terms of the
Creative Commons Attribution License
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provided the original author and source are credited.
13 Apr 2023
PONE-D-23-01109Urban-rural inequalities and spatial arrangement of informed
choice of FP in Ethiopia: further analysis of 2016 Ethiopian demographic health
survey.PLOS ONE
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Reviewer \#1: Review Comments to the Author
Using 2016 EDHS data the authors assessed urban-rural inequalities and spatial
arrangement of informed choice of family planning in Ethiopia. The paper
reported that spatial configuration of not informed choice was clustered and
magnitude of informed choice was 12 percent higher in urban residents compared
to rural residents.
Title:
It is not recommended to use abbreviation on the title: so better you write full
form.
Abstract: correct editorial error such as not capitalizing the first letter of
new sentence and capitalizing the middle word ‘’Descriptive’’. The same
editorial issues are there under discussion.
This sentence is incomplete and what authors want to say is not clear -More
specifically, place of FP offers i.e., private health facilities (-16%),
listening to the radio programs (+12%), age 35 to 49 years (-9.3%) and 25 to 34
years (+9.2%) (p\<0.05.
Check grammar for the following sentence:
In this study, visit private health facilities, women’s age group between 25 to
39 and visit to health facilities in the last 12 months factors that
significantly widen urban - rural gap of family planning.
You said: Besides, the spatial arrangement of not informed choice of family
planning is not regular. To express spatial distribution the term random/non-
random vs clustered are better rather than using not regular.
Introduction:
Check grammar issue in the following sentence:
•Informed choice of family planning means that when women choose a family
planning methods, all available information regarding side effects, what to in
case of side effects, and possible alternative methods
•Write full form followed by abbreviations in bracket first time abbreviations
appear in manuscript
Check grammar issue in the following sentence:
•Moreover, the current study identified the spatial distribution and identified
hotspots areas where women who had uninformed family planning choices.
Methods:
•Even if the authors used secondary data some sections of methods were absent.
Please add subsections on study area, design, population and data source
•Editorial issue: don’t capitalize letters in the middle of the sentence “Visit”
Results:
•In figure 1 title mention time component, you can use Year EDHS was released.
Similarly for table 1 title is incomplete address where and when component.
•Under the following section the authors wrongly cited Figure 1 replace it with
Figure 2 because there are no p value in Figure 1 “The spatial arrangement of
the uninformed choice of FP”
•Similarly in the paragraph under figure 2, omit figure 2 (wrongly cited)
Discussion
•Check grammar issue in the following sentence:
In this study, being in the age group 35 to 49 years was significantly narrowed
the urban and rural gap in the informed choice of family planning where as being
in the age group 25 to 34 found to widen the urban -rural gap of family planning
compared to age group 15 to 24 year.
•Try to include strength and limitations of this study
Conclusions
•The first sentence is unnecessary
Declarations
You specified only single abbreviations but there are a lot of abbreviations
inside document. So include all abbreviations-SNNP, NGO, FP……
References
Some references are too old (16=1996, 24=2007, 28=2001). Try to use recent
publications
Reviewer \#2: Dear editors.
Thank you very much for this opportunity to review this paper. This paper
assessed urban-rural disparities and spatial distribution of informed choice of
FP in Ethiopia, which is crucial for plociy makers and program managers to
identify intervention on inequality.
The following are my suggestions for the improvement of this manuscript:
General comments
The authors don't include page numbers and lines in the document, making it
difficult to give suggestions and comments by page and line
Make sure to leave space before each bracket or parenthesis
There is typo and spelling error thoughout the document
Introduction.
The introduction part must be rewritten orked and organised in a logical way.
The aouthors must highlights what lloks the condition in Africa, then in
Ethiopia. Incorporate national family planning strategy and initiatives, as well
as health service accessibility and distribution by residence. Describe the
efforts and gaps in providing access to quality family planning service to
everyone who needs it. It should be discussed that how much govt has spent on
family planning and benefit in terms of preventing unintended pregnancy and
highrsik fertility behaviors
FP: to be defined first
Method
EDHS is used two stage stratified study design so there is a need for adjustment
of cluster and weight while doing multivariate analysis. Need to elaborate the
design of the study and whether any type of stratification has been carried out?
In addition, the authors did a decomposition analysis but did not elaborate on
it in the method section. Which residence coded as “0” or “1”? it need more
details
Independent variables
Be consistent, for some variable athors mentioned the coatergy but form sone not
yet
Results
Present the frequency of Table 1 by residence, then the total and weighted
frequencies are enough.
Decomposition analysis
The authors stated that "the composition factors of region, religion, and place
of residence were not included in the multivariable composition analysis because
they had a p-value greater than 0.2 at the bivariable decomposition analysis".
How could residence be an explanatory variable here? It was an urban-rural
disparity study, hence the grouping variable in this case is residence.
In table one, I didn't notice a variable “place for the FP offer” to be
classified as government or private, but in the decomposed table, I did?????
Correct the FF typoerror
“in the f informed choice”
“urbanand rural”
“by 9.3%.. Oppositely”
Discusion
It may be helpful to have a short summary of important findings in the first
paragraph. The authors did not discuss limitations of the study such as the
self-reported nature of the outcome informed choice of FP.
It is surprising that the composition of private health facilities had a
narrowing effect, which indicates the composition of private health facilities
was higher in rural areas, which is far from reality. Most private health
facilities in Ethiopia are concentrated in urban areas.
Conclusion
State the the policy implications of this study based on the finding.
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Reviewer \#2: No
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10.1371/journal.pone.0289099.r002
Author response to Decision Letter 0
13 May 2023
Background: Ethiopia has made satisfactory progress in improving maternal and
child health over the past two decades. The introduction of family planning
through informed choice is one of the main strategies to improve maternal and
child health. However, this positive progress may have masked the significant
urban-rural disparities in informed choice for family planning.
Objective: To identify factor contributing to observed urban-rural disparities
and to determine the spatial distribution of informed family planning choices in
Ethiopia.
Methods: The study used information from 3,511 (1) women currently using
contraceptives (rural-2685 and urban-826)c from the most recent Ethiopian
demographic health survey cross-sectional data. Spatial and descriptive,
bivariable, and multivariable logit-based decomposition analysis methods were
used.
Results: The spatial configuration of uninformed choice was clustered. The
primary cluster (LLR=34.8, p-value\<0.001) was located at the southern portion
of Amhara region that covers east & west Gojjam, south Gondar and south Wollo
administrative zones. The magnitude of informed choice was 12 percent higher in
urban residents compared to rural residents. Urban-rural gap was attributed to
variations in characteristics (74%). Place of family planning offer i.e.,
private health facility, being aged between 35 and 49 years, and having visited
to health facility in the last 1 year are found decrease the urban-rural gap of
informed family planning choice by 15%, 9% and 5% respectively. Conversely,
being aged between 25 and 34 years, being a listener to radio has increased the
gap by 9% and 12% respectively.
Conclusion: The variables being private health facility visitors, being aged
between 25 and 39 years and having visited health facilities in the last one
year are found to increase the gap of informed family planning choices between
urban and rural residents Besides, the spatial distribution of uninformed family
planning choices is non-random.
10.1371/journal.pone.0289099.r003
Decision Letter 1
Simegn
Wudneh
Academic Editor
2023
Wudneh Simegn
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
5 Jul 2023
PONE-D-23-01109R1Urban-rural inequalities and spatial arrangement of informed
choice of Family planning in Ethiopia: further analysis of 2016 Ethiopian
demographic health survey.PLOS ONE
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Ethiopia: further analysis of 2016 Ethiopian demographic health survey”. After I
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the raised issue. However, see carefully the age group in the abstract section
particularly at result and conclusion section (35 to 49 VS 25 to 39).
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10.1371/journal.pone.0289099.r004
Author response to Decision Letter 1
6 Jul 2023
Authors’ response to reviews
Title: Urban-rural inequalities and spatial arrangement of informed choice of
family planning in Ethiopia: further analysis of 2016 Ethiopian demographic
health survey.
Authors:
Abiyu Abadi Tareke (<abiyu20010@gmail.com>)
Bayley Adane Takele
, Mohammedjud Hassen Ahmed
Masresha Derese Tegegne
Habitu Birhan Eshetu
Version: 2
Date: May 13, 2023
Point by point response for editors/reviewers’ comments
Manuscript number: PONE-D-23-01109
Dear editor/reviewer:
Dear all,
We express our profound appreciation for the insightful and productive feedback
that you have provided. Your invaluable comments have significantly enriched the
quality of the manuscript, and have greatly augmented our expertise in the realm
of scientific paper writing. The authors have diligently taken into account each
of the comments and queries raised by the editors and reviewers, and have
responded to them in a targeted manner. Our comprehensive point-by-point
rejoinders to all the comments and questions can be found in the subsequent
pages. In addition, an accompanying supplementary document has been enclosed,
which showcases the modifications made in detail, using the track changes
feature. We also made some change to fix grammatical error in some paragraphs.
Review Comments to the Author
Reviewer’s comment: see carefully the age group in the abstract section
particularly at result and conclusion section (35 to 49 VS 25 to 39).
Authors’ response: dear reviewer we are here because of your insight and deep
review to this manuscript. Sorry for this typographical error. After checking
the result written in the regression table, we changed the phrase “being aged
between 25 and 39 years” to “being aged between 35 and 49 years”.
10.1371/journal.pone.0289099.r005
Decision Letter 2
Simegn
Wudneh
Academic Editor
2023
Wudneh Simegn
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
12 Jul 2023
Urban-rural inequalities and spatial arrangement of informed choice of Family
planning in Ethiopia: further analysis of 2016 Ethiopian demographic health
survey.
PONE-D-23-01109R2
Dear Dr. Tareke,
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suitable for publication and will be formally accepted for publication once it
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Academic Editor
PLOS ONE
Additional Editor Comments (optional):
Reviewers' comments:
10.1371/journal.pone.0289099.r006
Acceptance letter
Simegn
Wudneh
Academic Editor
2023
Wudneh Simegn
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
11 Aug 2023
PONE-D-23-01109R2
Urban-rural inequalities and spatial arrangement of informed choice of Family
planning in Ethiopia: further analysis of 2016 Ethiopian demographic health
survey.
Dear Dr. Tareke:
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PLOS ONE
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Heat shock protein 70 (Hsp70) family members are present in all organisms and
are the most highly conserved heat shock protein family. Hsp70 proteins function
as molecular chaperones, and are involved in various cellular processes such as
protein folding and assembly of nascent polypeptides, refolding aggregated
proteins, protein translocation across membranes, protein degradation and
controlling the activity of regulatory proteins. Allosteric coupling between the
N-terminal ATPase domain and the C-terminal substrate binding domain is
essential for the function of Hsp70 and is mediated via the inter-domain linker.
ATP binding and hydrolysis leads to conformational changes in the two domains
and this regulates substrate affinity. The functions of Hsp70 are regulated by
co-chaperones, such as J-proteins (also called Hsp40 or DnaJ), and by GrpE-like
nucleotide exchange factors. Due to their crucial role in proteostasis, Hsp70
members are present in almost all cellular compartments.
The function of the mitochondria is critically dependent on mitochondrial Hsp70
(mtHsp70) and it plays a major role in the translocation of nuclear encoded
proteins across the mitochondrial membranes and folding of proteins in the
matrix. Ssc1, Ssq1 and Ssc3/Ecm10 are the Hsp70 proteins found in the
mitochondria of yeast, with Ssc1 being the major Hsp70. Protein folding by Ssc1
in the matrix is regulated by Mdj1 (the only Type I J-protein in the
mitochondria) which delivers substrate and stimulates the ATPase activity.
Unexpectedly, mtHsp70 has the propensity to self-aggregate and it requires an
additional essential regulator, Hep1 (<u>H</u>sp70 <u>e</u>scort
<u>p</u>rotein), to maintain its functional state. Hep1 orthologues are
conserved in many eukaryote species including protozoa, but not in prokaryotes.
Only Ssc1 and Ssq1 produce aggregation-prone conformers of the ATPase domain
that bind to Hep1. Yeast cells deprived of Hep1/Zim17/Tim15 accumulate insoluble
mtHsp70 and generally exhibit mitochondrial defects similar to those observed
upon mtHsp70 deletion.
Hep1 is a zinc-finger protein with one tetracysteine motif that is part of the
zinc finger domain. The binding of zinc ions to Hep1 in the mitochondria is
critical for its function. Hep1 only binds to nucleotide-free mtHsp70 and is
released upon nucleotide binding. The N-terminal ATPase domain of mtHsp70
in association with the interdomain linker is prone to aggregation, while the
ATPase domain and C-terminal substrate binding domain are both soluble when
expressed separately. The inter-domain linker attached to the ATPase domain is
the minimal binding entity required by Hep1 to keep mtHsp70 soluble and active.
The *P*. *falciparum* genome encodes six Hsp70 orthologues, and only PfHsp70-3
has been predicted to localise in the mitochondria. Plasmodial Hsp70 proteins
and their interactions with co-chaperones have received attention as potential
avenues for drug targeting as they play an integral part in the survival and
pathology of the parasite. Selective modulation by small molecules of *P*.
*falciparum* Hsp70 proteins has been demonstrated, as well the modulation of the
Hsp70/J-protein partnership. Little is known about the role of PfHsp70-3 in the
parasite, and a protein translocation model was previously described by van
Dooren et al.. More recently, an overview of the proposed roles of PfHsp70-3 and
its co-chaperones in the mitochondria was presented by Njunge et al.. The
putative co-chaperones of PfHsp70-3 which form part of the import machinery are
Tim44 (PF11_0265), GrpE (PF11_0258) and PfPam18 (PF07_0103). Other proposed co-
chaperones of PfHsp70-3 involved in protein refolding in the matrix are Pfj1 and
PFF1415c but these have not been experimentally validated. PfHsp70-3 is an
orthologue of Ssc1 and is predicted to localize in the parasite’s mitochondrion
and play a central role in the translocation of proteins into the mitochondria
and their subsequent folding in the matrix. Our results show that PfHsp70-3 is
indeed insoluble when heterologously produced in *E*. *coli* cells. We
identified a putative Hep1 orthologue in *P*. *falciparum*, and provide
experimental evidence that PfHep1 prevents the self-aggregation of PfHsp70-3
that is required for structural and functional activities. We also examined the
abilities of PfHsp70-3 and PfHep1 to function as holdases and suppress protein
aggregation, and demonstrate that the zinc ion in the zinc finger domain is
essential for stabilising the structure of PfHep1.
# Materials and Methods
## Primary structure sequence analysis and homology modelling of the zinc-binding domain (ZBD) of PfHep1
The protein domain mapping for PfHep1 was conducted using the online programs
SMART 7 (Simple Modular Architecture Research Tool; <http://smart.embl-
heidelberg.de/>;), and Prosite (<http://prosite.expasy.org/>;). In order to
predict the subcellular localisation of PfHep1 and PfHsp70-3 a number of online
programs that included NucPred (<http://www.sbc.su.se/~maccallr/nucpred/cgi-
bin/single.cgi>;), MitoPROT (<http://ihg.gsf.de/ihg/mitoprot.html>;), MultiLoc
(<http://abi.inf.uni-tuebingen.de/Services/MultiLoc>;, SignalP version 4.1
(<http://www.cbs.dtu.dk/services/SignalP/>;), and WoLF PSORT
(<http://www.genscript.com/wolf-psort.html>.;) were used. The primary amino acid
sequence of PfHep1 and other selected well-characterised Hep1 orthologues were
obtained from PlasmoDB v4.4 (<http://plasmodb.org/plasmo/>;), and the NCBI
database. Alignment was conducted using MAFFT
(<http://www.ebi.ac.uk/Tools/msa/mafft/>;). The zinc-binding domain structure
of PfHep1 was modelled using the online Swiss Model server. The solution
structure of Tim15c (yHep1) solved by NMR (PBD accession number 2EZZ) was used
as the template. The model was rendered using PyMol.
## Construction of *E*. *coli* expression plasmids encoding PfHsp70-3 and PfHep1
*E*. *coli* codon-optimized versions of the Hsp70-3 coding sequence (PlasmoDB
accession number: PF3D7_1134000) (41 – 622aa) and PfHep1 coding sequence
(PlasmoDB accession number: PF3D7_1420300) (15–302 aa), both lacking the
mitochondrial signal peptides, were synthesized by the GenScript Corporation
(Piscataway, New Jersey, USA) and inserted into a pQE30 expression vector
(Qiagen, Germany) to create the pQE30-PfHsp70-3 and pQE30-PfHep1, while the
pACYCDuet1 expression vector was used to create pACYCDuet1-PfHep1.
## Expression and purification of PfHep1
*E*. *coli* M15 (pREP4) cells were transformed with pQE30-PfHep1 and grown at
37°C in 2x YT medium supplemented with 100 μg/ml ampicillin and 50μg/ml
kanamycin and grown to mid-logarithmic phase (A<sub>600</sub> 0.4–0.6). Protein
production was induced by addition of 0.1 mM IPTG. Cells were harvested prior to
induction and at hourly intervals post induction for 5 hours and overnight.
Cells were harvested by centrifugation (13000×g; 2 min) and re-suspended in PBS
buffer. Protein production levels were evaluated using SDS-PAGE and Western blot
analysis. For recombinant protein purification, cells were harvested at the
fourth hour post induction and the harvested cells were re-suspended in lysis
buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 1 mg/ml
lysozyme) and frozen at -80°C overnight. The cells were then thawed on ice and
sonicated at 4°C in the presence of 3% sarcosyl (Sigma-Aldrich, Germany). The
lysed cells were centrifuged (16000×g, 40 min, 4°C) and the soluble supernatant
fractions were mixed with cOmplete His-tag purification resin (Roche, Germany)
and allowed to bind overnight at 4°C with gentle agitation. The resin was then
pelleted by centrifugation (5000×*g*; 2 min) to remove unbound proteins and
washed three times using wash buffer (100 mM Tris-HCl, pH 7.5, 300 mM NaCl, 50
mM imidazole) to remove non-specific contaminants. The bound protein was eluted
by re-suspending the resin in elution buffer (10 mM Tris-HCl, pH 7.5, 300 mM
NaCl, 750 mM imidazole). The eluted protein was extensively dialysed using
SnakeSkin dialysis tubing (Pierce-MWCO 10,000; Thermo Scientific, USA) into
dialysis buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.5 mM DTT, 10% (v/v)
glycerol, 50 mM KCl, 2 mM MgCl<sub>2</sub>), and concentrated against PEG 20000
(Merck, Germany). The efficacy of the purification process was assessed using
SDS-PAGE and western analysis using mouse monoclonal anti-His primary antibody
and HRP-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz
Biotechnology, USA). Chemiluminescence-based protein detection was achieved
using the Clarity<sup>TM</sup>Western ECL blotting kit (Bio-Rad, USA) as per the
manufacturer’s instructions, and captured with a Chemidoc chemiluminescence
imaging system (Bio-Rad, USA). The protein concentration for the purified
proteins was quantified using the Bradford’s assay (Sigma-Aldrich, USA) with BSA
as the standard. A fraction of the purified PfHep1 protein was dialysed
extensively against buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM DTT, and 1
mM PMSF) containing 200 mM EDTA. The protein was then further dialysed in buffer
without EDTA.
## Co-expression and co-production of PfHsp70-3 and PfHep1 in *E*. *coli*
Co-production of PfHep1 and PfHsp70-3 in *E*. *coli* was conducted in order to
functionally assess the escort activity of PfHep1. *E*. *coli* BL21 (DE3) cells
were transformed with pQE30-PfHsp70-3 in the presence of pACYCDuet1 (empty
vector) or pACYCDuet1-PfHep1 and grown at 37°C in 2x YT broth supplemented with
100 μg/ml ampicillin and 34 μg/ml chloramphenicol to mid-logarithmic phase, and
protein production was induced by adding 0.1 mM IPTG. Cells were harvested prior
to induction and at hourly intervals post induction for 5 hours and overnight.
The harvested cells were centrifuged (13000×g; 2 min) and re-suspended in PBS
buffer. Protein production levels of PfHsp70-3 in the presence or absence of
PfHep1 were evaluated using SDS-PAGE and western analysis. The procedure for
purification of the co-expressed PfHep1 and PfHsp70-3 was carried out as
described for PfHep1 except that cells were harvested 5 hours post induction.
For subsequent *in vitro* analyses, gel filtration was employed in order to
separate the co-produced proteins. The dialysed protein solution was filtered
through 0.2 μm filters and loaded into a HiPrep™ 16/60 Sephacryl™ S-200 HR
column driven by an ÄKTA fast-protein liquid chromatography system (GE
Healthcare, Biosciences, UK). The proteins were eluted at a flow rate of 0.5 ml/
min and 1 ml elution fractions were collected and analysed using SDS-PAGE and
western analysis. The eluted proteins were concentrated against polyethylene
glycol (PEG) 20000 (Merck, Germany).
## Suppression of thermally-induced PfHsp70-3 aggregation by PfHep1
An evaluation of the ability of PfHep1 to suppress the thermally-induced
aggregation of PfHsp70-3 was adapted and modified from Dores-Silva et al.. The
suppression of PfHsp70-3 aggregation by PfHep1 was monitored by light scattering
at 360 nm for 30 min at 50°C in assay buffer (50 mM Tris-HCl, 100 mM NaCl; pH
7.4). For this assay, 0.8 μM PfHsp70-3 was used with stoichiometric
concentrations of PfHep1 (dialysed in the presence and absence of EDTA). An
evaluation of PfHep1 and EDTA-treated PfHep1 (E-PfHep1) to self-aggregate under
the assay conditions was also conducted. Each assay was conducted in triplicate
and three independent experiments on independent batches of proteins were
conducted. Absorbance was plotted as percentage of PfHsp70-3 aggregation
subsequent to normalizing against assays with PfHsp70-3 alone.
## Suppression of thermally-induced MDH aggregation by PfHsp70-3 and PfHep1
An evaluation of the ability of PfHep1 and PfHsp70-3 to suppress the thermally-
induced aggregation of MDH was adapted from Burger et al.. Varying
concentrations of PfHsp70-3 (0.25–1 μM), PfHep1 (0.25–1 μM) and combinations of
these proteins in assay buffer (50 mM Tris-HCl, 100 mM NaCl; pH 7.5) were used
to assess the abilities of these proteins to suppress the aggregation of 0.72 μM
MDH by monitoring light scattering at 360 nm for 30 min at 45°C. PfHep1 alone
did not self-aggregate under the assay conditions (data not shown). Absorbance
was plotted as percent MDH aggregation over 30 min subsequent to normalizing
against assays with MDH alone. Each assay was conducted in triplicate and three
independent experiments on independent batches of proteins were conducted.
## Suppression of thermally induced citrate synthase aggregation by PfHsp70-3 and PfHep1
The ability of PfHep1 and PfHsp70-3 to suppress thermally induced aggregation of
citrate synthase was adapted and modified from Lee et al.. Different
concentrations of PfHsp70-3 (0.25 and 1 μM), PfHep1 (0.25–1 μM) and a
combination of these proteins in an assay buffer (100 mM HEPES-KOH, pH 7.5)
together with 0.15 μM citrate synthase from porcine heart (Sigma-Aldrich) were
used. Suppression of aggregation was determined by monitoring light scattering
at 320 nm for 30 min at 45°C Absorbance was plotted as percent over citrate
synthase aggregation subsequent to normalizing against assays with citrate
synthase alone. Each assay was conducted in triplicate and three independent
experiments on independent batches of proteins were conducted.
# Results and Discussion
## The functional domain of PfHep1 is conserved
Based on the observation that mitochondrial members of the plant zinc ribbon
(ZR) protein family show sequence similarities to Hep1 from yeast and humans, ZR
and Hep proteins were classified as part of a family consisting of five
subgroups: ZR1, ZR2, ZR3, Hep1 and Hep2. The classification was based on
sequence identity and sub-cellular localization of individual proteins within
the cell. The ZR subfamily refers to plant zinc finger proteins, ZR1 and ZR2
being plastidic and ZR3 residing within the mitochondria. The Hep subfamily
refers to non-plant zinc finger proteins with Hep1 being mitochondrial and Hep2
being plastidic. The putative mitochondrial targeting sequence and the zinc
finger domain (zf-DNL) of the hypothetical zinc finger protein from *P*.
*falciparum* (PF3D7_1420300) was identified, and based on its sequence identity
and predicted subcellular localization, it was denoted as PfHep1.
Hep and ZR proteins have been observed to play roles in suppressing the self-
aggregation of the respective mtHsp70 orthologues in humans, yeast, Leishmania,
green algae and in *Arabidopsis thaliana*. The primary structure of full-length
PfHep1 was aligned with its orthologues and the putative mitochondrial or
chloroplast signal peptides are highlighted for each sequence. The PfHep1
sequence is asparagine-rich, with a continuous stretch from residues 128–166. In
fact approximately a quarter of all amino residues found in PfHep1 are
asparagines. The asparagines repeats are characteristic of the *P*. *falciparum*
proteome and are often absent from heat shock proteins. PfHep1 is also larger
than its orthologues with 302 amino acid residues. Whilst the region upstream of
the zf-DNL is longer in PfHep1 than its orthologues, the C-terminal region is
slightly longer in both the hsHep1 and ScHep1 sequences. There is lack of
sequence conservation outside of the zf-DNL domain. No functional domains, with
the exception of the zf-DNL, were identified in the primary sequence of PfHep1.
However, the generation of truncation mutants in LbHep1 indicated that the
region upstream of the zf-DNL contributes to enhancing the solubility of
LbmtHsp70. The highest sequence identity was 38% between PfHep1 and CrHep2. This
is not surprising as the apicoplast, non-photosynthetic plastid, in *P*.
*falciparum* is of algal origin. There was approximately 25% sequence identity
between PfHep1 and the remaining orthologues. Despite the low overall sequence
identities between Hep proteins, the zinc-finger motifs (CXXC) that are part of
the zf-DNL were found to be conserved in all of the sequences including PfHep1
and are separated by 21 amino acids. Yeast mutants harbouring either a C75S or
C100S mutation in the tetracysteine motifs of Hep1 were found to be incapable of
rescuing growth defects in cells lacking Hep1.
Human Hep1 stimulated the ATPase activity of mtHsp70 but this was not observed
in yeast. Mutations of the key residues R81, H107, and D111 in human Hep1
decreased the binding affinities for HSPA9. Furthermore H107 played a critical
role in stimulating the ATPase activity of HSPA9. Interestingly two of these
residues are conserved in PfHep1 (H255 and D259), while R81 is replaced with
K229. In yeast Hep1/Zim17, residues R106, H107 and D111have been shown to play a
critical role in interactions with mtHsp70. Nuclear magnetic resonance (NMR)
structural elucidations have shown that Zim17 has an L-shape with the two zing-
finger motifs located at the end of the L sandwiched by two anti-parallel beta-
sheets. The zf-DNL of yeast Hep1 was used as a template to model the zf-DNL of
PfHep1 and the overall structure of PfHep1 resembled that of yeast Hep1 with the
cysteine residues found at the ends of two anti-parallel beta-sheets.
## Aggregation of PfHsp70-3 is prevented by PfHep1 co-expression
The coding sequences of PfHsp70-3 and PfHep1 were inserted into pQE30 expression
vectors without the mitochondrial signal sequences. PfHep1 was also inserted
into pACYCDuet1 for the purposes of co-expression with PfHsp70-3. Induction of
PfHsp70-3 with PfHep1 in *E*. *coli* cells was monitored over 5 hours and after
overnight growth; western analysis revealed that both proteins were produced
with lower levels of PfHep1 (data not shown). Examining the effect of PfHep1 co-
expression with PfHsp70-3 revealed that it substantially enhanced the solubility
of PfHsp70-3 (lane 5). PfHsp70-3 alone was virtually insoluble, as seen by the
prominence of the protein in the insoluble pellet (lane 3) compared to the
soluble fraction (lane 2). The solubility of PfHsp70-3 facilitated its native
purification using nickel affinity chromatography without the need for
denaturants such as urea. Some PfHsp70-3 was removed during the wash steps
(lanes 3–5) and a low concentration of PfHep1 (35 kDa) co-eluted with PfHsp70-3
(70.4 kDa) in elutions 1 and 2, however PfHep1 was not detected in elution 3
(lanes 6–8).
Human Hep1 displayed the features of a Type I J-protein and suppressed the
aggregation of rhodanese. To determine if PfHep1 performed a similar aggregation
suppression role, the recombinant protein was purified after heterologous
expression in *E*. *coli* \[pQE30-PfHep1\] cells. PfHep1 was initially found to
be insoluble (lanes 2 and 3), such that purification required the addition of
the ionic detergent sarcosyl to the lysis buffer to solubilise the protein,
prior to purification by nickel-affinity chromatography. Some PfHep1 was removed
during the wash steps (lanes 3–5) and PfHep1 was successfully eluted (lanes
6–8). To our knowledge, the orthologues of PfHep1 are soluble and can be
purified under native conditions. The insolubility of PfHep1 is probably due to
the challenges associated with the heterologous expression of soluble and
functional plasmodial proteins. Codon bias due to the AT-rich genome of *P*.
*falciparum* can lead to poor protein expression levels in *E*. *coli*. However,
codon optimisation does not ensure expression as a large scale screen involving
the heterologous expression of numerous *P*. *falciparum* genes revealed that a
quarter of the codon optimised proteins remained insoluble. The reason for this
failure to express in a soluble form is unclear but could be attributable to the
presence of long repeats of asparagine residues, resulting in a propensity of
these proteins to form insoluble aggregates when heterologously expressed.
## PfHep1 prevents thermally induced aggregation of PfHsp70-3
PfHsp70-3 aggregated at 50°C which enabled us to assess the ability of PfHep1 to
suppress its thermally induced aggregation. PfHep1 suppressed the aggregation of
PfHsp70-3 in a dose-dependent manner, with equimolar concentrations displaying
complete suppression of aggregation of PfHsp70-3. Similar results were obtained
for *Leishmania braziliensis* Hep 1 (LbHep1), which suppressed the thermal
aggregation of *L*. *braziliensis* mtHsp70 (LbmtHsp70). PfHep1 alone did not
aggregate, while EDTA-treated PfHep1 (E-PfHep1; zinc ions removed) did aggregate
under the assay conditions. E-PfHep1 failed to prevent the thermal aggregation
of PfHsp70-3, and an additive effect was observed as the percentage aggregation
was due to aggregated PfHsp70-3 and aggregated E-PfHep1. EDTA has been shown to
destabilize the structural integrity of LbHep1 probably by chelating zinc ions
that in turn weakens the secondary and tertiary structure of the protein.
E-PfHep1 aggregated at 50°C and this was probably due to disruption of the
structure after the removal of zinc ion. Zinc ions were found to be essential
for maintaining the overall secondary structure of yeast Hep1 and LbHep1.
## PfHep1 did not prevent thermally induced aggregation of malate dehydrogenate or citrate synthase
To determine if the prevention of aggregation by PfHep1 is specific to
PfHsp70-3, it was necessary to assess its ability to suppress the aggregation of
proteins that are known to aggregate thermally, such as MDH and citrate
synthase. The addition of varying concentrations of PfHep1 resulted in less than
10% aggregation suppression of MDH and citrate synthase, while PfHsp70-3
suppressed the aggregation of MDH and citrate synthase in a dose-dependent
manner. PfHsp70-3 did not aggregate under the assay conditions, neither did
PfHep1 (data not shown). PfHep1 did not enhance the ability of PfHsp70-3 to
suppress aggregation of either MDH or citrate synthase. Similarly, LbHep1 could
not prevent the aggregation of two model proteins, MDH and Luc, but was specific
for LbmtHsp70.
# Conclusion
We have shown for the first time that PfHep1 is required for maintaining the
solubility and thereby the activity of PfHsp70-3. Our study has indicated that
PfHep1 functions as a specialised co-factor that facilitates the folding of
PfHsp70-3. PfHep1 is larger than its orthologues and there is no sequence
conservation outside of the zf-DNL. The zinc binding domain of PfHep1 was
predicted to contain many of the conserved amino acid residues important for its
interaction with PfHsp70-3. Not surprisingly, PfHsp70-3 was insoluble when
heterologously expressed in *E*. *coli* cells, as many eukaryotic mtHsp70s are
insoluble. We did not anticipate that PfHep1 would be insoluble and this may be
due to plasmodial proteins being notoriously difficult to express heterologously
in a soluble form, even after codon-optimization. The co-expression of PfHep1
enhanced the expression and solubility of PfHsp70-3. The co-expression of PfHep1
and PfHsp70-3 facilitated the production of soluble and functional PfHsp70-3
that enables further biochemical characterisation of this chaperone in future
studies. At high temperatures, beyond that of the human host, PfHep1 suppressed
the aggregation of PfHsp70-3 but not other aggregation-prone proteins, such as
MDH or citrate synthase. Furthermore, PfHep1 did not enhance the aggregation
suppression activities of PfHsp70-3. Taken together, these data suggest that
PfHep1 is not a co-chaperone of PfHsp70-3, but rather a specific co-factor to
prevent its self-aggregation.
To our knowledge, human Hep1 is the only Hep protein that has been demonstrated
to display the features of a J-protein as it stimulated the ATPase activity of
mtHsp70 and functioned as a holdase by binding unfolded proteins such as
rhodanese. The sequence alignment revealed that the region downstream of the zf-
DNL or C-terminal sub-domain, is longer in both humans and yeast with a lack of
sequence identity between the two species. There is evidence to suggest that the
C-terminal sub-domain of human Hep1 is responsible for regulating the activity
of zf-DNL and conferring co-chaperone activity. An examination of the direct
functional effects of Zim17 on mtHsp70 in the cell indicated a novel role of
Zim17 in assisting its interaction with client proteins in a J co-chaperone-
dependent manner. There is also the proposition that Zim17 functions as a
“fractured” J-protein that provides a zinc finger domain to Type III J-proteins
for substrate binding. However, this remains to be experimentally elucidated.
The mechanism of action of human and yeast Hep1 appears to be different from
that of other orthologues, and further studies are required to understand this
differentiation. A greater understanding of Hep proteins from different
organisms is required to determine whether or not they have the properties of
*bona fide* co-chaperones. Thus future mechanistic studies on PfHep1-PfHsp70-3
could include determination of the ability of PfHep1 to stimulate the ATPase
activity of PfHsp70-3 and identification of potential J protein co-chaperones in
the mitochondrial matrix. In view of the fact that LbmtHsp70 plays a key role in
the adaptation of the parasite in the host, disrupting the interaction between
LbHep1 and LbmtHsp70 is a potential target for new therapies. Likewise,
abrogating the specific partnership between PfHep1 and PfHsp70-3 may also be a
target for new antimalarials.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: AB GLB HH DON. Performed the
experiments: DON LAMV SJB. Analyzed the data: AB DON GLB HH. Contributed
reagents/materials/analysis tools: AB HH. Wrote the paper: DON SJB AB. |
# Introduction
## Contemporary society and trends in adolescent mental health
The secular trends in adolescent mental health are, in many Western countries,
unclear due to changes in recognition, diagnosis, and how adolescents perceive
their health, and this perception appears to be deteriorating among adolescent
girls. In Norway, as in other Nordic countries, reports of mental health issues
have increased in recent cohorts of adolescents. The apparent increase in Nordic
adolescents affected by depressive symptoms and suffering from mental health
disorders must be considered in the context of social and cultural changes that
have occurred during the 21st century. Essential aspects of the past year’s
societal changes could have affected people’s mental health status through
psychological factors. These modern trends include (I) rising income inequality,
(II) changes in family consultations and dynamics, and (III) particularly among
adolescents, the growing use of modern online technology such as social
networking sites, which have created a significant arena for social comparisons
that could be psychologically harmful. Furthermore, secondary and tertiary
students commonly report high levels of academic-related stress, and feelings of
anxiety linked to schoolwork are common, particularly among girls, across OECD
countries. The social factors are presented at different levels of society:
individual, family, community, and national.
## Adolescence, social gradient in mental health, and family socioeconomic position (SEP)
The social gradient in mental health, as in health in general, is a widespread
phenomenon, and it has been well documented worldwide. Socioeconomic
inequalities affecting health emerge early on in life and occur across the life
course. Among adolescents, several studies have demonstrated a higher prevalence
of mental health issues with decreasing family SEP calculated using the parents’
income, education. and occupational levels. Low affluence may affect mental
health both directly and indirectly through a variety of mechanisms. Growing up
in families with limited socioeconomic resources may affect both access to
resources (e.g., time with parents, leisure activities, books and learning
materials, housing conditions) and psychosocial conditions among the parents
(e.g., stress, conflicts between parents, psychological difficulties,), which
together affect the children’s socio-emotional development. According to the
relative deprivation theory, it is also crucial how adolescents perceive their
situations relative to others. Being unable to afford goods and activities that
are considered affordable to most can be detrimental to mental health,
particularly in adolescence when peer influences are intense as the material
display of social status—how symbols of wealth are consumed and displayed (i.e.,
symbolic capital)—may be as important as the income itself.
Adolescence, particularly late adolescence, may well be the period of life with
the highest social equalization in health as adolescents earn independence from
their parents but are still in the process of determining their own
socioeconomic potential in terms of education, income, and job opportunities.
However, several studies demonstrate an association between subjective SEP and
adolescent health outcomes, particularly regarding mental health, although the
health inequalities are considered less clear and consistent than those for
adults. Lack of consistent findings may be due to geographical differences,
methodological factors, use of different measurements of SEP and mental health,
or the possibility that SEP may have different effects on various dimensions of
mental health and at different stages of adolescence. Measuring adolescents’ SEP
is an important methodological challenge in examining health disparities among
young people. School-aged children and youth are likely to still be attending
school and living with their parents, and consequently they lack their own
social and economic status. Material conditions in the family can also be
measured by the Family Affluence Scale (FAS) and used as an alternative to
uncertain estimates of household income, parental education level, and parents’
occupational status/position. Inequalities in self-reported health and
psychosomatic symptoms in adolescents have been revealed using FAS. However,
Amone-P’Olak et al. reported that adolescents in the low SEP category are at
risk of mental health problems, but family SEP accounts for a relatively small
proportion (≤ 5%) of the total variance in mental health. Another aspect to
consider is that objective SEP relates differently to adolescent health than
does subjective social status (SSS), and they are not equivalent indicators of
the same construct. It may be that SEP differences in adolescent health relate
more closely to psychosocial processes than to material inequality.
## Socioeconomic environment, psychological distress, and social comparison
Previous studies have emphasized different contextual domains that are likely to
affect mental health such as the structure and capacity of health care resources
in a community, local economic conditions, income inequality, social disruption,
and social capital. Lantz, Pritchard define socioeconomic environment “as a
place with geographically defined boundaries that also has economic,
educational, social, cultural, and political characteristics,” and it can be
measured within different units of geography (e.g., census tract, school
district, zip code, municipality, county, country). In addition to family-
related SEP, the socioeconomic environment shapes resources, opportunities, and
exposures that can both directly and indirectly influence health outcomes. For
example, in comparison to young people from wealthier neighborhoods, those from
areas with high levels of poverty and distress tend to have higher levels of
psychological distress and are at higher risk of suffering physical issues;
however, neighborhood deprivation is not necessarily a causal risk factor for
poor health nor for damaging health behaviors. Previous research suggests that
people living in more equal communities in terms of material standards report
better physical and mental health than do their peers in less equal communities.
However, it is not a straightforward process to determine which socioeconomic
conditions and community circumstances are considered beneficial for
adolescents’ mental health and which conditions have an adverse impact. From a
social interaction perspective, one might expect that adolescents living in high
socioeconomic communities achieve health benefits from living in an environment
that promotes an active and healthy lifestyle, having access to high-quality
health services when needed, and surrounded by people with hope in life and
faith in the future who facilitate (positive) learning, play, and growth.
Moreover, it could be argued that living in a safe neighborhood with high
materialistic standards has a positive psychological effect. Alternatively (and
worth considering even though the literature is sparse), adolescents residing in
high-level socioeconomic communities may be prone to creating harmful stress for
themselves as a result of being exposed to social comparison and pressure to
achieve. Being surrounded by ambitious and competitive fellow students from
well-educated families may, for example, produce high academic stress due to
educational expectations and pressure for academic achievement as well as
difficulties with social comparison making them feel inferior and worthless.
Other school-based social status dimensions, such as being attractive and
sporty, have also been associated with well-being and psychological distress,
and these might be reinforced in communities with high socioeconomic profiles.
A weakness of many previous studies is that family SEP is not seen in relation
to the socioeconomic standards of the local community. The potential interplay
between the effects of structural, family, and community resources on
adolescents’ depressive symptoms and well-being may manifest in various ways.
For example, in line with the contextual amplification hypothesis, we might
assume that the detrimental influence of familial factors and adverse community
conditions, including concentration of poverty, noxious environment, weak degree
of social integration, and collective socialization, reinforce one another. From
another perspective, we might assume a buffer effect brought about by the
beneficial resources at the family level which protect against adverse influence
on the community level, or vice versa. However, an alternative explanation may
be that the beneficial influence of family social resources on adolescents’
depressive symptoms decrease under adverse community conditions. Wickrama,
Bryant call this “moderation of a contextual dissipation” and argue that,
instead of buffering, the negative influence of family social resources on
adolescents’ depressive symptoms level off under adverse community conditions.
Several psychosocial factors could increase adolescents’ vulnerability to
symptoms of depression and anxiety. Perceiving oneself as having low social rank
compared to others has been demonstrated to be consistently linked to a higher
degree of depressive symptoms. Wetherall et al. stated in their review article
that “although markers of SES are consistently associated with depressive
symptoms (e.g., measures of social rank may have a stronger association as they
tap more psychosocial constructs than the objective indicators (Marmot and
Wilkinson, 2001).” Adolescents’ perceptions of social status may be two-
dimensionally rooted. One dimension is class identification based on parental
SEP and familial placement in society: adolescents in high-level socioeconomic
communities might live in families with relatively low or moderate socioeconomic
resources; these adolescents are particularly vulnerable to the adverse effects
of social comparison. Another dimension is the adolescents’ sense of personal
standing compared to peers/schoolmates when it comes to, among other things,
school performance and popularity in their school community. As adolescents age,
they undergo a process of cognitive maturation that may increase their self-
conception and ability to place themselves on a social status ladder. In Norway,
young adults without high school diplomas face a higher risk of receiving a
medically based disability pension (where the leading cause is mental illness)
before turning 40-years-old if they reside in a municipality with a high
socioeconomic profile.
## Study aims
As income and educational inequalities in Norway are rising, socioeconomic
disparities in adolescents’ mental health may be rising concurrently.
Furthermore, within a community, the average educational level should be
considered as a proper proxy measure of the socioeconomic profile of the area,
considering local economic conditions and other social community benefits. On
the other hand, there may be psychological mechanisms associated with well-
educated communities such as competitive-oriented environments, higher
expectations, and a rush for status that also impact (in a positive or negative
way) on adolescents’ mental health. Despite this, few studies have explored the
interactions between individual-level family affluence and community-level
socioeconomic profile. This study aims to explore, in the national context of
Norway, how the municipal socioeconomic indicators *education level* and *income
inequality* affect anxiety and depressive symptom scores among students in their
later teenage years, as well as to understand to what extent these associations
are conditioned by family affluence. This study asked the following three
research questions: (I) Do high school students (in general) achieve
psychological benefits by living in a municipality with a high average education
level? (II) Does the level of income inequality in a municipality affect the
psychological symptom load among students? (III) Is this hypothetical municipal
effect on high school students dependent on family affluence level?
# Methods
## Design and data sources
### The Ungdata survey
The studies involving human participants were reviewed and approved by the
Norwegian Centre for Research Data (NSD), <https://www.nsd.no/en/>. Informed
consent to participate in this study was obtained from the students, as well as
from legal guardian/next of kin if the student was under 16 years of age. This
cross-sectional study is based on questionnaire data collected in five waves
(2014–2018) of the Ungdata survey. Ungdata is a quality assured and standardized
system for local questionnaire surveys aimed at adolescents attending high
school in Norway. The Welfare Research Institute NOVA (at OsloMet) is, together
with Norway’s seven regional drug and alcohol competence centers (KoRus),
responsible for conducting the survey.
Participation in the survey is voluntary and based on the students’ informed
consent. The survey covers different aspects of the students’ lives encompassing
a wide range of thematic areas, and it is an important source of information on
young people’s health and well-being, both at the municipal and national levels.
The surveys take place during school hours and are carried out electronically.
The response rate varies between surveys, schools, and school years. The overall
response rate among senior high school students was 66% for surveys conducted in
2014–2016 and 69% for surveys conducted in 2016–2018. According to Bakken, the
data are considered nationally representative during a three-year period. Our
data are based on participants in the Ungdata survey during the period 2014 to
2018, and we thus consider that our study material in general gives a
representative picture of Norwegian youths. (See Frøyland for a detailed
description of the content and theoretical framework of the Ungdata survey.)
### Municipality state reporting (KOSTRA)
We combined the individual data with census information on individuals’ home
municipalities, sourced from Municipality-State-Reporting (KOSTRA) database
administered by Statistics Norway (SSB). KOSTRA is a national information system
that contains management information on municipal key activities including,
demography, economy and social services development.
## Study population
Our study sample consists of students who voluntarily undergo three years of
education at level 3 in the International Standard Classification of Education
(ISCED), which is the final stage of general and vocational secondary education.
In Norway, students generally begin ISCED level 3 at age 16 and complete it the
year they turn nineteen. Programs classified at ISCED level 3 may, for example,
be referred to as “upper secondary education” or “(senior) high school”; in the
present paper, we use the term “high school.”
In this study, all high school students completing the version of the Ungdata
survey containing questions on psychological distress, anxiety, and depression
in the period 2014–2018 were included (N = 144,239). We excluded individuals
with missing information on gender (n = 6,072), school year (n = 955), family
affluence (n = 687), and municipal residential identifiers (n = 31,923). The
final dataset with complete municipal identifiers contained 104,602 individuals.
The study sample was further reduced in the parametric estimations due to
individuals’ missing information on mean scores of psychological distress (n =
8,095), symptoms of depression (n = 7,142), and symptoms of anxiety (n = 7,459).
We found no statistical difference between included and excluded individuals for
mean symptom scores of psychological distress (1.91 vs. 1.91), depression (2.16
vs. 2.15), or anxiety (1.55 vs. 1.55). There was a noted higher share of first-
year students (54% versus 45%, p\<0.001) for excluded compared to included
groups.
The fact that first-year high school students are overrepresented in the study
population should be mentioned and may partly be explained by the fact that some
high schools only offered the youngest students the opportunity to participate
in the surveys. Moreover, it is more difficult to carry out surveys among the
oldest students due to exams and the fact that many of the students are
apprentices. A highly relevant explanation is also that the dropout rate in high
school increases with increasing age of students. We should therefore be aware
that the data, especially towards the end of high school, consists of a more
selected group of students compared to lower grade levels.
## Assessment of variables
### Psychological distress, and depressive and anxiety symptoms
Our dependent variables are adolescent’s psychological distress, and depressive
and anxiety symptoms. Psychological distress was measured using the 10-item
Hopkins Symptom Checklist, consisting of two subscales: a depression dimension
(six items that constitute the “Depressive Mood Inventory”) and an anxiety
dimension (four items). In addition, there is a total mean score (10 items).
The students reported how often they were bothered by each of the following
symptoms during the past week (the six first items relate to the depression
dimension of the scale): “felt that everything is a struggle”; “had sleep
problems”; “felt unhappy, sad, or depressed”; “felt hopeless about the future”;
“felt stiff or tense”; “worried too much about things”; “suddenly felt scared
for no reason”; “felt constant fear or anxiety”; “been nervous or felt uneasy”;
and “felt worthless”. Each item was answered on a four-point scale ranging from
“not at all” (1) to “very much” (4).
Separate measures for psychological distress (total mean) and depressive/anxiety
symptoms were constructed by adding up the scores (1 to 4) on all the items
covering each dimension (10 items in total: 6 for depression and 4 for anxiety)
and dividing the total by the number of completed items, given responses to at
least half the statements for each scale. The resulting mean symptom scale
scores—one for psychological distress, one for symptoms of depression, and one
for symptoms of anxiety—ranged from 1 to 4. In addition to the mean score, a
validated cut-off score of ≥1.85 was used to identify students reporting
moderate to high symptom loads related to overall psychological distress,
depression, and anxiety.
### Socioeconomic position (SEP)
The socioeconomic position (SEP) of the adolescents was measured using a
collective measure of SEP developed by Bakken et al. which includes, in addition
to four questions from FAS II, information on parental education levels and the
number of books in the home. The students answered the following four questions
retrieved from FAS II: “Does your family have a car?”; “Do you have your own
bedroom?”; “How many times have you travelled somewhere on holiday with your
family over the past year?”; and “How many computers or tablet computers does
your family have?” FAS II has been validated alongside other measures of
adolescents’ SEP and compared to measures in which adolescents report their
parents’ income, occupations, and education levels, and the scale has been found
to have better criterion validity and less susceptibility to non-response bias.
The goal with the collective measure of SEP is to capture three dimensions of a
family’s socioeconomic position—parents’ education level, number of books in the
home, and the family’s affluence level—and combine these into a collective
measure of the family’s socioeconomic status. Parents’ level of education,
number of books in the home, and family’s affluence level each have some clear
limitations as measures of a family’s socio-economic situation. A collective
index based on these three dimensions will probably provide a more robust and
valid measure of family socioeconomic status. Consideration of anonymity is the
main reason that the Ungdata surveys do not include questions about parents’
occupations or incomes. A critical review of each question included in the
collective affluence measure, as well as detailed information on how the measure
is developed, may be found in Bakken et al.. We calculated a mean sum score,
ranging from 0 to 3, for each study participant, and then the total study sample
was split into three equally sized groups ordered by increasing affluence level
from low to high (low, medium, and high).
### Municipal sociodemographic characteristics
In our descriptive analysis, we included municipal education level, median gross
household income, income inequality, unemployment rate, disability pension rate,
immigration rate, divorce rate, and life expectancy. The municipal education
level was defined as the percentage of municipal inhabitants aged 15–80 years
who completed tertiary education. In the descriptive analyses, the
municipalities were grouped into quartiles where Quartile 1 is the 25% of
municipalities with the lowest education level; Quartile 2 is the second lowest
education level group; Quartile 3 is the group of municipalities with the second
highest education level, and Quartile 4 is the 25% of municipalities with the
highest education level among the adult population.
The Gini coefficient, the most commonly used measure of income inequality, was
used to estimate income inequality within municipalities. We used the census
Gini coefficient (only available in the period 2014–2017) calculated for each
municipality by Statistic Norway. The Gini coefficient varies between 0 and 1,
where 0 corresponds to so-called “perfect income equality,” meaning that every
household has the same income and wealth, and 1 corresponds to perfect income
inequality, meaning that one household possesses the population’s entire income
and wealth. According to the OECD, in 2017 Norway was ranked as the sixth
country with the least income inequality, with a Gini coefficient of 0.262.
Statistics Norway excludes students and uses a different method when adjusting
for large households in their calculations of the Gini coefficient, which in
2017 was 0.252. The municipality characteristics enter the analyses as
continuous (percentages or median) census variables. In our parametric
estimations, we only included municipal education level and income inequality.
### Covariates
The individual variables in Ungdata used in this study are *gender*, *school
year* (proxy for age and categorized as follows: first, second, or third year of
high school), and *survey cycle* (survey conducted in 2014, 2015, 2016, 2017, or
2018). These variables were included because earlier studies show strong
associations with adolescents’ mental health. Introducing other individual or
contextual sociodemographic variables was considered problematic in the
modelling in regards to problems with potential statistical over-adjustment due
to uncertainty whether the covariates represent real cofounding issues; or
rather act as mediators, colliders or have bi-directionally roles.
## Statistical approach
First, descriptive analyses of percentages on individual variables and municipal
sociodemographic variables, by municipal education level were tested by chi
square tests and analysis of variance (ANOVA). Second, we investigated the
relevance of the residential context as well as the association between family
affluence, municipal education level/income inequality, and psychological
distress, depression, and anxiety symptoms among high school students and tested
the hypothetical interactions using multilevel models.
Linear multilevel models with individuals (level 1, n = 97,460) nested within
municipalities (level 2, n = 156) were estimated. A two-level random intercept
model was fitted using maximum likelihood estimation to distinguish the
individual and municipality sources of variation in adolescents’ mental health.
We modelled the prediction of adolescents’ mental health in five steps. First,
we estimated an intercept model, only including the random intercept, to
determine the impact of the municipality context on adolescents’ mental health.
Second, we included the individual and family level variables (gender and school
year) and family affluence. Third, as shown in, we extended the random intercept
model for the relationship between family affluence and adolescents’ mental
health to allow family affluence effect to vary across municipalities. A
likelihood ratio test (LR test) was used to compare the random intercept and the
random slope models’ goodness of fit. In the final steps, we included the
municipal education level (main effect model) and the interaction terms of
family socioeconomic status with municipal education level (interaction model)
for all three mental health outcomes: psychological distress, depressive
symptoms, and anxiety symptoms. Similarly, in municipal income inequality as
well as the interaction terms of family affluence with municipal income
inequality were included. In addition, supplementary analyses exploring the
association between municipal socioeconomic conditions and adolescent risk of
moderate-to-high psychological symptoms (main effect model) as well as analysis
of the hypothetical interactions with family affluence (interaction model) were
performed; the results are presented in – Tables.
Estimates for fixed effects are reported as coefficients with 95% confidence
intervals (CI). To quantify the influence of municipality of residence on
adolescents’ mental health, we computed the intraclass correlation coefficients
(ICCs) for each outcome. The ICC expresses the correlation in the outcomes
(i.e., psychological distress, depressive and anxiety symptoms) between two
individuals randomly selected from the same municipality: the larger the ICC,
the stronger the clustering of psychological distress within the municipality
and the larger the general contextual effect of the municipality. The Akaike
Information Criterion (AIC) and the Bayesian Information Criterion (BIC) were
used as measures of goodness of fit for our models. The model parameters were
estimated by a general linear model (GLM) mixed effects method using Stata/MP
software (version 13).
# Results
## Characteristics of the study population
presents descriptive information regarding the individual and municipality
contextual variables among high school students in Norway, both in total and by
municipal education level. In all, 50.4% females and 49.6% males participated in
this study. More first-year students (44.6%) compared to second- (33.7%) and
third-year (21.7%) students completed the questionnaire. Each family affluence
group was (approximately) equally represented in the study population, with
about one-third of the students each in high (36.3%), medium (30.8%), and low
(32.9%) affluence groups. The mean family affluence score was 1.9, and, as
expected, mean affluence score as well as share of high affluence students
increased with increasing municipal education (p\<0.001).
Inequalities in adolescents’ mental health were observed both between family
affluence groups and municipal education groups in all three mental health
domains, with increasing symptom load associated with decreasing affluence and
education level. Mean psychological distress, depressive, and anxiety symptoms
were higher (p\<0.001) in low affluence adolescents (1.95, 2.19, 1.58,
respectively) compared with their peers with medium (1.91, 2.16, 1.54,
respectively) and high (1.87, 2.11, 1.51, respectively) affluence levels
(results not shown in table). The prevalence of students with moderate-to-high
symptom loads showed similar patterns. Furthermore, mean psychological distress
(1.8 to 2.0, p\<0.001), depressive (2.1 to 2.2, p\<0.001), and anxiety (1.5 to
1.6, p\<0.001) symptoms increased with increasing municipal education level.
Similarly, the prevalence of students with moderate-to-high symptom loads
increased with increasing municipal education level in all three mental health
domains (psychological distress: 43% to 51%, depressive symptoms: 53% to 61%,
and anxiety symptoms: 23% to 28%).
We noted differences in other municipal characteristics across municipal
education levels when divided into quartiles. The mean share of municipal
inhabitants who have completed tertiary education increased from 22% in
municipalities with the lowest education levels to 51% in the most educated
municipalities. Income inequality (measured by the Gini coefficient) increased
incrementally from the 25% least educated municipalities up to the municipal
group in the top 25% education level. The most educated municipality
(75<sup>th</sup> percentile) had a larger population (528,217 vs. 169,047
inhabitants), higher median household income (648,476 NOK vs. 642,806 NOK), a
lower disability pension rate (5.2% vs. 8.7%), a higher immigration rate (30%
vs. 18.4%), and a slightly lower divorce rate (10% vs. 10.6%) compared to the
average of the municipalities. The unemployment rate was 0.2 lower in the
municipal group with the 25% lowest education level of the other groups (1.6 vs.
1.8). The life expectancy was 0.6 years higher in the municipal group “upper
middle education level” than in the other groups (81.8 years vs. 81.2 years).
## Individual- and municipal-level variation in adolescents’ psychological distress
In the first step of our parametric estimations, an intercept model containing
only the second random intercept was estimated. We found that the ICCs for
psychological distress, depressive symptoms, and anxiety symptoms among
adolescents were 0.017, 0.019, and 0.010, respectively. In other words, our
estimations suggest that about 2% of the variation in the students’
psychological distress and depressive symptoms could be attributed to
differences between municipalities. However, only 1% of the variation in
students’ anxiety symptoms could be attributed to differences between
municipalities.
shows the individual and family covariates of the three mental health outcomes:
psychological distress (Model 1), depressive symptoms (Model 2), and anxiety
symptoms (Model 3). Family affluence was negatively associated with each of the
outcomes, with decreasing symptom scores accompanying increasing affluence
levels. Being female and a third-year student was correlated with higher
psychological symptoms. The interaction terms with gender and school year are
negative and statistically significant, indicating that the positive association
between females and psychological distress and depressive/anxiety symptoms
decreases over time.
In, we extend the random intercept models for the relationship between family
affluence and a) psychological distress, b) depressive symptoms, and c) anxiety
symptoms to allow the impact of family affluence to vary across municipalities.
The two-level random intercept model, which is nested in the random slope model,
is rejected at the 5% significance level (using a likelihood ratio test),
suggesting that the impact of family affluence on adolescents’ anxiety and
depressive symptoms does vary between municipalities.
In relation to the municipal socioeconomic variables, we found that municipal
education level is associated with higher psychological distress and
depressive/anxiety symptoms among high school students in Norway. However, the
interaction models suggest that these associations are also conditioned by the
family affluence level of the students ( and). demonstrates that the predicted
depressive and anxiety symptoms increase with increasing municipal education
level among all high school students. Notably, among adolescents at the medium
affluence level, the adverse effect of high municipal education level is
statistically steeper than for students with high and low family affluence
levels. For example, for students that live in medium affluence families, the
predictive depressive symptom score is 2.12 in a municipality where 26% of the
inhabitants have completed a tertiary degree, compared to 2.24 in municipalities
with a tertiary education level of 46%.
The same impact of municipal education and family affluence and their
interactions were found on the predicted probability of moderate-to-high
depressive and anxiety symptom complaints. Students from families of medium
affluence who lived in one of the Norwegian municipalities with the lowest
percentage of residents with tertiary education (for example, 22%) had a 53%
chance of experiencing depressive symptoms. Students from families of medium
affluence living in municipalities where the tertiary education level was (as
high as) 50% had a predicted likelihood of 63% for moderate-to-high depressive
symptom complaints.
The effects of municipal income inequality and its interaction with family
affluence on psychological symptoms were also tested, with similar results as
with municipal education level. (main effect models) shows that psychological
distress and depressive/anxiety symptoms among high school students increase
with increasing income inequality. Notably, when adding the interaction terms
with family affluence, the associations with municipal income inequality and
psychological symptom load is most significant for high school students with
medium affluence levels (See).
# Discussion
## Key findings
This national representative study exploring the impact of interactions between
family affluence and municipal socioeconomic characteristics on psychological
distress among Norwegian high school students has produced findings that should
be highlighted. Overall, our results indicate substantial psychological symptom
loads among 16–18-year-old students in Norway. Inequalities in adolescents’
mental health between family affluence groups were evident, with increasing
symptom loads accompanying decreasing affluence levels. Females, particularly
those attending the third year of high school, showed higher psychological
symptom scores than males, mostly evidencing moderate-to-high psychological
symptom loads. The average psychological symptom load and prevalence of students
suffering from moderate-to-high symptom loads increased slightly with increasing
education levels and income inequalities in their residential municipalities;
this applies to adolescents in all three family affluence groups: low, medium,
and high. However, our parametric estimations suggest that these municipal
socioeconomic characteristics appear to have the highest impact on the mental
health of adolescents living in families at the medium affluence level. This
study deepens our understanding of how family affluence interacts with
residential contexts to promote or inhibit young people’s emotional development.
## Education level in municipality and other socioeconomic conditions
The first research question this study aimed to answer was whether high school
students in general achieve psychological benefits by living in a municipality
with a high average education level. Our parametric estimations suggest that the
predicted mean of depressive and anxiety symptoms among high school students to
some degree increases in line with higher proportion of municipal residents with
tertiary education. Although there are several plausible reasons why living in a
high socioeconomic environment with a high proportion of well-educated
inhabitants positively affects young people’s health and well-being, the
findings in the present study are contrary.
In our study, the Norwegian municipalities with high proportions of well-
educated people were typically characterized by high household incomes and low
disability pension rates, which have been used in comparison with lower educated
municipalities as proxy measures of local economic and neighborhood conditions
in multilevel analyses of health status. It is argued in the literature that
education creates social benefits to society beyond private returns through,
among other factors, civic participation, enhanced political behavior, and lower
crime rates. In accordance with these assumptions, we found that the education
level in a municipality was positively associated with the voting rate, whereas
it was inversely associated with the reported crime rate. Thus, the education
level in a municipality might be a contextual indicator not merely of the local
economic conditions, but also of social determinants of mental health such as
level of social support and community social capital. Despite that, we found a
positive association between municipality education level and adolescents’
psychological distress scores. This may indicate that contextual domains
typically related to community infrastructure and material living conditions are
either not of crucial relevance when considering differences in adolescents’
psychological distress between Norwegian municipalities, or that there are
psychological mechanisms that mask or balance out the beneficial effects
stemming from structural and materialistic factors. Elgar et al. indicated in
their study of 1,371 adolescents in seven European countries that socioeconomic
inequality in health is more closely related to psychosocial processes than to
material inequality. Living in a community with a high share of the adolescents
in socioeconomic advantageous families may, however, trigger some types of
psychological stress. For example, expectations can be an important source of
stress, and adolescents living in well-educated communities might perceive that
the people they are surrounded by demand and expect much from them, including
their parents, other family members, neighbors, classmates, teachers, training
partners, and team leaders, as well as society as a whole. Furthermore, during
adolescence the sense of social position is developing, and this psychological
mechanism might also influence adolescents’ health and well-being; perhaps this
source of stress is particularly prevalent in communities with high degrees of
symbolic capital. The evolutionarily based social rank theory (SRT) accounts for
the inferiority and submissiveness that is typical in depression. It might be
that well-educated communities are characterized by underlying factors that
increase the pursuit of social status and the chances of adolescents’ feelings
of inferiority. Societies that accentuate individual competitiveness will be
particularly vulnerable to the emotionally disturbing effects of social
comparison.
Furthermore, there are several other underlying contextual features that
potentially explain why the average depression and anxiety scores among our
study sample rise with increased municipal education level. It should be taken
into consideration that the immigration rate was nearly three times higher in
the most educated municipalities compared with the least educated
municipalities. Moreover, major differences were observed regarding the
municipalities’ per capita numbers. The least educated municipalities had the
smallest population size; in contrast, municipalities where the 60<sup>th</sup>
percentile of adult inhabitants had a tertiary education had the highest
populations. Differences in psychological distress between settlement types have
been shown in several other studies, but the results are mixed. Rural and urban
communities differ in environmental factors such as culture, socioeconomics, and
access to healthy diets and health care that may influence adolescents’
functioning and well-being. In many Western countries, urban residents have a
higher likelihood of suffering from psychological distress than rural residents.
However, across the degree of centrality, small geographical variations in
anxiety and depressive symptoms were reported in a previous study from Norway.
Although we found an effect of education level at the municipal level, it is
important to note that we found small variations between municipalities in
adolescents’ depressive and anxiety symptom scores which is in line with
previous Nordic population studies exploring geographical variations and rural-
urban differences in mental health, This might be because Norway has a high-
level welfare state which benefits all Norwegians no matter where in the country
they live. Norway is considered an egalitarian welfare country with generally
low income inequality (e.g., the Gini coefficient was 0.26 in 2017, compared to
a Gini coefficient of 0.32 across OECD countries and it imposes national
regulation on social and health services. The Nordic countries are leaders in
promoting health through public policy action. In 2018, 92.3% of high school
students attended public schools.
## Income inequality
The second research question in this study was whether the level of income
inequality in a municipality affects the psychological symptom load among
students. In line with other population-based studies, our findings linked
income inequality with poor mental health outcomes. According to Wilkinson,
Pickett, unequal societies become dominated by status competition and class
differentiation, and consequently they experience more health disadvantages. The
prominent and well-accepted phenomenon of income inequality/health association
is explained by hypotheses operating at different ecological levels, from the
individual up to the national. Rising income inequality in a society can lead to
a weakening of collective social capital and social integration, thereby
affecting the residents’ health. A second possibility, which overlaps with the
social capital mechanism, is that income inequality reinforces the negative
health effects of social comparison through a more prominent status hierarchy in
the local community. According to the status anxiety hypothesis, feelings of
social defeat and inferiority produce stress reactions that lead to poorer
health. However, the contextual mechanism behind the income inequality/health
association phenomena could also be interpreted without psychological
explanations, but rather based on distribution of material resources in a
society. The neo-materialist hypothesis argues that material conditions per se
and public and social infrastructure influence individual well-being and health.
A review study analyzing data from 27 European countries found the most support
for the status anxiety hypothesis and the social capital hypothesis, and the
author suggests that the mechanisms through which income inequality influences
mental well-being vary depending on the wealth of the country. Similarly, a
review of the inequality-depression relationship by Patel et al. suggests that
the main mechanisms at the local level are both the status anxiety hypothesis
and the social capital hypothesis. At the individual level, the inequality-
depression association is likely to be primarily mediated through psychological
stress. The heterogeneity of study findings across populations on the effect of
income inequality on depressive symptoms reflects the complexity of mechanisms
and pathways. The size and geographical unit of analysis matters when
considering the link between income inequality and mental health because
different geographical levels have different meanings and do not relate to the
same contextual characteristics. A study from The Netherlands, where income
inequality is almost as low as in Norway, found an association between municipal
income inequality and psychological distress (measured with the Kessler
Psychological Distress Scale) in the adult population. Our study found that
estimated income inequality had more effect on depressive symptoms than on
anxiety symptoms.
Notably, in the present study, well-educated municipalities were positively
associated with higher income inequality. This can be seen from an urban-rural
perspective. Both level of education and average income characterize
municipalities within a relatively large city. In geographic areas where there
are many knowledge-based jobs, many well-educated people will reside, and this
is reflected in the average income of the citizens. However, there is still a
need for low-income jobs in these areas, which apparently creates a connection
between education level and income inequality in Norwegian municipalities.
## Family affluence, and the case of medium affluent students
The third and final research question we raise in the present study is whether
the degree of association between socioeconomic municipal characteristics and
adolescents’ psychological distress depends on family affluence level. We found
that the psychological disadvantages resulting from increasing municipal
education levels and income inequalities were most significant among adolescents
living in families at the medium affluence level. Our parametric estimates
suggest that as the municipal education level and income inequalities increase,
the depressive and anxiety symptom mean scores in families of low and medium
affluence converge, while the differences between those in families of medium
and high affluence are slightly increased. It is somewhat puzzling why the
students in families of medium affluence (measured at the national level in
Norway) seem to be most affected by an increase in municipal education level.
This may partly be explained by the fact that students in families of medium
affluence residing in the least educated municipalities have proportionally more
peers at lower family affluence levels than themselves compared to students of
medium affluence living in the most highly educated municipalities in Norway.
Consequently, students in families of medium affluence may have more to lose
than affluent students in terms of socioeconomic rank by residing in highly
educated municipalities. A more open question asks why the difference in average
psychological symptom scores between students of low and medium affluence
gradually becomes smaller with increasing socioeconomic level. One speculation
is that students of medium affluence might be (somewhat) more concerned about
their social status than students of low affluence. In a study of adolescents
aged 14–17 years from six European cities, the socioeconomic inequalities in
adolescent health were more closely related to adolescents’ perceptions of
relative family SEP than to objective indicators of material family affluence.
Thus, our findings give some support to the assumption that late adolescents’
psychological symptom loads depend not only on their families’ affluence levels,
but also on the socioeconomic position that level of affluence confers in the
social hierarchy (see Subramanian, Kawachi for a brief review of linking income
inequality, relative income, and relative rank to health).
In general, our likelihood estimates show the highest psychological distress
among students in families with the lowest family affluence, while the most
affluent families had the fewest students suffering from anxiety and depressive
symptoms. By using the FAS, Elgar et al. demonstrated socioeconomic differences
in adolescents’ mental health in teenagers aged 11–15 years from high-income
countries. Our study indicates that the late period of adolescence (i.e., 16–18
years) is not exempt from a socioeconomic family gradient of mental health.
Given that people use conventional socioeconomic criteria to assign themselves
subjective status, we may assume that family affluence is involved in
adolescents’ assessments of their subjective SEP relative to their peers.
Adolescents who perceive themselves to be at a lower level in the socioeconomic
hierarchy compared to their peers are at higher risk of poor well-being and
health outcomes.
## Strengths and limitations
A main strength of the study is its relatively large and nationally
representative sample, with explanatory factors at the individual and family
levels linked to population-based municipal socioeconomic factors from national
administrative databases. Moreover, family affluence level and adolescents’
symptoms of depression were evaluated in a standardized manner using validated
measures. There are, however, several limitations of this study that should be
noted. First, the use of a cross-sectional design limits the interpretation of
our findings as we cannot draw inferences regarding the direction of the
observed associations or the risk factor status of explanatory variables. Nor
can our results reveal what may be the underlying causes of the associations we
found. Second, although overall response rates were relatively high,
approximately 30% of students were absent from the surveys, which inevitably
poses the possibility of non-response bias due to illness and truancy. Third,
while the items from the Depressive Mood Inventory have been validated in
clinical studies, the present combination of anxiety items has not been
validated. Thus, the reliability of the anxiety measure is uncertain, and the
use of exclusively validated instruments would have strengthened the study
findings. Fourth, the various high schools and municipalities included in the
study sample differ in each survey/study year. Fifth, using municipality as an
area-level when investigating how community environments may be related to
health outcomes poses methodological challenges. Other geographical areas and
social contexts (e.g., neighborhoods, school districts, peer groups, and other
types of communities that facilitate social interaction) may be more relevant
than municipalities for adolescents’ well-being and mental health outcomes.
Moreover, the socio-demographic distribution of the population varies between
Norwegian municipalities, and it is important to take this into account when
interpreting the results of this study. However, presenting and interpreting
estimates of effect measures for secondary risk factors (confounders and
modifiers of the exposure effect measure) from a single statistical model may
lead to several interpretative difficulties and may be misleading. Sixth and
finally, an ideal study examining contextual effects on various health outcomes
should include explanatory variables at multiple levels and allow the levels
(i.e., contexts) to change over time. Family background, neighborhood of
residence, and schools the children attended before high school are three highly
important levels, and adjustment for them would constitute a strong advancement
of the study. Ideally, this should be performed using a multiple-membership,
cross-classified, multilevel analysis that allows the levels (municipality,
neighborhood, school, and families) to change over time. However, our data do
not include information about school, neighborhood, or particular household the
child attended before entering secondary school, and thus this analytic
framework cannot be used in this case.
## Interpretations and conclusion
Living in a community with a high proportion of well-educated people is
associated with several benefits. One possible benefit is the opportunity to
learn and imitate healthy behaviors. Another benefit may be the sharing and
receiving of social capital as well as the materialistic facilities in these
types of communities. Despite these potential benefits of living in a well-
educated society, our study demonstrates that there must be other factors at
play which apparently are even more important for psychological well-being in
the high-income and egalitarian welfare state of Norway with its national
regulation of social benefits and health services. Thus, there may be
psychological mechanisms associated with well-educated communities that have
negative effects on adolescents’ mental health. The Norwegian municipalities
with high proportions of well-educated people were typically characterized by an
increase in income inequality compared to less educated municipalities. Living
in competitive-oriented environments with high degrees of demands and
expectations in several social arenas (such as at home, school, and sports
activities, and when socializing with friends both in the virtual world and when
physically present) might generate “status anxiety” and daily stress. We suggest
that this type of disadvantageous environment is amplified by increased
education level and income inequality in the Norwegian municipalities. Although
we identified a modest degree of association between adolescents’ socioeconomic
circumstances and psychological distress in Norway, the findings have
potentially important implications for population health and society at large
which should be considered when developing community planning and policy in
high-income countries facing social changes and within-region inequalities in
socioeconomic status.
# Supporting information
The Ungdata surveys were conducted by the Norwegian Social Research (NOVA)
institute in cooperation with regional centers for drug rehabilitation (KoRus).
We wish to thank them for their cooperation and for conducting the data
collection.
10.1371/journal.pone.0254033.r001
Decision Letter 0
Yang
Xiaozhao Yousef
Academic Editor
2021
Xiaozhao Yousef Yang
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
19 Apr 2021
PONE-D-21-02793
Psychological distress in late adolescence: The role of inequalities in family
affluence and municipal socioeconomic characteristics in Norway
PLOS ONE
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Reviewer \#1: I think this manuscript titled: "Psychological distress in late
adolescence: The role of inequalities in family affluence and municipal
socioeconomic characteristics in Norway" is of great interest and, in general,
is well organized and well written. The topic is exact and relevant in the
actual situation where mental health is more than ever valued. Besides, it
brings to the reader new insights about the variables to consider in terms of
youth, mental health, and social contexts and their associated features (family,
municipalities...). Although focused on psychological distress in adolescence,
it highlights several psycho-social aspects underlying a new vision and
understanding about youth mental health. I think this psycho-social focus on
mental health is one of the significant innovative contributions of the study.
The outcome is a study that contributes to deep analysis and understanding of a
psychological issue but going out from a psychologically perspective and thus
becoming a comprehensive tool for researchers and practitioners. Also of
relevance is the large and nationally representative sample used in the study.
Still, I have a few comments on strengths, and also suggestions about possible
changes to improve the article. Following the structure of the manuscript, they
are:
1\. INTRODUCTION
Very well organized and clear, straight to the point.
The authors did a good summary of fundamental literature to support the study's
aim and the problem. Although being targeted in Norway, the information is
sufficiently valuable and adequate to understand other countries through this
particular social organization's lens. Even related to just one country, the N
of participants is significantly large, making this a robust study with broad
implications and applications.
The introduction in subsections makes it easy to follow and incorporate main
conceptual aspects/issues and variables to consider.
Study aims and research questions are clear and emerging from the introduction's
contents.
2\. METHODS
\- Sample and data collection
The authors present a lot of pertinent information about participants and the
data collection. According to this, some more aspects could be pointed out in a
more explicitly way (e.g using subsections/subtitles), for example, if there
were any eligibility criteria to participate or if there was a differentiation
between the clinical and normative population, or if both were integrated into
the study or not. The sentence (see lines from 216-218): "We found no
significant differences between the study sample and excluded individuals in
terms of mean symptom scores of psychological distress (1.91 vs. 1.91),
depression (2.16 vs. 2.15), or anxiety (1.55 vs. 1.55)" is not clear, please add
the word "respectively" at the end of the sentence. Besides, giving some
information/characteristics about excluded individuals could help understand
reported values and compare excluded participants and the study sample.
\- Measures
This section requires a more attentive organization in terms of titles and
subtitles.
Some doubts emerged: are the titles of "Measures" and "Individual and family
level" and "Municipality level" at the same level, or all these points are about
measures and so "Individual and family level" and "Municipality level" should be
inside the section "Measures" as are the first subtitle: The outcome variables:
psychological distress, symptoms of depression and anxiety"? Please review this
organization of contents inside the subsection "Measures", even if authors
considered different measures under each level.
Besides, when reporting specific scales, I suggest informing the particular
version used because some scales have several different versions (with, for
example, different number of items); and/or if there were some changes in the
original ones to serve specific purposes in this study.
This means that more information on measurement instruments should be provided.
\- Statistical approach
I think the proposed statistical analysis is according to the study's aims and
data.
Besides this section, the authors also report some other specific analysis
inside other parts of the manuscript (e.g., family and municipality levels have
references to particular statistics). I suggest the authors write in this
section of the "statistical approach" that some other information on specific
statistics will be (or have already been) shown in specific other parts of the
article.
3\. RESULTS
I think this is a strong part of the manuscript.
According to study aims and methods, the authors obtained several types of
results. Tables and Figures are very elucidative, clear and readable,
aggregating significant results and improving readers' facility to follow all
the information.
4\. DISCUSSION
In the discussion, the authors summarize the main findings, showing how the
results support the conclusions. The authors highlighted the leading and
innovative findings produced by the study in an excellent way, supporting and
interpreting them clearly, through well-supported literature and previous
research. Although this study focus in a specific country, the new and
unexpected results and conclusions can inspire and support other studies on
youth mental health in general. Still, I ask if authors could explicitly re-
organize some of the research questions' findings. These questions defined at
the beginning (in the introduction) should be explicitly again in the discussion
in order to benefit the manuscript (just as a suggestion).
The authors also report the study's strengths and limitations, and they also
give suggestions to overcome some of these limitations in future studies. this
is a very relevant part in the discussion.
The list of references is complete and diverse, with very recent references
supporting the topics discussed along with the article.
In general, I think this is a relevant study demonstrating significant advances
in adolescent mental health.
I hope authors will reflect on comments made and improve the manuscript
following suggestions on few aspects.
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10.1371/journal.pone.0254033.r002
Author response to Decision Letter 0
27 May 2021
Dear editor and reviewer,
In the revised manuscript we have endeavored to meet PLOS ONE’s style
requirements and made some changes based on comments from reviewer.
Response to the reviewer.
Thank you for constructive suggestions for improvements in the method sections.
In the revised manuscript under “Methods” we have made the following revisions:
1\. Sample and data collection
\- renamed the "sample and data collection" into "Design and data sources" and
included the following subtitles "The ungdata survey". "Municiplaity state
reporting (KOSTRA)" and "study population", please see line 183-235.
\- rewritten the following sentences (line 216-219): "We found no statistical
difference between included and excluded individuals for mean symptom scores of
psychological distress (1.91 vs. 1.91), depression (2.16 vs. 2.15), or anxiety
(1.55 vs. 1.55). There was a noted higher share of first-year students (54 %
versus 45 %, p\<0.001) for excluded compared to included groups.
2\. Measures
\- Rewritten the section into "assessment of variables" with the following
subtitles: " Psychological distress, and depressive and anxiety symptoms", "
Family affluence as a proxy for socioeconomic position (SEP)", " Municipal
sociodemographic characteristics " and "Covariates"
\- Revised the sections “Psychological distress, and depressive and anxiety
symptoms” and “Socioeconomic position” – with clearer and more information of
the particular scales used.
3\. Statistical approach
\- We included the following sentence in the first part of the statistical
approach section (line 321-323): "First, descriptive analyses of percentages on
individual variables and municipal sociodemographic variables, by municipal
education level were tested by chi square tests and analysis of variance
(ANOVA)".
We are also very grateful for the reviewer's advice to reorganize parts of the
“Discussion” chapter. In the revised version of the manuscript, we have
highlighted our three research questions in separate sections. In each of these
sections, we first answer the respective research question by stating our
related main finding.
Data Availability statement
The data and materials from the Ungdata-surveys are closed and stored in a
national database administered by Norwegian Social Research (NOVA). The present
study and analysis of the Ungdata were approved by the Norwegian Centre for
Research Data (NSD). Norwegian legislation prohibits deposition of these data to
open archives. The data are freely available for research purposes upon
application. Details about the application process to NSD can be found at:
<https://nsd.no/nsddata/serier/ungdata_eng.html>.
Yours sincerely,
Tommy Haugan
10.1371/journal.pone.0254033.r003
Decision Letter 1
Yang
Xiaozhao Yousef
Academic Editor
2021
Xiaozhao Yousef Yang
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
18 Jun 2021
Psychological distress in late adolescence: The role of inequalities in family
affluence and municipal socioeconomic characteristics in Norway
PONE-D-21-02793R1
Dear Dr. Haugan,
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Additional Editor Comments (optional):
Reviewers' comments:
10.1371/journal.pone.0254033.r004
Acceptance letter
Yang
Xiaozhao Yousef
Academic Editor
2021
Xiaozhao Yousef Yang
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
23 Jun 2021
PONE-D-21-02793R1
Psychological distress in late adolescence: The role of inequalities in family
affluence and municipal socioeconomic characteristics in Norway
Dear Dr. Haugan:
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[^1]: The authors have declared that no competing interests exist. |
# Introduction
Controlled release of drugs into the epidermis often results in large amounts of
the drug remaining at the delivery site, whereas much smaller amounts of the
drug enter the body system. Although some transdermal delivery systems can be
efficient in supplying drugs that have a systemic effect, they are not practical
for controlling the delivery of drugs when the final target is the skin itself.
Thus, it would be useful to develop a delivery system that could maximize the
period of drug deposit, either in the epidermis or the dermis, while minimizing
its transdermal penetration into the body. Microsponge has been increasingly
investigated to reach the aim in recent years. Microsponges are a polymeric
delivery system consisting of porous microspheres, and they may enhance the rate
of dissolution of poorly water-soluble drugs by entrapping such drugs in
microsponge pores. These pores are very small, and the drug can be reduced to
microscopic particles in these pores, which significantly increase the surface
area of the drug and increase the rate of solubility. Hence, this system may
improve the efficacy and bioavailability of some poorly soluble drugs.
Microsponges are also designed to deliver active ingredients efficiently at
minimum dose and to enhance stability, reduce side-effects, and modify drug
release. Thus, the microsponge delivery system may be a promising drug-delivery
system in treating skin disease.
Paeonol is one of the main active components from the root bark of *Paeonia
suffruticosa* (the tree peony), which has been widely used in Asia and Europe.
Paeonol has potential for the treatment of neurodegenerative diseases in humans
by alleviating morphological damage , increasing neuron viability, and reducing
cerebral infarction. Paeonol also possesses anti-atherogenic and anti-arrhythmic
activities, and is widely used in cardiovascular diseases. Paeonol also has
anti-tumor and anti-inflammatory activity, as well as the ability to inhibit
melanin. Recently, it has been reported that paeonol has anti-anaphylactic
activity through regulating histamine and tumor necrosis factor-α, and these
effects could be exploited in treating eczema. However, paeonol is hydrophobic
and has a low aqueous solubility, with an oil/water partition coefficient of
2.21. Consequently, it is likely that it will be unable to penetrate the stratum
corneum, and using enhancers may cause the drug to reach the blood too easily,
but it is not desirable for the side effect. We know that, for any drug,
including paeonol, to be effective in treating eczema, there must be a
sufficient concentration of the drug in the epidermis. It is likely that paeonol
would not be able to have an effect in treating skin disease, not because of the
inefficacy of the drug itself, but because the drug cannot reach its site of
action. Furthermore, increasing the amount of paeonol in the plasma might induce
production of drug-metabolizing enzymes by the liver, which would affect the
metabolism of other drugs. Therefore, formulation of a drug-delivery system to
increase the rate of solubility of paeonol and its deposition in the epidermis,
as well as to reduce its systemic action in patients with eczema, is of
interest.
Previous authors have developed paeonol-loaded liposomes to increase paeonol
bioavailability in skin tissues. However, liposomes are very expensive and
difficult to formulate in the final product and their manufacturing processes
are very complex, thus it is difficult to carry out large-scale production with
liposomes. <u>Rui-guang et al</u> did not report whether the prepared liposomes
were stable over a long period at room temperature. Complexing paeonol with
β-cyclodextrin was shown to increase its solubility, but this formulation could
not control the release rate of the active agent by itself. Recent studies have
shown that microsponge formulations were able to deliver drugs to the colon and
then release the drug while retained in the colonic lumen, indicating that this
formulation could be a new approach for colon-specific drug delivery,. In
studies of a microsponge intra-dermal drug-delivery system, researchers used
mupirocin, hydroxyzine hydrochloride, and benzoyl peroxide as the model drugs to
evaluate the characteristics of this formulation, and showed that the main
mechanism of drug release was diffusion and that the microsponge formulation
could enhance the rate of drug release,. Generally, microsponges were prepared
mainly by a quasi-emulsion solvent-diffusion method and free-radical suspension
polymerization. However, free-radical suspension polymerization requires
irradiation, high temperature, or catalysis to activate the monomers, and thus
the manufacturing processes are very complex. In our project, the authors used
an emulsion solvent-diffusion method to prepare the paeonol microsponges.
Generally, the microsponges were stable over a wide range of pH values (1 to 11)
and temperature (up to 130°C), and were compatible with most vehicles and
ingredients. Because of the small pore diameter of the microsponges, bacteria
cannot penetrate inside and reach their contents, thus, unlike liposomes and
β-cyclodextrin, microsponges do not require any preservative compounds.
It has been shown that some microsponge formulations can significantly maximize
the time that the active ingredient remains in the skin, while minimizing its
penetration through the dermis into the body. In addition, the controlled
release of the drug from the microsponge formulation into the epidermis means
that the drug remains primarily localized, with only a restricted amount
entering the systemic circulation, and thus can be used as a means of
controlling side-effects. Therefore, development of a microsponge delivery
system for paeonol could improve drug efficacy in the local region and reduce
side-effects.
In this study, we prepared paeonol microsponges using an emulsion solvent-
diffusion method. Scanning electron microscopy (SEM), production yield, loading
efficiency, and particle size distribution were used to evaluate the
characteristic of the paeonol microsponges thus formed. The microsponges were
then incorporated into a cream base and we carried out *in vitro* and *in vivo*
drug-release studies.
# Materials and Methods
## Materials
Paeonol was purchased from Sigma Co. Ltd. (Tianjin, China). Ethyl cellulose-M70,
polyvinyl alcohol (PVA) 1788, white beeswax, and stearic acid were obtained from
Aladdin Chemistry Co. Ltd. (Shanghai, China). Triethanolamine and liquid
paraffin were obtained from Tianjin Fuyu Chemical Reagents (Tianjin, China). All
other chemicals and solvents were analytical grade.
## Animals
Female Wistar rats weighing about 200 g and female nude mice aged 6 to 7 weeks
were purchased from the Center of Experimental Animals, Southern Medical
University (Guangzhou, China). All animal experiments were performed according
to the guidelines of the Experimental Animal Ethics Committee of Southern
Medical University. And the protocol was approved by the Experimental Animal
Ethics Committee of Southern Medical University (Permit Number for rats:
4402101707, Permit Number for nude mice: 4402102090). All surgery was performed
under 10% chloral hydrate anesthesia, and all efforts were made to minimize
suffering.
## Preparation of paeonol microsponges
Preparation was optimized based on production yield, loading efficiency and
sorting coefficient (data not shown). The preparation procedure was as follows.
The paeonol microsponges were prepared using a quasi-emulsion solvent-diffusion
method with external and internal phases. To prepare the internal phase, paeonol
7 g and ethylcellulose 1 g were dissolved in dichloromethane 20 ml. In this
procedure, dichloromethane was an effective solvent for dissolving both the drug
and the polymer. The external phase, which contained the emulsifying agent PVA 3
g dissolved in 100 ml of distilled water, was placed in the vessel, and stirred
with a propeller-type agitator at 1000 rpm, and then the internal phase was
gradually added into the stirring external phase. The mixture was then stirred
at 1000 rpm for 4 h at room temperature to remove the dichloromethane from the
reaction flask. After that, the formed microsponges were filtered through filter
paper with a pore size of 0.45 µm (Millipore, Maidstone, Kent, UK), washed with
distilled water, and dried at room temperature.
## Analytical system
The quantitative determination of paeonol in microsponges was carried out using
reverse-phase high-performance liquid chromatography (HPLC) using a
chromatograph equipped with a quaternary pump (both model series 1100; Agilent
Technologies Inc., Wilmington, DE, USA), on-line vacuum degasser, autosampler,
column temperature controller, diode array, and multiple-wavelength detectors,
along with an analytical workstation. The chromatographic separations were
performed using an HPLC column 250×4.6 mm with a particle size of 5 µm
(Syncronis C18; Thermo Fisher Scientific Inc., Rockford, IL, USA). A mixture of
methanol and distilled water (65∶45) was used as the mobile phase. The filtered
mobile phase was pumped at a flow rate of 1 ml/min, and the wavelength was set
at 276 nm. All the determinations were performed at 25°C. The retention time for
paeonol was found to be about 8.3 min, and the total run time was 10 min. A good
linear relationship was found between the peak areas for various concentrations,
from 0.004 mg/ml to 0.02 mg/ml (R<sup>2</sup> = 0.9996). External
standardization by peak area was used for quantitative determination of the
paeonol microsponges. The developed method had good precision (0.114%) and
accuracy (2.76%). Each determination was calculated in triplicate, and the mean
of the values were reported.
## Characterization and evaluation of microsponge formulations
### Scanning electron microscopy
The morphology and appearance of the microsponges were studied using SEM with a
system (H-3000N, Japan) operating at 10 kV. The samples were dusted onto double-
sided tape on a metal stub and coated with gold/palladium alloy under vacuum.
The obtained photograph was recorded at ×400 magnification.
### Determination of production yield
The production yield of the microparticles was calculated according to the
following equation:where M<sub>ms</sub> is the final weight of the microsponges
obtained, and M<sub>rm</sub> is the initial weight of the raw materials (polymer
and drug). All the experiments were performed in triplicate and the mean of the
values were reported.
### Determination of drug content and loading efficiency
The quantitative determination of paeonol in microparticles was carried out
using HPLC as described above. The drug-loaded microsponges were weighed
accurately and treated with ultrasonic waves for 30 min with methanol as the
extraction reagent. Before injection into the HPLC, the samples were filtered
through a nylon membrane filter (0.45 µm). The actual drug content and loading
efficiency were calculated according to the following equation :where
M<sub>act</sub> is the actual quantity of paeonol in the weighed quantity of
microparticles, M<sub>ms</sub> is the weighed quantity of the microsponges, and
M<sub>the</sub> is the theoretical amount of paeonol in the microsponges.
### Particle size distribution analysis
The particle size distribution of the microsponges was determined by a laser
light-scattering technique (Mastersizer 2000; Malvern Instuments Ltd., Malvern,
Worcestershire, UK). Before measurement, samples were dispersed in distilled
water. The particle size range was set to 0.02 to 2000 µm, and the particle
refractive index was set to 1.520. Sorting coefficient (σ) was calculated to
evaluate particle uniformity, and particle size distribution by volume of the
paeonol microsponge was calculated internally.
## Preparation of paeonol microsponge creams
Creams were prepared using a standard reverse-emulsification method, using an
oil phase and an aqueous phase in order to carry out convenient administration
of the microsponges to the skin. The aqueous phase, contained 3.7 g
triethanolamine, and the oil phase consisted of 7 g stearic acid, 3.5 g liquid
paraffin, and 1 g white beeswax. Both phases were heated to 65°C in a water
bath. The aqueous phase was then added dropwise into the oil phase while the
mixture was stirred using a magnetic stirrer, while being allowed to cool to
room temperature, thus forming a cream. Once the cream was complete, we added
either paeonol alone or the paeonol-containing microsponges to the cream, and
thoroughly mixed the compound to ensure a homogenous preparation. As a
comparison, a saturated solution of paeonol, using normal saline as solvent, was
also prepared. In our study, the content of paeonol in the microsponge cream or
paeonol cream was set at 50 mg/g to keep the cream stable (data not shown),
while it reached 505.52 µg/ml in the saturated solution.
## *In vitro* drug-release studies
The *in vitro* release studies were carried out using a recirculating water bath
and three Franz diffusion cells, with a receptor compartment volume of 15 mL and
an effective area of 3.14 cm<sup>2</sup>. The permeation membrane was mouse
skin, obtained from female nude mice aged 6–7 weeks. After the mouse was
euthanized, the whole skin was excised, the subcutaneous fat was carefully
removed with forceps, and then the skin was washed with normal saline and
examined for integrity. The skin was clamped between the donor and the receptor
chambers. The receptor chamber was filled with normal saline and set at 37°C,
and then the solution in the receptor chambers was stirred continuously at 300
rpm. The respective formulation (paeonol cream 1.0 g, paeonol microsponge cream
1.0 g, or saturated aqueous solution 1 ml) was gently placed in the donor
chamber. At 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8
h, 9 h, 10 h, 11 h, and 12 h, samples of 1 ml were withdrawn from the receiver
compartment and replaced immediately with an equal volume of normal saline kept
at 37°C. The collected samples were then analyzed by HPLC. The cumulative curve
was plotted of the total amount of paeonol that permeated at each time interval
*vs.* time. The release kinetics of the paeonol cream and paeonol microsponge
cream were calculated, and their release patterns were analyzed using different
mathematical modes.
## *Ex vivo* drug-deposition studies
For determination of the amount of drug deposited in the skin, we used Franz
diffusion cells as described above for the *in vitro* release studies. Skin
obtained from female nude mice was clamped between the donor and the receptor
chambers, with the respective formulations (paeonol cream 1.0 g, paeonol
microsponge cream 1.0 g, or saturated aqueous solution 1 ml) placed gently in
the donor chamber. The permeation membrane was dismantled after 4, 8, 12 and 24
h, respectively. The remaining cream outside the skin was carefully removed, and
then the skin was weighed and cleaned with 10 ml distilled water each time (five
times in total). The treated skin was then cut into small pieces, and the drug
in the skin was extracted by homogenization with 5 ml methanol. Finally, the
tissue samples were centrifuged at 10,000 rpm for 30 min. The supernatants were
collected and analyzed using HPLC.
## *In vivo* microdialysis
Female Wistar rats weighing about 200 g were anesthetized with 10% chloral
hydrate 0.35 ml/100 g, given as an intraperitoneal injection with supplementary
injections of half the dose every 90 min if needed. For intra-dermal
microdialysis, the abdominal fur of the rats was carefully shaved, and then the
skin was incised over the dermis, followed by intra-dermal insertion of an
introducer, assembled by inserting a stainless needle into the tubing. After
placing the tubing into the dermis, the needle was withdrawn, followed by
insertion of a microdialysis probe (CMA 20 Elite, 10 mm in membrane length, 20
kDa cut-off; CMA Microdialysis AB, Solna, Sweden) and then the tubing removed.
After placement, the probe was perfused with normal saline using a syringe pump
(CMA 402; CMA Microdialysis AB, Solna, Sweden) at a constant flow rate of 5
µl/min. While the rat was still under anesthetized condition, the jugular vein
was isolated. For plasma microdialysis, the microdialysis probe described above
was placed in the jugular vein, and perfused with normal saline by the
microdialysis pump at a constant flow rate of 5 µl/min. The system was
equilibrated for 1 h, and then 1.0 g paeonol cream or paeonol microsponge cream
was applied to an area of skin 3.14 cm<sup>2</sup> in size. The intra-dermal and
plasma dialysates were collected for HPLC analysis, using a refrigerated
fraction collector (MAB 85; Microbiotech/se AB, Stockholm, Sweden) every 20 min
for 12 h.
In order to obtain absolute tissue concentrations from dialysate concentrations,
*in vivo* relative recovery (R*<sub>in vivo</sub>*) was carried out using
retrodialysis prior to the microdialysis studies. In this experiment, one
microdialysis probe was inserted into the dermis as indicated above, while the
other probe was inserted into the jugular vein. Probes were perfused with 6.15
µg/ml paeonol at a constant flow rate of 5 µl/min, and the respective dialysates
were collected at 20-min intervals and analyzed using HPLC. Recovery was
determined from six consecutive dialysis samples per probe. R*<sub>in
vivo</sub>* was calculated as:where C<sub>d</sub> is the drug concentration in
the dialysate, and C<sub>p</sub> is the initial perfusate concentration of
paeonol. Prior to the PK analysis, concentrations for all dialysate samples were
corrected using the following equation:where C<sub>dermis/blood</sub> is the
unbound drug concentration in the intradermal fluid or plasma.
## Statistical analysis
Data in all experiments are presented as means ± SD. Statistical differences
were tested by one-way ANOVA and the independent samples *t*-test. *P*\<0.05 was
considered significant.
# Results
## Characterization of the microsponge formulation
The morphology of the microsponges was studied by SEM. The typical shape and
surface characteristics of the microsphere are shown in. The microsponges were
finely spherical and uniform in shape, and porous in nature, with no drug
crystals on the surface.
The production yield, actual drug content, and loading efficiency was calculated
according to to. The production yield was 72.20±3.59% (n = 3), the actual drug
content was 77.40±1.06% (n = 3) and the loading efficiency of the paeonol
microsponge was 55.90±3.27% (n = 3).
Using the laser light-scattering technique, a particle size distribution map by
volume of the paeonol microsponges was determined, which showed that the
specific area, surface diameter and diameter by volume of the particles were
0.65 m<sup>2</sup>/g, 9.5 µm and 22.4 µm, respectively. The particle size
distribution of d(0.1), d(0.5) and d(0.9) were 6.3 µm, 15.8 µm and 37.4 µm
respectively, and the sorting coefficient was 0.569.
## *In vitro* release studies
The *in vitro* release profiles of paeonol cream, paeonol microsponge cream, and
saturated aqueous solution are shown in. In order to have a better comparison
between the release profiles of the three formulations, the slopes (flux) of the
linear portion of the release profiles were calculated, as was the steady state
flux (Jss), based on the cumulative amount of drug permeated per unit area
plotted against time, and the estimated as steady state flux (J<sub>ss</sub>).
Based on these results, the *in vitro* release profile of paeonol microsponge
cream could be best expressed by zero order kinetics, as the cumulative drug
release *vs.* time were found to be linear (R<sup>2</sup> = 0.997).
## *Ex vivo* drug-deposition studies
The amount of paeonol deposited in the skin from different formulations at
different time intervals was determined by HPLC. As shown in, the amount of
paeonol deposited in the skin was much higher for the paeonol microsponge cream
than for the paeonol cream, especially at 4 h (0.5675±0.0394 mg/cm<sup>2</sup>
*vs.* 0.3194±0.0091 mg/cm<sup>2</sup>) and 24 h (1.3627±0.0699 mg/cm<sup>2</sup>
*vs.* 0.9988±0.0801 mg/cm<sup>2</sup>). Therefore, the highest amount of paeonol
deposited by the microsponge cream at the same dose was found after 24 h,
indicating that the microsponge cream improved drug residence in the skin.
## *In vivo* microdialysis
The average percentage recovery of paeonol by the retrodialysis method over 120
min was 52.44±3.67% (n = 6) in the intradermal fluid and 51.70±3.75% (n = 6) in
plasma. The dialysis membrane showed steady loss of paeonol for 120 min in the
subcutaneous liquid and in plasma through the CMA-20 probe.
The corrected dialysate concentrations of paeonol in plasma and intradermal
fluid after topical application of paeonol cream and paeonol microsponge cream
over the time interval of 12 h are shown in. The pharmacokinetic parameters for
unbound paeonol were calculated.
As shown in, the paeonol absorption was significantly higher with the paeonol
microsponge cream than with the paeonol cream, which is in accordance with *in
vitro* drug-release studies. Likewise, in the intradermal fluid, the area under
the curve (AUC) for concentration versus time (AUC<sub>0∼t</sub>) value was much
higher for paeonol microsponge cream (2396±258.2 µg/ml/min) than for paeonol
cream (1587±308.0 µg/ml/min). Maximum time (T<sub>max</sub>) was 220 min for
paeonol microsponge cream and 480 min for paeonol cream, while t<sub>1/2</sub>
for paeonol microsponge cream (935.1 min) was almost twice that of the paeonol
cream (548.6 min). By contrast, the AUC<sub>0∼t</sub> and C<sub>max</sub> values
of the paeonol microsponge cream were less than half that of the paeonol cream
(270.1±14.10 µg/ml/min *vs.* 656.7±153.2 µg/ml/min and 1.145±0.2126/ml *vs.*
0.6359±0.04150 µg/ml, respectively).
# Discussion
## Characterization of microsponge formulations
SEM images showed that the paeonol microsponges were porous and had a spherical
shape. The pores were caused by the diffusion of the solvent from the surface of
the microsponges. Thus, the volume of dichloromethane has a key role to play in
the preparation of microsponges.
## *In vitro* release studies
It is considered that microsponge formulations are too large to pass through the
stratum corneum, hence they would be expected to remain on the skin surface,
gradually releasing their contents over time. However, our results showed that
preparing paeonol in microsponges could increase the permeation rate of the
drug, compared with paeonol cream and saturated aqueous solution. This showed
that the particle size has a significant effect on the drug release, and
probably accounts for the discrepancy between our study and that of Jelvehgari
et al, who used a pore size of about 300–400 µm, which is more than 10-fold
larger than that used in our study. Our results indicate that microsponge
formulations are able to increase the permeation rate of some liposolubility
drugs formulated as small particles. Furthermore, the *in vitro* release
profiles shown in indicated that paeonol microsponge cream was able to show a
sustained release for up to 12 h compared with paeonol cream. The mechanism of
drug release from microsponges may be associated with its porous surface, as
this enables easy penetration of the release media and accessibility to the
entrapped drug molecule. As the release media penetrated into the porous of the
microsponge, the drug was then dissolved into it, thus drug released from the
microsponge. As the release media first gained access to the surface of the
microsponge and then gradually into the internal, the drug release measured over
the first few hours was due to the presence of non-encapsulated paeonol on the
surface of the microsponge, then as the release media gained access to the
pores, there was release of the drug entrapped in the pores, resulting in
sustained drug release for up to 12 h.
## *Ex vivo* drug-deposition studies
Effective topical drug therapy requires a sufficient amount of drug to be taken
up into the skin over a particular period of time to allow maximal
pharmacological activity. Thus, the larger amount of drug deposited in the skin
from paeonol microsponge cream indicates greater drug bioavailability in the
topical area, which is in agreement with an earlier report. The higher drug
retention by the paeonol microsponges for all time points may be partly
explained by the occlusive effect, as the microparticles produced a film on the
skin surface, which reduced transepidermal water loss. And then increased the
hydration state of the stratum corneum, and finally leading to increased drug
penetration into the skin. Moreover, the high lipophilicity of the drug
microsponge formulation prevented drug diffusion from the skin into the receiver
fluid, thus maintaining efficacious local drug levels for a long period of time.
## *In vivo* microdialysis
As shown in, the observed T<sub>max</sub> for paeonol microsponge cream was 220
min, compared with 480 min for paeonol cream. This may be due to the microsponge
formulation producing an occlusive layer on the skin surface and thus reduced
the transepidermal water loss and leading to high drug penetration, which was in
accordance with the *in vitro* release studies. The average t<sub>1/2</sub> for
the paeonol microsponge cream and paeonol cream was 935.1 min and 548.6 min,
respectively, thus the microsponge formulation showed good distribution into the
dermis. As shown in, the drug concentration of paeonol microsponge cream was
very stable compared with that of paeonol cream, showing that the microsponge
could act as a drug reservoir within the upper layers of the stratum corneum,
allowing the drug to be released in a controlled fashion. For treating skin
disease, the drug (in this case, paeonol) should stay in the treated areas and
there should be no absorption to other areas. The AUC measured by microdialysis
represents the total amount of drug that penetrated through the stratum corneum,
into the epidermis and finally into the dermis after 12 h. Therefore the higher
values of AUC<sub>0∼12 h</sub> for paeonol microsponge cream in the subcutaneous
liquid showed that the microsponge formulations could deliver significant
amounts of paeonol to the dermis, thus leading to high drug bioavailability, but
with high inter-individual variability. However, the lower values of
AUC<sub>0∼12 h</sub> and C<sub>max</sub> for paeonol microsponge cream in plasma
showed that the amount of drug absorbed into the plasma was not significant,
thus this should result in reduced side-effects. This reduced absorption may be
due to the high lipophilicity of the drug formulation preventing drug diffusion
from the skin into the interstitial fluid. The results indicated that preparing
paeonol as a microsponge formulation may be an effective drug-delivery system
for skin targeting in treating skin disease.
Major limitations of the microdialysis method are the low recoveries of
molecules with large molecular weights (\>20 kDa) and high lipophilicity or high
protein binding of some drugs. To determine the appropriate perfusate for
paeonol, we also measured the average recovery and protein binding. The
hydrophilicity (2.21 of partition coefficient), low molecular weight (166.18),
high recovery (52.44% in intradermal fluid and 51.70% in plasma) and low protein
binding (33.01±5.75) of the drug, showed microdialysis method was particularly
appropriate for paeonol with saline as perfusate.
There are controversial results regarding the influence of the actual position
of the probe on recovery. The different probe depth into the dermis may
influence the drug concentration in intradermal fluid. All the probes were
inserted into nearly the same depth by the same person and the histological
analysis was used to confirm that. And Esther et al. has reported that nearly
consistent depths can be achieved if the probes are inserted by the same trained
person. However the exact value in the dermis depth was not available. But in a
skin penetration study of salicylic compounds, there was no significant
correlation between probe depth and drug concentration in the range from 0.7 to
1.1 mm. The same conclusion had been found in the report of Muller et al. and
Hegemann et al.. And Esther had also reported that there would be no significant
correlation between probe depth and drug concentration when the variation in
probe depth was very small.
# Conclusions
In this study, we first found that the microsponge delivery system could not
only increase paeonol permeation rate but also minimize transdermal penetration
of the drug into the body, which should increase drug bioavailability at the
level of the skin and reduce side-effects when treating skin disease.
Additionally, microsponges were able to improve the drug residence in skin and
allowed sustained drug release for up to 12 h, resulting in a long active time
for the drug in topical treatment of the skin. These properties indicate that a
microsponge delivery system could be a useful strategy for a new generation of
pharmaceutical and cosmetic treatments. However, the level of drug distribution
in the stratum corneum, epidermis, and dermis and the mechanism of drug
penetration is still unknown, thus further studies into these aspects are
needed.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: QL SSL. Performed the
experiments: SSL XJ BZ ZGL XLL TZ. Analyzed the data: SSL XJ. Contributed
reagents/materials/analysis tools: LDW LL. Wrote the paper: SSL GFL. |
# Introduction
In domains ranging from the economy to national security, large-scale decisions
often involve judgments about the machinations of a group agent, such as a
terrorist organization, government, or corporation. Sometimes, judgments about a
group agent simply reduce to judgments about one or more of its individual
members (for example, thinking about whether or not a *country* is hiding
nuclear weapons may primarily involve consideration of that country's *leader*).
However, people also sometimes appear to make judgments about a group by
treating it as an entity in and of itself. Individuals assign moral blame and
punishment to whole organizations, interpret laws by looking for the
‘intentions’ of the legislature, may get into financial trouble by reasoning
about the ‘mind’ of the market, and, in a recent decision by the United States
Supreme Court, extended rights typically granted to individuals to a corporation
as a whole.
Although an abundance of research has investigated the effects of group
membership on how people perceive and reason about the minds of individual
people (for recent reviews, see, less is known about how perceivers reason about
the ‘mind’ of a group agent itself. To investigate this question, the present
work uses a combination of behavioral and fMRI approaches to examine the extent
to which understanding the ‘mind’ of the group as a whole shares important
properties and processes with understanding the minds of individuals.
Specifically, we ask (1) to what extent people sometimes reason about the
beliefs and intentions of a group agent separately from those of the groups’
members and (2) to what extent brain regions associated with understanding
individuals also support understanding group agents.
In order to predict or understand the behavior of a single individual,
perceivers often appeal to that individual’s *mental states* (i.e., his or her
thoughts, beliefs, intentions, desires, and feelings). This capacity to ascribe
mental states to others—that is, to *mentalize*, or engage *theory-of-mind*,
—reveals itself in the words perceivers use when talking about other people. For
example, we can say that Dick *thought* he was aiming for a partridge and never
*intended* to shoot his friend. Words like *think, believe, feel, intend, want*,
and *plan* all refer to the inner contents of other minds, allowing perceivers
to speak about the purported underlying causes of others’ behavior even as they
diverge from that behavior itself. In turn, inferences about these internal
causes guide moral decisions about how others should be treated, including the
extent to which they deserve praise or punishment.
Over the past two decades, an abundance of neuroimaging research has linked
mentalizing or theory-of-mind to a consistent set of brain regions, including
the medial prefrontal cortex (MPFC), temporo-parietal junction (TPJ), and
precuneus/posterior cingulate, sometimes collectively called the ‘theory-of-mind
network’. Using carefully controlled tasks that aim to isolate theory-of-mind,
these regions show preferential engagement when people are thinking about humans
versus other entities – and when people are thinking about humans' minds versus
their other aspects, such as their physical attributes,. Although much of this
evidence has been correlational, recent work using TMS has demonstrated a causal
role for the Right TPJ (RTPJ) in the use of mental state information for moral
judgment, and research on individuals with damage to MPFC and TPJ has
demonstrated a role for those regions in the ability to make inferences about
others' mental states.
Intriguingly, mental state words pervade perceivers' statements not only about
individuals but also about groups. In recent news reports, we learn that “Apple
thinks carefully about its entire product lineup”, that “Apple wants owners to
sell their old iPhones back to the company for a discount on a new phone”, and
that “Apple intends to work with record labels to identify and promote up and
coming artists”. In cases like these, people apply words normally associated
with the psychological states of an individual person—words like ‘thinks’,
‘wants’, and ‘intends’—to a corporation as a whole. These same expressions can
also be applied to other sorts of group agents. People talk about what a
government agency ‘intends’, what a religious organization ‘thinks’, or what a
sports team ‘loves’ or ‘hates’. Indeed, archival studies show that people speak
about groups using mental state words spontaneously, even outside the context of
an experiment, and cross-cultural studies document the use of mental state words
in descriptions of groups not only in the West, but also in East Asian cultures.
Does the use of such language indicate that people understand governments and
other organizations by attributing mental states to a group? Critically, there
are two different senses in which one might think about ‘groups’ and,
accordingly, two different senses in which one might investigate the processes
perceivers use to understand groups. On one hand, one could think about a
‘group’ as referring to the *members* of groups. If each group member is a human
being, then the group is simply a collection of human beings. A first sense in
which one might investigate how perceivers understand groups, then, is to
investigate how people understand collections of human beings. On the other
hand, one could think about a ‘group’ as referring to a *group agent*. A group
agent itself is not merely a collection of separate human beings but, instead,
an entity with whatever sort of status attaches itself to corporations, nations,
and sports teams. Thus, a second sense in which one might investigate how
perceivers understand groups is to investigate how people understand not
collections of individuals, but group agents.
An example highlights the distinction between a group in the sense of a
collection of individuals and a group in the sense of a group agent. Consider
the sentence “The employees and stockholders of Acme Corp. are all in debt.”
This sentence says something about the financial condition of various individual
human beings while making no claims about the financial condition of the
corporation with which they are associated. In other words, the sentence
ascribes a property to the members without ascribing that property to the group
agent itself. By contrast, consider the sentence, “Acme Corp. is in debt.” This
sentence says something about the financial condition of a corporation, but it
makes no claims at all about the financial condition of any individual human
beings. (The corporation itself could be in debt even if all of the employees
and stockholders were in excellent financial shape.) Thus, this sentence
ascribes a property to a group agent without ascribing that same property to any
of the members.
Existing work already provides some evidence for the claim thinking about groups
in the first sense—i.e., thinking about collections of human beings—shares
properties and processes with thinking about individual people. Behaviorally,
the vast literatures on stereotypes and intergroup relations show that people
are willing to ascribe psychological attributes to whole collections of others,,
and studies indicate that some of the same principles that apply to the
ascription of properties to individual agents also appear in the ascription of
properties to whole collections of agents. Moreover, a recent neuroimaging study
observed activation in brain regions associated with theory-of-mind—MPFC, TPJ,
and precuneus—when participants evaluated the applicability of certain
preferences both to individual people and to collections of individuals,
compared to a non-mental control condition. Taken together, these behavioral and
neuroimaging studies provide support for the view that people can ascribe
psychological attributes not only to individual human beings but also to
collections of human beings, and that they may use similar processes to do so
(even if the outcomes of those processes may sometimes differ).
Yet studies like these still leave open the question of how people understand
groups in the second sense—i.e., how they understand group agents. As we saw
above, people can ascribe a non-mental property to all of the members of a group
agent without ascribing that property to the group agent itself (“All of the
employees and stockholders are in debt”). Similarly, perhaps people can ascribe
a mental property (i.e., a mental state) to all of the members of a group
without in any way ascribing these states to the group agent itself (“The
employees and stockholders all love Jeopardy!”). We have also seen that people
can ascribe a non-mental property to a group without ascribing that property to
the individual members (“Acme Corp. is in debt.”). Similarly, perhaps people can
ascribe mental states to a group agent without ascribing that state to any of
the members. Indeed, recent research suggests that the more people perceive a
‘group mind’, the less they tend to perceive the minds of the members of that
group.
With this in mind, the current studies investigate how perceivers understand
group agents by examining the extent to which understanding group agents shares
important properties and processes with understanding individuals. Experiment 1
examines behaviorally the extent to which people ascribe mental states to group
agents over and above attributions of mental states to their individual members.
Experiment 2 uses fMRI to investigate the extent to which understanding and
predicting the behavior of group agents recruits brain regions associated with
understanding and predicting the behavior of individuals—i.e., brain regions
associated with theory of mind.
# Experiment 1: Ascriptions to group agents vs. ascriptions to group members
When people use sentences that appear to ascribe mental states to a group agent,
are they actually ascribing something to the group agent, or are they merely
attributing something to the group's members? For example, consider the
sentence, “United Food Corp. believes that the new policy is morally
unacceptable.” At least on the surface, this sentence appears to attribute a
mental state (the belief that the policy is morally unacceptable) to a group
agent (United Food Corp). However, it is possible that this is just a linguistic
shortcut, and that when people use or hear sentences like this one, they are
really attributing mental states to the members of the group, not to the group
itself.
Existing research demonstrates that people sometimes do use sentences that
appear to attribute a property to a group when referring to its members,
specifically when the members of the group have the particular property in their
roles as group members. For example, if each member of the Sigma Chi fraternity
gets drunk, and if each of them does so in his role as a Sigma Chi member,
people tend to agree with the sentence, “The Sigma Chi fraternity got drunk”.
This sentence appears on the surface to be ascribing a property to the
fraternity itself—the actual organization— but is in fact just a shorthand way
of ascribing a property to the individual members in their roles as members.
In Experiment 1, we examine whether apparent mental state attributions to group
agents can involve attributions of a property to a group agent itself, or
whether they reduce to attributions to individual group members. To the extent
that perceivers genuinely attribute a property to the group agent itself,
attributions to group agents should sometimes diverge from attributions to the
members of those groups. That is, we should observe (a) cases in which
perceivers attribute a mental state to all of the members of the group without
attributing that state to the group agent itself and (b) cases in which
perceivers attribute a mental state to the group agent without attributing that
state to any of the group's members. In contrast, to the extent that apparent
attributions to group agents are merely shorthand for attributions to the group
members, participants should not attribute properties to the group agent that
they do not also attribute to the members of the group. Thus, finding that
individuals attribute mental states to a group agent without attributing that
state to any of the group's members would be the most unambiguous evidence that
perceivers can apply mental states to group agents themselves.
## Method
### Participants
116 Yale students and faculty (33% female; age range 18-54, mean age 21 years)
were recruited outside a dining hall to fill out a questionnaire for payment.
### Ethics statement
This study was approved by the Institutional Review Board at Yale University.
All participants provided written informed consent.
### Materials and Procedure
This experiment used a 2 (mental state: individual-only or group-only) × 3
(question: any member, each member, group) design in which target was
manipulated within-subject and question type was manipulated between subjects.
Each participant received eight vignettes in counterbalanced order. Four
vignettes were designed in such a way that it would be logically possible to
ascribe a particular mental state to each of the individuals in the group
without ascribing that state to the group itself (*Individual-only* condition).
For example, one vignette described an organization devoted to fighting the
death penalty. All of the members of this anti-death penalty organization are
also interested in antebellum American history, so they decide to form a
separate organization, with exactly the same members, called the Shady Grove
Antebellum Historical Society (SGAHS), which meets to discuss historical
questions. If participants are willing to ascribe a mental state to all of the
individual members without ascribing that mental state to the group as a whole,
participants should report that all of the members of SGAHS want to fight the
death penalty but that the SGAHS itself *does not* want to fight the death
penalty. On the other hand, to the extent that attributions to a group simply
reduce to the attributions made to the individual members, participants should
report that SGAHS *does* want to fight the death penalty.
The other four vignettes were designed such that that it would be logically
possible to ascribe a mental state to the group itself without ascribing that
state to any of the individual members (*Group-only* condition). For example,
one vignette described a large organization that was commissioned to build a
space shuttle. Some members of the organization put together the software,
others build the exterior, still others are in charge of the fuel, and so forth.
But there is no single person who works on every aspect of the project. To the
extent that people are willing to ascribe a property to a group agent over and
above its members, participants should say that the organization knows how to
build a space shuttle, but the individual members do not. In another vignette, a
Community Association needs to choose music for an upcoming event. Some members
really want to play punk music and can't stand classical, others really want to
play classical music but strongly dislike punk, so in the end, the Association
selects a third option: classic rock. If people are willing to attribute
properties to group agents over and above their members, participants should say
that the Community Association itself preferred playing classic rock but that
none of the individual members shared this preference. On the other hand, to the
extent that attributions to the group simply reduce to the attributions made to
the individual members, participants should report either that most or all of
the individual members prefer playing classic rock or that the group itself does
not prefer playing classic rock. For full texts of the vignettes, see.
Each participant was randomly assigned to one of three question conditions: ‘any
member,’ ‘each member,’ or ‘group.’ Participants in the ‘any member’ condition
received after each vignette a question about whether any individual member of
the group had a particular mental state (‘Do any of the members of the Community
Association prefer the idea of playing classic rock to the idea of playing every
other type of music?’). Participants in the ‘each member’ condition were asked
whether *each* member had the relevant state (‘Do each of the individual members
of the Community Association prefer…?’). Finally, participants in the ‘group’
condition received questions about whether the group itself had the relevant
state (‘Does the Community Association prefer…?’). Each question was answered on
a scale from 1 (‘No’) to 7 (‘Yes’).
## Results
Two participants failed to complete all items of the questionnaire. We
calculated the mean response to ‘group’, ‘any member’, and ‘each member’
questions in the ‘Members Only’ vignettes and the ‘Group Only’ vignettes for the
remaining participants. To the extent that participants attributed purported
mental states to group agents themselves, we should observe both cases in which
participants attribute a state to all of the members of the group without
attributing that state to the group itself and, most critically, cases in which
participants attribute a state to the group itself without attributing that
state to any of the individual members. See for complete dataset.
For the Members-Only vignettes, a one-way ANOVA revealed a significant effect of
question condition, *F*(2, 114) = 41.2, *p* \<.001, η<sup>2</sup> = .42, such
that participants were willing to attribute states to some or all of the members
of a group without attributing those states to the group itself. Tukey's posthoc
tests showed that participants agreed less with ascriptions in the ‘group’
question condition than in either the ‘any member’ question condition, *p*
\<.001, or the ‘each member’ question condition, *p* \<.001, suggesting that
attributions to the group did not simply reduce to attributions to the group's
members.
Critically, for the Group-Only vignettes, a one-way ANOVA again revealed a
significant effect of question condition on participants' responses, *F*(2, 114)
= 91.6, *p* \<.001, η<sup>2</sup> = .62, such that participants were willing
to attribute states to the group itself that they did not attribute to any of
the members of the group. Tukey's posthoc tests showed that participants agreed
more with ascriptions in the ‘group’ question condition than in either the ‘any
member’ question condition, *p* \<.001, or the ‘each member’ question condition,
*p* \<.001. Moreover, participants' responses in the group question condition
were significantly above the neutral midpoint of the scale, *p* \<.001,
indicating that participants were genuinely endorsing sentences ascribing mental
states to group agents. These results suggest that attributions to the group
agent were made over and above the attributions made to individual members.
This study explored the relationship between ascribing states to group agents
and their members. We observed cases in which participants attributed a state to
all of the members but did not attribute that state to the group itself and also
cases in which participants attributed a state to the group itself but did not
attribute the state to any of the members. Together, these results demonstrate
that mental state ascriptions to a group agent can diverge from those made to
the group's individual members, suggesting that perceivers can attribute a
property of some sort to the group agent itself.
# Experiment 2: Neural processes supporting mental state ascriptions to group agents
Experiment 1 suggests that that when people use expressions of the form ‘United
Food Corp. wants.’, they appear to be ascribing something to the group itself,
rather than to the members of the group. However, a further question concerns
the processes supporting these ascriptions. That is, although such statements
clearly involve the same linguistic expressions that people use when applying
theory-of-mind to individual human beings, to what extent do they also involve
the same cognitive processes?
To investigate the processes supporting attributions of purported mental states
to group agents, we scanned participants using fMRI as they considered the
mental states of individuals and groups. In one task, participants read
sentences that referred explicitly to the mental states of groups and
individuals (along with matched, non-mental control sentences). In a second
task, participants carried out a procedure that relied on mental state
ascription incidentally, without the use of mental state words: making
predictions about what an individual or group would do in a variety of
situations. To the extent that perceivers rely on processes associated with
understanding individuals when they understand and predict the behavior of
groups, brain regions associated with theory-of-mind should be active both when
thinking about individuals and when thinking about group agents, and they should
be active to a similar degree. On the other hand, to the extent that perceivers
rely on different processes to understand group agents, we should observe
reduced activation in brain regions associated with theory-of-mind—RTPJ, MPFC,
and precuneus—during consideration of groups versus individuals. In the design
of this study, steps were taken to (a) minimize, as much as possible, the
likelihood that participants would simply consider the minds of individual group
members when considering group agents and (b) test sensitively the degree to
which brain regions associated with theory of mind are engaged during
consideration of group agents. Unlike past studies, no individuals were
mentioned or shown in the group condition, and both directed and spontaneous
theory of mind tasks were included. Moreover, the results of Experiment 1 show
that perceivers do interpret sentences about group mental states as ascribing
mental states to the group agent itself.
Although MPFC, TPJ, and precuneus have all been associated consistently with
theory-of-mind, finer-grained differences in the response profiles of these
regions facilitate predictions about their involvement during consideration of
group agents. Recent neuroimaging research has increasingly revealed that, even
when mental state attributions to individuals are concerned, MPFC, TPJ, and
precuneus do not all respond in the same ways under the same circumstances. In
particular, there are at least two ways in which the processes associated with
purported mental state reasoning about group agents may differ from those
associated with individual people. One is that certain properties of the *type*
of mental state content being attributed may differ. The other is that certain
properties of the *target* to whom that content is being attributed may differ.
The RTPJ consistently demonstrates sensitivity to the *type* of mental state
being ascribed. Specifically, a series of studies has demonstrated that RTPJ is
selective for processing representational mental states, such as beliefs –; see
for review. The RTPJ response is high when participants read stories that
describe a character's true or false beliefs but low during stories containing
other socially salient information, such as a character's physical appearance,
cultural background, or even internal sensations such as hunger or fatigue.
Similarly, activation in RTPJ is higher during inferences about an individual's
beliefs than during closely matched inferences about an individual's preferences
regardless of whether such inferences are more or less constrained by external
information—a response profile that is not shared by other regions associated
with social cognition, such as MPFC. Moreover, activation in the RTPJ
consistently tracks with thinking about mental contents, not merely seeing
mental state words. RTPJ becomes engaged when participants think about others'
mental states even in the absence of any mental state words, such as when they
view non-verbal cartoons or read descriptions of actions that imply a particular
mental state. Conversely, mental state words alone do not elicit activation in
the RTPJ; for review see. Thus, mental state words are neither necessary nor
sufficient for eliciting RTPJ activation. Instead, RTPJ activation during social
cognition appears to be associated with the ascription of representational
mental state content; for discussion see. Thus, to the extent that perceivers
attribute representational mental states to group agents, we should observe
similar levels of RTPJ activation during consideration of group agents and
individuals, both of which should exceed that associated with a non-mental
control condition.
In contrast, MPFC appears to be especially sensitive to the *target* of mental
state ascription. In particular, thinking about oneself, a similar individual, a
familiar individual, or an individual whose perspective one has taken earlier is
associated with more MPFC activation than thinking about more distant others.
MPFC also appears to be sensitive to the target of consideration when theory-of-
mind is not explicitly called for. For example, this region exhibits less
activation during consideration of “dehumanized” than “humanized” individuals
and responds more during consideration of one's own versus another person's
physical attributes. Although it remains open to further inquiry whether lower
MPFC response in these cases genuinely indexes a difference in the degree to
which mental states are attributed or rather the use of an alternative process
for doing so, the sensitivity of MPFC to the target of judgment suggests that
group agents may be particularly likely to be associated with lower activation
than individuals in this region.
## Method
### Participants
Nineteen right-handed, native English speakers (10 female; age range 19-25, mean
age 21 years) with no history of neurological problems participated for payment.
All participants had normal or corrected-to-normal vision.
### Ethics statement
This study was approved by the Committee on the Use of Humans as Experimental
Subjects (COUHES) at the Massachusetts Institute of Technology. All participants
provided written informed consent.
### Stimuli and Behavioral Procedure
#### Directed theory-of-mind task
During fMRI scanning, participants completed an individual vs. group agent
theory-of-mind task in which they read short statements about everyday events.
Participants were instructed to read each statement and were told that they
would be asked a series of questions about the statements later on in the
experiment. Inanimate (control) statements communicated information without
reference to people (e.g., “Although there wasn't much real data on agricultural
production, the statistics showed that rutabaga production was consistently
going down.”). Based on each control statement, an *individual* statement and a
*group* statement were constructed. *Individual* statements concerned a single
person's mental state (e.g., “Although there wasn't much real data on
agricultural production, George Hailwood was sure that rutabaga production was
going down.”). *Group* statements concerned the ‘mental state’ of a group agent
(e.g., “Although there wasn't much real data on agricultural production, United
Food Corp. was sure that rutabaga production was going down.”). No participant
viewed more than one version of the same base statement.
In each run of this task, participants read statements organized around a single
theme (e.g., one run concerned George Hailwood, United Food Corp., and food
production, whereas another concerned Stephanie Ann Majors, a record company,
and music sales). For full texts of the stimuli, see. Participants completed ten
functional runs of eighteen statements each (six per condition), totaling 180
trials. Statements were displayed in random order within each run and remained
onscreen for 8 s. Trials were separated by a variable inter-stimulus interval
(2–16 s) during which participants passively viewed a black screen.
#### Spontaneous theory-of-mind task
Following each run of the directed theory-of-mind task, participants were asked
to make a series of predictions about the individual and group about which they
had just read (e.g., “The asparagus might be contaminated by bacteria. Would
George Hailwood \[United Food Corp.\] be more likely to (a) recall all of the
asparagus or (b) cover up the whole incident?”). This task elicited mental state
reasoning indirectly by asking participants to formulate predictions about
behavior, such that no mental state words were presented to participants at any
point. Each question remained onscreen for 12 s, and participants were obliged
to respond during that time by pressing one of two buttons on a button box held
in the left hand. Each run comprised eight trials (four per condition) separated
by 10 s. Each participant answered each question either for the individual or
the group, but not both (question assignment randomized across participants).
#### Theory-of-mind localizer
In order to facilitate region-of-interest (ROI) analyses focusing on brain
regions associated with theory-of-mind, participants also completed a functional
localizer task in which they read short narratives and made inferences about
individual protagonists' beliefs (e.g., concerning the location of a hidden
object) and inferences about physical representations (e.g., the contents of an
outdated photograph). Each narrative was displayed for 10 s and was followed by
a statement that participants judged as true or false (e.g., Belief story:
“Sarah thinks her shoes are under the dress”; Physical story: “The original
photograph shows the apple on the ground”) which remained onscreen for 4 s.
Participants were obliged to respond during that time by pressing one of two
buttons. Trials were separated by 12 s fixation. Participants completed four
runs, each of which comprised eight trials (four per condition), for a total of
32 trials.
### Imaging Procedure
fMRI data were collected using a 3 Tesla Siemens scanner. Functional imaging
used a gradient-echo echo-planar pulse sequence (TR = 2 s; TE = 30 ms; flip
angle = 90°, 30 near-axial slices, 4 mm thick, in-plane resolution = 3×3 mm,
whole brain coverage). These sequences used PACE online motion correction for
movement \< 8 mm. fMRI data were preprocessed and analyzed using SPM2 (Wellcome
Department of Cognitive Neurology, London, United Kingdom) and custom software.
Data from each subject were motion corrected and normalized into a standard
anatomical space based on the ICBM 152 brain template (Montreal Neurological
Institute). Normalized data were then spatially smoothed (5 mm full-width-at-
half-maximum \[FWHM\]) using a Gaussian kernel.
Statistical analyses were performed using the general linear model in which the
event-related design was modeled using a canonical hemodynamic response function
and other covariates of no interest (a session mean and a linear trend). After
these analyses were performed individually for each participant, the resulting
contrast images for each participant (i.e., *individual \> control, group \>
control*) were entered into a second-level analysis in which participants were
treated as a random effect. Data were thresholded at *p*\<.001, *k*\>10,
uncorrected.
For the directed theory of mind task, conjunction analysis was performed
following the procedure described by Cabeza, Dolcos, Graham, & Nyberg. Whole-
brain statistical maps were created from the *individual* \> *control* and
*group* \> *control* contrasts separately to identify voxels activated by each
condition (thresholded individually at *p* \<.01), making for a conjoint
threshold of *p* \<.001.
ROIs were defined for each subject individually based on a whole-brain analysis
of the theory-of-mind localizer in three regions: RTPJ, precuneus, and MPFC.
Regions were defined as 10 or more contiguous voxels that were significantly
more active (*p* \< 0.001, uncorrected) during stories about mental states than
during control stories about physical representations. The average responses
relative to rest during the individual and group conditions were then estimated
in these regions. Within each ROI, the mean percent signal change (PSC = 100 ×
raw BOLD magnitude for \[condition − rest\]/raw BOLD magnitude for rest) was
calculated for each condition at each time point (averaging across all voxels in
the ROI and all trials of the same condition) and averaged across seconds 6–10
to account for hemodynamic lag. Individual subject means for each condition of
each task are available as. The full fMRI dataset is available upon request.
## Results
### Directed theory-of-mind task
In order to assess the extent to which common cognitive processes subserve
thinking about the minds of individuals and groups, we first conducted whole-
brain, random effects analyses of BOLD signal. In whole-brain analyses,
activation when participants contemplated the mental states of both individuals
and groups (compared to control) was observed in brain regions associated with
theory-of-mind, including MPFC, RTPJ, and precuneus. The direct comparisons
between the individual and group conditions (individual \<\> group) yielded no
areas of differential activation in regions typically associated with social
cognition. To the extent that overlapping BOLD activation reflects the
engagement of overlapping cognitive processes, these initial observations
suggest that thinking about individuals and groups may draw upon shared theory-
of-mind processes.
Next, to test more directly the extent to which overlapping regions of cortex
were recruited during contemplation of the mental states of individuals and
groups, we conducted a conjunction analysis on the individual \> control and
group \> control contrasts. This analysis revealed conjoint activation
specifically in brain regions associated with theory-of-mind–MPFC, right and
left TPJ, and precuneus–suggesting further that thinking about individuals and
groups draw upon shared processes.
Although the foregoing analyses suggest that similar processes subserve thinking
about individuals and groups as compared to a control condition, they leave open
the possibility that thinking about individual and group agents may recruit
theory-of-mind processes to different degrees. In order to evaluate the degree
to which processes associated with theory-of-mind were recruited when thinking
about individuals versus groups, we conducted independent region-of-interest
(ROI) analyses within the regions of MPFC, RTPJ, and precuneus identified by the
independent theory-of-mind localizer. Because the mental states in the localizer
task were attributed to individual protagonists, this analysis technique
provides a particularly stringent test for whether thinking about group agents
genuinely recruits processes associated with thinking about individuals.
Consistent with previous research, the theory-of-mind localizer (belief \> photo
contrast) yielded activation in MPFC (17/19 participants), RTPJ (19/19
participants), and precuneus (19/19 participants);. First, ROI analyses of the
main task confirmed that each of these regions showed greater activation in the
individual condition than in the control condition (MPFC, *t*(16) = 2.28, *p*
\<.04, *d* = 0.57; Right TPJ, *t*(18) = 2.43, *p* \<.03, *d* = 0.57;
precuneus, *t*(18) = 5.99, *p* \<.0001, *d* = 1.41). Second, ROI analyses
further revealed that each of these regions showed greater activation in the
group condition as compared to control (MPFC, *t*(16) = 2.22, *p* \<.04, *d* =
0.55; Right TPJ, *t*(18) = 2.39, *p* \<.03, *d* = 0.56; precuneus,
*t*(18) = 6.32, *p* \<.0001, *d* = 1.49). Finally, no significant differences
were observed between the responses to individuals versus groups in any of these
regions, (MPFC, *t*(16) = 0.69, *p* = .5; Right TPJ, *t*(18) = 0.09, *p*
= .93; precuneus, *t*(18) = 1.51, *p* = .15;). Together, these analyses
suggest that brain regions associated with theory-of-mind are recruited to a
highly similar degree during the contemplation of individuals and groups.
### Spontaneous theory-of-mind task
The design of the previous task raises the possibility that activation during
the individual and group conditions may have differed from the control condition
due to the explicit use of mental state words (e.g., thinks, believes, wants) in
the individual and group conditions. To explore whether common theory-of-mind
processes subserve attributions to individuals and groups even when no mental
state terms are used, we analyzed data from the portion of the study during
which participants made predictions about the behavior of individuals and
groups. Specifically, we compared activation during the individual and group
conditions of the prediction task in the same regions of RTPJ, MPFC, and
precuneus identified by the theory-of-mind localizer. Results replicated those
from the directed theory-of-mind task. Consistent with the hypothesis that
thinking about the minds of individuals and groups recruit similar theory-of-
mind processes, activations above baseline were observed across the network in
both the individual, *t*(19) = 2.84, *p* \<.02, *d* = 0.65, and the group
condition, *t*(19) = 2.23, *p* \<.04, *d* = 0.51 (averaging across regions),
and no differences were observed between the individual and group conditions in
RTPJ (*M*<sub>ind</sub> = −.004 *M*<sub>group</sub> = −.019, *t*(19) =
0.86, *p* \>.39), MPFC (*M*<sub>ind</sub> = .197 *M*<sub>group</sub> = .180,
*t*(19) = 0.36, *p* \>.72), or precuneus (*M*<sub>ind</sub> = .266
*M*<sub>group</sub> = .231, *t*(19) = 1.64, *p* \>.12). For individual
subject data, see. These results suggest that the similar patterns of activation
in the individual and group conditions observed in the first task are not simply
due to the common use of mental state terms in those conditions. Here, when no
mental state terms were presented, making predictions about individual and group
agents' behavior also recruited the theory-of-mind network to an
indistinguishable degree.
## Discussion
In describing corporations, government agencies and other organizations, people
sometimes use sentences of the form ‘Apple thinks…’ or ‘The CIA wants…’ The aim
of the present investigation was to help illuminate how people think about group
agents. The results of Experiment 1 indicate that sentences like these are
ascribing something to the group agent itself. Perceivers used expressions like
‘believes’ and ‘wants,’ not merely to talk about some or all of the individual
members of a group, but to talk about the group agent. Thus, attributions to the
group sometimes diverged from attributions to the individual members:
participants were willing to attribute a state to the group itself even when
they were not willing to attribute that state to any of the individual members,
and they were willing to attribute a mental state to all members of a group even
when they were not willing to attribute that state to the group itself. In turn,
the results of Experiment 2 reveal that that such ascriptions recruit brain
regions associated with thinking about the minds of individuals, i.e., brain
regions associated with theory-of-mind, both when theory-of-mind use is called
for explicitly and when it arises spontaneously.
Past research has demonstrated consistent engagement of a particular network of
regions, including MPFC, RTPJ, and precuneus, during inferences about the minds
of individual people, i.e., during theory-of-mind. Across two tasks, we observed
activation in this network when participants read or made predictions about
group agents. In the *directed theory-of-mind task*, participants read about the
states of individuals, group agents, and inanimate objects. In the *spontaneous
theory-of-mind* task, participants made predictions about what individual or
group agents would do in particular situations. In both cases, activation
associated with groups was indistinguishable from that associated with
consideration of individuals. Whole-brain analyses, conjunction analysis, and
ROI analyses all support the conclusion that cognitive processes associated with
thinking about the minds of individuals were also recruited when participants
thought about the ‘mind’ of a group agent. However, it is worth noting the
possibility that participants may have been thinking to some degree about the
minds of individual group members, and that this may have accounted for the
observed activation in theory-of-mind regions during consideration of group
agents. This possibility is weakened, but not completely ruled out, by (a) the
fact that, unlike past studies, no individuals were mentioned or shown in the
group condition and (b) the observation that perceivers interpret sentences
about group mental states as ascribing mental states to the group agent itself
in Experiment 1, and (c) the recent observation that the more perceivers think
about the ‘mind’ of the group, the less they think about the minds of its
members.
Past research has documented the selectivity of the RTPJ for attributing
representational mental content, such as beliefs and intentions, to others,
compared to other sorts of attributions, such as those concerning a person's
physical appearance, preferences, or personality traits. In this research,
neither the mere presence of a person nor the need to make other types of
inferences about that person was associated with as much activation in this
region as attributing representational mental states. Accordingly, the fact that
the RTPJ activated indistinguishably during consideration of individuals and
groups (but distinguished both from the inanimate control condition) is an
especially compelling suggestion that participants used similar processes for
understanding the representational mental states of individuals and group
agents.
Although the specific contributions of MPFC to social cognition remain
uncertain, this region has been observed to be sensitive to the *target* of
mental state ascription. In particular, greater MPFC activation has been
associated with interpersonally close others, and with humanized others,
compared to those who are more distant or dehumanized. Accordingly, it would not
have been surprising to observe reduced MPFC response to group agents compared
to individuals. However, the current study observed indistinguishable engagement
during consideration of group agents and individuals in a region of MPFC
involved in attributing mental states to individuals, as identified by the
theory-of-mind localizer, and similar to regions of MPFC associated with
mentalizing or theory-of-mind in past studies (according to Neurosynth).
Moreover, the individual condition and group condition were associated with
greater MPFC activation than the inanimate control condition, suggesting that
MPFC's contributions to individual-oriented social cognition are also present
during social cognition concerning group agents.
More generally, an abundance of past research has observed greater engagement of
brain regions associated with theory-of-mind when perceivers think about certain
types of target entities (humans and, to some degree, other animals) than when
they think about other types of target entities (computers, food, furniture);
for reviews, see. Here, we find just as much activation in brain regions
associated with theory-of-mind when people think about group agents as when they
think about individual humans, yet a group agent is something very different
from a human being or animal, or even from a collection of human beings.
Accordingly, the current results are consistent with the possibility that
perceivers apply theory-of-mind generally to things that conform to a certain
kind of abstract structure, and that group agents turn out to be among the
things that conform to that structure. This possibility draws further support
from recent research observing activation in brain regions associated with
theory-of-mind during consideration of other non-human agents that display
human-like properties – and is broadly consistent with the observation that
brain regions engaged when people construct representations of others' mental
states are also engaged when people construct other types of representations
that are removed from their current, first-person experience, such as
representations of the past or future.
In sum, people appear in certain respects to treat groups as ‘entities’. They
assign moral blame to whole organizations as a whole, treat whole financial
markets as though they have minds of their own, and give corporations many of
the legal rights enjoyed by individual human beings. In the current studies, we
observed that perceivers were willing to attribute mental states to group agents
that they did not attribute to the individual members of those groups, and that
attributing mental states to group agents was associated with activation in the
same brain regions that support ascriptions of mental states to individual
people (as confirmed by an independent localizer task). Taken together, these
results suggest that in order to understand the striking ways in which people
reason about corporations, governments, and other group agents, it may be
important to consider the possibility that perceivers sometimes attribute mental
states such as beliefs, desires, and intentions not only to the members of such
groups but also to the group agent itself.
# Supporting Information
The authors thank Rebecca Cox for assistance with data collection. Imaging data
were collected at the Athinoula A. Martinos Center for Biomedical Imaging at
MIT.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: ACJ RS JK. Performed the
experiments: ACJ DDF. Analyzed the data: ACJ DDF JK. Contributed to the
writing of the manuscript: ACJ DDF RS JK. |
# 1. Introduction
Schizophrenia, a severe psychiatric disorder characterized by positive and
negative symptoms and cognitive deficits, has long been hypothesized as a
disorder of brain connectivity. Disrupted brain development can lead to abnormal
neural connectivity or network, which may cause abnormal information processing
and integration, and clinical symptoms including psychosis\[–\]. It has been
well established that the insula cortex is an anatomical gateway between the
visual, olfactory, auditory, somatosensory cortices and the limbic structures.
Secluded deep within the lateral sulcus of the human brain, the insular cortex
is part of an extended network of neuronal pathways connecting to the anterior
cingulate cortex, temporal lobe, parietal lobe, hippocampus, amygdala, olfactory
cortex, and thalamus. The ventromedial prefrontal cortex, posterior
cingulate cortex, bilateral inferior parietal cortex and middle temporal lobe
are parts of default mode network (DMN). The central executive network (CEN)
consists of mainly the dorsolateral prefrontal cortex and posterior parietal
cortex. The salience network (SN) includes primarily the anterior insular cortex
and dorsal anterior cingulate cortex. Apparentlythe insular cortex
functions as a multimodal sensory integration region. In addition, the insular
cortex plays an important role in emotion processing including interoceptive
awareness, anticipation, evaluation of emotional stimuli, self-awareness,
episodic memory, executive function, attention, and saliency
processing.
Recently, the insula cortex has attracted significant attention in schizophrenia
research. The aberrant functional and structural alterations in insular
cortex have been frequently reported in schizophrenia including reduced gray
matter volume, thickness, and surface area, decreased white matter integrity
(measured by fractional anisotropy or mean diffusivity), and altered
functional activity under various tasks or during resting state.
Changes in the SN may be one of the most important findings among all insular
cortex related networks in schizophrenia. Recently, the anterior insular
cortex within the SN has been demonstrated to be crucial to modulate DMN /CEN
interactions in patients with schizophrenia. Manoliu et al. also found that
the dependence of CEN and DMN interactions on SN’s right anterior insular
activity is altered in patients with schizophrenia during acute psychosis or
psychotic remission. Furthermore, insular cortical dysfunction might be
associated with core symptoms of schizophrenia. Aberrant salience network
activity in the insular and cingulate cortices has been implicated in the
development of positive symptoms of schizophrenia such as delusions and
hallucinations due to an inappropriate assignment of salience to stimuli that
would normally be considered irrelevant. Patients with schizophrenia are
usually suffer from impaired insight; both awareness and mental state
attribution, two core components of insight, are associated with the function of
insular cortex.
Previous studies have provided compelling evidence supporting the critical role
of insular cortex in schizophrenia. However, most studies examining the
functional connectivity between insular cortex and other brain areas were in
schizophrenia patients treated with antipsychotics. The potential confounding
effect of antipsychotic treatment on the brain functional connectivity has been
well established. The purpose of the present study was to examine the
insular cortical functional connectivity and its relationship with the severity
of clinical symptoms in drug naïve, first episode schizophrenic patients.
# 2. Methods
## 2.1. Subjects
The study was approved by the Ethics Committee of the First Affiliated Hospital
of Zhengzhou University. All subjects provided written informed consent to
participate in the study. Subjects were recruited from the consecutive
admissions to the inpatient unit between November 2011 and December 2012.
Inclusion criteria included: 1) diagnosis of schizophrenia according to the
criteria of DSM-IV; 2) 18–45 years old; 3) in the age rangenever treated with
antipsychotic medications or other psychotropics. The diagnosis of schizophrenia
was confirmed by a research psychiatrist (X.S.) using the Structured Clinical
Interview for DSM-IV Axis I Disorders (SCID-IV). Exclusion criteria were: 1)
history of alcohol or other substance use; 2) history of brain injury; 3) any
ongoing significant medical conditions. Healthy control subjects were recruited
from the local community through advertisements. The same research psychiatrist
(X.S.) conducted a comprehensive clinical interview to rule out any psychiatric
conditions in healthy controls. A complete medical history, physical
examination, and routine laboratory tests were obtained from all subjects to
rule out possible medical conditions. All subjects were right-handed.
## 2.2. Clinical symptom measurement
Symptoms of schizophrenia were assessed for all patients using the Positive and
Negative Syndrome Scale (PANSS), which includes 3 subscales: positive symptoms,
negative symptoms and general psychopathology. The PANSS was administered
by the same rater (J.G.) throughout the study.
## 2.3. Image acquisition
On the same day after clinical assessment and before taking any antipsychotic or
other psychotropic medications, all participants were scanned on a 3.0 Tesla
Scanner (Signa HDxt 3T GEHCGEHC) at the Magnetic Resonance Center of the First
Affiliated Hospital of Zhengzhou University. A 6 minute ‘resting-state’
functional MRI scan was obtained, comprising of 180 time points of whole-brain
functional (EPI) volumes (TR = 2000 ms; TE = 30ms; flip angle = 90; 34
contiguous AC–PC aligned axial slices; matrix = 64×64; FOV = 22 cm; acquisition
voxel size = 3.4mm×3.4mm×4 mm). During this scan that acquired all neuroimaging
data, participants were instructed to rest without moving. The wakefulness of
the subject was verified by self-report at the end of the scan. T1-weighted
Spoiled Gradient Echo (SPGR) images were also collected for the purposes of
anatomical localization. The acquisition protocol included the following pulse
sequence and parameters: repetition time (TR) = 12ms, echo time (TE) = 4.5ms,
inversion time (TI) = 1100ms, flip angle 7°, field of view (FOV) = 256×256×220
mm<sup>3</sup>, matrix size 256×256, slice thickness 1mm contiguous, and scan
time 17 min.
## 2.4. Image processing
Imaging data were coded and catalogued before being transferred to the Psychotic
Disorders Program of the University of Massachusetts Medical School (UMMS) for
blinded analysis. AFNI was used for image preprocessing
(<http://www.afni.nimh.gov/afni>), performing slice timing correction for
interleaved acquisition (using Fourier interpolation), motion correction (by
aligning each volume to a “base” image \[middle volume\] using Fourier
interpolation) and de-spiking (detection and reduction of extreme time series
outliers using a hyperbolic tangent function). All other data processing was
carried out using FMRIB Software Library (FSL) (<http://www.fmrib.ox.ac.uk>),
including spatial smoothing (FWHM = 6 mm), mean-based intensity normalization of
all volumes, temporal bandpass filtering (highpass temporal filtering: Gaussian-
weighted least-squares straight line fitting, with sigma = 100.0 s; Gaussian
lowpass temporal filtering HWHM 2.8 s), and pre-whitening. Each individual’s
time series were spatially normalized by registration to the MNI152 (Montreal
Neurological Institute) template with 2mm<sup>3</sup> resolution, using a 12
degree of freedom affine transformation. Nine nuisance covariates (time series
for global signal intensity, white matter, cerebrospinal fluid, and six motion
parameters) were regressed out of the data to minimize the contributions of
artifactual physiological signals (e.g., cardiac and respiratory cycles) using
the general linear model implemented in FSL program FEAT. The FreeSurfer
software (<http://surfer.nmr.mgh.harvard.edu/>) was used to segment T1-weighted
SPGR images into cortical and subcortical gray and white matter regions, as well
as total intracranial volume, for each subject.
Functional connectivity was examined using a seed-based approach. The left
and right insular seed regions were generated using the Harvard-Oxford atlas,
co-localized to the MNI space used in this study. For each participant, we
calculated the mean time series of each seed by averaging across all voxels
within the seed. A voxel-wise correlation map was generated to each of these
seed-based time course reference signals. This correlation map was r-to-Z
transformed in order to generate a standardized representation. Each of the
remaining cortical and subcortical gray matter regions of the Harvard-Oxford
atlas were then used as a mask to determine the average resting state
correlation (Z-score) between the insular cortex and each ipsilateral gray
matter region in each subject.
## 2.5. Statistical analysis
The data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL). Demographic and
clinical characteristics were reported using descriptive statistics. The
Shapiro-Wilk test was used to check the normality of the data. Group comparisons
were performed using the independent samples t-test for continuous variables and
Chi-square or Fisher’s exact test for categorical variables.
Connectivity values (Z-scores) of the insular cortex with ipsilateral cortical
ROIs were extracted for group comparison, using the independent samples t-test.
Bonferroni corrections considering the number of ROIs were used to define the
significance level: *p*\<0.05/48 = 0.001. Insula connectivity maps (Z-score
maps) were subjected to voxel-based morphometry (VBM) analysis. To further
explore the regions that show group difference in insular connectivity. A false
discovery rate (FDR) corrected *p*\<0.05 was used to define the overall
significant level.
Peasson correlation analysis was used to examine the relationship beteween
insular connectivity and clinical symptoms as measured by the PANSS; the
significance level was set at *p*\<0.05.
# 3. Results
The original study sample included 46 patients with schizophrenia (SZ) and 30
healthy control (HC) subjects. One SZ and 2 HC individuals were excluded due to
the poor quality of their T1 scans; one SZ and 1 HC individuals were excluded
due to the poor quality (missing time series) of the resting-state scans; Seven
SZ and 2 HC individuals were excluded because the imaging values were defined as
outliers using the criteria of Mean±2SD. The final study sample used for data
analysis was thus composed of 37 SZ subjects and 25 HC subjects.
shows demographic and clinical characteristics of the study sample. There were
no significant differences between the patient group and the HC group in age,
gender, education, and intracranial volume (ICV) (*p*’s \> 0.05).
## 3.1. ROI-based analysis
shows the complete results of the seed-based analysis for each hemisphere. In
the left hemisphere, no regions met the p \<.001 criterion (with Bonferroni
corrections) for between-group differences. However, seven regions including
caudate, Heschl’s gyrus, posterior cingulate, hippocampus, anterior and
posterior parahippocampal gyrus and central operculum showed a trend-level
difference (p’s \<.05). In the right hemisphere, three regions met the
significance criterion (Heschl’s gyrus, anterior cingulate and caudate), and
five regions showed a trend-level difference (central operculum, putamen, planum
polare, planum temporale and thalamus). shows the group differences in average
connectivity for the regions of right insular cortical connectivity that had
significant group difference. The regions include the right insular cortical
connectivity to the right anterior cingulate cortex (ACC), right Heschl’s gyrus,
and right caudate nucleus. Each of these regions showed a significant decrease
in the SZ group compared with the HC group.
## 3.2. Confirmatory voxel-based analysis
shows the results of the VBM analysis for the right hemisphere insula-seeded
correlation maps. A FDR—corrected significant cluster was observed in Heschl’s
gyrus. An additional significant cluster was observed in the superior temporal
gyrus. FDR trend-level clusters were also observed in the anterior cingulate
gyrus and caudate nucleus. Thus, of the three ROI-based regions in the right
hemisphere with significant betwee-group difference, only the Heschl’s gyrus
difference was confirmed in the VBM analysis.
## 3.3. Correlation with clinical symptoms
The relationship between the resting state average Z-scores of insula-Heschl’s
gyrus connectivity and the measures of clinical symptoms were examined in both
hemispheres. As shown in, there was a significant negative correlation between
the left insula-Heschl’s gyrus connectivity and the PANSS negative symptoms
subscale scores (r = -0.406, p = 0.019), and a significant positive correlation
between the right insula—Heschl’s gyrus and the PANSS general psychopathology
subscale scores (r = 0.384, p = 0.019).
# 4. Discussion
Minimizing the potential confounding effect of antipsychotics, the present study
provides new evidence regarding changes in insula-Heschl gyrus connectivity in
drug naïve, first episode schizophrenia patients. In this study, we found that
there was significant reduction in the insular cortical connectivity with
Heschl’s gyrus, anterior cingulate cortex (ACC), and caudate nucleus in the
right hemisphere. We also found that the insula-Heschis connectivity might be
associated withclinical symptoms of schizophrenia.
Reduced insular cortical connectivity with Heschl’s gyrus in the right brain
hemisphere was found in the seed-based analysis and was confirmed in the VBM
analysis. Similar to insular cortex, Heschl's gyrus (transverse temporal gyrus)
may also play an important role in the neural mechanism of schizophrenia. It
has been reported that first-episode schizophrenia patients have bilateral
Heschl’s gyrus gray matter volume reduction, and decreased left Heschl’s
gyrus gray matter volume over time. Heschl's gyrus contains the primary auditory
cortex (PAC), which is the first cortical structure to process incoming auditory
information. The PAC is in close spatial proximity to the posteroinferior
insular cortex. The auditory information related to prosodic processing may be
conveyed to the posterior insular cortex directly through links with the primary
auditory and association areas. These findings suggest that deficits in the
right insula-Heschl’s functional connectivity may be engaged in the prosody
dysfunction in patients with schizophrenia.
Reduced right insula-caudate connectivity is another finding in the present
study based on the seed-based analysis. Caudate nucleus is related to successful
goal-directed action. Studies have found that schizophrenia and their relatives
have a reduced caudate volume and compromised white matter integrity. Using
functional magnetic resonance imaging to study brain activation during a
Monetary Incentive Delay task, Mucci et al. found that avolition in patients
with schizophrenia might be related to dorsal caudate hypo-activation. Another
research team has reported an abnormal hemispheric specialization of caudate
nucleus connectivity in schizophrenia. Reduced insula-caudate connectivity found
in our study may play an important role in the development of schizophrenia.
We also found significantly decreased right insula connectivity with ACC using
the ROI-based analysis. The insula and ACC together constitute the SN, an
intrinsic large-scale network showing strong functional connectivity. Menon and
Uddin have recently proposed that the primary role of this network is to
segregate the most relevant stimuli among internal and extra-personal stimuli,
to assist target brain regions and generate appropriate behavioral responses to
salient stimuli, and to enable switching between the default mode and task-
related states of brain connectivity. Abnormal connectivity of SN has been
reported to be associated with the negative symptoms, hallucinations, delusions
and other psychotic symptoms of schizophrenia\[–\]. Previous studies have
suggested that functional and/or structural alterations within the insular
cortex might contribute to aberrant salience processing, leading to the
development of schizophrenia symptoms.
In the present study, our correlation analysis within the patient group showed a
significant negative relationship between left insula-Heschl’s gyrus
connectivity and the PANSS negative symptoms subscale scores, suggesting the
impairment in this particular functional connectivity might be related to the
development of negative symptoms of schizophrenia. In addition, the correlation
analysis found a significant positive relationship between the right insula-
Heschl gyrus connectivity and the PANSS general psychopathology subscale scores,
suggesting that an abnormally increased connectivity might contribute to the
manifestation of clinical symptoms such as depression and anxiety observed in
patients with schizophrenia. Symptoms of schizophrenia have been attributed to a
failure of functional integration or aberrant connectivity among regions or
systems of the brain. Manoliu et al. found that the right anterior insular
cortical dysfunction was associated with positive symptoms of schizophrenia
during the acute phase of psychosis; they further suggested that the specific
SN/DMN/CEN reorganization with distinct insular cortical pathways might be
related to different symptom domains of schizophrenia. Another study reported
that structural alterations of the insular cortex might be related to negative
symptoms during psychotic remission, which is consist with our report about the
negative correlation between the insular cortex-Heschl’s gyrus connectivity in
the left brain hemisphere and the PANSS negative symptoms. Lee et al. reported
that progressive gray matter volume reduction in both insular cortex and
temporal pole in first episode psychosis was inversely associated with changes
in the overall Brief Psychiatric Rating Scale symptom scores. Some studies
suggested that disturbances in emotional prosody is due to the impairment in
early auditory sensory processing, which may contribute to later impairment in
attention-dependent processes in schizophrenia. It is also reported that the
cortical surface area and local white matter volume of posterior insula might
play an important role in insight impairment in schizophrenia. Both attention
deficit and insight impairment might be related to the general psychopathology
symptoms (depression, anxiety etc.) observed in patients with schizophrenia.
The present study has several limitations: 1) Some meaningful information may
have been lost by removing global signal in preprocessing of the imaging data.
2) The patient subjects were recruited from an inpatient unit; the overall
symptom severity of the study sample might be higher than patients in the
outpatient setting. Therefore the findings from this study, especially the
correlation between functional connectivity and clinical symptoms, may not be
generalizable to all patients. 3) The correlation analysis between functional
connectivity and clinical symptoms was not corrected for multiple correlations;
4) Several software tools were used for pre-processing and processing of imaging
data; 5) The PANSS was the only rating scale used to assess clinical symptoms.
In summary, our study demonstrates the abnormal functional connectivity of
insula, insula-Heschl gyrus connectivity in particular, in drug naïve, first
episode schizophrenia patients. These abnormalities might contribute to the
development of schizophrenia. Future studies with a larger sample size and in
combination with other techniques such as diffusion tensor imaging (DTI) are
needed to further assess the regions of insular connectivity in relation to the
development and treatment of schizophrenia.
# Supporting Information
Funding for this study was provided by the National Natural Science Foundation
of China (No.81571318 to X-QS; No.81371472 to L-XL; No.81401110 to XL), Science
and Technology Planning Project of Health and Family Planning Commission
(No.201501015 to X-QS), International Science and Technology Cooperation Program
of Henan (No. 162102410061 to X-QS), Science and Technology Planning Project of
Guangdong Province (No. 2013B021800085 to Q-LW), the Fundamental Research Funds
for the Central University (No. 14ykpy28 to Q-LW) and the Youth Fund of the
First Affiliated Hospital of Zhengzhou University (to XL, L-JP).
[^1]: The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** XF XS. **Data curation:** DK MQ XL. **Formal
analysis:** XF DK. **Funding acquisition:** LL XS. **Investigation:** LP QW
JG YY. **Methodology:** DK MQ HL. **Project administration:** LL XS.
**Resources:** XL XS. **Supervision:** LL XS. **Validation:** DK XF.
**Visualization:** LP DK QW. **Writing – original draft:** LP MQ. **Writing
– review & editing:** LL XL XF XS.
[^3]: ‡ These authors share co-first authorship on this work. |
# Introduction
Metabolic profiling is essential to ensure the quality, consistency, safety and
efficacy of herbal medicine products; especially for the injection dosage form.
Water extraction or decoction is the most favored method of preparation of
herbal medicine injections (HMIs). Saccharides, amino acids, organic acids, and
other primary metabolites are unavoidably extracted along with targeted
secondary metabolites during the process of HMIs, such as Qingkailing injection,
Danshen injection, Guanxinning injection, and Shuxuetong injection. In general,
amino acids are considered to provide tonic activities and act as key regulators
of nutrient metabolism, and polysaccharides are believed to be one of the most
important constitutes in some herbal materials for pharmacological activities.
Some monosaccharides have the suppressive effect on cell-mediated immune
reactions. However, these primary metabolites in HMIs are often ignored to
detect and set corresponding quality criteria in China Pharmacopeia and national
standard. Due to the strong polarity and hydrophilicity, conventional reversed-
phase performance liquid chromatography (HPLC) fingerprint, and other analytical
methodologies, including anion exchange chromatography, gas chromatography, and
capillary electrophoresis, are limited to separate and detect primary
metabolites unless with complicated pretreatment, derivatization reagents and
laborious preparation procedures. To delineate various class metabolites in
HMIs, different types of fingerprints are necessary for a holistic quality
evaluation, which are difficult to realize during the practical industry
processing. Therefore, a simple and fast approach is required to be capable of
detecting saccharides, amino acids, and organic acids, together with mainly
bioactive secondary metabolites in one fingerprinting spectrum simultaneously.
Proton nuclear magnetic resonance (<sup>1</sup>HNMR) spectroscopy provides
access to detect all proton-bearing compounds, almost irrespective of the
chemical compound class. Because of the signal intensity directly proportional
to the number of nucleus contributing to a specific resonance, <sup>1</sup>H NMR
method achieved the identification and quantification of metabolites in a one-
step acquisition. With simple sample preparation, <sup>1</sup>H NMR facilitates
high-throughput analysis for metabolic studies and quality control of various
food – and herbal materials. As a universal technique with the simplicity and
rapidity of implementation, <sup>1</sup>H NMR has expansive prospect of
application in profiling of polar metabolites of HMIs.
Since the complexity of chemical composition, HMIs cannot be completely
represented by a limited number of certain bioactive compounds. To extract the
feature variables, principal component analysis (PCA) and independent component
analysis (ICA) are classical tools to reduce the dimension of multivariate. The
components (PCs) in PCA method are mutually orthogonal, while ICA method
contains the components to be statistically independent. ICA has been found to
be a successful alternative to PCA in eliminating the overlapping information
between the components. However, ICA faces some limitations due to some
instability, the choice of number of components to extract and high
dimensionality. As a consequence, independent principal component analysis
(IPCA) was proposed by Yao et al. in 2012 to use PCA as a pre-processing step to
reduce the dimension of the data, and then use ICA as a denoising process of PCA
to separate relevant information. On simulation studies and real data sets, IPCA
offered a better visualization of the data than ICA and with a smaller number of
components than PCA. Owing to the benefit to generate denoised the loading
vectors, we attempted to employ IPCA method to construct χ<sup>2</sup> and
Hotelling T<sup>2</sup> control charts for multivariate statistical analysis.
Danhong injection (DHI) is a patent injection made from the extracts of *Radix
Salviae Miltiorrhizae* and *Flos Carthami*. It has been widely used for the
prevention and treatment of cardiovascular and cerebrovascular diseases in
clinic. In our previous work, ultra-performance liquid chromatography (UPLC)
coupled with UV detection was adopted to identify 11polyphenolic acids in DHI.
However, the total weight of identified constituents accounted for only a low
proportion (about 10%) of the solid content in DHI.
In this study, we describe a strategy to detect more hydrophilic primary
metabolites in DHI based on quantitative <sup>1</sup>H NMR spectroscopy. The
absolute concentration of identified metabolites was calculated by using
internal standard method. Linearity, precision, repeatability, stability and
accuracy were carried out to validate the method. The contents of polar
metabolites were further evaluated by the multivariate analysis tool IPCA to
establish χ<sup>2</sup> and Hotelling T<sup>2</sup> control charts.
# Materials and Methods
## Materials and Chemicals
Thirty-six batches of DHI manufactured in 2011, 2012 and 2013 were provided by
Heze Buchang Pharmaceutical Co. Ltd (Heze, China). The standards of valine,
threonine, alanine, pyroglutamate, procatechuic aldehyde and asparagine were
purchased from the National Institute for Food and Drug Control (Beijing,
China). Salvianic acid and procatechuic acid were obtained from Zhongxin Innova
Laboratories (Tianjin, RP China). Succinate and malonate were obtained from Dr.
Ehrenstorfer GmbH (Augsburg, Germany). Fructose and glucose were purchased from
Sigma (Aldrich, America). Rutinose was purchased from Hazard Communication
(Tokyo, Japan).The purities of the compounds were all above 98%, using NMR
analysis. Deuterium oxide (D<sub>2</sub>O, 99.9%) and sodium 3-trimethylsilyl
\[2,2,3,3-*d*<sub>4</sub>\] propionate (TSP) were purchased from Cambridge
Isotope Laboratories (Miami, FL, USA). D<sub>2</sub>O was used as internal lock;
TSP was as internal standard for chemical shift calibration and quantification.
## Ethics
No specific permission was required for the described field studies. The field
locations are neither privately owned nor protected, and neither endangered nor
protected species were involved.
## Sample preparation
All the injection samples were subjected to freeze-drying. The dried powders (18
mg) were accurately weighed and dissolved with 600 µL of D<sub>2</sub>O
containing 0.58 mM TSP. Exactly 500 µL of sample solution was transferred into a
standard 5 mm NMR tube (Vineland, NJ, USA). No buffer was used due to the stable
pH of injection.
## NMR measurements
<sup>1</sup>H NMR spectra were acquired at 298 K on a Bruker AV III 600 MHz NMR
spectrometer (600.23 MHz for proton frequency) with a 5 mm broadband BBFO
probehead. All pulse sequences were from Bruker pulse program library. A
standard one dimensional composite pulse sequence (zgcppr) was employed to
suppress the residual water signal. The 90° pulse width was adjusted to about 13
µm for each sample. Sixty-four scans were collected into 32k data points using a
spectral width of 12335 Hz, a relaxation delay of 1.0 s and an acquisition time
of 2.66 s. A 0.3 Hz line-broadening function was applied to all spectra for
Fourier transformation (FT) followed by phasing and baseline correction.
For proton signals assignment purposes, a set of two dimensional (2D) spectra,
including <sup>1</sup>H-<sup>1</sup>H correlation spectroscopy (COSY),
<sup>1</sup>H-<sup>1</sup>H total correlation spectroscopy (TOCSY),
<sup>1</sup>H *J*-resolved (*J*-res), and <sup>1</sup>H-<sup>13</sup>C
heteronuclear single quantum coherence (HSQC), were acquired for selected
samples and processed with similar parameters as described previously.
Spin-lattice relaxation time (*T*<sub>1</sub>) values of the quantified protons
of individual constituent and TSP were measured using a classical inversion
recovery pulse sequence with 10 relaxation delays (*τ*) ranging from 0.01 to 20
s.
## Quantification of the metabolites
Because the intensity of a given <sup>1</sup>H NMR signal is directly
proportional to its contributing number of protons, the amount of metabolites in
DHI can be measured by the signal areas of given metabolites and an internal
reference with known concentration. Thirteen metabolites in DHI were selected
for quantification. The important parameters for data acquisition and processing
of <sup>1</sup>H NMR spectra must be set appropriately to obtain accurate and
precise measurements. Most of all, the relaxation delay *τ* should be long
enough to ensure complete relaxation for all the signals of
interest.<sup>1</sup>H NMR measurements are done in a longer acquisition time by
choosing *τ*≥5× longest *T*<sub>1</sub>. For shortening the acquisition time,
<sup>1</sup>H NMR spectra can be acquired in an incompletely relaxed condition,
and the absolute concentrations should be calculated taking the *T*<sub>1</sub>
values in consideration. With the effective magnetization reading pulse of 90°,
the quantification of chemical constituents in this study can be performed by
using the following equation: where *P<sub>X</sub>* and *P<sub>TSP</sub>* are
the mass concentrations of metabolite and TSP, *A<sub>X</sub>* and
*A<sub>TSP</sub>* are the integral areas for targeted signal of metabolite and
for methyl groups of TSP, *N<sub>X</sub>* and *N<sub>TSP</sub>* are the proton
numbers of metabolite and of methyl groups of TSP, *M<sub>X</sub>* and
*M<sub>TSP</sub>* are the molar masses of metabolite and TSP, and are the spin-
lattice relaxation times for proton X and methyl protons of TSP, respectively;
*t* is total relaxation time (relaxation delay plus acquisition time).
The quantitative <sup>1</sup>H NMR method was checked for linearity, precision,
repeatability, stability, and accuracy. Precision, repeatability and stability
were calculated as the relative standard deviation (RSD). Recovery test was
employed to determine the accuracy, and four typical compounds, alanine,
glucose, salvianic acid and procatechuic aldehyde, were chosen to evaluate the
average recovery.
## Assay for multivariate quality control
36 batches of DHI samples were split into two phases, Phase I and Phase II.
Phase I as training set included 25 batches of qualified products manufactured
continuously in the years of 2012 and 2013. Phase II as testing set included 6
batches of qualified products in 2012 and 2013, and 5 batches of expired
products manufactured in 2011.
The quantitative resulting data of 13 metabolites was imported to R 3.0.2
software loaded with packages of MVA, MSQC and mixOmics
([www.r-project.org](http://www.r-project.org)) for multivariate statistical
analysis. For simplifying the multivariate problem, principal component analysis
(PCA) and independence principal component analysis (IPCA) were performed to
reduce the dimensionality of data. The scores of principal components
characterized the whole data were then imported to χ<sup>2</sup> and hotelling
T<sup>2</sup> control charts to calculate the upper control limits (with 99%
confidence ellipsoids). Moreover, as one of the requisites in control chart is
the independence of the data, the independence of selected components in PCA and
IPCA models were validated by autocorrelation function (ACF). The out-of-control
samples in Phase II were examined by the upper control limits achieved from
Phase I.
# Result and Discussion
## Proton signal assignments and chemical identification
A representative <sup>1</sup>H NMR spectrum of DHI was shown in. The resonance
signals were assigned to 30 metabolites based on the elucidation with extensive
2D NMR experiments (<sup>1</sup>H-<sup>1</sup>H COSY,
<sup>1</sup>H-<sup>1</sup>H TOCSY, <sup>1</sup>H *J*-resolved, HSQC),the
literature data in our former work , and in-house database. In the range of *δ*
3.2–5.8, the spectrum is dominated by 5monosaccharides and 2 disaccharides,
including glucose, galactose, arabinose, fructose, rhamnose, rutinose, and
rutinulose. In the high-field region (*δ* 0.5–3.2), 8 amino acids (isoleucine,
leucine, valine, threonine, alanine, proline, pyroglutamate, asparagine) and 3
organic acids (acetate, succinate, malonate) were observed. In the low-field
region (*δ* 5.8–10.0), 7 polyphenolic acids, including salvianic acid,
salvianolic acid B, salvianolic acid A, rosmarinic acid, lithospermic acid,
procatechuic acid and procatechuic aldehyde, together withuridine and
5-(hydroxymethyl)-2-furaldehyde (5-HMF), were identified. Moreover, 3 organic
acids (4-hydroxybenzoic acid, 4-hydroxycinnamic acid, and formate) were observed
as well in the low-field region. The chemical shifts of the identified 30
metabolites by<sup>1</sup>H NMR were listed in.To our knowledge, 7 saccharides
and 6 organic acids were reported for the first time in DHI. Without the need of
any sample pretreatment or pre-column derivatization, the established
<sup>1</sup>H NMR method provided an approach to determine 7 saccharides, 6
organic acids, 8 amino acids, 1nucleoside, 1 carbohydrate derivatives (5-HMF)
and 7 polyphenolic acids in DHI simultaneously.
## Quantitative <sup>1</sup>H NMR analysis and method validation
Due to the narrow chemical shift range of <sup>1</sup>H NMR and frequent signal
overlap, it is a challenge to quantify all the constituents in a mixture. In our
study, 13 metabolites with fully separated signals were selected for
quantification. In order to improve the efficiency of <sup>1</sup>H NMR method,
the spectra were acquired in an incompletely relaxed state. As a consequence,
the spin-lattice relaxation time (*T*<sub>1</sub>) value must be accurately
measured and taken into account for quantitative analysis. The *T*<sub>1</sub>
values were determined by the inversion-recovery experiments. Accordingly, the
absolute concentrations of the 13 metabolites were calculated from three
parallel samples of each batch (Table S1).
### Linearity
<sup>1</sup>H NMR as method itself is linear and no calibration is necessary for
the determination of molar ratios of mixtures. Thus, the 13 metabolites in five
different molar ratios confirmed the linearity of NMR spectroscopy. Good
linearity was achievable, as indicated by the equations and satisfactory
correlation coefficients (*r*<sup>2</sup>).
### Precision
The intraday and interday precision was determined by analyzing six replicates
on the same day and on three consecutive days respectively. The intraday
precision for the contents of 13 metabolites ranged from 0.20% to 0.89%, and the
interday precision ranged from 0.29% to 1.49%. The RSD values were adequate and
indicated the suitability of the method.
### Repeatability
Six samples prepared from the same batch showed RSD values ranging from 1.46% to
2.75%, indicating a high repeatability.
### Stability
One sample was analyzed to determine stability on three consecutive days. The
RSD values of the analytes were in the range of 0.37% to 2.21%.
### Accuracy
Considering the limited volume of NMR tube and the cost of using deuterium
reagents to dilute sample continuously, four metabolites of different types were
employed, including alanine, glucose, salvianic acid and procatechuic aldehyde.
The recovery was calculated as the ratio of the response of the selected four
compounds in the spiked DHI samples against that of the standards at the same
levels. The average recoveries were found to be 106.6% (±1.2), 106.6% (±2.5),
98.3% (±2.1) and 91.3% (±4.0) for alanine, glucose, salvianic acid and
procatechuic aldehyde, respectively, indicating acceptable recovery.
According to the results, the concentration of glucose was extremely high in DHI
as shown in, and the amount of saccharides, amino acids and organic acids
represented about 60% of the total solid content of DHI (Table S1).
## Control Charts based on PCA and IPCA
With the enhancement in quality control of DHI, the analysis should be performed
through a multivariate approach, that is, the above 13 metabolites must be
analyzed together, not independently. The concentrations of 13 metabolites were
mean centered and unit-variance scaled before being analyzed by PCA and IPCA. To
avoid the loss of significant information, the percent specified of the
principal components (PCs) cumulative proportion of explain variance is normally
fixed on 80% in PCA model. Thus, the first three PCs in Phase I were selected to
construct χ<sup>2</sup> and Hotelling T<sup>2</sup> control charts. Since the
independence of the data is one of the requisites in control chart, we assessed
the marginal independence of each necessary PC to indicate the model validation.
Correlograms showed that PC1 fell outside of the confidence bands, which
indicated that there was an evidence of autocorrelation or dependence of PC1,
and PCA model using Phase I data was not achieved.
In order to remove the autocorrelation effects of PCA, we employed IPCA to
generate denoised and independent loading vectors. The kurtosis measure of
loading vectors was used to decide the number of independent principal
components (IPCs). The kurtosis of all extracted IPCs was plotted in, whereas
the kurtosis of IPC7 was close to zero. By using the first 7 components of IPCA,
the exactly choosing number of IPCs was obtained. Since the kurtosis of IPC3 was
close to zero, the first 2 components were sufficient with IPCA. The presence of
autocorrelation was assessed as shown in (IPC1 and IPC2), which indicated that
there was no evidence of relation between the adjacent observations. Therefore
the original 13 dimension of our data had been reduced to a two-dimensional
problem.
Then the first two IPCs were taken to establish the in-control state (Phase I).
According to χ<sup>2</sup> and Hotelling T<sup>2</sup> control charts with the
99% confidence region, the upper control limits (UCL) were determined as 9.21
and 11.56, respectively. The first two IPCs were consequently controlled through
2D ellipsoids.The χ<sup>2</sup> control ellipse (UCL = 9.21) could be used as
process region, and the T<sup>2</sup> control ellipse with less restrictive
(UCL = 11.56) was used as tolerance region. In both cases, all the points in
Phase I fell inside the confidence ellipsoids. The samples in Phase II (Table
S2) were monitored by employing the UCLs of both χ<sup>2</sup> and T<sup>2</sup>
charts obtained from Phase I, and the points of Phase II were added into 2D
ellipsoids. The seventh sample fell outside the 99th confidence ellipsoids of
both the process and tolerance regions, indicating the presence of out-of-
control sample (batch 110402). The decomposition of T<sup>2</sup> value showed
that the out-of-control variability was associated to the IPC1 since *p*-value
was equal to 0.0032. The same result was also obtained through IPCA loading
plots as shown in. Although the loading plot could not exactly determine which
metabolites were responsible for the variation, it still showed the contents of
succinate, malonate, glucose, fructose, salvianic acid and protocatechuic
aldehyde made more contributions to the independent loading vectors of IPC1.
# Conclusion
Based on quantitative <sup>1</sup>H NMR analysis, a reliable approach for
simultaneous determination of amino acids, organic acids, saccharides, and
botanic secondary metabolites of HMIs in one fingerprinting spectrum has been
developed and validated by using Danhong injection as a model. The method had
taken*T*<sub>1</sub> values into account when calculated the contents of feature
metabolites, which allowed the assay with good linearity, precision,
repeatability, stability and accuracy. Unlike HPLC fingerprinting methods, the
<sup>1</sup>H NMR approach has the significant advantages of less analysis time
(about 5 min) without chromatographic separation, and no requirement of standard
materials used to quantitative analysis. In combination with IPCA, two kinds of
multivariate control charts (χ<sup>2</sup> and Hotelling T<sup>2</sup>) were
also successfully carried out for detecting off-test HMI samples by employing
the independence principle components. The decomposition of T<sup>2</sup> value
and IPCA loading vectors can reflect the significance variations of overall
metabolite profiling, although it cannot decide the mutative metabolites
exactly. The established multivariate models have the prospects for extracting
sufficient characterization to monitor more feature metabolites in HMIs.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: MJ YW BZ LJ YZ XG. Performed
the experiments: MJ YJ LX. Analyzed the data: MJ YJ YW. Contributed
reagents/materials/analysis tools: BZ LJ YZ XG HP. Wrote the paper: MJ YJ YW
YZ XG MW. |
# Introduction
The spatial distribution of a species is one of the fundamental aspects of its
ecology and is a component of many conservation and management plans. For marine
fishes, understanding a species’ spatial distribution is necessary for
delineating Essential Fish Habitat (EFH), which is important for an ecosystem-
based approach to fisheries management, and is part of the Magnussen-Stevens Act
(as amended in 1996). In the United States, the National Marine Fisheries
Service (NMFS) and regional fisheries councils are required to identify EFH \[,
\]. However, broadly defined as “those waters and substrate necessary to fish
for spawning, breeding, feeding and/or growth to maturity,” EFH quickly comes to
include most waters within the exclusive economic zone
(<https://www.fisheries.noaa.gov/resource/map/essential-fish-habitat-mapper>)
when EFH is summed across species, making the prioritization of conservation and
management actions difficult.
While a species may occupy many habitats or areas, some areas are likely to be
more ecologically important than others to the long-term persistence and
productivity of the species. For example, protecting core adult habitat for
adult rockfishes *Sebastes* spp. has been shown to lead to increases in
reproductive output for these populations. Alternatively, restoration of
juvenile habitats for red drum *Sciaenops ocellatus* has been shown produce
larger increases in population growth rate and adult abundance than would
protection and restoration of adult habitat. These ‘essential’ EFHs are termed
Habitat Areas of Particular Concern (HAPC) due to their increased importance for
the species’ population dynamics. Locating areas that support high densities of
juvenile fishes is a first step towards understanding EFH and HAPC and being
able to apply spatial management plans. Nursery areas are juvenile habitats that
contribute disproportionately more individuals to the adult population than
average (usually defined in terms of production per unit area) due to higher
juvenile: (1) density, (2) growth rate, (3) survival, and (4) movement to adult
habitat. Here, we focus on the first criteria: density of juvenile fishes.
Strong recruitment events can determine age structure and stock size in marine
fishes, suggesting that the location, quality, and availability of juvenile
habitat are essential to the health of these populations. For example,
recruitment to five populations of sole *Solea solea* in the northeast Atlantic
is related to the area of suitable juvenile habitat, and the abundance of age-0
cod *Gadus morhua* is correlated with the availability of eelgrass habitat.
While there has been a substantial focus on nearshore and estuarine habitats,
less work has gone into quantifying the spatial distribution of juvenile habitat
for deeper marine species across large spatial scales.
Starting with an areal delineation of a species juvenile habitat is useful
because bottom type alone does not necessarily define high quality areas. Other
factors, such as larval supply, food availability, and connectivity with adult
habitats can be important. For example, there are substantial seagrass beds that
appear to be suitable habitat for juvenile queen conch (*Stombas gigas*) in the
Bahamas. However, settlement occurs to specific areas where tidal circulation
concentrates larvae, and conch growth is highest in areas where macroalgal
production exceeds surrounding areas. In other cases, nursery habitat can be
ephemeral and temporally variable when species recruit to biogenic habitats or
respond to changes in oceanographic or climate drivers like temperature. In
fact, shifts in juvenile distributions related to climate or other factors may
hamper management efforts.
Quantifying the spatial distribution and abundance of juvenile fishes allows us
to identify juvenile habitats and potential nursery areas as well as to begin
developing hypotheses concerning the factors controlling the spatial
distribution of juvenile fishes (e.g., current regimes that may entrain larvae).
Here, we used data from the Northwest Fisheries Science Center’s US West Coast
Groundfish Bottom Trawl Survey \[WCGBTS, \] to quantify the spatial distribution
of juvenile fishes for 13 species (five flatfishes and eight roundfishes) in
waters off the west coast of the United States. We used vector-autoregressive
spatio-temporal modeling \[VAST, \] to estimate species distribution models and
quantify spatial and temporal patterns in juvenile density and identify juvenile
habitat (in terms of spatial distributions) for each species. We ask: (1) are
there juvenile hotspots along the coast for each of the species or are juveniles
distributed homogeneously? (2) Is there spatial fidelity over time in juvenile
hotspots for each of the species? And (3) are spatial patterns of juvenile
biomass stable through time? Finally, (4) we also estimate an annual, coast-wide
index of juvenile biomass for each species, the species effective area occupied,
and its center of gravity (CoG).
# Materials and methods
## Data source
We used data for 2003–2018 from the Northwest Fisheries Science Center’s (NWFSC)
U.S. West Coast Bottom Trawl Survey of Groundfish Resources off Washington,
Oregon, and California \[WCGBTS, \]. The survey is takes depth-stratified,
random samples that span approximately 32–48.58°N and 55–1280 m and is conducted
in two passes from May to October with over 600 trawls per year \[see, for a
detailed description of the sampling design\]. Survey vessels start at Newport,
Oregon and initially head north to Cape Flattery before heading south to San
Diego. The survey uses a standard Aberdeen-net with 25.9-m headrope, 31.7-m
footrope, and an additional 3.8-cm liner extending from the middle of the net
through the codend, to retain smaller fish and invertebrates. The net was towed
at \~2.2 knots for a nominal 15 minutes \[an average of 20 minutes on bottom
time including lift-off lag, \] and swept area ranged from 0.8 to 4.5 ha
(median: 1.7). We included only those hauls deemed acceptable for stock
assessment.
## Data selection and processing
Data selection and processing involved two issues. First, ideally we would have
analyzed only age-0 or the youngest observed age class fish to provide estimates
of recruitment distributions and abundance. However, the survey trawls often do
not catch small juvenile fish in high numbers, resulting in a large number of
tows with zero catch for the smallest age classes. For some species, we had to
include multiple age classes in the analysis (sum biomass across age classes) in
order to achieve a positive-definite Hessian and assure that the VAST model
converged (see *Statistical analyses* below). Thus, for some species (lingcod,
sablefish, arrowtooth flounder, and English sole), the analysis examined a
single juvenile age class (either age-0 or age-1) and the resulting pattern and
index can be considered an index of annual recruitment. However, for the
remainder of the species, the analysis quantifies juvenile density because we
had to combine several juvenile age classes. Hereafter, we refer to both cases
as juvenile density.
Second, while the survey data include subsamples of individual fish lengths,
weights, and age structures–with ages determined from otoliths (except for
Lingcod for which dorsal fin rays are used)–there are always more length
measurements than ages due to limited processing capacity. Since the size
distributions for a multiple ages can overlap, we used length-at-age
relationships from the aged individuals to set a maximum length for inclusion in
a given age class for those individuals that were not aged. We then pooled all
individuals of this maximum length or below for the statistical analyses, which
do not distinguish age classes within the statistical model.
To set the maximum length for each species, we examined the length distributions
of aged individuals by 1-cm bins. The longest length bin where younger
individuals outnumbered older individuals was set as the maximum length for the
younger age class. For example, the length distribution for age-0 and age-1
sablefish overlapped between 26 and 31cm (Table A). However, 29 cm is longest
length bin where there were more age-0 fish (ten) than age-1 fish (nine). At 30
cm there were more age-1 than age-0 fish. Therefore we set a maximum length of
29 cm for inclusion of unaged sablefish in the age-0 group.
We then extrapolated the full tow weight for the lengths selected based on their
weighted proportion in that species’ subsample that was measured for length. If
the extrapolated tow-level species weights from the initial multispecies sample
were already in the database, they were used as given. The length-weight
relationship for each species was calculated from the available data and used
when a weight was missing for an individual fish. We also used the length-weight
relationships to check for weights that were outliers, for example, in cases
where a basket holding the specimen was not correctly tared. In such cases, the
calculated weight was used instead. The implied assumption that the length is
the correct measurement and not the weight. This assumption is justified given
the ease of taking length measurements using the digital length boards employed
by the survey. The trawl survey data did not contain age-structure information
for Pacific grenadier and the two thornyhead species. Instead, we used age-at-
length information from Andrews, Cailliet \[; Pacific grenadier\], Taylor and
Stephens \[, shortspine thornyhead\], and Stephens and Taylor \[, longspine
thornyhead\] as a guide. For each species, lengths (in 1 cm intervals) were
added to the analysis until we obtained acceptable model fits, and based on the
published information, we estimated the maximum age of individuals in the
analysis.
## Statistical analyses
We used vector-autoregressive spatio-temporal modeling \[R package VAST, version
4.1, \] to quantify the spatial and temporal trends of juvenile biomass density
(hereafter, density for brevity) for each of the 13 species. VAST is a species
distribution modeling approach that effectively reproduce species distributions
and provide good estimates of abundance and associated error. VAST performs well
with linear covariates, but has limited ability to include non-linear
relationships beyond general quadratic ones compared to generalized additive
models or branched regression trees. We use VAST here because we are primarily
interested in estimating species distributions and an annual index of abundance.
Catch per unit effort (CPUE) was the dependent variable, calculated as the
extrapolated abundance estimate of juveniles divided by the swept area of the
net. The year of capture, pass, vessel, tow location (latitude and longitude),
and depth were the predictor variables. The trawl survey is done in two passes
between May and October. Pass therefore accounts for intra-annual variation the
time of sampling, and the inclusion of pass is consistent with VAST models used
to produce biomass indices for west coast stock assessments. Since the goal was
to model distributions and not test for explanatory variables, we did not
include additional environmental predictors in the models. We applied one common
intercept across years, which allowed spatial variation to be explained by
spatial and spatio-temporal variation terms, both of which were included in the
model. This parameterization prevents the model from forcing biomass to increase
or decrease coast-wide in a given year (thereby potentially overestimating
recruitment in some areas) as would be the case for yearly intercept. We used
gamma-distributed errors for the positive catch rates with a Poisson-link
function. This approach approximates a Tweedie distribution, which has positive
mass at zero but is otherwise continuous, but is more computationally efficient.
Previous work has shown that this approach generally provides a better fit to
the WCGBTS data than does a conventional delta model. The extrapolation grid or
stratum area was defined based on the data extent for each species separately
(Region = ‘Other’ in VAST, Table B). Trawls with zero catch for the target
species and within the stratum area were included in the analysis. We used 600
knots \[locations at which spatial processes are estimated by VAST, used for
computational efficiency; \] for all model runs. See Thorson for more detail on
VAST and Table B in for more detail on the VAST parameterization used here.
## Hotspots
We arbitrarily defined juvenile hotspots as areas where estimated juvenile
density (biomass) derived from VAST was in the top 20% of its maximum density
(80%+) over the study period (red areas the following distribution figures). We
define secondary hotspots as areas where juvenile density was in the top third
(66% +) of its maximum density over the study period (orange areas in the
following distribution figures). Additionally, we examined hotspots at two
temporal scales: within years and across years (‘All Years’ on the following
distribution figures). All Years was the average juvenile density from
2003–2018. Therefore, areas that consistently had moderate juvenile density may
appear as a hotspots across all years, though they may not necessarily qualify
as a hotspot at an annual scale.
# Results
We observed three general, qualitative patterns of juvenile density (biomass)
along the West Coast: (1) species with distinct, spatially-limited hotspots that
were consistent through time; (2) species with distinct, spatially-limited
hotspots that spatially varied through time; and (3) species with large regional
hotspots that spanned a broad latitudinal range but were depth limited.
Additionally, we estimated the time series of juvenile density for each species,
which illustrated that the prevalence and intensity of juvenile hotspots was
often related to density. Finally, we also report the effective area occupied
and CoG for each species.
Dover sole, English sole, Pacific grenadier, shortspine thornyhead, and
splitnose rockfish had distinct and spatially limited hotspots of juvenile
density (shortspine thornyhead, ; others Figs A-E in ; see for diagnostic plots
including QQ-plots and plots of the spatial residuals for the presence/absence
portion of the model). For example, the density of juvenile shortspine
thornyheads was fairly consistent through time but peaked in 2009–2010, after
which it declined. There was a single, distinct across-years hotspot at
approximately 45° N in waters between 160–625 m, just north of an area with an
expansive area of shallower shelf habitat that includes Heceta, Perpetua, and
Stonewall Banks (Fig F in). The hotspot north of the banks had primary or
secondary hotspots in most years until approximately 2014, after which juvenile
density was very low, but it was most obvious in 2010 when juvenile density was
highest. There was a small, secondary, across-years hotspot adjacent to the
Columbia River mouth, with a primary hotspot at this location in 2010 when the
density of shortspine juveniles peaked.
Pacific hake and darkblotched rockfish (Fig G) also showed distinct hotspots of
juvenile density, but there was more temporal variation in the location of these
hotspots. For example, Pacific hake showed peaks in juvenile density in 2006 and
2009. Juveniles (age-0 and age-1) were caught between 50–700 m. Within a given
year, they showed localized areas of high density, but the location of these
hotspots varied from year to year. While the location of the hotspots did not
appear related to the density of juveniles, hotspots were most apparent in 2006
and 2009 when the density of juvenile hake showed peaks. For example, in 2006
hotspots occurred north and south of Cape Blanco and around Point Conception.
However, in 2009 there was a hotspot to the north on either side of the Columbia
River outflow. Juvenile density was consistently moderate south of Cape
Mendocino resulting in across-years hotspots north of San Francisco Bay and
around Point Conception (‘All Years’).
For juvenile arrowtooth flounder, sablefish, lingcod, longspine thornyhead,
petrale sole, and Pacific sanddab, the areas of high juvenile density were more
expansive in area (sablefish, ; lingcod, ; others Fig H-K). These spatial
patterns were generally consistent through time. For example, the density of
juvenile sablefish was variable with peaks in density in 2004, 2008, 2010, 2013
and 2016. Spatially, across years, age-0 sablefish had large, primary hotspots
north of Cape Blanco and on either side of San Francisco Bay with a smaller
hotspot around Point Conception. These hotspots to the north of Cape Blanco were
most obvious in years with high coast-wide density of age-0 fishes (2004, 2008,
2013, and 2016, and to some extent 2017), suggesting that successful coast-wide
recruitment relies to a large extent on successful recruitment in northern
waters. The exception was 2010 when juvenile density was high in the absence of
the northern hotspot, but density was high in the southern region. Age-0
sablefish were found on shelf and upper slope waters (50–475 m).
Additionally, age-0 lingcod had a large, primary hotspot in shelf waters (50–240
m) located from Point Arena to the south of Point Conception. However, there
were some small secondary hotspots just north of Cape Mendocino and Cape Blanco.
Within years, the prevalence of the large southern hotspot varied and was
related to overall density of age-0 lingcod. Again, hotspots were most apparent
in years with high juvenile density. For lingcod, the density trend indicated
longer-term patterns with low density from 2003–2007, followed by higher
juvenile density from 2008–2012, followed again by lower density from 2013 to
2018.
Effective area occupied varied for all species, but there were no Temporal
trends except for Dover sole, which showed a decline in effective area occupied
through time ( and Fig L). Area occupied was negatively correlated with
abundance for most species (Fig M).
Two species, Pacific hake and sablefish, showed larger variations in their CoG
of approximately 1000 km ( and Fig N). The remaining species tended to show
fluctuations in the range of 200–400 km, but there were no strong directional
trends over time except for Dover sole; its CoG shifted south over time (Fig N
in. Relationships between CoG and abundance varied among species (Fig O), as did
the relationship between effective area occupied and the CoG (Fig P). For
example, the effective area occupied by Dover sole juveniles decreased as the
CoG shifted south over time. However, for longspine thornyhead, higher area
occupied was associated with more northerly CoG.
# Discussion
We quantified the temporal variation in the spatial distribution of the juvenile
abundance for 13 groundfish species. The observed spatial patterns provide
important temporal and spatial information for fisheries managers that will
improve our understanding of each species, which was formerly only based on
adult distributions. Additionally, we estimated annual coast-wide indices of
juvenile density. While evaluating the causes of the observed spatial and
temporal patterns was beyond the scope of this paper, we suggest potential
ecological and physical habitat mechanisms that likely play a role in the
observed temporal and spatial patterns, and we describe how our results have
direct application for fisheries management.
Mesoscale geographic features like headlands and submarine canyons can alter
local circulation, in particular the development of eddies, which may lead to
increased retention of larvae and increased productivity, resulting in higher
settlement and better food resources. Such processes may explain the location of
distinct juvenile hotspots for some particular species and the presence of
locations that are hotspots or areas of high juvenile density for many species.
For example, multiple species (Dover sole, English sole, hake, lingcod, Pacific
sanddab, sablefish and splitnose rockfish) showed high juvenile density near
Point Conception, an area of significant eddy formation. Similarly sub-mesoscale
eddies form north of San Francisco Bay between Pt. Reyes and Pt Arena where
several species also had high juvenile density. Water circulation on wider,
shallower sloping areas of the shelf off of the Washington coast is slower
leading to higher residence times, and potentially higher retention, which may
also help to explain areas of high juvenile density in areas with extensive
shelf habitat. Transport mechanisms will obviously interact with an
individual species’ life history, in particular the location of any spawning
habitats and those advantages to larval biology.
Horizontal transport and water temperature during the pelagic larval stage prior
to recruitment to demersal habitat also likely drive the inter-annual variation
in juvenile densities seen here. For example, inter-annual recruitment variation
around the stock-recruitment relationship for sablefish and petrale sole is
associated with horizontal transport and water temperature during specific
phases in the pelagic life-history of these species. The current results for
sablefish are consistent with stronger age-0 recruitment when conditions favored
the onshore transport of eggs and northerly transport of yolk-sac larvae.
Recruitment variation in bocaccio rockfishes, *Sebastes paucispinus*, is related
to basin scale processes and density dependence. For stocks with weak stock-
recruitment relationships and persistent low spawning, inter-annual climate
variability is probably the most important driver of recruitment variation.
However, for species like lingcod that have experienced recovery in spawning
density during the period studied, increases in juvenile density over time were
also likely due to increased spawning biomass.
Depth is clearly an important factor (as a proxy for other variables such as
light, pressure, temperature or oxygen saturation) in determining assemblage
structure in marine fishes, and this pattern was supported by our analyses. Some
species, such as English sole, Pacific sanddab, petrale sole, and lingcod had
fairly restricted depth distributions of 100–200 m for juveniles on the shelf.
Others like arrowtooth flounder, Dover sole, sablefish and shortspine
thornyheads had broader juvenile depth ranges across the shelf and upper slope.
Pacific grenadier and longspine thornyhead had broad depth ranges that
encompassed deeper slope waters.
While not examined here, bottom type, biogenic habitat, temperature, frontal
boundaries, and other physical characteristics are known to be important in
influencing species distributions, community structure, and the location of
juvenile habitats. For demersal fishes early, post-settlement mortality is often
high, density-dependent and mediated via competition for shelter. Bottom depth
and bottom temperature are important predictors of recently settled juvenile
Kamchatka flounder *Atheresthes evermanni*, and the size and location of the
cold pool largely determines the distribution of arrowtooth flounder in the
eastern Bering Sea. In waters off the U.S. west coast, groundfish assemblages on
the slope shift to deeper waters moving north to south likely tracking changes
in bottom temperature, and geographic features like Point Conception, Cape
Mendocino, and Cape Blanco are known transition zones for biogeographic regions
in the California Current.
For semi-pelagic or mid-water species like Pacific hake, characteristics of the
water column are habitat, and factors like temperature, currents, and distance
from the shelf break may be important in determining both spawning location and
the distribution of pelagic juveniles. Immature Pacific hake have higher biomass
in cooler than average temperatures, while the response of adults varies with
latitude. Additionally, hake tend to be found in areas with poleward drift,
which may aid in their annual migration to northern waters. Temporal variability
in these factors may explain the variability in the location of hotspots for
juvenile hake seen here.
The distribution of adult habitat may also explain some of the observed juvenile
hotspots in that juvenile fishes need access to adult habitats as they age. Many
groundfishes recruit to shallow habitats, potentially to avoid cannibalism or
predation by other species, but move to deeper areas as they grow and age. For
example, sablefish show ontogenetic increases in depth distributions. The
location of juvenile hotspots for sablefish were inshore of adult areas,
especially the shelf and upper slope waters between Cape Mendocino and the
Columbia River outflow. Successful coast-wide recruitment, in terms of total
abundance, appears reliant largely on recruitment to this northern hotspot,
which is consistent with onshore transport during the egg stage and northerly
long-shore transport during the yolk-sack larval stage in the region being
correlated with stronger recruitment. Additionally, the sablefish assessment,
which incorporates age structure data across multiple age classes to estimate
recruitment history, also captured the high age-0 density years seen in the
WCGBTS data, suggesting that these northern recruitment pulses were important to
stock size. Current habitat maps of EFH for juvenile sablefish show the majority
of juvenile EFH in the same area, but in slightly deeper waters farther off
shore. Our results suggest that the EFH definition for juvenile sablefish could
be revised to include to shallower waters in this northern area.
Several species showed large-scale spatial discrepancies between the
distribution of juveniles and adults, raising the question of what ecological
processes (or potentially methodological mechanisms) establish adult
distributions. For example, shortspine thornyheads had a single, high-density
northern juvenile hotspot that was consistent across the study period. However,
adults are relatively evenly distributed along the coast at mid- to deeper-
depths much further south than this northerly area with a high density of
juveniles. Two potential mechanisms could lead to the distribution of adult
thornyheads. First, dispersal of young fish from this northern hotspot could
lead to a much wider distribution of adult fish farther south. Second, juvenile
density was not zero to the south, and consistently lower density in the south
could lead to lower density dependence resulting in higher survival in more
southerly waters. Other factors such as lower predator densities or differing
trawling intensity may also influence the distribution pattern. Likewise, while
the density of juvenile Dover sole was highly restricted to several hotspots in
the south, adults were widely distributed along the coast. Similarly, splitnose
rockfish juveniles occupied several hotspots around and to the south of Point
Conception, while adults were abundant around and to the north of Point
Conception as well as around and to the north of San Francisco Bay, where
juvenile density was particularly low.
Contrasting patterns in juvenile and adult biomass were also apparent for
lingcod, which are currently assessed as two separate stocks off the US west
coast. The most recent stock assessments estimated higher stock biomass off the
coasts of Washington and Oregon (21,976 mt) compared to the stock off the coast
of California (6,509 mt). However, the distribution of age-0 lingcod in the
WCGBT survey was consistently higher south of Cape Mendocino. Longshore movement
of age-1+ lingcod does not appear to explain the difference in the distributions
of age-0 and adult lingcod as genetic analyses identified genetic
differentiation between lingcod north and south of Cape Mendocino. Lingcod
initially settle to shallow, vegetated habitats, which the trawl survey (limited
to trawlable, non-rocky habitats and depths \>55 m) does not sample. So the
survey may not adequately describe the distribution age-0 fish if the
distribution diverges in these shallower habitats. Alternatively, these
genetically dissimilar sub-stocks may have different ecology in the north and
south with different levels of recruitment and survival of juvenile fish. Since
lingcod move to deeper habitats as they grow and age, the observed distributions
are to some extent the result of processes like post-settlement migration,
predator avoidance, prey availability and habitat connectivity.
Long-term changes in the latitudinal distribution of fish biomass (CoG) on in
the California Current have varied among species. For example, from 1977–2013,
the CoG for lingcod, rex sole *Glyptocephalus zachirus*, spotted ratfish
*Hydrolagus colliei*, darkblotched rockfish, greenstriped rockfish *S*.
*elongatus*, and slender sole *Lyopsetta exilis*, all shifted north—a response
that might be expected due to long-term warming—while the distribution of
Pacific hake and sablefish biomass moved south. Here, while the juveniles of
some species like Pacific hake and sablefish showed large fluctuations in their
CoG form 2004–2018, only Dover sole had a significant shift in biomass of
juvenile fishes to the south. Instead, the variability in the latitudinal
distribution appeared related to the fluctuation in the intensity of juvenile
hotspots. For example, when sablefish had high age-0 abundance in the north, the
CoG shifted north.
The species distribution maps presented here should be interpreted in relation
to methodological factors like gear type, survey design, and catchability. The
trawl survey samples between approximately 55–1280 m, and any conclusions are
limited to trawlable (non-rocky) habitat in this range. For some species, like
lingcod discussed above, this limited depth range may explain mismatches between
juvenile abundance patters and adult biomass. Additionally, because the trawl
survey does not sample complex, rocky habitats, abundance estimates may be
biased for some species, especially if there is density dependence.
For many species, the effective area occupied was negatively correlated with
abundance, albeit the relationship was not significant in all cases. The
relationship does not mean that the latitudinal or depth range of the species
contracted at higher juvenile biomass. Effective area as calculated by the VAST
package is the area needed to contain the population at average biomass-density.
Therefore, this relationship would be expected as densities increase in hotspots
more quickly relative to other areas, thus concentrating biomass–a pattern we
might expect if juveniles follow ideal free distributions. Rockfish, for
example, are generally more abundant on complex, rocky habitat. Our species
distribution maps likely, therefore, miss or underestimate biomass in juvenile
hotspots associated with untrawlable habitat like rocky reefs or rocky
pinnacles. Temporally, if juvenile abundance follows an ideal free, trawl-
derived abundance indices may underestimate abundance at low densities because
densities will increase in the primary and unquantified habitat (untrawlabe)
before increasing in secondary, sampled habitat (trawlable). Conversely, at
higher levels, juvenile density will continue to increase in trawlable areas
after density in untrablable areas has stabilized, potentially overestimating
abundance. Nevertheless, for species like sablefish, the observed temporal
patterns largely match model derived trends for age-0 abundance in the stock
assessments.
Gear selectivity in relation to fish body size may also influence the results to
some extent. For many species, we had to combine age classes to obtain enough
data to produce acceptable model fits. Growth of recently settled individuals
over the sampling period may influence the distributions as larger fish become
more selected. However, the survey is conducted in two passes between mid-May
and October, and we account for this effect in part by including pass in the
VAST model. Including pass also accounts to some extent for variability in the
within-year timing of settlement. These effects are most likely more important
for species where we analyzed a single age class. For each pass, vessels begin
in Newport, Oregon and initially work north to Cape Flattery; vessels then head
south to San Diego. The timing of sampling within year could, therefore, miss
late-season settlement in northern waters if settlement occurs after the vessels
have passed. Natural mortality may lead to lower estimates of abundance within
years for southern waters, as fishes in the south well have been subjected to
more predation or starvation risk when they are caught comparted to more
northerly fish.
Catchability, the product of the efficiency of the sampling gear and the
availability of taxa to that gear, likely varies among depths, bottom types, and
the size of individuals among other factors. Since we have not adjusted for
catchability, the estimates of abundance here should be considered indices and
not absolute measures of biomass. While the effect of different sediment sizes
within the range of trawlable habitats may not be large, the spatial patterns
may be influenced by these factors and the distribution maps should be
interpreted with this issue effect in mind.
Species distribution modes have been increasingly used to define essential fish
habitat in regions managed by the U.S. National Marine Fisheries Service, as
mandated by the Magnuson-Stevens Act. For example, Laman et al. estimated
habitat-based models for Alaskan species. Our analyses increase the information
available to fisheries management practices of the 13 stocks we analyzed. The
spatial and temporal results of this study can be used as leading indicators of
potential future changes in stock size for both stock and integrated ecological
assessments or for investigating environmental drivers of recruitment variation,
particularly for those results that capture age-0 and age-1 fish. Delineation of
juvenile habitats can also help to parameterize dispersal models and understand
dispersal patterns and connectivity. If fact, one of the motivators for the
present study was the need to identify juvenile habitats for petrale sole for
use in an individual-based dispersal model.
Fishery managers can use the spatially explicit information on juveniles from
our analyses to further refine and improve essential fish habitat and nursery
habitat definitions and delineations. Identifying juvenile habitats allows for
more targeted spatial management and conservation, allows for better prediction
of responses to environmental events, and forms the foundation for a more
complete understanding of a species ecology. For example, fisheries closures
implemented to protect adult rockfish and adult habitats led to increased
spawning output near closed areas. In the North Sea, managers implemented a
closed area to protect the nursery habitat of North Sea plaice *Pleuronectes
platessa* from trawling. However, the closure of plaice nursery habitat to
trawling was unsuccessful because the distribution of juvenile plaice shifted.
The locations of juvenile hotspots for the species we analyzed were generally
persistent through time, suggesting that closures or gear restrictions to
protect juveniles and juvenile habitat might be effective mechanisms to protect
juveniles for these species. Since many species show ontogenetic shifts to
deeper waters, such spatially explicit and targeted closures might not adversely
affect adult fishing effort, although adverse impacts will be dependent on gear
type and the species’ life history, since one species’ adult habitat may be
another’s juvenile habitat. Likewise, the multi-species nature of the fishery
may also complicate spatial management, but good information on the HAPC for
target species is essential for resolving such issues.
Our characterization of the spatial distribution of juvenile habitats improves
our understanding of how climate or physical conditions affect juvenile
survival. For example, there is a large oxygen minimum zone (OMZ) in mid-depth
(500–720 m) slope waters off the NW Pacific Coast of the U.S.. This OMZ affects
both assemblage structure and diversity, and there is evidence that dissolved
oxygen (DO) concentrations are decreasing in the OMZ. Shoaling of these low DO
waters can lead to fish kills and alter the behavior and occurrence of fishes.
Knowledge of the location of juvenile habitats will aid in assessing the
vulnerability of these species to low DO events.
# Conclusions
Our analysis represents a refinement of information available for the
delineation of essential fish habitat for west coast groundfishes by
identifying, more specifically, the geographic locations and temporal stability
of juvenile habitats (and potential nursery areas). As we address only the
density of juvenile fishes, the areas we identify are best termed juvenile
habitat rather than nursery habitat. Nevertheless, these areas represent
potential, if not likely, nursery habitat for some species. Further work such as
tagging studies or growth analyses will help us to better understand whether
these areas function as nurseries and their importance to the population
dynamics of each species.
# Supporting information
We thank two anonymous reviewers, B. Feist, K. Marshall, C. Harvey, and J.
Samhouri for comments on the manuscript. We also thank A. Shelton, L. Barnett,
and J. Thorson for discussions regarding VAST. Special thanks to the trawl
survey crew for collecting the data and to M. C. Longino and J. Boulogne for
inspiration regarding wearing gloves and masks, and maintaining distance.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Astrocytomas are the most common primary brain tumors. The World Health
Organization (WHO) classifies astrocytomas into four malignant grades: grade I,
or pilocytic astrocytoma; grade II, or low-grade astrocytoma (AGII), grade III,
or anaplastic astrocytoma (AGIII); and grade IV astrocytoma or glioblastoma
(AGIV or GBM). Diffusely infiltrative astrocytomas (AGII-GBM) have the ability
to invade the surrounding normal brain tissue, hampering tumor resection. GBM,
the most malignant and frequent brain tumor in adults, can be divided into two
subgroups: primary GBM, which arises *de novo*, and secondary GBM, which results
from the progression of a lower grade astrocytoma. Interestingly, mutations in
the gene that encodes isocitrate dehydrogenase 1 (IDH1) have been reported in
diffuse gliomas, including WHO grades II and III astroglial and oligodendroglial
lineages. *IDH1* mutations are strong predictors of a more favorable prognosis
and serve as a highly selective molecular marker of secondary GBM that
complements clinical criteria for distinguishing secondary GBM from primary GBM.
Lysyl oxidase (LOX), a copper-dependent amine oxidase, catalyzes the enzymatic
stage of collagen and elastin cross-linking by oxidizing primary amines into
reactive aldehydes. These reactions are essential for stabilization of collagen
fibrils and for the integrity and elasticity of mature elastin to ensure normal
functionality of connective tissue, embryonic development and adult tissue
remodeling. Importantly, biologically active compounds, hydrogen peroxide and
ammonia are generated as by-products during these catalytic reactions. LOX also
has intracellular functions and is involved in the regulation of cell
differentiation, motility/migration and gene transcription. Aberrant expression
of the *LOX* gene has been reported in multiple tumors.
LOX is synthesized by several cell types as a 48 kDa protein. After signal
peptide cleavage and N-glycosylation, the resulting 50 kDa proenzyme is secreted
and converted into a mature, active 30 kDa form as a result of proteolytic
processing by procollagen C proteinase/bone morphogenic protein-1 (BMP1). The
catalytic activity of *LOX* can be specifically and irreversibly inhibited by
beta-aminopropionitrile (BAPN). LOX has been identified as an important
regulator of the hypoxia-induced tumor progression pathway through a
HIF-1α-dependent mechanism in numerous cancer types, such as breast, head and
neck, prostate and renal cell carcinomas. LOX is involved in the hypoxic
upregulation of *HIF1A*, and LOX and HIF-1α potentiate each other to foster
tumor progression in the colon through the PI3K-Akt signaling pathway. Secreted
LOX is responsible for the invasive properties of hypoxic cancer cells,
including astrocytomas, through the activation of focal adhesion kinase
(FAK)/paxillin. LOX has been implicated in tumor angiogenesis *in vitro* and *in
vivo* by increasing vascular endothelial growth factor (VEGF) expression and
secretion as well as blood vessel formation.
Recently, it was demonstrated that HIF-1α-responsive genes essential for cell
growth, including *LOX*, were underexpressed in gliomas with *IDH1* mutation.
Therefore, we aimed to investigate *LOX*, *BMP1* and *HIF1A* mRNA expression
levels in a large series of astrocytomas of different malignant grades and
compare these results between cases with wild type *IDH1* and cases with mutated
*IDH1*. *LOX* knockdown by siRNA was performed for functional studies *in
vitro*, and LOX protein was also analyzed in tumor samples. These data suggest
that *LOX* expression increases according to malignancy grade in astrocytomas
and represents a potential therapeutic target, especially for cases without
*IDH1* mutation.
# Methods
## Tissue Samples
The samples used in this study consisted of 153 astrocytomas (grades I to IV).
Tumors were graded according to the WHO classification into AGI (n = 23; mean
age at diagnosis, 19.4±9.7 years; 14 males and 9 females), AGII (n = 26; mean
age at diagnosis, 34.0±8.1 years; 15 males and 11 females), AGIII (n = 18; mean
age at diagnosis, 35.0±12.3 years; 11 males and 7 females) and GBM (n = 86; mean
age at diagnosis, 54.0±13.9 years; 58 males and 28 females). The non-neoplastic
control group consisted of samples from individuals undergoing temporal lobe
resection during epilepsy surgery (n = 22; mean age at diagnosis, 38.0±7.6
years; 10 males and 12 females). All samples were collected during surgical
procedures by the Neurosurgery Group of the Department of Neurology at the
Hospital das Clinicas of School of Medicine, University of Sao Paulo, Brazil.
Fresh surgical samples were immediately snap-frozen in liquid nitrogen upon
surgical removal. Before RNA extraction, a 4-μm-thick cryosection of each sample
was stained with hematoxylin, and necrotic and non-neoplastic areas were removed
from the frozen block of tumoral tissue by microdissection. Grey matter was
avoided in the control non-neoplastic samples. Written informed consent was
obtained from all patients according to the ethical guidelines approved by the
Ethics Committee of the School of Medicine, University of São Paulo (0600/10).
The Ethical Commission for Research Projects Analysis (CAPPesq) from the
Clinical Board of Hospital das Clinicas and School of Medicine, University of
São Paulo, in council session taken place at 2012, August 8th, approved the
research protocol entitled: “Expression and role of lysyl oxidase family genes
in astrocytomas”, presented by the Department of Neurology.
## Total RNA extraction and cDNA synthesis
Total RNA was extracted from frozen tissues (tumor and non-neoplastic) using an
RNeasy Mini kit (Qiagen, Hilden, Germany). The RNA concentration and purity were
evaluated by measuring the absorbance at 260 and 280 nm. A 260/280 ratio ranging
from 1.8 to 2.0 was considered satisfactory purity. Denaturing agarose gel
electrophoresis was used to assess the quality of the samples. A conventional
reverse transcription reaction was performed to yield single-stranded cDNA.
First-strand cDNA was synthesized from 1 μg of total RNA that was previously
treated with 1 unit of DNase I (FPLC-pure, GE Healthcare, Uppsala, Sweden) using
random and oligo(dT) primers, RNase inhibitor, and SuperScript III reverse
transcriptase according to the manufacturer’s recommendations (Life
Technologies, Carlsbad, CA). The resulting cDNA was subsequently treated with 1
unit of RNase H (GE Healthcare, Uppsala, Sweden), diluted with TE buffer, and
stored at −20°C until later use.
## Quantitative real time PCR (RT-qPCR)
The relative expression levels of *LOX*, *HIF1A* and *BMP1* were analyzed by RT-
qPCR using the SYBR Green approach. Quantitative data were normalized to the
geometric mean of three reference genes suitable for the analysis: hypoxanthine
phosphoribosyltransferase (*HPRT*), beta glucuronidase (*GUSB*) and TATA box
binding protein (*TBP*). The primer sequences were as follows (from 5’ to 3’):
LOX F: CCTACTACATCCAGGCGTCCA; LOX F R: CATAATCTCTGACATCTGCCCCTGT; HIF1A F:
CATCCAAGAAGCCCTAACGTGT; HIF1A R: CATTTTTCGCTTTCTCTGAGCAT; BMP1 F:
CTCGTAAGTCCTCCATCAAAGCT; BMP1 R: CTCTCCATCTCCCACAGGCTC; HPRT F:
TGAGGATTTGGAAAGGGTGT; HPRT R: GAGCACACAGAGGGCTACA; GUSB F:
GAAAATACGTGGTTGGAGAGCTCATT, GUSB R: CCGAGTGAAGATCCCCTTTTTA; TBP F:
AGGATAAGAGAGCCACGAACCA, TBP R: CTTGCTGCCAGTCTGGACTGT. The primers were
synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). The
minimum primer concentrations necessary were determined to give the lowest
threshold cycle (Ct) and maximum amplification efficiency while minimizing non-
specific amplification. The primer concentrations used were 200 nM for *LOX*,
*HIF1A*, *BMP1*, *HPRT* and *TBP* and 400 nM for *GUSB*. Standard curves were
established to ensure amplification efficiency, and an analysis of melting
curves demonstrated a single peak for all PCR products. Additionally, agarose
gel electrophoresis was employed to check the size of the PCR products
amplified. SYBR Green I amplification mixtures (12 μl) contained 3 μl of cDNA, 6
μl of 2x Power SYBR Green I Master Mix (Life Technologies) and primers. The PCRs
were run on an ABI Prism 7500 sequence detector (Life Technologies) as follows:
2 min at 50°C, 10 min of polymerase activation at 95°C, and 40 cycles of 15 s at
95°C and 1 min at 60°C. Quantitative data were normalized relative to the
internal housekeeping control genes. The equation 2<sup>−ΔCt</sup> was applied
to calculate the expression of *LOX*, where ΔCt = Ct of the target
gene—geometric mean of the Ct of the reference genes. The RT-qPCR reactions were
performed in duplicate for each sample and repeated when the Ct values were not
similar. The results are presented on a log10 scale for better visualization.
*LOX* expression was scored according to the median expression values of each
astrocytoma grade. For statistical analysis, scores equal to or higher than the
median values were defined as *LOX* overexpression. For functional analysis of
*LOX* knockdown after siRNA transfection, the same procedures were followed,
except that only *HPRT* was used as a reference gene. The expression values were
calculated relative to the scrambled non-target control (NTC).
## DNA extraction and IDH1 mutational analyses
DNA was extracted from frozen tumor samples using a QiaAmp DNA Micro kit
(Qiagen). Polymerase chain reaction (PCR) followed by DNA sequencing was applied
to detect mutations in IDH1, as previously described. The sequences of primers
(5’-3’) synthesized by IDT for PCR amplification of exon 4 were as follows:
CCATCACTGCAGTTGTAGGTT and CATACAAGTTGGAAATTTCTGG. PCR products were generated in
a 25 μL reaction mixture including 100 ng of DNA, 50 mM KCl, 50 mM of each dNTP,
10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl<sub>2</sub>, 10 pmol of each primer and 1
unit of Taq DNA polymerase (GE Healthcare). The PCR was performed with an
initial denaturating step at 94˚C for 5 min, followed by 35 cycles consisting of
94˚C for 30 s, 54˚C for 30 s and at 72˚C for 30 s. After the final cycle, an
extension period of 10 min at 72˚C was performed. The PCR products (436 bp) were
purified with a GFX column (GE Healthcare) and sequenced on an ABI Prism 3130
DNA automated sequencer using the Big Dye Terminator Cycle Sequencing Ready
Reaction Kit version 3.1 (Life Technologies). The primers used for sequencing
were the same as those used for PCR.
## Cell culture conditions and transient transfection
The human malignant astrocytoma cell lines U87MG and A172 were routinely
cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies)
supplemented with 10% fetal bovine serum (FBS) (Life Technologies), 100 IU/ml
penicillin and 100 μg/ml streptomycin in an atmosphere consisting of 5%
CO<sub>2</sub> in air at 37°C in a humidified incubator. Dicer substrate small
interfering RNA (siRNA) duplexes for *LOX* knockdown
(5′-GUAAUUACAGAAUUGAAACACUGUGUU-3′) were diluted in buffer according to the
manufacturer’s recommendations (IDT). U87MG and A172 cells (1 × 10<sup>5</sup>
cells/well) were seeded in a six-well plate; after 24 h, they were transfected
with Lipofectamine RNAiMax (Life Technologies). Control cells were transfected
with a scrambled non-target control (NTC) siRNA from IDT. siRNAs for both LOX
and NCT were used at a concentration of 1 nM, and *LOX* knockdown as well as the
effect of LOX silencing were evaluated at 2, 4, and 7 days after transfection by
RT-qPCR and Western blotting.
## Cell migration and invasion assay
The migration and invasion abilities of the cells were assessed by determining
the ability of the cell lines to cross a membrane with a pore size of 8 m in
non-coated (for migration) and matrigel-coated (for invasion) Transwells (BD
Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ), according to
the manufacturer's recommendations. After 48 hours of transfection, the cells
were maintained in DMEM supplemented with 1% FBS for 2 hours. To determine the
effect of LOX inhibitor treatment on migration, the cells were treated with 100
mM β-aminopropionitrile (BAPN Sigma-Aldrich, Saint Louis, MO) for 24 h prior to
the migration assays and for an additional 4 h during the assay. This
concentration of BAPN has no cytotoxic effects, as demonstrated previously.
After trypsinization, a total of 2.5 x 10<sup>4</sup> cells were suspended in
0.5 ml DMEM with 1% FBS and added to the upper compartment of the Transwell. The
bottom chamber contained 10% FBS, and the cells were incubated for 18 hours at
37°C in an atmosphere containing 5% CO<sub>2</sub>. Non-migrating and non-
invading cells were wiped away from the upper surface of the membrane with a
cotton swab. The cells were fixed with 4% paraformaldehyde, stained with 0.2%
crystal violet (Sigma-Aldrich) in 20% methanol, and analyzed by inverted
microscopy (40x magnification). The results of the migration and invasion assays
were quantified by counting 18 random fields from each of the experimental
inserts in duplicate. Data were generated from two independent experiments.
Migration values were expressed as the average number of migrated cells per
microscope field.
## Cell proliferation and anchorage-independent cell growth assay
Proliferation of U87MG and A172 cells was evaluated after 2, 4, and 7 days of
siRNA *LOX* transfection in duplicate experiments. The cells were stained with
Trypan blue and counted using an automatic counter (Countess, Life Technologies)
for determination of live cells. Anchorage-independent cell growth was analyzed
via a soft agar colony formation assay. Two days after transfection, 1 x
10<sup>3</sup> cells were resuspended in 0.3% agar on a layer of 0.6% agar in
DMEM (1x) in a six-well plate and incubated in a humidified atmosphere in the
presence of 5% CO<sub>2</sub> at 37°C. After 10 days, the colonies were fixed
with formaldehyde and stained with 0.005% crystal violet in phosphate-buffered
saline (PBS) for 1 h. The number of colonies was recorded for each well. Two
independent experiments were performed in duplicate.
## Western blot analysis
After transfection with control siRNA (NTC) and siRNA specific for *LOX*, total
protein lysates were prepared from cell cultures with RIPA lysis buffer and
protease inhibitor cocktail (Sigma-Aldrich) on ice. The protein concentration
was determined using the Bradford reagent (Bio-Rad Laboratories, Richmond, CA).
The absorbance was measured at 595 nm using a microplate spectrophotometer
(Multiskan Spectrum; Thermo Labsystems, Helsinki, Finland). All assays were
conducted in duplicate, and calculations were carried out with a standard curve
constructed using different concentrations of bovine serum albumin (2 mg/ml to
0.125 mg/ml). Total protein lysates (30 mg) were separated by 12% SDS
polyacrylamide gel electrophoresis (TGX Mini Protean, Bio-Rad) with Tris-glycine
running buffer. The proteins were transferred to a nitrocellulose membrane using
the iBlot dry blotting system (Life Technologies). The membrane was blocked with
5% skim milk and incubated with rabbit polyclonal primary anti-LOX diluted
1:1,000 (Sigma-Aldrich). The membrane was also incubated with mouse monoclonal
anti-β-actin (1:5,000, clone AC-74, Sigma-Aldrich) as a protein loading control.
The secondary antibodies used were anti-rabbit (1:1,000) and anti-mouse IgG
(1:5,000) conjugated to peroxidase (Sigma-Aldrich). The immune complexes were
visualized using enhanced chemiluminescence reagent (Western Lightning
Chemiluminescence Reagent Plus, Perkin Elmer, Waltham, MA) and detected with
ImageQuant LAS4000 (GE Healthcare).
## Immunohistochemistry
For immunohistochemical detection, 4-μm formalin fixed paraffin embedded tissue
sections were routinely processed and subjected to antigen retrieval. Briefly,
slides were immersed in 10 mM citrate buffer (pH 6.0) and incubated at 122°C for
3 min using an electric pressure cooker (BioCare Medical, Walnut Creek, CA).
Specimens were then incubated for endogenous peroxidase blocking (Novolink
system, Novocastra, Newcastle-upon-Tyne, UK) and further incubated with a
polyclonal antibody raised in rabbits against human LOX (ab31238, 1:100
dilution; Abcam, Cambridge, UK) at 16–20°C for 16 hours. The reaction was
developed with a commercial kit (Novolink; Novocastra, Newcastle-upon-Tyne, UK)
at room temperature using diaminobenzidine and Harris hematoxylin for nuclear
staining. Optimization using a positive control suggested by the manufacturer
(breast carcinoma) was performed to obtain the optimal dilution. The staining
intensity of tissue sections was evaluated independently by two observers (SKNM
and RS). A semi-quantitative scoring system considering both the intensity of
staining and percentage of cells was applied as follows: for intensity of
staining, 0 = negative, 1 = weak, 2 = moderate and 3 = strong; for cell
percentage, 0 = no cells stained, 1 = 10–25%, 2 = 26–50%, 3 = 51–75% and 4 =
76–100%. The LOX immunohistochemistry labeling score (ILS) was obtained by
determining the product of the staining intensity and the percentage of cells
stained. Digital photomicrographs of representative fields were captured and
processed using PICASA 3 (Google, USA).
## Statistical analysis
Kolmogorov-Smirnov normality test was used to analyze the distribution of gene
expression data and also the effect of *IDH1* mutations on gene expression. The
Kruskal- Wallis test was used for analysis of the differences in gene expression
between non-neoplastic tissues and astrocytomas of different grades of
malignancy. Coexpression of genes was analyzed using the Spearman-rho test. A
correlation coefficient (r) of ≥0.7 was interpreted as a strong correlation,
0.3≤r\<0.7 was interpreted as a moderate correlation, and r \<0.3 was
interpreted as a slight correlation. The Mann-Whitney test and *t* test were
used to compare *IDH1* mutational status and gene expression for non-parametric
and parametric distributions, respectively. The Mann-Whitney test was also used
for functional assays. When the Kruskal-Wallis test was used for gene expression
as well as ILS analysis among the different groups and the results were
significant, Dunn multiple comparison post hoc test was applied. The Kaplan-
Meier survival curves were analyzed using the log rank test (Mantel Cox),
excluding 9 cases of tumor relapse in GBM patients. To perform the analysis, the
patients were divided into two groups characterized by high (above the median)
and low (below the median) gene expression. The statistical significance was set
at *p*\<0.05. All analyses were performed using SPSS version 15.0 (Chicago, IL),
and scatter plots were constructed using the program GraphPad Prism version 5.0
(GraphPad Software, Inc., San Diego, CA).
# Results
## *LOX*, *BMP1* and *HIF1A* Expression Levels in Astrocytomas of Different Malignant Grades
*LOX*, *BMP1* and *HIF1A* expression analysis by qRT-PCR showed great
variability in astrocytomas of all malignant grades when compared to non-
neoplastic samples. The GBM cases had higher *LOX* expression levels relative to
non-neoplastic cases, with a statistically significant difference. No difference
was found in *LOX* expression level between AGI, AGII and AGIII when compared to
non-neoplastic samples. On the other hand, *BMP1* expression levels were
significantly higher in AGI and GBM groups when compared to non-neoplastic
samples. *HIF1A* expression levels increased with the malignant grade of
astrocytomas, with statistically significant values for all malignant grades of
astrocytomas when compared to control samples. Coexpression of the three genes
was compared for astrocytomas of all malignant grades. Interestingly, Spearman
analysis demonstrated that *LOX* mRNA levels were positively correlated with
*BMP1* and *HIF1A* in GBM (*r* = 0.217 and *p* = 0.045, ; *r* = 0.367 and *p* =
0.001, respectively). *BMP1* expression also presented a positive correlation
with *HIF1A* in GBM (*r* = 0.389; *p*\<0.001) and in AGI (*r* = 0.477; *p* =
0.033). *LOX*, *BMP1* and *HIF1A* expression levels were correlated with the
clinical outcome of the GBM cases. The analysis of overall survival of GBM cases
with high and low expression did not reveal a significant correlation between
the gene expression data and the prognosis of GBM patients.
## Association of *LOX*, *BMP1*, and *HIF1A* mRNA Expression Levels and *IDH1* Mutation Status
The frequency of IDH1 mutation was 80.8% in AGII (21 out of 26), 61.1% in AGIII
(11 out of 18) and 12.8% in GBM (11 out of 86) (, respectively), which was also
described in our previous study of the frequency of *IDH1* mutations in a series
of GBM patients. Our GBM cases were composed mainly by primary GBMs, which
explains the low frequency of *IDH1* mutation. Of all the mutations, R132H was
the most common in glioma. summarizes the comparison of the median expression
levels of cases with wild-type and mutated *IDH1*. *IDH1*-mutated AGII and GBM
cases showed lower *LOX* expression levels when compared to wild-type *IDH1*
AGII (*p* = 0.049) and GBM (*p* = 0.008) cases. On the other hand, AGIII cases
with wild-type *IDH1* presented lower *HIF1A* expression levels when compared to
*IDH1*-mutated cases. (*p* = 0.038). No difference was found for *LOX*
expression in AGIII groups with mutated and wild type *IDH1*. Similarly, *BMP1*
expression between wild-type and mutated *IDH1* cases was not different
regardless of astrocytoma group, and *HIF1A* expression was not different for
AGII and GBM.
## Effect of *LOX* Knockdown in GBM Cells
*LOX* knockdown was evaluated after 2 days of transfection of U87MG and A172 GBM
cell lines with siRNA targeted against *LOX* or non-targeted control (NTC)
siRNA. The efficiency of transfection was analyzed by RT-qPCR and western
blotting for each of the duplicate experiments. Both cell lines presented an
approximate 80% decrease in LOX mRNA expression when compared to NTC. Protein
expression was also confirmed to be diminished after *LOX* knockdown with siRNA.
Transfected cells were maintained under cell culture conditions for 2, 4 and 7
days after transfection to evaluate the involvement of *LOX* in the
proliferation of A172 and U87MG cells. There was no difference in the
proliferation rate of cells after *LOX* knockdown when compared to NTC in both
cell lines analyzed (data not shown). To test the hypothesis that *LOX*
expression was correlated with the migratory ability of tumor cells, U87MG and
A172 cell lines were evaluated after knocking down *LOX* expression by siRNA and
after inhibiting the active form of LOX with a specific drug (BAPN). There was a
reduction in the migration ability of U87MG and A172 cells after either
transfection with siRNA for LOX or treatment with BAPN, as shown in. The
differences between the *LOX* siRNA and NTC groups were statistically
significant in both U87MG (*p*\<0.001) and A172 (*p*\<0.0001) cells. Inhibition
of LOX by BAPN significantly inhibited the migration of both U87MG and A172
cells when compared to non-treated cells (*p*\<0.0001 for both cell lines). LOX
involvement in the invasion of GBM cell lines was also observed for U87MG and
A172 cells (*p*\<0.0001 for both).
Anchorage-independent growth assays were subsequently used to examine the role
of *LOX* in the colony formation ability of U87MG and A172 cells. No reduction
in colony numbers was observed for U87MG cells after knockdown of *LOX* when
compared to NTC. On the other hand, there was a significant decrease in the
number of colonies of A172 cells transfected with *LOX* RNAi compared to NTC
(*p*\<0.0001).
## LOX Immunohistochemistry Analyses
Expression of LOX at the protein level was investigated by immunohistochemistry
of non-neoplastic brain tissues and astrocytomas of different malignant grades,
as shown in. LOX expression was observed in the cytoplasm and nucleus of all
tissues with variable intensity. Endothelial cells were stained specifically in
neoformed vessels of GBM cases. The immunohistochemistry results were analyzed
semi-quantitatively by the ILS, as described in Methods. The cytoplasmic ILS did
not vary significantly among the different astrocytoma groups and control
samples. Nevertheless, a high nuclear ILS was found in non-neoplastic samples,
with staining of glial cells and neurons, while the nuclear ILS in astrocytomas
increased with increasing degree of malignancy. AGI group presented statistical
differences to non-neoplastic, AGIII and GBM samples. Additionally, endothelial
cells were predominantly stained for LOX in GBM cases, with significant
differences of this tumor group and non-neoplastic and AGI, AGII and AGIII
groups. Interestingly, GBM cases with mutated *IDH1* presented lower LOX nuclear
and endothelial staining, confirming the results of *LOX* transcript expression
and *IDH1* mutation status analyses.
# Discussion
In the present study, we investigated the expression of *LOX*, *BMP1* and
*HIF1A* in astrocytomas of different malignant grades. Additionally, the effect
of *IDH1* mutation on gene expression was also evaluated as well as the role of
*LOX* in the behavior of GBM cell lines.
LOX is a secreted amine oxidase that plays a key role in modifying the primary
tumor microenvironment by crosslinking collagens and elastin in the ECM thereby
causing stiffening of the matrix and enhancing the invasive and metastatic
properties of the tumor. The stiffness of the ECM is particularly enhanced by
the active form of LOX (, and BMP1 is responsible for processing LOX into
the active form.
We demonstrated significantly higher *LOX* mRNA expression levels in GBM cases,
when compared to non-neoplastic brain tissue samples, as reported by others in
different types of tumors. Furthermore, higher *BMP1* expression that was
positively correlated with *LOX* expression was also detected in GBM cases. Such
findings are in agreement with the fact that GBMs are the most malignant and
invasive astrocytomas. In fact, we used functional analysis to demonstrate that
knocking down *LOX* with siRNA or inhibiting LOX with BAPN led to a reduction in
the migration and invasion of U87MG and A172 GBM cell lines. LOX knockdown and
BAPN treatment have similar effect on migration in U87MG cell line. On the other
hand, the same was not observed for A172 cells even though reduction of LOX
expression by siRNA treatment was confirmed. Those results might reflect the GBM
heterogeneity. The mutational profile of U87MG and A172 is different according
to data of COSMIC cell lines project
(<http://cancer.sanger.ac.uk/cancergenome/projects/cell_lines/>) shows that
U87MG has as main consensus genes mutated *PTEN*, *NF1* and *ATRX*, while A172
has *EGFR*, *RB1* and *PTCH1*, among others.
Similar results were obtained by Lackzo et al. using other GBM cells (U251 and
U373) treated with BAPN and catalase. These authors associated the role of
active LOX in the migration/invasiveness of GBM cell lines with FAK/paxillin
activation through hydrogen peroxide generated by LOX catalytic reactions.
Intracellular hydrogen peroxide in excess can facilitate the phosphorylation and
activation of Src, which then phosphorylates FAK, with consequent induction of
various signaling pathways involved in the regulation of cell adhesion and
migration. FAK phosphorylation may also explain our finding of a decrease in the
number of colonies of A172 cells in which *LOX* expression was knocked down, as
suggested by others~~)~~. The absence of a similar effect in the colony
formation assay with U87MG cells corroborates the heterogeneity observed in this
type of tumor.
The irreversible LOX inhibitor BAPN was initially used to treat disorders such
as hypertension, decreased wound healing, and peripheral blood mononuclear cell
chemotaxis by preventing the formation of a highly cross-linked form of vascular
collagen. Later, BAPN was proposed to also be useful in the treatment of cancers
with LOX hyperexpression, such as melanoma, head and neck carcinoma and breast
carcinoma. Recently, BAPN was used in the treatment of *in vivo* tumor models of
pancreatic ductal adenocarcinoma in mice, and it was able to stabilize
senescence, delay tumorigenesis, and increase survival. Moreover, another
inhibitor of LOX, magnolol, presented a similar effect in a breast cancer model
*in vitro*. In addition to BAPN, d-penicillamine, which depletes intracerebral
copper, also exhibited antiangiogenic effects on GBM tumor growth in mice. LOX
is essential for stimulating endothelial cells and angiogenesis by increasing
VEGF expression, and the increase in matrix stiffness also upregulates VEGF
expression. Interestingly, LOX is expressed in GBM endothelial cells, as
demonstrated by immunohistochemistry. This result corroborates a recent report
of LOX expression in tumor endothelial cells. The expression of LOX by tumoral
and endothelial cells in GBM may be controlled by a positive feedback loop
mechanism, although this will require further investigation. Taken together,
these results reinforce the notion that LOX may be a target in the treatment of
tumors.
The high proliferative capacity of GBM leads to the development of hypoxic areas
and ultimately necrosis. The reduction in oxygen availability activates hypoxia-
inducible factor-1, which in turn activates the transcription of target genes,
including *LOX*.
Indeed, our results demonstrated that *HIF1A* is highly expressed in GBM cases,
and its expression is associated with *LOX* and *BMP1* expression.
Interestingly, the median expression levels of both *LOX* and *BMP1* were higher
in AGI samples than in AGII samples. In contrast, the median expression of
*HIF1A* in AGI cases was lower than that in AGII. AGI is non-invasive tumor,
while AGII is infiltrative. Additionally, AGI presents vascular proliferation
and shows greater contrast on neuroimaging compared to AGII. Taking these
phenotypic characteristics together, we speculate that *HIF1A* upregulation in
AGI activates a hypoxia-driven angiogenic pathway. Additionally, BMP1 can
activate various other substrates that are important regulators of extracellular
matrix production and quality as well as of antiangiogenic responses by
producing a factor from the basal membrane compound. In contrast, stepwise
upregulation of *HIF1A* observed in our cases of diffusely infiltrative
astrocytomas associated with high expression of *LOX* and *BMP1* suggest
progressive activation of both angiogenic and invasive pathways.
Another interesting finding of the present study was the association of *IDH1*
mutation with low expression of *LOX*. A list of underexpressed genes responsive
to HIF-1, including *LOX*, has been recently reported in *IDH1* mutant gliomas
and brain tumor stem cells. In fact, in the present series, *LOX* expression was
significantly lower among AGII (p = 0.049) and GBM (p = 0.008) cases presenting
*IDH1* mutations when compared to cases with wild-type *IDH1*. For GBM, a
decreased level of LOX immunostaining in the nucleus was observed with the
presence of *IDH1* mutations. Mutated *IDH1* is a common feature in lower grade
gliomas and secondary GBMs. Our series of GBM presented only 12% of IDH1
mutation, consisting mainly of primary cases, as previously reported. The
presence of mutated *IDH1* is strongly correlated with a CpG island methylator
phenotype in gliomas. Therefore, promoter methylation might explain the
decreased LOX expression in the presence of such a mutation. Indeed, *LOX*
inactivation by methylation has already been demonstrated in gastric cancers as
well as colon, lung and ovarian cancer cell lines.
Using an antibody against the C-terminus of LOX that detects only the 50 kDa
pro-LOX or the processed active 32 kDa LOX, we have unequivocally detected a
high level of LOX nuclear staining, particularly in the most malignant
astrocytomas (grades III and IV). Previous studies have described only intense
perinuclear and cytoplasmic staining of astrocytomas of different malignant
grades; nuclear staining has not been mentioned. Intriguingly, nuclear staining
was also detected in our normal controls, including glial and neuronal cells, as
described by others. These findings were in agreement with previous reports of
LOX nuclear localization in smooth muscle and proliferating cells. LOX can
oxidize nuclear proteins such as histone H1, leading to epigenetic effects on
DNA-histone and histone-histone interactions, with consequent effects on DNA
transcription analogous to the effects resulting from histone acetylation.
Further studies are needed to clarify differential LOX function in normal and
tumor tissues as well as in distinct intracellular compartments.
In summary, our results confirmed that LOX plays an important role in migration
and angiogenesis in diffusively infiltrative astrocytomas, especially GBMs.
Moreover, *LOX* expression is influenced by *IDH1* mutational status, which
provides new insights for the design of targeted therapies to control these
tumors.
# Supporting Information
We are very thankful for all the neurosurgeons of the Division of Neurosurgery
of the Department of Neurology at Hospital das Clinicas of School of Medicine,
University of São Paulo for the therapeutic and follow-up procedures of all
patients included in this study. We particularly acknowledge Thais F. Galatro
for the valuable immunohistochemistry reactions.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: SMOS. Performed the
experiments: RDS. Analyzed the data: MU. Contributed
reagents/materials/analysis tools: SKNM. Wrote the paper: RDS SMOS SKNM. |
# Introduction
The oral detection of sugars is presumed to occur by activation of a class 1
taste receptor heteromer, TAS1R2-TAS1R3, sometimes referred to as a ‘sweetener
receptor’. Notions of how human sweet taste operates are largely based on mouse
sweetener perceptual genetics and physiology. By genetic homology, binding to
and activation of this receptor is believed to stimulate perceived sweetness in
humans as well. In support of this idea, mutations in regulatory regions of the
human T1R receptor genes are associated with slightly altered sensitivity to
sucrose. However, more recent genetic studies of human sweet taste perception
and intake have not replicated these observations. Based on the assumption that
the human preference for sugary foods and beverages is driven by stimulation of
sweet taste orally, the design and creation of artificially sweetened beverages
occurred over 100 years ago by Hyman Kirsch in 1904 who made No-Cal ginger ale
with calcium cyclamate to treat diabetics. Despite the century-long refinement
of no-calorie or low-caloric sweeteners in beverages, diet sodas have never
captured a major share of the beverage market. Reasons for this are presently
unknown, but have been attributed to: 1) non-sucrose-like sweetness, 2) non-
sucrose-like bitterness and other side tastes, 3) non-sucrose-like temporal
profile of sweetness (lingering taste), 4) customer fears of artificial
ingredients in foods (naturalism bias), and 5) concerns for increased risk of
medical pathologies from use, such as cancer. We hypothesize an additional
explanation that sugars may engage a second oral signaling pathway for calories
that noncaloric sweeteners fail to engage.
Recently, mouse taste bud cells were found to contain many of the same molecular
components as do pancreatic beta islet of Langerhans cells, which use a multi-
step metabolic signaling pathway to detect glucose in the blood. Beta islet
cells indicate increases in blood glucose via: A) the transport of glucose into
the cells via molecular carriers such as the sodium-glucose co-transporters
(SGLTs) along with other transporters, such as GLUTs, B) the oxidation of
glucose to produce several ATP molecules via i) glycolysis involving glucokinase
and the production of ATP and pyruvate and ii) oxidative metabolism of the
pyruvate via Krebs cycle in mitochondria to yield additional ATP, and lastly, C)
the closing of potassium channels that are gated by ATP (K<sub>ATP</sub>
channel). The presence of glucose transporters, glucokinase, and the ATP gated
potassium channels K<sub>ATP</sub> (Kir6.1 and SUR) have all been identified in
mouse taste bud cells. Thus, it appears that taste bud cells in the mouth of
mice are capable of identifying when a “sweetener” is metabolizable. This system
appears to have functionality in mice, as oral stimulation with sugars in the
absence of T1R2-T1R3 taste receptors continues to elicit anticipatory insulin
responses to sugar.
Whether a similar glucose metabolic signaling pathway exists and is functional
within human oral taste cells and whether it impacts sugar perception has not
been determined. Were such a signaling system to exist and be functional in the
human mouth, it would have implications for oral signaling of the metabolizable
sugar glucose and could help explain preference for sugared beverages over non-
caloric sweetener beverages (cf.). In the present study, we conducted several
psychopharmacological experiments to identify whether a second signaling pathway
exists in human mouths for sugars such as glucose, but not for non-caloric
sweeteners, such as sucralose, and whether this pathway may involve the sodium-
glucose-linked co-transporters (SGLTs) as an initial step in this additional
signaling pathway. Support for the role of the specific SGLT transporters in
glucose taste come from our use of added sodium, which is co-transported with
glucose, use of the SGLT inhibitor phlorizin, use of the sugar fructose, which
is not transported by SGLT, and use of the glucose analog α-methyl-D-
glucopyranoside (MDG) which is co-transported with sodium by SGLT, but is not
metabolized to produce ATP.
# Materials and methods
## Experiment 1
### Hypothesis
We hypothesize that glucose engages a dual oral signal comprised of i) T1R
receptor-based signaling and ii) a form of oral metabolic-pathway signaling that
involves transport of glucose by an SGLT, whereas sucralose engages only the T1R
receptor signaling. Therefore, we predicted that inhibiting the T1R2-T1R3 taste
signal with the T1R inhibitor Na-lactisole would have a greater impact (raising
the threshold concentration) on sucralose than it would on glucose detection
thresholds.
### Ethics statement
All research was conducted according to the principles expressed in the
Declaration of Helsinki, and approved by an Institutional Review Board at the
University of Pennsylvania (IRB \#701334). Subjects provided written, informed
consent on forms approved by the Institutional Review Board prior to
participation.
### Subjects
Twelve healthy adults (7 female, 5 male; mean age = 39 years, S.D. = 13)
participated. Subjects were recruited from the surrounding community, and paid
for their time.
### Stimulus materials
Stimuli included filtered water (Milli-Q Water Purification System); serial
dilutions of D-(+)-glucose (\> 99.5%, Sigma-Aldrich, USA) in filtered water in
1/8 log steps ranging from 0.73–73 mM; serial dilutions of sucralose (\> 98.0%,
Sigma-Aldrich, China) in filtered water in 1/8 log steps ranging from 2.0–200
μM; serial dilutions of glucose in filtered water in 1/8 log steps ranging from
0.73–412 mM with the addition of 2 mM Na-lactisole (Sodium 2-\[4-methoxyphenoxy
propionate\], Endeavor Specialty Chemicals, UK); serial dilutions of sucralose
in filtered water in 1/8 log steps ranging from 2.0–1124.7 μM and 2 mM Na-
lactisole; serial dilutions of glucose in filtered water in 1/8 log steps
ranging from 0.73–73 mM with the addition of 2mM NaCl (\>99.0% sodium chloride,
Fisher Scientific, USA); serial dilutions of sucralose in filtered water in 1/8
log steps ranging from 2.0–200 μM and 2 mM NaCl; 0.8 mM Na-lactisole used as a
rinse. Stimuli were presented at room temperature.
### Detection threshold method
Subjects completed six conditions each with a replicate. The conditions measured
absolute detection thresholds for i) glucose and ii) sucralose alone, iii & iv)
each with 2 mM Na-lactisole added, and v & vi) each with 2 mM NaCl to control
for the sodium associated with lactisole. In these conditions, subjects rinsed
with water before and between samples.
Each subject was instructed to refrain from smoking, eating, chewing gum, and
drinking anything, except water, for one hour before participation. At the start
of each session subjects rinsed their mouths with water 4 times for 30 seconds
each and then expectorated for a total rinse time of 2 minutes. They were
presented with two 10 ml samples. In the glucose condition the samples were
water and glucose. While wearing nose clips, they put the whole sample in their
mouth and after 1–2 seconds expectorated and then rinsed with water. Next, they
repeated the tasting with the second sample. Their task was to select the sample
that is different from water in a two-alternative forced-choice (2-AFC) trial.
If unsure, they were instructed to guess. For the glucose + Na-lactisole
condition, the samples were water + 2 mM Na-lactisole and glucose + 2 mM Na-
lactisole. For the glucose + NaCl condition the samples were water + 2 mM NaCl
and glucose + 2 mM NaCl. For these conditions their task was to identify which
sample was stronger. Again, if unsure they were instructed to guess. The same
scenario was repeated using sucralose in place of glucose comprising three
glucose conditions and three sucralose conditions.
Detection thresholds were measured using a modified staircase method. Starting
with the average threshold for each test sample (7.33 mM for glucose, 20 μM for
sucralose) subjects made their selection (either different from water or the
stronger sample depending on the test condition). If their response was correct,
they were presented with the same concentration until they gave 4 correct
responses. If their response was incorrect, the next higher concentration was
presented in ascending order until 4 correct responses were given in a row at
the same concentration. After 4 correct responses, a descending order of
concentrations was presented until an incorrect response was given. This pattern
was followed until subjects completed 5 reversals in concentration direction
(ascending to descending or descending to ascending). If the 5 reversals spread
over more than 3 concentration-steps, however, testing continued until the 5
reversals remained within a three concentration-step range in order to clamp
variability and avoid random walks on the staircase. The concentrations of the
last 4 reversals were averaged to calculate each subject’s absolute detection
threshold.
### Sweet water taste control
Rinsing with Na-lactisole may result in what is known as “sweet water taste”.
That is, after Na-lactisole treatment, plain water rinses are sometimes
perceived as sweet. To determine if our procedure gave rise to this phenomenon,
we measured subject’s detection thresholds for both glucose and sucralose under
three conditions: 1) when presented against water, 2) with the addition of 2 mM
Na-lactisole, and 3) with 0.8 mM Na-lactisole rinses between samples to inhibit
sweet water taste. The same twelve subjects (7 female, 5 male; mean age = 39
years, S.D. = 13) that participated in Experiment 1 were tested in these three
conditions in duplicate. The concentration of 0.8 mM Na-lactisole for the rinse
was selected in preliminary studies to prevent sweet water taste from 2 mM Na-
lactisole treatment. See.
A repeated measures analysis of variance for the glucose detection thresholds
revealed a significant effect of condition F (2, 66) = 31.50, p \<.00001. Post-
hoc Tukey HSD analyses revealed the detection thresholds for all three
conditions (water, Na-lactisole, and Na-lactisole rinse) were significantly
different from one another, p \<.001, with the detection threshold for glucose
without treatment as the lowest and the detection threshold with the 0.8 mM Na-
lactisole rinses between stimuli as the highest. Therefore, there was no
evidence for an effect of sweet water taste following Na-lactisole treatment in
this procedure. There was no effect of replication, nor an interaction between
condition and replication. The repeated measures analysis of variance for the
sucralose detection thresholds also revealed a significant effect of condition F
(2, 66) = 21.42, p \<.00001. Post-hoc Tukey HSD analyses revealed the detection
thresholds for Na-lactisole treatment and the Na-lactisole treatment with rinse
were significantly higher than the detection threshold for sucralose alone, p
\<.001. Again, there was no evidence for a sweet water taste following Na-
lactisole treatment in this procedure. There was no effect of replication, nor
an interaction between condition and replication.
## Experiment 2A & B
### Hypothesis
Based upon the outcomes of Experiment 1, we hypothesized that if the additional
signaling pathway for glucose involves transport into cells via the sodium
glucose co-transporter (SGLT), then adding NaCl to both glucose and MDG should
enhance detection (lower detection threshold), but not enhance sucralose
detection. MDG is transported by SGLT but is not metabolizable into ATP. Thus,
MDG will determine whether movement of sodium with MDG by an SGLT is sufficient
to enhance detection.
### Subjects
The same subjects who participated in Experiment 1, participated in the
detection threshold experiment comparing glucose to sucralose (Experiment 2A). A
different group of subjects participated in the experiment comparing detection
thresholds of glucose and MDG (Experiment 2B). Eleven healthy adults (7 female,
4 male; mean age = 44 years, S.D. = 12) participated. Subjects were
recruited from among the surrounding community, and paid for their time.
### Stimulus materials
Stimuli included filtered water (Milli-Q Water Purification System); serial
dilutions of D-(+)-glucose (\> 99.5%, Sigma-Aldrich, USA) in filtered water in
1/8 log steps ranging from 0.73–73 mM; serial dilutions of D-(+)-glucose in
filtered water in 1/8 log steps ranging from 0.73–73 mM with 20 mM NaCl (\>99.0%
sodium chloride, Fisher Scientific, USA) added; serial dilutions of sucralose
(\> 98.0%, Sigma-Aldrich, China) in filtered water in 1/8 log steps ranging from
2.0–200 μM; serial dilutions of sucralose in filtered water in 1/8 log steps
ranging from 2.0–200 μM with 20 mM NaCl added; serial dilutions of α-methyl-D-
glucopyranoside (MDG) (\> 99%, Sigma-Aldrich, China) in filtered water in 1/8
log steps ranging from 0.73–73 mM; serial dilutions of MDG in filtered water in
1/8 log steps ranging from 0.73–73 mM with 20 mM NaCl added. Stimuli were
presented at room temperature.
### Detection threshold method
For Experiment 2A, subjects completed 4 conditions each with a replicate. The
conditions measured detection thresholds for glucose and sucralose each with and
without the addition of 20 mM NaCl. The same threshold measurement protocol
described in Experiment 1 was followed for Experiment 2A. Detection thresholds
were measured using a modified staircase method with 5 reversals. In the glucose
condition, the sample pairs were deionized filtered water versus glucose or
deionized filtered water with 20 mM NaCl added versus glucose with 20 mM NaCl
added. In the sucralose condition, the sample pairs were deionized filtered
water versus sucralose or deionized filtered water with 20 mM NaCl added versus
sucralose with 20 mM NaCl added. The subject’s task was to identify which sample
in the pair was stronger. If unsure, they were instructed to pick one.
For Experiment 2B, subjects also completed 4 conditions each with a replicate.
The conditions measured detection thresholds for glucose and MDG each with and
without the addition of 20 mM NaCl. The same threshold measurement protocol
described in Experiment 1 was followed for Experiment 2B. Detection thresholds
were measured using a modified staircase method with 5 reversals. In the glucose
condition, the sample pairs were deionized filtered water versus glucose or
deionized filter water with 20 mM NaCl added versus glucose with 20 mM NaCl
added. In the MDG condition the samples were deionized filtered water versus MDG
or deionized filtered water with 20 mM NaCl added versus MDG with 20 mM NaCl
added. The subject’s task was to identify which sample in the pair was stronger.
If unsure, they were instructed to pick one.
## Experiment 3
### Hypothesis
Based upon the outcome of Experiments 2A and 2B, we hypothesized if oral glucose
signaling involves an SGLT, then the pharmacological SGLT inhibitor, phlorizin,
should increase absolute detection thresholds for glucose and MDG, but not for
sucralose, and have no effect on fructose that is transported by GLUT5 instead
of SGLT.
### Subjects
Eleven of the same subjects that participated in Experiment 1participated in the
glucose and sucralose segments of Experiment 3 (6 female, 5 male; mean age =
39 years, S.D. = 13). For the MDG segment subjects were the same individuals
that participated in Experiment 2B (7 females and 4 males with a mean age = 44
years with S.D. = 12). Finally, for the fructose segment subjects included 7
females and 5 males with a mean age = 36 years with S.D. = 12.
### Stimulus materials
Stimuli included filtered water (Milli-Q Water Purification System); serial
dilutions of D-(+)-glucose (\> 99.5%, Sigma-Aldrich, USA) in filtered water in
1/8 log steps ranging from 0.73–73 mM; serial dilutions of D-(+)-glucose in
filtered water in 1/8 log steps ranging from 0.73–73 mM with 20 mM NaCl (\>99.0%
sodium chloride, Fisher Scientific, USA) added; serial dilutions of
D-(+)-glucose in filtered water in 1/8 log steps ranging from 0.73–73 mM with 20
mM NaCl and 0.2 mM phlorizin (\>98.0%, Cayman Chemical Company, USA) added;
serial dilutions of sucralose (\> 98.0%, Sigma-Aldrich, China) in filtered water
in 1/8 log steps ranging from 2.0–200 μM; serial dilutions of sucralose in
filtered water in 1/8 log steps ranging from 2.0–200 μM with 20 mM NaCl added;
serial dilutions of sucralose in filtered water in 1/8 log steps ranging from
2.0–200 μM with 20 mM NaCl and 0.2 mM phlorizin added; serial dilutions of
α-methyl-D-glucopyranoside (MDG) (\> 99%, Sigma-Aldrich, China) in filtered
water in 1/8 log steps ranging from 0.73–73 mM; serial dilutions of α-methyl-D-
glucopyranoside (MDG) in filtered water in 1/8 log steps ranging from 0.73–73 mM
with 20 mM NaCl added; serial dilutions of α-methyl-D-glucopyranoside (MDG) in
filtered water in 1/8 log steps ranging from 0.73–73 mM with 20 mM NaCl and 0.2
mM phlorizin added; serial dilutions of D--fructose (\> 99%, Sigma-Aldrich, USA)
in filtered water in 1/8 log steps ranging from 0.56–56 mM; serial dilutions of
D--fructose in filtered water in 1/8 log steps ranging from 0.56–56 mM with 20
mM NaCl added; serial dilutions of D--fructose in filtered water in 1/8 log
steps ranging from 0.56–56 mM with 20 mM NaCl and 0.2 mM phlorizin added.
Stimuli were presented at room temperature.
### Detection threshold method
There were four segments in Experiment 3. One for each sweetener: glucose,
fructose, MDG, and sucralose. In each segment, subjects completed 3 conditions
each with a replicate. The conditions measured detection thresholds for the
sweetener alone, with the addition of 20 mM NaCl, and with the addition of 20 mM
NaCl and 0.2 mM phlorizin. The same protocol described in Experiments 1 and 2
was followed for Experiment 3. Detection thresholds were measured using a
modified staircase method with 5 reversals. For each sweetener type (glucose,
fructose, MDG, and sucralose the sample pairs were filtered water versus
sweetener alone, filtered water with 20 mM NaCl versus sweetener with 20 mM
NaCl, filtered water with 20 mM NaCl + 0.2 mM phlorizin added versus sweetener
with 20 mM NaCl + 0.2 mM phlorizin added. The subject’s task was to identify
which sample was stronger. If unsure, they were instructed to pick one.
# Results
All analyses were conducted using Statistica software (Version 13.5.0.17,
Tibco), using an alpha value of \<0.05 for allowing Type I errors.
## Experiment 1
Factorial repeated-measures analysis of variance (ANOVA) were conducted to
compare the main effects of condition (water, Na-lactisole, NaCl), and
replication (1, 2) and the interaction effect on detection thresholds for both
glucose and sucralose. There was a main effect of condition for each sweetener
such that the Na-lactisole treatment significantly raised threshold levels. For
glucose F (2, 66) = 24.86, p \<.0001, post-hoc Tukey HSD analyses showed the
detection threshold with Na-lactisole (mean = 41.8 mM, S.D. = 25.9) was
significantly higher than both glucose alone (mean = 13.3 mM, S.D. = 5.5) and
glucose with NaCl (mean = 13.8 mM, S.D. = 4.6), p \<.001. For sucralose F (2,
66) = 33.00, p \<.0001, post-hoc Tukey HSD analyses showed the detection
threshold with Na-lactisole (mean = 100.0 μM, S.D. = 73.7) was significantly
higher than both sucralose alone (mean = 11.9 μM, S.D. = 5.4) and sucralose with
NaCl (mean = 11.9 μM, S.D. = 5.0), p \<.001. The water and NaCl conditions did
not differ from each other for either sweetener. The 2 mM NaCl was added to
control for the effects of 2 mM sodium from the Na-lactisole. There was no
effect of replication nor an interaction between condition and replication for
either sweetener. As expected, the Na-lactisole treatment decreased sensitivity
to the sweeteners glucose and sucralose (See).
Also as expected the Na-lactisole treatment had a significantly larger impact on
sucralose than on glucose. After Na-lactisole treatment and with water rinsing
between samples, the ‘fold’ increase in threshold concentration for glucose was
x 3.1 and for sucralose x 10.0, t (11) = -2.99, p = 0.012 (See). Overall, our
hypothesis is supported by these data; Na-lactisole interfered with detecting
sucralose approximately three times more than it interfered with glucose
detection.
## Experiment 2A
Factorial repeated-measures ANOVA were conducted to compare the main effects of
condition (with and without NaCl), and replication (1, 2) and the interaction
effect on detection thresholds for both glucose and sucralose. For each
sweetener there was a significant effect of condition. For glucose the addition
of NaCl significantly lowered the detection threshold (glucose mean = 13.3 mM,
S.D. = 5.5 while glucose + NaCl mean = 7.2 mM, S.D. = 1.6), F (1, 44) = 22.71, p
\<.0001. Conversely, for sucralose the addition of NaCl significantly raised the
detection threshold (sucralose mean = 11.9 μM, S.D. = 5.4 while sucralose + NaCl
mean = 19.1 μM, S.D. = 7.7), F (1, 44) = 13.88, p \<.001. Thus, the addition of
20 mM NaCl enhanced sensitivity to glucose (lowered threshold concentration) by
46% on average, whereas the addition of 20 mM NaCl diminished sensitivity to
sucralose (raised threshold concentration) by 161% on average. There was no
effect of replication nor an interaction between condition and replication for
either sweetener (See).
The hypothesis that NaCl would enhance sensitivity to glucose but not sucralose
is supported by these data. These data are consistent with SGLT participating in
glucose signaling by moving glucose and sodium together into the cells.
## Experiment 2B
A factorial repeated-measures ANOVA was conducted to compare the main effects of
sweetener (glucose or MDG), condition (with and without NaCl), and replication
(1, 2) and the interaction effects on the detection thresholds. The ANOVA
revealed there was not a significant difference between the sweeteners,
thresholds are similar, but there was a main effect of condition F (3, 80) =
6.28, p \<.001. Post-hoc Tukey HSD analyses revealed the addition of NaCl
lowered the detection thresholds for each sweetener, glucose mean = 12.6 mM,
S.D. = 4.2 while glucose + NaCl mean = 9.3 mM, S.D. = 2.8, p \<.01; MDG mean =
11.7 mM, S.D. = 2.9 while MDG + NaCl mean = 9.0 mM, S.D. = 1.8, p \<.05. There
was no effect of replication nor any significant interaction effects (See). The
lowering of the detection thresholds for both glucose and MDG by the addition of
NaCl is further evidence that SGLT is involved in moving glucose and its analog
into the cell, suggesting a second signaling pathway. Furthermore, it suggests
that movement of MDG with sodium by SGLT is sufficient to alter thresholds and
ATP generation is not required.
## Experiment 3
Factorial repeated-measures ANOVA were conducted to compare the main effects of
condition (water, NaCl, and phlorizin), and replication (1, 2) and the
interaction effects on detection thresholds for glucose, MDG, fructose, and
sucralose. The results for glucose revealed a significant main effect for
condition F (2, 60) = 58.27, p \<.00001. Post-hoc Tukey HSD analyses showed the
detection threshold for glucose with NaCl and phlorizin added (mean = 39.9 mM,
S.D. = 16.8) was significantly higher than the detection threshold for glucose
alone (mean = 13.5 mM, S.D. = 5.7) and for glucose with NaCl (mean = 7.2 mM,
S.D. = 1.6 mM), p \<.001. Similar to glucose, there was a main effect for
condition for MDG, F (2, 60) = 62.05, p \<.00001. Post-hoc Tukey HSD analyses
showed the detection threshold for MDG with NaCl and phlorizin added (mean =
37.6 mM, S.D. = 15.4) was significantly higher than the detection threshold for
MDG alone (mean = 11.7 mM, S.D. = 2.9) and for MDG with NaCl (mean = 9.0 mM,
S.D. = 1.8), p \<.001. For fructose, there were no significant differences in
condition. Finally, for sucralose there was a main effect for condition F (2,
60) = 3.45, p \<.05. Post-hoc Tukey HSD analyses showed the detection threshold
for sucralose with NaCl (mean = 18.3 mM, S.D. = 7.5 mM) was significantly higher
than sucralose alone, (mean = 12.2 mM, S.D. = 5.6 mM) p \<.05, but no
significant differences for the phlorizin condition (mean = 16.2 mM, S.D. = 9.6
mM). There was no effect of replication nor an interaction between condition and
replication for any of the sweeteners (See).
Phlorizin increased the absolute detection thresholds for both glucose and MDG
in 100% of the subjects (11 of 11) by approximately a 3-fold increase on
average. Whereas, phlorizin had no effect on absolute detection thresholds for
fructose or sucralose. The pharmacological SGLT inhibitor phlorizin had a
profound impact on impeding every subject’s ability to detect glucose and MDG
but did not impede their detection of fructose nor sucralose, further supporting
the idea that oral glucose signaling involves a second pathway involving SGLT.
# Discussion
These rinse-and-expectorate experiments point to the ability of humans to sense
glucose orally via a signaling pathway that includes the sodium-glucose
cotransporters (SGLTs). The SGLT is an initial step in what is described as the
glucose metabolic signaling pathway, and our data suggest this exists in
parallel to the traditional T1R sweet taste pathway for oral glucose sensing in
humans. In the first experiment, we used the T1R (class 1 taste receptor)
inhibitor Na-lactisole to block sweet taste. Na-lactisole had a much larger
impact on sucralose detection thresholds than it did on glucose thresholds.
illustrates that detection thresholds for sucralose increased in concentration
(indicating decreased sensitivity) by more than 8-fold in the presence of Na-
lactisole, whereas glucose thresholds only increased by 3-fold. We interpret
this as supporting the idea that there are two sugar sensing coding channels in
the mouth: one for signaling the sweet taste of sugars and a second that we
hypothesized is based on the metabolic signaling pathway for saccharides.
Sucralose only engages the T1R-sweet taste receptor, so inhibition of this
receptor has a larger impact on its detection. Glucose, however, engages both
signaling channels and in the presence of Na-lactisole continues to be signaled
by a second pathway.
In a second experiment, we tested whether the SGLT glucose transporters are
involved with the initial step of moving the saccharide into the cell. Since the
SGLTs must move sodium together with glucose, we hypothesized that adding sodium
(NaCl) at levels comparable to the detection thresholds for glucose (\~20 mM at
the high end) would lower the threshold for glucose but not for the non-caloric
sweetener sucralose by enhancing glucose transport into the cells. The SGLT1
transporter moves two sodium ions for each glucose molecule and the SGLT2
transporter moves one sodium ion for each glucose molecule; therefore, this
concentration of sodium would improve movement for both transporters. We found
that glucose thresholds decreased (indicating increased sensitivity) in the
presence of 20 mM NaCl by approximately 50%, but sucralose thresholds did not
decrease with added NaCl (See). In fact, the addition of NaCl interfered with
sucralose sensitivity and raised detection threshold concentrations modestly.
This is most likely due to the weak salty taste of 20 mM NaCl cognitively
suppressing detection of sucralose by acting as a sweet-taste masking agent.
This influence of the salty taste would also have occurred for glucose, but the
impact of added Na on the SGLT mechanism overcame this masking effect.
In the third experiment, we used phlorizin as an SGLT inhibitor to interfere
with glucose signaling in the mouth. Whereas the addition of sodium was expected
to enhance glucose signaling and decrease detection thresholds of glucose, the
addition of phlorizin was predicted to interfere with glucose signaling and
increase glucose detection thresholds. We found the addition of 2 mM phlorizin
increased glucose detection thresholds compared to the glucose + NaCl condition
by approximately 5-fold, whereas sucralose detection thresholds were unaffected
by the addition of phlorizin (See). Furthermore, there was no effect on
fructose thresholds of the addition of phlorizin. This is consistent with
glucose taste signaling utilizing an SGLT transporter, but fructose does not use
SGLT. Phlorizin is a natural phenolic glucoside chalcone common in apple trees,
which when dimerized and oxidized is responsible for the yellow-brown color of
apple juice and cider. It is a non-selective inhibitor of the SGLTs, so we
cannot conclude from the present data whether one particular SGLT (SGLT1 or
SGLT2) is more important for glucose signaling. Note that there are currently
six known forms of SGLT transporters, although SGLT3 in humans is thought to be
a glucose receptor, not a transporter. There is recent evidence that phlorizin
may also inhibit the GLUTs in addition to SGLTs. We do not believe this is
reflected in our results, however, as phlorizin had no impact on fructose,
supporting the involvement of SGLTs but not GLUT2, GLUT5, or GLUT8. We also saw
NaCl enhance glucose thresholds (See), further indication of a role for SGLT
and not GLUTs in this effect. Regardless, we have established that we can
manipulate human oral sensitivity to glucose positively with the addition of
sodium and negatively with the addition of phlorizin, all without similar impact
on the sweetener sucralose. This bidirectional psychopharmacological approach
has strongly implicated the SGLTs as participating in the first step of sugar
transport into the cell. Logically, it is likely that other sugars besides
glucose, such as galactose, can be transported by SGLT in taste tissue, and
sugars that do not engage SGLT, such as fructose, may be transported via GLUT2
and GLUT8 in taste tissue and oxidized in taste tissue to produce ATP and close
K<sub>ATP</sub> channels.
The glucose metabolic signaling pathway combined with the sweet taste pathway
creates a striking parallel between the glucose signaling abilities of the
pancreatic beta islet cells of Langerhans and the signaling abilities of the
oral cavity (most likely taste tissue). It has previously been reported
that mice possess within oral taste buds: 1) sugar transporters, 2) kinases
required to convert sugars into ATP, and 3) an ATP sensor in the form of an ion
channel. Whether any of these exist in humans has been previously unknown. In
the present work, we also included MDG as a taste stimulus. This glucose analog
can be transported by SGLT but does not get oxidized to produce ATP, as it
cannot be metabolized. This allows us to distinguish whether the first step in
the metabolic signaling pathway can produce oral signals independently of the
remainder of the traditional metabolic signaling pathway, such as ATP generation
and the closure of K<sub>ATP</sub> channels. We found that MDG oral detection
thresholds were enhanced by the addition of NaCl and impaired by the addition of
phlorizin (See). This indicates that the transport of sodium by SGLT with
glucose and MDG can activate cells irrespective of whether ATP acts on
K<sub>ATP</sub> channels. Logically, the closure of K<sub>ATP</sub> channels in
this signaling pathway may also further activate cells.
Collectively, these data allow us to screen enhancers of glucose signaling via
stimulation of the SGLT pathway for their impact on oral sugar signaling. These
may involve any form of pharmacological SGLT enhancement including the addition
of sodium to glucose, SGLT pharmacological modulation (allosteric or otherwise)
to better transport glucose across the membrane, and any glucose mimetic that
can be transported into cells via SGLT. We speculate that it may be possible to
reduce sugar levels in beverages and foods by enhancing glucose via the
metabolic signaling pathway, especially at the level of SGLT transport. We note
that SGLT signals may contribute to sweet taste, but may also contribute to an
independent non-sweet signal that conveys the presence of glucose. This idea is
suggested by work in T13-Knock-Out mice which showed anticipatory insulin
release to a glucose load, but did not show a behavioral preference for glucose
in water. It is possible that oral SGLT contributes to glucose reward in humans
either directly or indirectly via anticipatory metabolic regulatory reflexes,
but note that oral SGLT in mice does not appear to contribute to preference
In summary, the experiments presented here utilizing the T1R inhibitor Na-
lactisole, the co-transport agent for SGLT added NaCl, and the SGLT inhibitor
phlorizin support the idea that SGLTs are involved in the oral perception of
glucose, but not the perception of sucralose or fructose. These studies show
that a non-T1R oral signaling pathway profoundly affects absolute detection of
glucose but not sucralose or fructose. Future studies will determine the utility
of this SGLT-linked oral glucose signal in human psychology and physiology.
# Supporting information
10.1371/journal.pone.0256989.r001
Author response to previous submission
17 May 2021
10.1371/journal.pone.0256989.r002
Decision Letter 0
Glendinning
John I.
Academic Editor
2021
John I. Glendinning
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
8 Jun 2021
PONE-D-21-16275
Evidence that Human Oral Glucose Detection Involves A Sweet Taste Pathway and A
Glucose Transporter Pathway
PLOS ONE
Dear Dr. Breslin,
Thank you for submitting your manuscript to PLOS ONE. After careful
consideration, we feel that it has merit but does not fully meet PLOS ONE’s
publication criteria as it currently stands. Therefore, we invite you to submit
a revised version of the manuscript that addresses the points raised during the
review process.
Dear Dr. Breslin and coauthors,
You have done a superb job addressing most of the concerns raised by the
reviewers. In particular, I commend you for conducting the additional
experiments. As you note, they strengthen your study and provide robust support
for the central claim of your study--that SGLT1 is involved in oral glucose
detection in humans. This is clearly a novel and important finding.
Overall, I believe that you have addressed the majority of the reviewers'
original concerns. You will see, however, that Reviewer 1 has some remaining
concerns that need to be addressed in your revision. I also have a few editorial
suggestions.
1\. Your current title is: "Evidence that Human Oral Glucose Detection Involves
A Sweet Taste Pathway and A Glucose Transporter Pathway." This title implies
that you have identified two pathways for detecting glucose in the oral cavity.
This is not the case. What you have established is that one can systematically
alter glucose detection thresholds by modulating the activity of SGLT1. While I
do not mean to diminish the importance of this finding, you currently do not
know whether modulating the activity of SGLT1 alters downstream voltage-
sensitive events in the T1r2-r3 pathway or whether it activates a
T1r2-r3-independent pathway. Thus, I recommend that you scale back the scope of
your title.
2\. End of first paragraph of Introduction: You state: "Clearly sugars engage a
second signaling pathway for calories that noncaloric sweeteners fail to engage
post-prandially." I do not think that this statement follows logically from the
five reasons you provide for why "...diet sodas have never captured a major
share of the beverage market." Reviewer 1 has raised additional concerns about
this statement.
3\. Line 1, second paragraph of Introduction: Change "mice" to "mouse"
4\. Line 4, second paragraph of Introduction: The following phrase seems
awkward: "...the transportation of glucose". I think that it is more
conventional to say "...the transport of glucose"
5\. Line 5, second paragraph of Introduction: Why don't you refer directly to
the GLUTs? That would seem to be clearer than a vague reference to "...other
transporters".
6\. Lines 5-6, second paragraph of Introduction: I think that the following
wording is accurate: "...the conversion of glucose into several ATP
molecules..." Glucose is not actually converted into ATP molecules. The energy
in the glucose molecule is used by glycolysis, krebs cycle and oxidative
phosphorylation to add high-energy phosphate bonds to AMP and ADP.
7\. Line 10, second paragraph of Introduction: I recommend changing "mouse
tissue" to "mouse taste bud cells."
8\. Lines 10-13, second paragraph of Introduction: I do not think that the prior
literature has established that "taste bud cells in the mouth of mice are
capable of identifying when a “sweetener” is metabolizable." Instead, I think
this literature has established that murine taste bud cells may be able to
detect elevations in salivary glucose via a T1r2+r3-independent mechanism.
9\. In the last paragraph of the Introduction, you state: "Were such a signaling
system to exist and be functional in the human mouth, it would have implications
for oral signaling of metabolizable sugars and could help explain why humans
overwhelmingly prefer sugared beverages to non-caloric beverages." I recommend
that you dial-back this statement a bit for two reasons. First, because your
paper addresses threshold (not suprathreshold) effects of SGLT1, you are
creating false expectations in your readers. Second, the use of "metabolizable
sugar" is a bit confusing. As noted by Reviewer 1, fructose may not be
metabolized in taste cells, but it is certainly metabolized in the liver. Thus,
fructose is a metabolizable sugar that does not activate SGLT1.
10\. I was surprised that you did not incorporate the findings from a recent
mouse paper \[Yasumatsu K, Ohkuri T, Yoshida R, Iwata S, Margolskee RF, Ninomiya
Y (2020) Sodium-glucose cotransporter 1 as a sugar taste sensor in mouse tongue.
Acta Physiologica 230: e13560. doi: 10.1111/apha.13529\], as it is highly
relevant and supportive of your general hypothesis.
Please submit your revised manuscript by Jul 23 2021 11:59PM. If you will need
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Kind regards,
John I. Glendinning, PhD
Academic Editor
PLOS ONE
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Reviewer \#1: This revised manuscript is substantially enhanced by the addition
of MDG and fructose experiments which support the involvement of SGLT1 sensing
in oral glucose detection in humans. However, the characterization of this oral
SGLT1-glucose sensing pathway as a "caloric" or " metabolic signaling pathway"
is misleading. The SGLT1 pathway does not respond to fructose which is a
"caloric" and "metabolizable" sugar. It is also unclear how much sucrose
activates the SGLT1 taste pathway given that sucrose must be first hydrolyzed to
glucose in the mouth to have this action. The authors should revise their
description of the SGLT1 taste pathway. Also, for completeness, the authors
should mention that galactose is a ligand for SGLT1 although, given the
relatively low amount of free galactose in foods, this sugar may have minimal
effects on the oral SGLT1 pathway.
The authors describe MDG as a ligand for SGLT1 but don't mention that it is also
a ligand for the T1R2/T1R3 receptor and thus has a sweet taste to humans as it
apparently has to mice as judged by their avidity for this glucose analog. The
authors speculate that the SGLT1 taste pathway may contribute to the sweetness
(or palatability) of sugars in humans and contribute to their preference for
sugars over non-nutritive sweeteners. However, this does not appear to be the
case in mice. SGLT1 KO and WT mice showed identical preferences for MDG over
water in 3-min, 2-bottle tests (Sclafani, Koepsell and Ackroff, 2016).
Furthermore, SGLT1 KO and WT mice showed similar preferences for a glucose +
saccharin solution over a saccharin in a 1-h choice test. The KO mice consumed
less glucose and MDG than WT mice which was attributed to their failure to
normally absorb these sugars in the intestine. The authors should acknowledge
that the oral glucose-sensing SGLT1 pathway does not appear to mediate oral
sugar preference in mice although, as discussed next, it is essential for post-
oral glucose preference conditioning. This does not preclude them from
speculating about a role of SGLT1 taste pathway in sugar preference in humans.
The following statement in the Introduction should be supported with citations:
"Clearly sugars engage a second signaling pathway for calories that noncaloric
sweeteners fail to engage post-prandially." Presumably, the authors are
referring to rodent and human studies showing that the post-oral actions of
glucose and glucose-containing carbohydrates (sucrose, maltodextrin) condition
preferences. However, describing this post-prandial pathway as "for calories" is
questionable given the many rodent studies showing that glucose is much more
effective than fructose in activating the post-prandial pathway. Of particular
relevance here are studies showing that mice learn to prefer glucose but not
isocaloric fructose over non-nutritive sweeteners (sucralose, Ace K), an outcome
thought to be mediated by intestinal SGLT1 signaling (e.g., Sclafani & Ackroff,
2017; Tan et al., 2020). The effectiveness of post-prandial fructose in
conditioning sugar preferences in humans is uncertain because the few published
human conditioning studies all used maltodextrins.
\*\*\*\*\*\*\*\*\*\*
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10.1371/journal.pone.0256989.r003
Author response to Decision Letter 0
17 Aug 2021
1\. Your current title is: "Evidence that Human Oral Glucose Detection Involves
A Sweet Taste Pathway and A Glucose Transporter Pathway." This title implies
that you have identified two pathways for detecting glucose in the oral cavity.
This is not the case. What you have established is that one can systematically
alter glucose detection thresholds by modulating the activity of SGLT1. While I
do not mean to diminish the importance of this finding, you currently do not
know whether modulating the activity of SGLT1 alters downstream voltage-
sensitive events in the T1r2-r3 pathway or whether it activates a
T1r2-r3-independent pathway. Thus, I recommend that you scale back the scope of
your title.
We respectfully disagree. We can take out T1R2/3 with lactisole and still have
glucose transduction occur. We can also take out SGLT with phlorizin and still
have sucralose be sweet. Therefore, the T1R2/3 and SGLT participate forin
glucose transduction and sweet taste transduction with inhibition of the other
and are, therefore, independent. They may converge at some point, but at least
initially they are able to function are two independently of each other as taste
systems.
2\. End of first paragraph of Introduction: You state: "Clearly sugars engage a
second signaling pathway for calories that noncaloric sweeteners fail to engage
post-prandially." I do not think that this statement follows logically from the
five reasons you provide for why "...diet sodas have never captured a major
share of the beverage market." Reviewer 1 has raised additional concerns about
this statement.
We changed to: We hypothesize an additional explanation that sugars may engage a
second oral signaling pathway for calories that noncaloric sweeteners fail to
engage..
3\. Line 1, second paragraph of Introduction: Change "mice" to "mouse"
OK
4\. Line 4, second paragraph of Introduction: The following phrase seems
awkward: "...the transportation of glucose". I think that it is more
conventional to say "...the transport of glucose"
OK
5\. Line 5, second paragraph of Introduction: Why don't you refer directly to
the GLUTs? That would seem to be clearer than a vague reference to "...other
transporters".
We changed to…..Other transporters, such as GLUTs.
6\. Lines 5-6, second paragraph of Introduction: I think that the following
wording is accurate: "...the conversion of glucose into several ATP
molecules..." Glucose is not actually converted into ATP molecules. The energy
in the glucose molecule is used by glycolysis, krebs cycle and oxidative
phosphorylation to add high-energy phosphate bonds to AMP and ADP.
Changed to "...the oxidation of glucose to produce several ATP molecules..."
7\. Line 10, second paragraph of Introduction: I recommend changing "mouse
tissue" to "mouse taste bud cells."
OK
8\. Lines 10-13, second paragraph of Introduction: I do not think that the prior
literature has established that "taste bud cells in the mouth of mice are
capable of identifying when a “sweetener” is metabolizable." Instead, I think
this literature has established that murine taste bud cells may be able to
detect elevations in salivary glucose via a T1r2+r3-independent mechanism.
We are asked to switch the verb capable to the verb able. Given our wording that
the "system appears capable" we believe our wording is still appropriate.
9\. In the last paragraph of the Introduction, you state: "Were such a signaling
system to exist and be functional in the human mouth, it would have implications
for oral signaling of metabolizable sugars and could help explain why humans
overwhelmingly prefer sugared beverages to non-caloric beverages." I recommend
that you dial-back this statement a bit for two reasons. First, because your
paper addresses threshold (not suprathreshold) effects of SGLT1, you are
creating false expectations in your readers. Second, the use of "metabolizable
sugar" is a bit confusing. As noted by Reviewer 1, fructose may not be
metabolized in taste cells, but it is certainly metabolized in the liver. Thus,
fructose is a metabolizable sugar that does not activate SGLT1.
We changed sugars to glucose and emphasize preference here as an implication of
our work rather than suprathreshold sweetness.
Changed to: Were such a signaling system to exist and be functional in the human
mouth, it would have implications for oral signaling of the metabolizable sugar
glucose and could help explain preference for sugared beverages over non-caloric
sweetener beverages.
10\. I was surprised that you did not incorporate the findings from a recent
mouse paper \[Yasumatsu K, Ohkuri T, Yoshida R, Iwata S, Margolskee RF, Ninomiya
Y (2020) Sodium-glucose cotransporter 1 as a sugar taste sensor in mouse tongue.
Acta Physiologica 230: e13560. doi: 10.1111/apha.13529\], as it is highly
relevant and supportive of your general hypothesis.
We cite this paper now.
2\. Thank you for stating the following in the Acknowledgments Section of your
manuscript:
"This research was funded in part by a grant from the Suntory Global Innovation
Center Limited
to PASB. The funder consulted on the general conception of the study and
provided support in
the form of salaries for the authors (PASB, AT, LJF) and a scientist from
Suntory (AI) helped
collect data under the supervision of the Monell Center. The specific roles of
the authors are
articulated in the “Author Contributions” section. The funder did not play a
role in the specific
study design, data analyses, decision to publish, or preparation of the
manuscript."
We note that you have provided funding information that is not currently
declared in your Funding Statement. However, funding information should not
appear in the Acknowledgments section or other areas of your manuscript. We will
only publish funding information present in the Funding Statement section of the
online submission form.
Please remove any funding-related text from the manuscript and let us know how
you would like to update your Funding Statement. Currently, your Funding
Statement reads as follows:
"The funder did not play a role in the specific study design, data analyses,
decision to publish, or preparation of the manuscript. "
The revised funding statement should read:
This research was funded in part by a grant from the Suntory Global Innovation
Center Limited to PASB. The funder consulted on the general conception of the
study and provided support in the form of salaries for the authors (PASB, AT,
LJF), and a scientist from Suntory (AI) helped collect data under the
supervision of the Monell Center. The funder did not play a role in the specific
study design, data analyses, decision to publish, or preparation of the
manuscript.
Add to online-submission form
Additionally, because some of your funding information pertains to commercial
funding, we ask you to provide an updated Competing Interests statement,
declaring all sources of commercial funding.
In your Competing Interests statement, please confirm that your commercial
funding does not alter your adherence to PLOS ONE Editorial policies and
criteria by including the following statement: "This does not alter our
adherence to PLOS ONE policies on sharing data and materials.”
as detailed online in our guide for authors
<http://journals.plos.org/plosone/s/competing-interests>. If this statement is
not true and your adherence to PLOS policies on sharing data and materials is
altered, please explain how.
Please include the updated Competing Interests Statement and Funding Statement
in your cover letter. We will change the online submission form on your behalf.
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
5\. Review Comments to the Author
Please use the space provided to explain your answers to the questions above.
You may also include additional comments for the author, including concerns
about dual publication, research ethics, or publication ethics. (Please upload
your review as an attachment if it exceeds 20,000 characters)
Reviewer \#1: This revised manuscript is substantially enhanced by the addition
of MDG and fructose experiments which support the involvement of SGLT1 sensing
in oral glucose detection in humans. However, the characterization of this oral
SGLT1-glucose sensing pathway as a "caloric" or " metabolic signaling pathway"
is misleading. The SGLT1 pathway does not respond to fructose which is a
"caloric" and "metabolizable" sugar. It is also unclear how much sucrose
activates the SGLT1 taste pathway given that sucrose must be first hydrolyzed to
glucose in the mouth to have this action. The authors should revise their
description of the SGLT1 taste pathway. Also, for completeness, the authors
should mention that galactose is a ligand for SGLT1 although, given the
relatively low amount of free galactose in foods, this sugar may have minimal
effects on the oral SGLT1 pathway.
We now state near the end of the discussion:
Logically, it is likely that other sugars besides glucose, such as galactose,
can be transported by SGLT in taste tissue (Harada et al., 2012), and sugars
that do not engage SGLT, such as fructose, may be transported via GLUT2 and
GLUT8 in taste tissue (Sukumaran et al, 2016) and oxidized in taste tissue to
produce ATP and close KATP channels (Berger et al, 2020).
The authors describe MDG as a ligand for SGLT1 but don't mention that it is also
a ligand for the T1R2/T1R3 receptor and thus has a sweet taste to humans as it
apparently has to mice as judged by their avidity for this glucose analog. The
authors speculate that the SGLT1 taste pathway may contribute to the sweetness
(or palatability) of sugars in humans and contribute to their preference for
sugars over non-nutritive sweeteners. However, this does not appear to be the
case in mice. SGLT1 KO and WT mice showed identical preferences for MDG over
water in 3-min, 2-bottle tests (Sclafani, Koepsell and Ackroff, 2016).
Furthermore, SGLT1 KO and WT mice showed similar preferences for a glucose +
saccharin solution over a saccharin in a 1-h choice test. The KO mice consumed
less glucose and MDG than WT mice which was attributed to their failure to
normally absorb these sugars in the intestine. The authors should acknowledge
that the oral glucose-sensing SGLT1 pathway does not appear to mediate oral
sugar preference in mice although, as discussed next, it is essential for post-
oral glucose preference conditioning. This does not preclude them from
speculating about a role of SGLT1 taste pathway in sugar preference in humans.
We now acknowledge that SGLT1KO mice have sugar preference unaffected but still
speculate about human oral influences on preference for sugars. We write: It is
possible that oral SGLT contributes to glucose reward in humans either directly
or indirectly via anticipatory metabolic regulatory reflexes, but not that oral
SGLT in mice does not appear to contribute to preference (Sclafani et al.,
2016).
The following statement in the Introduction should be supported with citations:
"Clearly sugars engage a second signaling pathway for calories that noncaloric
sweeteners fail to engage post-prandially."
We now cite the paper showing that T1R KO mice have no responses to non-caloric
sweeteners but continue to show responses to glucose and sucrose. Margolskee et
al.
Presumably, the authors are referring to rodent and human studies showing that
the post-oral actions of glucose and glucose-containing carbohydrates (sucrose,
maltodextrin) condition preferences. However, describing this post-prandial
pathway as "for calories" is questionable given the many rodent studies showing
that glucose is much more effective than fructose in activating the post-
prandial pathway. Of particular relevance here are studies showing that mice
learn to prefer glucose but not isocaloric fructose over non-nutritive
sweeteners (sucralose, Ace K), an outcome thought to be mediated by intestinal
SGLT1 signaling (e.g., Sclafani & Ackroff, 2017; Tan et al., 2020). The
effectiveness of post-prandial fructose in conditioning sugar preferences in
humans is uncertain because the few published human conditioning studies all
used maltodextrins.
In this context, we avoid discussing post-prandial feedback mechanisms in
humans.
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
10.1371/journal.pone.0256989.r004
Decision Letter 1
Glendinning
John I.
Academic Editor
2021
John I. Glendinning
This is an open access article distributed under the terms of the
Creative Commons Attribution License
, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
20 Aug 2021
Evidence that Human Oral Glucose Detection Involves A Sweet Taste Pathway and A
Glucose Transporter Pathway
PONE-D-21-16275R1
Dear Dr. Breslin,
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# Introduction
Cells recognize and respond to external mechanical signals in their surrounding
microenvironment such as an extracellular matrix (ECM). There have been various
reports into the effects of mechanical forces *in vitro*, including applied
substrate strain and substrate stiffness, on cell behaviors. Various cell types
such as endothelial cells, fibroblasts, and smooth muscle cells exhibit
morphological changes and migration that are dependent on substrate stiffness
although the preferential stiffness range is dependent on cell type. Recent
studies have revealed that neural cells including several types of neurons and
glial cells, which are the main constituents of the central nervous system
(CNS), respond to mechanical cues. There have been various reports on how the
mechanical properties of ECM affect neuronal development, growth, disorders and
regeneration.
One example is traumatic injury to the CNS, which changes the mechanical
properties of the surrounding environment. Injury to the CNS induces the
formation of glial scars, which are mainly composed of glial cells that are
responsible for the local immune response and wound healing processes. It has
been reported that a glial scar prevents neurons from regenerating and
elongating their neurites due to many obstacles including an injury environment
filled with deleterious factors. Although the role of the glial scar is under
discussion, a change in its mechanical properties seems to act as a mechanical
barrier to axon regeneration. As another example, devices implanted into the
body become encapsulated due to a foreign body reaction. In the CNS, this can
lead to loss of functionality in electrodes since a mechanical mismatch between
nervous tissue and the devices induces the formation of glial scars. From the
above, mechanical matching between nervous tissue and a lesion or implant should
be considered as regards developing neuronal regeneration and bio-interfaces.
Although there have been reports on neurite elongation and the dependence of its
molecular pathways on the mechanical properties of the surrounding environment,
less is known about how the mechanical properties affect the first morphological
step of neurite formation. Neurite extension from a cell body is the first step
in forming a functional nervous system and realizing neuronal regeneration. When
neurons generate and grow, they initially attach themselves to the surrounding
ECM and sprout neurites from a spherical cell body. Neurite extension, which is
differentiated in the axon and dendrites, forms the basis of proper neuronal
connectivity and brain function. Morphological changes in hippocampal neurons
from neurite initiation to neurite extension have been well studied *in vitro*.
The neuronal development stages can be classified as follows. First, immature
neurons with a localized bud from a cell membrane are classified as stage 1. The
transformation of a bud into neurites of approximately equal lengths is
classified as stage 2. Neurons that possess one neurite considerably longer than
the rest are classified as stage 3. Neuritogenesis occurs during stage a 1 to 2
transition. First, the rearrangement of F-actin assembly in the bud induces the
leading edge of the cell body to protrude. Next, microtubules and other
components must be engaged with the F-actin assembly; specifically, F-actin
bundles can facilitate microtubule elongation. Finally, the proper consolidation
of the proteins leads to neuritogenesis. Actin and microtubules are the main
components of neurites, and the proper organization of F-actin assembly occurs
prior to microtubule organization in the process of neurite formation. Although
a variety of neuronal shapes appear during development, the initial sprouting of
neurites seems to follow the morphological criteria described above. When
neurons are cultivated on a substrate *in vitro*, they initially attach
themselves to the substrate and form lamellipodia and filopodia, which are a
sheet-like extension of a crosslinked F-actin meshwork at the leading edge of
cells and a thin protrusion of F-actin-bundles at the periphery of cells and
growth cones, respectively. F-actin organization plays an important role in
initiating neurites from the cell body.
Here, we investigated how the mechanical properties of the microenvironment
affected neuronal morphologies and the F-actin structures of neurons during
neurite initiation by preparing polyacrylamide gel substrates with the various
stiffness ranging from similar to greater than brain stiffness. We hypothesized
that the substrate stiffness can affect neurite initiation if injury-induced
mechanical changes in the surrounding environment are related to the suppression
of axonal regrowth or regeneration in the CNS. Since the F-actin cytoskeleton is
an important structure for neurite initiation as described above, we focused on
the organization of the F-actin cytoskeleton of neurons on gel substrates. A
detailed investigation of the structures of the F-actin cytoskeleton reveals
that the F-actin organization depends on the substrate stiffness, and the
stiffness-dependent F-actin structures regulate neuritogenesis.
# Materials and methods
## Materials
The sources of the materials, chemicals and antibodies used in this work were as
follows. Coverslips (No. 1, 18 mm x 18 mm) were purchased from Matsunami Glass,
Japan. Acrylamide (AAm), N,N’-methylenebisacrylamide (Bis), and
3-(trimethoxysilylpropyl) methacrylate were purchased from Tokyo Chemical
Industries, Japan. A photoinitiator, lithium
phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was synthesized in accordance
with a previous report. Poly-*D*-lysine hydrobromide (PDL) and triton X-100 were
obtained from Sigma-Aldrich. Blebbistatin (BS) and cytochalasin D (CD) were
purchased from WAKO Pure Chemicals, Japan. ProLong Diamond as a mounting
reagent, Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-
SANPAH), *L*-glutamine, trypsin, glutamate, gentamycin, neurobasal medium, and
B27 supplement were obtained from Thermo Fisher Scientific, USA. Mouse anti-β-
III tubulin and phalloidin labeled with green fluorescence dye were purchased
from R&D Systems, USA and Cytoskeleton, USA, respectively. Wistar rats were
obtained from Charles River Laboratories, Japan.
## Preparation of hydrogel substrate
Hydrogel substrates were prepared using the following procedures. All cover
glasses were used after overnight treatment with 0.1 M NaOH followed by
O<sub>2</sub>-plasma treatment. The cleaned cover glasses were immersed in
silane coupling solution containing 0.5% 3-(trimethoxysilylpropyl) methacrylate
for 2 hours at room temperature. The silanized cover glasses were annealed at
110 <sup>o</sup>C for 30 min. We prepared two types of hydrogel precursor
solutions that contained 5% w/v AAm with 0.05, 0.1, 0.15, 0.2, 0.25, and 0.5%
w/v Bis for soft gels and 0.5% w/v Bis for stiff gels. For photo-initiated
polymerization, LAP was added as a photoinitiator to the precursor solutions at
a final concentration of 1 mM. The precursor solution was dropped on the
silanized coverslip and sealed with a cleaned coverslip with 11-μm-thick
spacers. The substrate was irradiated with 360 nm wavelength UV light at 10
mW/cm<sup>2</sup> for 10 min. After the light irradiation, the substrates were
immersed in PBS and gently agitated at 50 rpm and 25 <sup>o</sup>C over 3 days.
For comparison with previous reports about the neuritogenesis of neurons on
glass substrates, we used poly-D-lysine as adhesive molecules. To display the
PDL on the surface of the hydrogel, sulfo-SANPAH was used as a crosslinker.
After a 10-min UV irradiation of the sulfo-SANPAH solution on the hydrogel, 1
mg/mL of PDL was applied to the hydrogel surface.
## Cell culture
All animal experiments were approved by Biological Safety and Ethics Committee
of NTT Basic Research Laboratories (approval ID 2014–04). Wistar rats (embryo
day 18) were used to obtain hippocampal cells. They were used immediately after
receipt, and anesthetized with a gas mixture of 1–3% isoflurane and air during
preparation. Every effort was made to minimize suffering. The hippocampus was
extracted from rat brain, and then treated with 2.5 mg/ml trypsin for 10 min at
37 <sup>o</sup>C. The cells were then centrifuged at 1000 rpm for 5 min and
triturated with a pipette. The culture was carried out with a neurobasal medium
that consisted of 74 μg/ml *L*-glutamine, 25 μM glutamate, 50 μg/ml gentamycin
and 2% B27 supplement. The cell suspension was applied to gel substrates with an
initial cell number of 15000 cells/substrate, and cultured at 37 <sup>o</sup>C
and in 5% CO<sub>2</sub>, with saturated humidity.
## Measurement of elastic modulus of hydrogel substrates
A customized atomic force microscope (AFM) equipped on an upright optical
microscope (Eclipse FN1, Nikon, Japan) was used to measure the Young’s modulus,
*E*, of the gel substrates. A rectangular cantilever (BioLever mini, BL-
AC40TS-C2, Olympus, Japan) with a nominal spring constant of less than 0.1 N/m
was used. The loading force was determined using Hook’s law by multiplying the
cantilever deflection by the spring constant calibrated using a thermal
fluctuation method. The force curve measurements were examined in a 300 μm × 300
μm scan region at a maximum loading force of around 1.5 nN. *E* was estimated
from the observed force-distance curves with the Sneddon’s modulation of a
Hertzian contact model as a conical indenter, which is expressed as: $$F =
\frac{2\tan\alpha}{\pi}\frac{E}{(1 - \nu^{2})}\delta^{2},$$ where *F* is the
loading force, *δ* is the indentation depth, α is the half angle of the conical
probe of 17.5°, and *ν* is the Poisson’s ratio of the gel substrate, assumed
here to be 0.5.
## Fluorescence staining
To count neurons without neurites on a polyacrylamide gel substrate, we used
calcein-AM and propidium iodide (PI) to stain living and dead neurons,
respectively. These fluorescent dyes were directly added to samples in a culture
medium. After 30 minutes’ incubation at 37 <sup>o</sup>C in a 5% CO<sub>2</sub>
atmosphere, the cells were observed with a fluorescent microscope. The cells
were immunofluorescently stained as follows. All the samples were fixed with 4%
paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, and then
blocked with a mixture of 5% NBS and 1% BSA in PBS at room temperature. After
the blocking treatment, primary antibodies were applied to the cells followed by
washing and incubation with appropriate secondary antibodies bound to
fluorescent dyes. For F-actin staining, the cells were treated with 0.5%
TritonX-100 in PBS, and then stained with phalloidin bound to fluorescent dyes.
Fluorescently labeled samples were mounted with a mounting reagent. To
distinguish neurons from the other hippocampal cells, neuron-specific marker
β-III tubulin with anti-β-III tubulin antibody and 500 times dilution. It was a
marker for neuritogenesis because it is the main component of a microtubule in
neurites. The antibodies were labeled with species-specific secondary antibodies
conjugated with Alexa 568 with 400 times dilution.
## Fluorescent imaging
Fluorescent images were obtained using a fluorescent microscope (Eclipse TE3000,
Nikon, Japan) with a CMOS camera (ORCA flash 4.0, Hamamatsu Photonics, Japan),
and a laser scanning confocal microscope (LSM510, Carl Zeiss, Germany or IX81,
Olympus, Japan). The microscope setup for the spinning disc superresolution
microscope (SDSRM) is based on a disk-scanning confocal microscope system, which
includes an IX81 and a disc-scanning unit (IX2-DSU, Olympus, Japan). To
investigate neuritogenesis and F-actin bundle formation, acquired images were
analyzed with ImageJ (NIH, <http://rsb.info.nih.gov/ImageJ>).
## Statistical analysis
All experiments were performed using at least three independent donors and three
replicate gel substrates. To obtain a statistical analysis of neuritogenesis, we
classified neurons that had protrusions with a diameter less than that of the
cell body as stage 1. Protrusions whose diameter exceeded that of the cell body
were classified as neurites. Neurons with neurites of approximately equal length
were defined as stage 2. Neurons that possessed one neurite at least twice as
long as the rest were classified as stage 3. Statistical comparisons were
performed using an independent t-test when filopodia density and length were
compared, and one-way analysis of variance (ANOVA) with Bonferroni’s *post hoc*
testing was used to make pairwise comparisons between multiple groups. The
statistical significance was set at *p* \< 0.05.
# Results
## Characterization of gel substrates
A hydrogel substrate with a homogeneous elastic modulus and defined thickness is
required if we are to investigate how substrate stiffness affects
neuritogenesis. To characterize the gel substrate, the Young’s modulus, *E*, was
measured with AFM with different crosslinker concentrations. The results were
shown in. E for 0.05% BIS concentration was 1.7 × 10<sup>2</sup> (± 0.3 ×
10<sup>2</sup>) Pa, which was comparable to *in vivo* brain stiffness. E for
0.1% BIS concentration was 2.2 × 10<sup>3</sup> (± 0.3 × 10<sup>2</sup>) Pa, and
E for more than 0.15% BIS concentration was 3.2 × 10<sup>3</sup> (± 0.5 ×
10<sup>3</sup>) Pa, which was one order of magnitude higher than the brain’s
stiffness.
## Neuritogenesis on polyacrylamide gel substrates
As described in the Introduction, neuronal developmental stages *in vitro* can
be divided from neuronal morphologies. To obtain a statistical analysis of
neuritogenesis on the gel substrates, we defined neurons with the protrusions
that were less than the diameter of the cell body as stage 1 and elongation of
the protrusions equal to the diameter of the cell body as neurite initiation or
neuritogenesis. We counted the number of neurons in stage 1 from fluorescent
images of neurons stained with calcein-AM and PI, which allowed us to
distinguish living neurons from dead ones in stage 1. Fluorescence observations
showed that the cellular viability on each gel substrate was not statistically
significant. The neurons in stage 1 as a percentage of the total number of cells
on the gel substrates are shown in. The graph shows that the percentage of
neurons in stage 1 on stiff substrates at 26 hours after plating was 17%±1.8%
for 0.05% BIS, 19%±3.2% for 0.1% BIS, 55%±2.8% for 0.15% BIS, 66%±2.5% for 0.2%
BIS, 65%±2.8% for 0.25% BIS, and 66%±1.0% for 0.5%BIS. The results indicate that
the neuritogenesis on the gel substrates with elastic moduli more than 3.0 kPa
is significantly suppressed. To investigate the stiffness dependent suppression
of neuritogenesis, we hereafter used gel substrates prepared from 0.05% BIS as
the standard substrate (soft substrate) and 0.5% BIS as the stiffer gel
substrate (stiff substrate). We statistically analyzed the further development
of neurons on soft and stiff substrates at 20 hours after plating. For the
analysis, protrusions that exceeded the diameter of the cell body were
classified as neurites. Neurons with neurites of approximately equal length were
defined as stage 2. Neurons that possessed one neurite at least twice as long as
the rest were classified as stage 3. The percentages of neurons in stage 2 were
48% ± 10% for the soft substrate and 23% ± 4% for the stiff substrate, whereas
the percentages in stage 3 were 26% ± 14% for the soft substrate and 2.6% ± 3%
for the stiff substrate. The differences are statistically significant. The
results suggested that substrate stiffness affected neurite elongation and
development as well as neuritogenesis.
To investigate neuronal morphologies, the neurons were immunofluorescently
labeled with phalloidin and anti-βIII-tubulin for F-actin and microtubule,
respectively. Fluorescent images of neurons on soft and stiff substrates at 20
hours after plating are shown in, and. respectively. On soft substrates,
fluorescent images revealed the co-existence of neurons with and without red
fluorescence from anti-βIII-tubulin, indicating the formation of neurites. The
neurons had thin and short protrusions with green fluorescence from phalloidin,
indicating F-actin fibers (black arrow head). Compared with the soft substrates,
fewer neurons had protrusions with red fluorescence, and F-actin meshworks with
green fluorescence were formed at the cell periphery on stiff substrates (white
arrow head). These fluorescent images indicated that the stiffening of the
surrounding area induces neuronal morphological changes, especially F-actin
organization.
## F-actin cytoskeleton structure
To assess whether F-actin organization induced by substrate stiffness is
involved in neurite initiation, the F-actin structures were observed in detail
with a spinning disk superresolution microscope (SDSRM). show representative
fluorescent images of neurons on polyacrylamide gel substrates. At developmental
stage 1, neurons on stiff substrates had larger areas of circumferential F-actin
meshworks as shown in compared with the soft substrates shown in. Some neurons
on the stiff substrates had a F-actin structure with two distinct parts. One
part indicated by a black arrowhead consisted of F-actin meshworks and the other
indicated by a white arrowhead consisted of condensed F-actin meshworks, known
as F-actin arcs. F-actin arcs were not observed on the soft substrates with our
experimental setup. At developmental stage 2, neurons on the stiff substrates
had larger growth cones at the tips of neurites and larger areas of F-actin
meshworks than soft substrates. On the other hand, fewer F-actin bundles
sprouted from the leading edge of a cell body and neurites on the stiff
substrates. The fluorescent images showed that F-actin bundle formation was
suppressed on stiff substrates. To analyze the density and length distribution
of F-actin bundles statistically, we counted the number and measured the length
of F-actin bundles protruding from the perimeter of neurons on a soft or a stiff
substrate. The graph in, shows the number of F-actin bundles per 10 μm at the
perimeter of a neuron. Neurons on stiff substrates have F-actin bundles with
significantly lower densities than those on soft substrates. In addition, the
average length of the F-actin bundles on stiff substrates (2.5±1.6 μm) are
significantly shorter than those on soft substrates (4.4±2.5 μm). It has been
reported that the prevention of filopodia formation suppressed neuritogenesis
because filopodia can guide microtubule elongation during neuritogenesis. Our
results imply that the suppression of F-actin bundle formation on stiff
substrates is involved in neuritogenesis. Moreover, stiffness of the gel
substrates induced a change in the cytoskeletal F-actin organization including
the formation of a larger area of F-actin meshworks and F-actin arcs. Therefore,
we hypothesized that F-actin organization induced by substrate stiffness can
play a role in regulating neuritogenesis.
## Blebbistatin treatment
We investigated the way in which the organization of the F-actin cytoskeleton,
and in particular the circumferential F-actin arcs and the suppression of the
F-actin bundles, are associated with neuritogenesis. We used blebbistatin (BS),
which is known to be a myosin II inhibitor and inducer of filopodia formation.
Since myosin II activity contributes to the compaction of F-actin meshworks and
the consequent formation of F-actin arcs, BS treatment can suppress F-actin arc
formation. BS was exposed at a concentration of 100 μM, which was sufficient to
attenuate the interaction between F-actin and myosin II, immediately after cell
plating. The neuritogenesis of neurons on gel substrates after BS exposure was
analyzed statistically by counting the number of neurons in stage 1 at 20 hours
after plating. As control experiments, neurons on gel substrates were exposed to
dimethyl sulfoxide (DMSO), which did not affect neuritogenesis or the F-actin
structures. The graph shows that the percentage of neurons in stage 1 on both
the soft and stiff substrates decreased significantly after the BS (from 36% to
17% for soft substrates, and from 55% to 29% for stiff substrates). Although the
BS treatment improved neuritogenesis, the percentage of neurons in stage 1 on
stiff substrates was higher than that on soft substrates, indicating the
suppression of neuritogenesis. To investigate BS-induced neurite protrusions, an
SDSRM was used for a detailed observation of the F-actin structures after BS
treatment on the stiff substrate. We could not find significant structural
changes of F-actin organization on the soft substrates after BS treatment when
compared with neurons without BS exposure. On the other hand, on stiff
substrates, although the circumferential actin arcs disappeared, F-actin
meshworks were observed at the edge of a cell body. The F-actin meshworks were
segmented, and the widely spread growth cones observed before BS treatment were
barely seen. Parallel arranged F-actin organization was observed in the
segmented F-actin area. These F-actin structures were not observed on the soft
substrate. These results indicate that F-actin structures induced by the
attenuation of myosin II activity are associated with neuritogenesis, although
the myosin II activity is strongly involved in neurite initiation.
## Cytochalasin D treatment
Although the exposure of neurons on gel substrates to BS revealed that F-actin
arcs at the leading edge of a cell body suppressed neuritogenesis, it remained
unknown whether the other aspects of F-actin organization, which was observed
after the BS treatment, were related to neuritogenesis. Therefore, we attempted
to completely suppress F-actin organization by treating neurons with
cytochalasin D (CD), which induces the disruption of actin filaments and
prevents actin polymerization by binding to the barbed end. CD was exposed at a
concentration of 100 nM, which was sufficient to disrupt the F-actin structures
at the leading edge of the cell body, immediately after cell plating. To assess
whether CD treatment affected neuritogenesis, we counted the neurons without
neurites on the hydrogel substrates. The control results were the same as those
in. The exposure of CD to neurons on soft substrates significantly impaired
neuritogenesis whereas it significantly improved neuritogenesis on stiff
substrates (36% to 41% for soft substrates and 55% to 40% for stiff substrates).
In addition, the percentages of the soft and stiff substrates after CD treatment
had no statistical significance. To investigate the way in which CD treatment
affected the F-actin organization of the neurons on the soft and stiff
substrates, the structure of the F-actin cytoskeleton was observed in detail
with an SDSRM. Neurons on both soft and stiff substrates had similar
morphologies after CD treatment. There was no F-actin bundle formation and no
meshwork on the gel substrates, indicating that the CD concentration in was
sufficient to disrupt the F-actin structures at the leading edge of the cell
body. The suppression of F-actin organization induced the suppression of
neuritogenesis on the soft substrates but the acceleration of neurite initiation
on the stiff substrates. As F-actin bundles can accelerate neuritogenesis, the
suppression of F-actin bundle formation on the soft substrates resulted in the
suppression of neuritogenesis. On the other hand, whether or not CD treatment
improved neuritogenesis on the stiff substrates remains controversial. CD-
induced changes in the F-actin organization on the stiff substrates consisted of
the disruption of F-actin meshworks and arcs at the leading edge as well as the
suppression of F-actin bundles. This suggests that F-actin structures formed on
the stiff substrates help prevent the suppression neuritogenesis.
# Discussion
It has been reported that the mechanical properties of ECM in various tissues
varied during several events such as external stimuli, aging, and diseases. With
respect to the CNS, understanding the relationship between ECM mechanical
properties and neural functions is an important issue in the field of tissue
engineering and neuroscience. This study demonstrates the effects of substrate
stiffness on the neuritogenesis of hippocampal neurons because neuritogenesis is
the first step towards development and regeneration. Also, we investigated
structure of the F-actin cytoskeleton as proper arrangements of the F-actin
assembly are required prior to organization of the other proteins related to
neurites.
To accomplish this, we employed a polyacrylamide gel as a substrate with a
mechanical property ranging from similar to a hippocampus to a stiffer one. As
an adhesion molecule, we used PDL which can act as a non-selective focal
adhesion activator. Previous study reported that adhesion molecules derived from
ECM proteins such as laminin, fibronectin and collagen, initiate integrin-
mediated cell binding via the formation of focal adhesions, which is a key
structure as regards regulating mechanotransduction and activating signaling
pathway of neuritogenesis. For example, laminin can facilitate neurite
initiation and axonal outgrowth compared with fibronectin. Moreover, laminin can
rescue the neuritogenesis of neurons whose neurite initiation is genetically
inhibited whereas collagen and fibronectin have no effect. These findings
indicate that distinct ECM proteins activate distinct signaling pathways related
to neuritogenesis. On the other hand, PDL modulates cell adhesion via an
electrostatic interaction between a negatively charged cell membrane and a
positively charged PDL. The PDL-mediated non-selective activation of focal
adhesion allows us to eliminate the possibility that specific activation of a
certain signaling pathway induced by ECM proteins affects suppression or
acceleration of neritogenesis.
We found that a stiff substrate with an elastic modulus exceeding 3.0 kPa
changed the neuronal morphologies and organization of the F-actin cytoskeleton,
and suppressed neuritogenesis. The further development of neurons from stage 2
to 3 was suppressed on the stiff substrate. Noteworthy features of the
morphologies of neurons on the stiff substrates were that they had fewer F-actin
bundles and the formation of more circumferential F-actin meshworks and arcs at
the leading edge. According to a previous report stating that sprouts of F-actin
bundles are required for neuritogenesis, the suppression of F-actin bundle
formation on stiff substrates prevents microtubules from invading and protruding
from the leading edge. Together, F-actin meshworks and arcs on stiff substrates
appeared to suppress neurite protrusions. Regarding the further development from
stage 2 to 3, the neurite elongation at the leading edge of the growth cones can
be suppressed by the stiffer substrate-induced F-actin structures.
To assess our assumption that stiffer substrate-induced F-actin organization
plays a critical role in neuritogenesis, we treated neurons on polyacrylamide
gel substrates with BS to inhibit the formation of actin arcs. The disruption of
F-actin arcs is observed on stiff substrates after the BS treatment. It is known
that neurons, which genetically lack the ability to form filopodia, fail to
initiate neurites, and they form F-actin arcs at the leading edge. In the
report, BS treatment disrupts the F-actin arcs and rescues neuritogenesis. These
results are similar to ours. Although we must analyze protein expression if we
are to discuss the relationship between previous reports and our results,
substrate stiffness may regulate protein expression relating to cytoskeletal
organization. Also, BS treatment accelerates F-actin bundle formation and
improves neuritogenesis regardless of the substrate stiffness. Since an F-actin
bundle assists microtubule protrusion at the leading edge of the cell body as
described above, the acceleration of F-actin bundle formation can help to
improve neuritogenesis.
After BS treatment, neuritogenesis on stiff substrates was suppressed compared
with that on soft substrates even though the formation of actin arcs was
prevented and F-actin bundle formation was accelerated. This suggests that the
F-actin structure at the leading edge of neurons on the stiff substrates after
BS treatment prevents microtubule protrusion. To prevent the F-actin
organization at the leading edge, neurons on the gel substrates were treated
with cytochalasin D. After the CD treatment, the percentages of neurons in stage
1 on the soft and stiff substrates are similar, indicating the suppression of
neuritogenesis on soft substrates and the improvement on stiff substrates.
Fluorescent images revealed that the structures of F-actin cytoskeletons on
stiff substrates were similar to those on soft substrates after the CD
treatment. The suppression of neuritogenesis on the soft substrates strongly
supports the view that F-actin bundles play a beneficial role in sprouting
neurites. On the other hand, neuritogenesis on the stiff substrates was improved
even though the formation of the F-actin bundles was suppressed. This supports
the idea that the F-actin meshworks and arcs prevent microtubules from sprouting
from the leading edge. It is known that the elongation process in the growth
cones takes place preferentially where the actin meshwork is unstable, whereas
the stable actin meshwork tends to impair microtubule protrusion.
We propose a neuritogenesis mechanism that is dependent on substrate stiffness
as shown in. F-actin cytoskeletons such as F-actin meshworks and arcs at the
leading edge of the cell body were preferentially organized on the stiff
substrates, whereas F-actin bundle formation was suppressed. The F-actin
structures on the stiff substrates are obstacles to the protrusion of
microtubules from the leading edge of the cell body. Therefore, on a stiff
substrate, the disruption of F-actin organization by CD had an effect on
neuritogenesis even though F-actin bundle formation was suppressed.
# Conclusions
We investigated how substrate stiffness affected neuritogenesis. The high-
resolution microscopy observations showed that stiff substrates significantly
suppressed neuritogenesis due to their stronger myosin II-based contractility
and stabilized the F-actin cytoskeleton. To our knowledge, this is the first
observations regarding the organization of neuronal F-actin cytoskeleton
regulated by the mechanical properties of the surrounding environment. The
stiffness-induced F-actin structures played a key role in regulating
neuritogenesis. Our findings suggest that the suppression of neuronal
regeneration at a glial scar could be influenced in part by a stiffening
microenvironment, which suppresses neuritogenesis and development to the later
stage. Further investigations are required to understand the neuronal
development because various proteins are properly associated and dissociated
during neurite development. Of the several processes, the interaction of
multiple proteins with F-actin assembly can be important as regards protrusions
maturing into neurites and developing neurons from stage 2 to stage 3,
corresponding to axon formation.
In this study, we qualitatively discuss the circumferential F-actin meshworks
and arcs because we did not observe the structures on the soft substrates with
our experimental setup. It is known that the low contractile force at the
interface between a cell and a substrate suppresses the formation of stable
adhesion and cytoskeleton assembly at the interface. This is why no F-actin
meshworks were observed on the soft substrates. We must investigate the dynamics
of F-actin structures and other proteins such as microtubules at the leading
edge during neuritogenesis by time-lapse imaging to verify our proposed model.
The 3D cultivation of neurons on artificial platforms such as extracted ECM,
collagen, and biocompatible polymers has been reported. However, less is known
about how the mechanical properties of ECM affect neuronal development in 3D
cultivation. Although our experimental methods cannot be applied to 3D
experiments due to the toxicity of gel monomers, our findings suggest that ECM
stiffness can affect the F-actin structure of neurons in 3D cultivation. This
study provides an insight not only for developing a scaffold for neuronal
regeneration, but also for designing a compliant interface between tissue and a
device such as a brain-machine interface.
# Supporting information
We thank Ms. Yuriko Furukawa for preparing the samples, Dr. Misaki Takahashi for
helpful artwork, and members of the Molecular and Bio Science Research Group at
NTT Basic Research Laboratories. We also thank Mr. Kohei Hirono for experimental
assistance, and members of the Cellular and Tissue Engineering Laboratory at
Hokkaido University for fruitful comments and discussions.
[^1]: NTT does not alter our adherence to PLOS ONE policies on sharing data
and materials. |
# Introduction
Chronic hypertension is a relatively common disorder occurring in approximately
1–5% of pregnant women; rates depending on the population studied and the
criteria used for the diagnosis. Because of increasing maternal age, obesity,
and type 2 diabetes worldwide, it is expected that the prevalence of chronic
hypertension in pregnancy will continue to increase. The study ENNS (National
Nutrition Health Survey) cross-sectional survey in France between 2006 and 2007
reveals a prevalence of chronic hypertension of 4.1% in women between 18 and 34
years and of 8.3% between 35 and 44 years. Hypertension was known to the patient
in 22.3% of cases between 18 and 34 years and in 55.5% of cases between 35 and
44 years. Pregnancies complicated by chronic hypertension are at increased risk
of superimposed preeclampsia, abruptio placenta, fetal growth restriction,
preterm delivery, and perinatal death,.
In women with chronic hypertension, the risk of superimposed preeclampsia is
increased in black ethnic origin, raised body mass index (BMI), smoking, booking
systolic blood pressure of 130 to 139 mm Hg, and diastolic blood pressure of 80
to 89 mm Hg, and in women with chronic hypertension ≥4 years. Conflicting
results have been published on the relationship between a history of
preeclampsia and the rate of superimposed preeclampsia in subsequent
pregnancies. These differences might be related to inclusion of heterogeneous
population of women with hypertension; in some studies women had only essential
hypertension whereas in others women had all forms of hypertension. In addition,
most of the studies included women who were diagnosed with chronic hypertension
on the basis of either having hypertension prior to pregnancy or during the
first 20 weeks gestation. Moreover, none of the studies reported to date have
included only women who received antihypertensive medication prior to
conception.
The objective of this study was to identify risk factors for superimposed
preeclampsia at first prenatal visit in women with essential chronic
hypertension receiving antihypertensive medication prior to conception.
# Methods
This retrospective study included women with chronic hypertension delivered
between 1 January 2004 and 31 December 2007 who were identified from the
hospital computer databases of two university hospital centers (CHI Creteil and
AP-HP Cochin Port-Royal Paris). Every medical chart was reviewed to collect the
data. The criteria used to select women with chronic hypertension was a
diagnosis of hypertension that needed a treatment before the onset of the
pregnancy. Exclusion criteria were: women with multiple pregnancies, women with
secondary hypertension, women with proteinuria at less than 20 weeks’ gestation,
women considered as having a chronic hypertension but without any treatment at
first prenatal visit, women transferred from other maternities, pregnancies
complicated by fetal malformations.
The data collected from medical records included: age, pre-pregnancy BMI,
parity, ethnic origin, tobacco use during pregnancy, duration of hypertension,
past obstetric history, antihypertensive treatment, treatment with low dose
aspirin, maternal systolic and diastolic blood pressure at booking, presence or
absence of proteinuria at first prenatal visit, maternal and neonatal outcomes.
The blood pressure was obtained with automated device, patient in sitting up
position. The mean arterial pressure was calculated from brachial systolic and
diastolic blood pressure (BP), according to the following formula: Mean arterial
pressure = \[systolic BP+(2\*diastolic BP)\]/3. Superimposed preeclampsia was
defined as a new onset proteinuria (0.3 g of protein or more in a 24-hour
specimen) after 20 weeks’ gestation and without proteinuria early in pregnancy
(less than 20 weeks’ gestation). Fetal growth restriction (FGR) was defined as a
birth weight \<5th percentile. Abruptio placenta was diagnosed according to
clinical findings and/or placental examination. HELLP syndrome (hemolysis,
elevated liver enzymes, low platelet count) was defined according to Sibai’s
criteria.
We analyzed the risk factors that may influence the rates of superimposed
preeclampsia. Categorical variables are presented as percentage, and continuous
variables as mean and SD. Categorical variables were compared with
*X<sup>2</sup>* square or Fisher’s exact test and continuous variables with a
two-tailed student *t* test. Variables with a p\<.1 were included in a logistic
regression analysis. Data are expressed as odds ratio (OR) with 95% confidence
interval (CI). P\<.05 was considered as significant. Positive likelihood ratio
with 95% CI was calculated for the prediction analysis. The statistical software
Statview 5.0 (SAS Institute) was used for statistical analysis, and REALbasic
(2002) for Receiver Operative Characteristics (ROC) analysis.
The evaluation of chronic hypertensive women did not need approval of our
ethical committee because it was a retrospective analysis of data in women who
received standard management at both hospitals. Therefore, our ethical committee
has waved requirement for approval.
# Results
The study population consisted of 362 women with chronic hypertension. Women
transferred from other centers (n = 61), women with secondary hypertension or
with chronic hypertension and pre-existing proteinuria before 20 weeks’
gestation (n = 20), women with fetal anomalies (n = 4), and women not receiving
antihypertensive medication prior to pregnancy (n = 66) were excluded. The
analysis concerned exclusively the 211 women with essential chronic hypertension
who were receiving antihypertensive therapy prior to conception.
describes maternal characteristics at booking (first visit) and summarizes
overall maternal and perinatal outcomes. No maternal death occurred among the
women.
The rate of superimposed preeclampsia was 23.2%. All women with HELLP syndrome
had preeclampsia. shows perinatal outcomes according to the occurrence of
superimposed preeclampsia. The following variables obtained at first prenatal
visit: black ethnicity, nulliparity, previous preeclampsia, previous FGR,
systolic and diastolic blood pressure at booking, mean arterial blood pressure
≥95 mmHg at booking, and duration of hypertension ≥4 years were significantly
associated with the occurrence of superimposed preeclampsia in univariate
analysis. In contrast, the rate of superimposed preeclampsia was not affected by
maternal age, BMI, tobacco use during pregnancy, antihypertensive medication
(one drug only versus several drugs), and aspirin treatment.
We have chosen to include mean arterial blood pressure ≥95 mmHg at multivariate
analysis and to analyze its predictive status, rather than systolic or diastolic
blood pressure, because mean arterial blood pressure had the best prediction
analysis for superimposed preeclampsia \[area under curve at ROC analysis of.72
(p\<.0001) for mean arterial blood pressure.69 (p\<.0001) for diastolic blood
pressure, and.68 (p = .0001) for systolic blood pressure\] and 95 mmHg was the
best cut-off for sensitivity and specificity. Multivariate analysis showed that
previous preeclampsia, and mean arterial blood pressure ≥95 mmHg were
independently associated with the occurrence of superimposed preeclampsia.
We analyzed the prediction for the occurrence of superimposed preeclampsia using
the variables that were selected in logistic regression for superimposed
preeclampsia at first prenatal visit: previous preeclampsia and mean arterial
blood pressure. The ROC analysis of mean arterial blood pressure and previous
preeclampsia showed an area under curve at of.77 (p\<.0001). The prediction
analysis of previous preeclampsia and MAP≥95 mmHg is shown in. When both
variables were present, sensitivity, specificity, positive predictive value,
negative predictive value, and likelihood ratio for superimposed preeclampsia
were 43%, 94%, 70%, 85%, and 7.71 (95% CI: 3.20–18.57), respectively.
Of the 49 patients with superimposed preeclampsia, 19 occurred at less than 34
weeks’ gestation. Of all variables analyzed, only SBP at booking was associated
with increased risk of early onset compared to late onset of preeclampsia
(SBP≥140 mmHg: 74% vs. 27%, SBP 130–140 mmHg: 21% vs. 40%, and SBP\<130∶5% vs.
33%, respectively, p = .004).
# Discussion
The main goal of this study was to analyze factors obtained in the first half of
the pregnancy that may be associated with superimposed preeclampsia in women
with essential chronic hypertension that needed a treatment before the onset of
pregnancy. In contrast to other studies, we have focused on essential chronic
hypertension women receiving antihypertensive therapy prior to pregnancy for two
reasons: 1- the diagnosis of chronic hypertension was already made, 2- the
results are not biased by the primary disease as it may be observed in secondary
chronic hypertension related to nephropathy or auto-immune disease. In addition,
only two studies have focused on women with chronic essential hypertension, one
of these studies did not specify whether or not patients were treated by
antihypertensives, and in the second one almost half of the women had no
antihypertensives before the onset of pregnancy.
The main findings of the study were: 1- superimposed preeclampsia occurred in
23.2% of women, 2- previous preeclampsia, and mean arterial blood pressure ≥95
mmHg at booking was associated with increased risk of superimposed preeclampsia.
The rate of superimposed preeclampsia of 23.2% observed in our study is in
agreement with that reported in other studies, ranging from 17% to 34.9%.
In several of these studies, conflicting results concerning the relationship
between a history of previous preeclampsia and the recurrence of preeclampsia in
a subsequent pregnancy have been reported. Sibai et al. showed that the risk of
superimposed preeclampsia was greater in chronic hypertensive women who had
preeclampsia during a previous pregnancy (32% vs. 23%; OR: 1.6; 95% CI:
1.1–2.3), as well as Chappell et al. (OR = 1.95, 95% CI: 1.25–3.04). A recent
study, however, reported that in women with chronic hypertension a history of
preeclampsia was not associated with increased risk of superimposed preeclampsia
(OR = 1.28, 95% CI: 0.78 to 2.1). This latter study, however, had only 61% power
to detect differences in rate of superimposed preeclampsia between groups. In
our study, among the women who developed preeclampsia 53.3% had a history of
preeclampsia in a previous pregnancy, and previous preeclampsia was clearly
associated with superimposed preeclampsia in subsequent pregnancy in the
logistic regression analysis. Along with others, blood pressure at first
prenatal visit (systolic, diastolic, and mean arterial) was associated with
increased risk of superimposed preeclampsia at univariate analysis. The
prediction of a history of preeclampsia, and mean arterial blood pressure ≥95
mmHg for superimposed preeclampsia in our population were encouraging,
particularly, when both variables were present at first prenatal visit. Although
sensitivity was only of 43%, positive predictive value of 70% was extremely
high, and the likelihood ratio to have a superimposed preeclampsia was 7.71-fold
increase. These two variables simply obtained early during the pregnancy may
help to select women at very high risk of superimposed preeclampsia that might
be eligible for low dose aspirin treatment. Although the usefulness of low dose
aspirin in the prevention of superimposed preeclampsia is not established in
women with chronic hypertension, this may be related to the late start (after 20
weeks’ gestation) of the treatment; and the use of low dose aspirin in this
subgroup could be beneficial, particularly if started before 16 weeks’
gestation.
Maternal age and parity had no significant effect on the risk of superimposed
preeclampsia in our study. A similar lack of effect of maternal age in women
with chronic hypertension was found by Sibai and colleagues. This is in contrast
to recent reports where nulliparity, and maternal age were associated with
increased risk of preeclampsia in unselected women. Therefore, it is not clear
whether multiparity or lower maternal age are protective in pregnant women with
essential chronic hypertension, highlighting if necessary that surveillance
should be heightened for these women.
Duration of hypertension prior to pregnancy of more than 4 years has been
associated with increased risk of superimposed preeclampsia. Our study, however,
did not confirm this finding, and is in agreement with a recent one.
The prevalence of black ethnicity (53.6%) in chronic hypertensive women is quite
high and it does not reflect our general population. It is, however, important
to note that ethnicity was not independently associated with superimposed
preeclampsia in our study.
In several studies, pregnant women with essential chronic hypertension had high
rates of fetal growth restriction (\<5th birth weight percentile) without
superimposed preeclampsia. In our study where 44 babies were \<5th birth weight
percentile, only 12 (20.7%) were observed in women having superimposed
preeclampsia whereas the latter 32 (17.2%) were found in women without
superimposed preeclampsia (non significant difference). These findings show that
women with essential chronic hypertension are at risk for severe fetal growth
restriction irrespective of the occurrence of superimposed preeclampsia. The
explanation may be an overcorrection of blood pressure suggested by some
authors, and in our study, treatment was stopped for 16 patients because of a
SBP under 120 mmHg and DBP under 70 mmHg. A recent meta-analysis, however, did
not find such relationship.
Our study has some limitations. The number of patients is relatively small. It
is to note, however, that our population concerned exclusively women with
essential chronic hypertension who had a treatment at first prenatal visit, and
this is not the case of the majority of published studies. Another limitation is
related to the retrospective structure of the study, for instance, we did not
make any confirmation on past obstetric history that were mentioned in the
medical charts, and this may induce some errors in the analysis. In addition, an
important number of women transferred from other centers (n = 61), and not
followed in both maternities since the beginning of their pregnancy were
excluded from the study because the management of antihypertensive treatment in
these women could have been different. Moreover, almost all these patients had
an adverse pregnancy outcome, and therefore the results would have been biased.
Finally, gestational age at first prenatal visit is relatively late (18.1 wks).
This result, however, reflects our clinical practice and it still remains
possible to introduce some preventive treatment such as aspirin and to plan a
close monitoring of the pregnancy.
## Conclusion
In essential chronic hypertensive women treated before the pregnancy, previous
preeclampsia, and mean arterial blood pressure ≥95 mmHg are associated with
increased risk of superimposed preeclampsia. Together, these two variables may
select women at extreme high risk of superimposed preeclampsia.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: EL BH. Performed the
experiments: EL BH. Analyzed the data: EL BH VT FG DC BS. Contributed
reagents/materials/analysis tools: VT FG DC BS. Wrote the paper: EL BH. |
# Introduction
The receptor tyrosine kinase EGFR (ErbB1) plays a crucial role in both cancer
initiation and progression and is discussed as a promising target for cancer
therapy. New insights into EGFR biology have established that EGFR signals
through two distinct pathways: (i) canonical membrane-associated signaling and
(ii) non-canonical nuclear signaling to regulate gene expression, DNA
replication and DNA damage repair.
The most intriguing step associated with nuclear EGFR signaling represents the
physical translocation of the EGFR protein from the cell membrane to the
perinuclear/nuclear location in response to cellular stress or stimulation with
EGF. All four members of the ErbB family have been reported to undergo nuclear
translocation. Several distinct functions have been described for nuclear EGFR.
Nuclear EGFR (nEGFR) is reported to act as a co-transcriptional activator for
cyclin D1. Furthermore, nEGFR controls proliferating cell nuclear antigen (PCNA)
activity during DNA replication. In the context of DNA repair, EGFR regulates
DNA-PK activity. In addition, recent data suggests a role for nEGFR in
regulating mRNA stability and protein translation. Mechanistically, nEGFR
regulates the ribonuclease activity of polynucleotide phosphorylase through DNA-
PK-mediated phosphorylation, and consequently, the expression of the *c-myc*
mRNA is increased. Moreover, the same study reported an inhibitory effect of
EGFR-mediated argonaute 2 (AGO2) phosphorylation on miRNA maturation in response
to hypoxia.
Importantly, miRNA-mediated RNA silencing represents an effective pathway to
prevent the active translation of mRNAs. This process is performed by the RISC
(RNA-induced silencing complex) in the perinuclear cytoplasm. Major elements of
the RISC are AGO2 and GW182 (TNRC6), which are organized in protein complexes
within the nucleus, where these RNAi factors are presumably involved in
regulating gene transcription and mRNA splicing. The GW182 protein acts as a
platform that recruits and activates the deadenylase complex CCR4-NOT to the
miRNA-directed target mRNA and facilitates the removal of the poly(A) tail. This
process promotes mRNA degradation by AGO2 and inhibition of translation.
Deadenylation is the major step triggering mRNA decay in eukaryotic cells. The
poly(A) tail and associated poly(A)-binding protein (PABP) interact with the 5’
m<sup>7</sup>G-cap/cap-binding complex to form a closed loop that enhances
translation initiation and protects mRNA ends from nuclease attack.
Consequently, deadenylation represents an important control point for both mRNA
degradation and translational silencing.
As shown in the present study, nEGFR is part of the miRNA-directed cNOT1
deadenylase complex and regulates the stability and translation of mRNAs through
its kinase activity. In summary, we propose a mechanism by which nEGFR triggers
a fast and efficient switch in protein translation in a miRNA-directed manner.
# Material and methods
## Cell culture and irradiation
Experiments were performed using the A549 human bronchial carcinoma cell line
(ATCC CCL-185, Manassas, Virginia, USA), the FaDu head and neck tumor cell line
(ATCC, HTB43), and HSF7 normal fibroblasts. Irradiations were performed using
the X-ray cabinet RS 225 (X-Strahl, Surrey, United Kingdom). The voltage was set
to 200 kV, with a current of 15 mA (dose rate = 1 Gy/min at a 49 cm distance
from the X-ray tube). The X-ray beam was hardened with a 0.5 mm removable copper
filter. Dosimetry was performed with a farmer chamber (PTW, Freiburg, Germany).
Irradiation was conducted at 37°C. Erlotinib was purchased from Selleck
(Houston, Texas, USA) and EGF was purchased from Sigma-Aldrich (St. Louis,
Missouri, USA).
## Subcellular protein fractionation
Cytoplasmic and nuclear extracts were prepared using the NE-PER<sup>®</sup> kit
(Thermo Scientific, Waltham, Massachusetts, USA), according to the
manufacturer’s instructions.
## Quantification of mRNAs enriched in complexes containing nuclear EGFR
EGFR-IgG or IgG was covalently bound to an agarose support (direct IP-kit,
Thermo Fisher Scientific/Pierce, \#26148). Nuclear proteins were isolated from
cells treated with the indicated compounds and EGFR complexes were precipitated
by direct IP, in which the EGFR antibody (clone 13, BD Biosciences) was directly
linked to agarose beads. We proved a lack of nonspecific complex formation by
binding a nonspecific mouse IgG (isotype IgG1, Sigma) to agarose. Enriched mRNA
templates were primed with oligo(dT)<sub>12-18</sub> primer (Thermo Fisher
Scientific) and transcribed into cDNAs using ImProm-II reverse transcriptase
(Promega) in the presence of <sup>33</sup>P-dATP. Free <sup>33</sup>P-dATP was
separated from labelled cDNAs using an Illustra NICK G50 Column (GE Healthcare).
The incorporation of <sup>33</sup>P-dATP in eluted cDNAs was quantified using a
scintillation counter. Incorporated radioactivity was normalized to the nuclear
EGFR protein concentration.
## Western blot analysis and immunoprecipitation
After irradiation, cells were lysed and proteins were resolved by SDS-PAGE.
Western blotting was performed using standard procedures. All primary antibodies
were diluted 1:1000 and purchased from the following sources: anti-EGFR (BD
Transduction Laboratories, clone 1F4); anti-EGFR-pY992 (Abcam, ab81440), anti-
lamin B1 (Biozol, clone ZL-5), anti-NFATC4 (Abcam, ab3447), anti-cNOT1 (Sigma,
HPA046577), anti-GW182 (Biorbyt, orb183979), anti-AGO2 (Cell Signaling
Technology, clone C34C6), anti-AGO2-pY393 (ECM Bioscience, AP5311), and anti-
Actin (Sigma, A2066). Quantification was performed with the LI-COR detection
system (LI-COR, Odyssey Fc). Immunoprecipitations were performed using the
Pierce direct IP kit (#26148), according to the manufacturer’s instructions.
Cell lysates were pre-cleared with nonspecific IgG bound to agarose beads.
## Expression of recEGFR-GST 3000–4000 containing the epitope for binding to the EGFR antibody clone 1F4
Total mRNAs were isolated from A549 cells and transcribed into cDNAs by RT-PCR.
The GST coding sequence was amplified using the forward primer (Sall) ``
5`-ACGCGTCGACATAGTCGCCCAAAGTTCCGTGA-3` `` and the reverse primer (Not1) `` 5`-
ATAAGAATGCGGCCGCATGCTACCAGCAAGCTTCTTCC-3´ ``. After digestion at the restriction
sites, the product was cloned in frame into the pGEX-4T vector (GE Healthcare)
and transformed into competent *E*. *coli* cells. Positive colonies were
selected and the proper orientation and correct reading frame were confirmed by
sequencing. The expression of GST fusion protein and affinity purification were
performed using standard procedures.
## DNA microarray analysis
A549 cells were irradiated with 4 Gy of radiation or sham irradiated, and nuclei
were isolated from both samples 24 h after irradiation. Nuclei were lysed and
EGFR or IgG immunoprecipitation was performed with a direct IP kit (Thermo-
Fisher/Pierce, \#26148). RNA was eluted from biological triplicates, transcribed
into cDNAs, and hybridized to the Human Gene 2.1 ST Array (Affymetrix). The DNA
microchip analysis was performed in cooperation with the Microarray Facility
Tübingen (MFT, now c.ATG <http://www.c-atg.de>), and the statistical analysis
was conducted in cooperation with the Quantitative Biology Center Tübingen
(QBIC, <https://portal.qbic.uni-tuebingen.de/portal/>). Bioinformatics analyses
of the microarray data were performed in the statistical language R (R version
3.1.1). For QC and data normalization, the R package ‘oligo’ (version 1.28.3)
was used to quantitatively normalize probe intensities across all samples using
the robust multi-array average (RMA) procedure. The ‘genefilter’ package
(version 1.46.1) was used to remove probes with very low variability between
samples. Differential expression was analyzed using the package ‘Limma’ (version
3.20.9,). For the Limma analysis, a linear model was fitted to the log2
expression data for each probe using the following formula: expr \~ construct \*
treatment. This model examines the effect of the factors construct
(immunoprecipitation with EGFR versus immunoprecipitation with IgG) and
treatment (non-irradiated versus irradiated) and their interaction on gene
expression (expr). This model allowed us to extract coefficients/ratios for
immunoprecipitation with nEGFR versus immunoprecipitation with IgG in cells that
were not treated (contrast 1), the treatment effect for IgG (contrast 2) and the
difference between contrast 1 and contrast 2 (the interaction term, contrast 3).
Contrast 3, the interaction term in the model, equals the treatment effect for
nEGFR. A post hoc interaction analysis was then performed with Limma and
p-values were adjusted for multiple tests using. For each of the three above-
mentioned coefficients, gene expression was considered significantly different
at an adjusted p value \< 0.05. A log fold change cut-off was applied afterwards
to only look at genes with a fold change \> +1 or \< -1. Raw data and metadata
from the project were deposited into Gene Expression Omnibus (GEO) with the
identifier GSE92428. Pathway analyses were performed using the DAVID functional
annotation software.
## RT-PCR
A549 cells were pretreated with erlotinib (2 micromolar) for 2 h and
subsequently irradiated with 4 Gy of radiation or sham irradiated. Twenty-four
hours after irradiation, RNA was isolated from cells using an RNeasy Mini Kit
(Qiagen). The cDNAs were generated with an RT2 First Strand Kit (Qiagen).
Quantitative PCR was performed with the RT<sup>2</sup> Profiler<sup>™</sup> PCR
Array for Human VEGF Signaling (Qiagen, \#330231), according to the
manufacturer’s instructions.
## Quantification of deadenylase activity
Nuclear fractions were isolated and the CCR4-NOT deadenylase complex was
enriched by immunoprecipitation with a cNOT1 antibody, as described above. The
assay was performed using a previously described method, with the following
modifications. High-performance liquid chromatography-purified oligonucleotides
were purchased from Eurofins. We used a 16-mer RNA substrate oligonucleotide
(`5’-CCU UUC CAA AAA AAA A-3’`) containing a 5’-CY5 label, the 15-mer (`Cy5-CCU
UUC CAA AAA AAA-3’`) and the 14-mer (`Cy5-CCU UUC CAA AAA AA-3’` as a marker of
deadenylation. In a standard reaction, cNOT1 precipitates were dissolved in 5
microliter of reaction buffer (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, 2 mM
MgCl<sub>2</sub>, 10% glycerol, and 1 mM beta-mercaptoethanol) containing 1.0
microliter RNA substrate in nuclease-free water. For gel-based detection and
quantification of deadenylase activity, reactions were incubated at 30°C for 60
min, stopped by the addition of 12 microliter of RNA loading buffer (95%
formamide, 0.025% bromophenol blue, 0.025% sodium dodecylsulfate and 5 mM
ethylenediaminetetraacetic acid (EDTA)), and heated for 3 min at 85°C. A 3
microliter sample of the RNA mixture was analyzed by denaturing PAGE using a 20%
acrylamide:bisacrylamide (19:1) gel containing 50% (w/v) urea. Intact mRNA
labelled with the fluorescent dye Cy5 and deadenylation products were visualized
and quantified using the LI-COR detection system.
## miRNA-mediated inhibition using the MISSION synthetic microRNA inhibitor
Inhibition of hsa-miR-1180-5p (NFATC4) was performed by transfecting A549 cells
with the MISSION synthetic microRNA inhibitor (Sigma). Cells were seeded in
24-well plates at densities of 1 × 10<sup>5</sup> cells in DMEM containing 10%
FBS on the day before transfection. Cells were then transfected in triplicate
with Lipofectamine 2000 and SYN MIRNA INHIB HUM hsa-mir-1180-5p
(`UUUCCGGCUCGCGUGGGUGUGU`) or SYN MIRNA INHIB NEG. CONTROL 1
(`GGUUCGUACGUACACUGUUCA`) (25 nM). After 24 h, transfected cells were irradiated
with 4 Gy of radiation, and after an additional 24 h, cell lysates were prepared
for western blot analysis with an antibody against NFATC4.
## siRNA treatment
For EGFR or NFATC4 silencing, cells were treated with a specific siRNA for 72
hours before irradiation (ON-TARGETplus SMARTpool L-003114-00-0005 human EGFR,
or ON-TARGETplus SMARTpool LQ-009584-00-0002, human NFATC4 siRNA, Dharmacon) and
with on-TARGETplus Non-Targeting Pool siRNA (Dharmacon) as a control.
Transfections were performed with Lipofectamine 2000 transfection reagent
according to the manufacturer’s protocol (Invitrogen).
## Statistics
All data represent the means ± s.d. of three independent experiments. All
statistical analyses, with the exception of the analysis of the microarray data,
were performed using two-sample t-tests and Bonferroni’s correction for multiple
testing. All raw data are given in supplementary file.
# Results
The observation that nEGFR is detected in a complex with mRNA binding proteins
suggests a potential role for nEGFR in mRNA processing. We synthesized cDNAs
from mRNAs enriched in nEGFR complexes that were immunoprecipitated from nuclear
extracts to elucidate the role of nEGFR in this process.
Immunoprecipitation with an anti-EGFR antibody enriched the EGFR protein in
nuclear fractions. The addition of a recEGFR-GST-fragment (68 kda) containing
the epitope that binds the EGFR antibody reduced the precipitation of endogenous
EGFR (180 kda), indicating the specificity of the applied antibody. As described
in a previous study, several cellular stress treatments increased the expression
of the nuclear EGFR protein.
Immunoprecipitates were used to transcribe mRNAs complexed with nuclear EGFR
into cDNAs in a reaction containing poly (TT) primers and radioactive dATP. Free
radioactivity was separated from labelled cDNAs by gel filtration and the
incorporation of radioactively labelled dATP into cDNAs was quantified. After
normalization to the concentration of the nuclear EGFR protein, the expression
of mRNAs enriched in complexes with immunoprecipitated nEGFR was increased
compared to the NRT-IP-EGFR-control. However, irradiation and cisplatin
treatments decreased the relative amount of enriched mRNAs complexed with nEGFR
compared to untreated cells. Notably, an immunoprecipitation with nonspecific
IgG was also positive for mRNA enrichment–as presented in —, although to a much
smaller extent. Enrichment of mRNAs in complex with nEGFR was not only observed
in A549 cells but also in the FaDu head & neck tumor cell line and in HSF7
normal skin fibroblasts.
We performed an immunoprecipitation of nEGFR and the IgG control at time 0
without irradiation (0 Gy, non-irradiated) and also at 24 h with irradiation (4
Gy, irradiated) to elucidate the functional relevance of complexes between nEGFR
and mRNAs. The mRNAs were extracted after immunoprecipitation from the complexes
and characterized with the help of Affymetrix microarrays. After quality control
of the microarray data, quantile normalization of the probe intensities was
performed. Subsequently, normalized intensities were used to assess differential
expression (DE) of genes and determine whether genes respond differently to
immunoprecipitation with nEGFR versus immunoprecipitation with IgG in the
absence of irradiation (contrast 1) or whether genes respond differently to
radiation in IgG-precipitated samples (contrast 2). An interaction term was also
added to the linear model (contrast 3, see the section) to determine whether a
difference in gene expression was observed in nEGFR samples in response to
radiation that differs from IgG samples. Following the DE analysis, we observed
a strong effect on contrast 1, as 15068 probes of 53617 probes were DE (p \<
0.05), when considering only oligos referring to genes. 8920 out of these 15068
probes have in addition a log fold change either \> +1 or \< -1 (S1_Tab.xls). No
genes were DE with a multiple adjusted p value \< 0.05 for contrast 2 and the
interaction term (contrast 3). This finding indicates a strong influence of
immunoprecipitation with either nEGFR or IgG on gene expression (contrast 1).
However, a treatment/radiation effect that differed between IgG and EGFR samples
was not observed (contrast 2 and 3). Heatmap-based visualization of the log2
normalized expression values for the 8920 DE probes in contrast 1 revealed the
differentially expressed genes when either immunoprecipitated with nEGFR (two
left columns) or IgG (two right columns). The heatmap also indicated that most
DE genes in contrast 1 were expressed at rather low levels (indicated by the
green color).
We performed a functional annotation of all 8920 identified mRNAs in complex
with nEGFR that were significant different in non-irradiated cells compared to
IgG complexes (p\<0.05) using the DAVID Gene Ontology Analysis to further
evaluate the binding of these mRNAs to nEGFR. DAVID matched 5515 of those mRNAs
and the analysis resulted in the following top scoring KEGG pathway groups:
Protein processing in the endoplasmic reticulum, Endocytosis, Proteasome,
Lysosome, Ribosome, Cell cycle and HIF-1A signaling pathway
According to our previous study, nEGFR is involved in regulating HIF-1A and VEGF
expression; therefore, we focused in a first approach on mRNAs involved in
HIF-1A /VEGF signaling. We isolated mRNAs from irradiated or non-irradiated A549
cells 24 h after irradiation and performed a quantitative RT-PCR to determine
the expression of a panel of 84 mRNAs associated with HIF-1A /VEGF signaling in
response to irradiation. We correlated mRNA expression quantified by RT-PCR with
constitutive enrichment of distinct mRNAs at nEGFR measured using an Affymetrix
chip analysis.
Eighty-three of 84 mRNAs transcribed from the genes assigned to the
RT<sup>2</sup> Profiler<sup>™</sup> PCR Array for Human VEGF/ HIF-1A -signaling
(Qiagen) were detected in a constitutive complex with nEGFR in the absence of
irradiation. We isolated mRNA from cells 24 h after irradiation and performed a
quantitative RT-PCR for this panel of RNAs to elucidate the relevance of the
formation of this complex. Fifty-three of the 84 mRNAs showed increased
expression in response to irradiation (mean expression 1.68 ± 0.86) (Tab 1,
column 2). Furthermore, treatment with the EGFR kinase inhibitor Erlotinib
reduced the radiation-induced increase in the expression of 45 of 53 mRNAs (mean
expression 1.1 ± 0.3, , column 3). Hence, we propose that nEGFR and its kinase
activity are involved in regulating the increased expression/stability and
translation of mRNAs associated with VEGF signaling in response to irradiation.
We also determined the expression of representative proteins encoded by 10 of
the 84 genes assigned to the RT<sup>2</sup> Profiler<sup>™</sup> PCR Array for
Human VEGF/HIF-1A signaling to further elucidate the relevance of the formation
of the complex between nEGFR and mRNAs. The increased mRNA expression observed
in response to irradiation correlated with increased protein expression.
Moreover, we detected over 1000 different miRNA species in complex with nEGFR
(GEO identifier GSE92428).
Notably, the amount of mRNA-specific miRNAs within the complex with nEGFR
decreased in response to irradiation, whereas the amount of the corresponding
mRNA and protein increased.
Based on this observation, the net amount of mRNA-specific miRNAs in the complex
with nEGFR negatively regulates the corresponding mRNA levels and protein
translation. In summary, we hypothesize that nEGFR must be part of the P-bodies,
which regulate mRNA translation and degradation in a miRNA-regulated manner.
Consequentially, we immunoprecipitated nEGFR and assessed the presence of the
GW182 protein, a marker of RISC and P-bodies.
In addition to a radiation-induced increase in nEGFR expression, we observed the
formation of a complex with the AGO2, PABPC1 and cNOT1 proteins, which all
belong to the RNA-induced silencing complex (RISC). The relative amounts of
these proteins in the EGFR-complexes were approximately equivalent, suggesting
that the radiation-induced increase in nEGFR expression is associated with
increased formation of the complex with RISC proteins. None of these proteins
were detected in the precipitate with the nonspecific IgGs. Neither the RNAse
nor DNAse treatment resolved the protein complex, which argues for a direct
protein interaction. These observations strongly suggest the functional
relevance of nEGFR in the context of regulating mRNA stability.
As further proof of the hypothesis that EGFR kinase is involved in AGO2
phosphorylation in response to irradiation, as has previously been reported for
hypoxia, we detected increased amounts of AGO2 within the nuclear fraction,
which was phosphorylated at residue Y393. Y393 phosphorylation is a well
characterized phosphorylation event performed by EGFR kinase. Importantly,
blockade of EGFR kinase activity by pretreatment with erlotinib, as visualized
by reduced auto-phosphorylation of nEGFR at Y992, reduced AGO2 phosphorylation
at Y393 in response to irradiation.
The deadenylase cNOT1 is critically important in the regulation of mRNA
stability. The cNOT1 protein shortens the poly-A tails of mRNAs and initiates
mRNA degradation. We applied an in vitro deadenylase assay to investigate the
role of nEGFR in cNOT1 activity. We immunoprecipitated cNot1 from nuclear
protein extracts and incubated it with a recombinant mRNA molecule terminally
labelled with the Cy5 dye. Deadenylated products were separated by urea-PAGE gel
electrophoresis and quantified using LICOR.
Deadenylation by cNOT1 was detected in the nuclear protein preparation, which
included proteins from the perinuclear region. Irradiation increased deadenylase
activity with a maximum at 6 hours and decreased 24 hours after irradiation.
Deadenylation was visualized by detecting smaller, deadenylated versions of the
Cy3-labelled recombinant mRNA, and the quantified activity was inversely
correlated with the amount of residual intact mRNA substrate. Bands were
allocated to deadenylation products using specific markers, and cNOT1 antibody
specificity was confirmed by incubating the substrate with a precipitate with
nonspecific IgG. The specificity of the deadenylation reaction was confirmed by
knocking down cNot1 expression with a specific siRNA.
Incubation of recRNA-polyA(8) with cNot1 immunoprecipitates for 1 h resulted in
the appearance of a band corresponding to the first deadenylation product. This
deadenylation reaction was blocked by the addition of poly A-oligonucleotides.
Incubation of the intact recRNA-polyA(8) template with the control IP for 1 h
did not produce a deadenylated product.
Pretreatment with erlotinib, which blocks EGFR kinase activity, strongly
increased radiation-induced cNot1 activity, whereas EGFR knockdown with a
specific siRNA inhibited deadenylase activity. Moreover, pretreatment of cells
with EGF for 16 hours also induced cNot1 deadenylase activity. Likewise,
irradiation induced cNot1 deadenylase activity in several tumor cell lines,
e.g., FaDu, SAS, and PC3 cells, and in normal fibroblasts.
We transfected A549 cells with an inhibitor of a miRNA, hsa-miR1180-5p, which is
enriched in the nEGFR complex and was validated to regulate stability and
translation of the NFATC4 mRNA, to examine the hypothesis that nEGFR regulates
mRNA stability and translation in concert with the cNot1 deadenylase and miRNAs.
We selected this miRNA as a target, since hsa-miR1180-5p was the only miRNA
within the nEGFR complex that targets the NFATC4 mRNA. NFATC4 regulates
HIF-1A/VEGF signaling and acts as a transcription factor that regulates cell
survival, differentiation, angiogenesis, invasive migration, and the tumor
microenvironment.
Inhibition of hsa-mir1180-5p increased the expression of NFATC4 protein to a
maximal level and prevented a further radiation-induced increase. Since NFATC4
expression responds to irradiation, we tested for a possible role for NFATC4 in
the radiation response. Clonogenic survival after radiation treatment was
determined to elucidate the effect of increased levels of the NFATC4 protein
induced by hsa-mir1180-5p inhibition. Increased NFATC4 protein expression
reduced the plating efficiency and increased radiosensitivity of A549 cells.
Comparable results for NFATC4 protein expression were achieved after EGFR
expression was knocked down with a specific siRNA. EGFR knockdown resulted in
increased expression of the NFATC4 protein, which was accompanied by a lack of a
radiation-induced increase in NFATC4 expression, similar to the control siRNA.
Since hsa-miR1180-5p does not exclusively target the NFATC4 mRNA, we directly
knocked down NFATC4 expression with a specific siRNA. The knockdown of NFATC4 in
A549 cells was efficient and protein expression was reduced. This knockdown
resulted in a radioprotective effect on both A549 and FaDu cells.
# Discussion
Here, we described a mechanism by which nEGFR regulates the stability of mRNAs
associated with VEGF signaling in a miRNA-directed manner. We immunoprecipitated
EGFR from nuclear protein preparations and identified bound proteins using
MALDI-TOF to elucidate the molecular function of nEGFR. Under control
conditions, nEGFR had already complexed with heterogeneous nuclear
ribonucleoproteins (HNRNP) and PML bodies, which are involved in regulating gene
transcription and mRNA translation during the cellular stress response,.
Indeed, based on published results from our group and other researchers, nuclear
translocation of EGFR is induced by cellular stress, e.g., radiation, hypoxia,
cisplatin, as well as its natural ligand, EGF. Interestingly, the binding of
mRNAs to EGFR was reduced significantly in response to radiation and cisplatin
treatment. As shown in previous studies and the present study, nuclear EGFR
translocation was accompanied by the activation of the tyrosine kinase.
Importantly, EGFR tyrosine kinase activity seemed to negatively correlate with
mRNA enrichment. In addition, mRNA binding to nEGFR was observed not only in
tumor cell lines but also in normal skin fibroblasts, suggesting a general role
for nEGFR in regulating mRNA stability.
Using a microarray analysis, we were not able to identify a radiation-induced
enrichment of mRNAs bound to nEGFR at 24 h that differed from IgG (control) in
the present study. However, these results do not exclude the possibility that
irradiation might alter mRNA binding to nEGFR compared to IgG at different time
points. Although a treatment effect was not observed during the microarray
analysis, we detected a significant enrichment of mRNAs in the EGFR complex
compared to control IgG samples in the absence of stress treatment. Differential
expression (DE) of 15068 of a total of 53617 mRNAs tested was detected. 8920 of
them code for genes and have in addition a log fold change either \> +1 or \< -1
and were therefore estimated as potential biological relevant. We hypothesize
that this large biological variation might only partially be related to the
differential binding of mRNAs to nEGFR. Among other factors, nonspecific binding
to nEGFR, genetic polymorphisms, and changes in mRNA levels due to age and
genotype-environment interactions most likely also contribute to the observed
high rate of differential expression.
The observation that mRNAs are also expressed in IgG-precipitated samples is
consistent with previously published data and also indicates that many tumor
cells, including A549 cells, produce endogenous IgGs, which are involved in
regulating tumor growth. The hypothesis that the list of DE genes contains
specific and nonspecific signals is supported by gene ontology analysis, which
pointed towards the regulation of terms such as cell cycle and ribosome. These
categories are enriched in many experiments, indicating that these GO terms are
not specific and do not need further interpretation. In contrast, the GO term
HIF-1A/VEGF signaling pathway is potentially attributed to binding to nEGFR, as
a link to nEGFR was observed in previously published data. An RT-PCR analysis of
83 DE genes related to the HIF-1A/VEGF signaling pathway and the expression of
proteins encoded by 10 selected genes from this group confirmed microarray data.
Preliminary data also revealed a potential link between nuclear EGFR and mRNA
stability, as the expression of genes involved in the HIF-1A/VEGF signaling
pathway was slightly increased in response to irradiation, which was abrogated
again by the EGFR kinase inhibitor erlotinib. However, based on the results from
the microarray analysis, the radiation-induced increase in the expression of the
candidate genes is not due to altered binding to nEGFR in response to
irradiation. Therefore, we propose that additional factors play roles. In this
context, a potentially important observation is that the radiation-induced
increase in the expression of mRNAs associated with VEGF signaling and protein
translation was associated with a simultaneous loss of miRNAs from the nEGFR
complex. This loss was not significant for a single miRNA species, as determined
by the array analysis. However, when we summed up the radiation-induced losses
of different miRNAs validated to target specific mRNAs (up to 339 species per
mRNA) from the nEGFR complex, the loss was apparent. Nevertheless, the interplay
between nEGFR, mRNAs and miRNAs has not yet been completely resolved yet. Our
data indicate that nEGFR may participate in a central process that negatively
regulates mRNA stability and translation in a miRNA dependent manner.
Based on these observations and the perinuclear location of EGFR in complex with
mRNAs and miRNAs, we hypothesized that nEGFR is part of the P-bodies. P-bodies
are involved in mRNA storage and RISC-regulated mRNA degradation. Therefore, we
assessed the localization of EGFR within P-bodies.
Indeed, we identified the RISC proteins cNot1, AGO2, GW182 and PABPC1 in complex
with EGFR. All proteins are located in P-bodies or GW-bodies in cells from
higher eukaryotes and govern miRNA-mediated silencing to inhibit the active
translation of mRNAs.
Translational repression of mRNAs is initiated by mRNA sorting to P-bodies for
decapping-dependent decay or sequestration. Alternatively, active translation of
mRNAs is inhibited by miRNA-mediated RNA silencing in the presence of GW182, the
endonuclease AGO, and the CCR4-Not deadenylation complex. However, although AGO
1–4 isoenzymes are capable of loading miRNA and exhibit endonuclease activity,
RNAi-dependent gene silencing is exclusively executed by AGO2 (RISC).
An interaction between AGO2 and nEGFR in response to hypoxia has been
described. Mechanistically, hypoxia activates EGFR kinase activity, which
phosphorylates AGO2 at residue Y393, inhibits AGO2 activity and subsequently
blocks miRNA maturation and loading to the miRNA-induced silencing complex
(miRISC). Our data also suggest in addition to an effect on miRNA maturation, a
role for the AGO2/EGFR interaction in the regulation of mRNA stability by the
miRISC. This complex is a miRNA-recruited protein complex that regulates mRNA
stability and subsequent protein translation. The core of this complex consists
of miRNA-loaded Argonaute and GW182 proteins. In mammalian cells, RISC contains
both poly(A)-binding proteins (PABP) and a deadenylase complex, which initiates
the mRNA degradation process by inducing deadenylation.
The mechanism by which protein translation is repressed is still not defined.
The CCR4-Not protein complex recruited by the GW182 protein was recently shown
to release PABP from the poly(A) tail, thereby disrupting mRNA circularization
and facilitating translational repression and deadenylation.
As shown here, nEGFR complexes with GW182 and cNot1 proteins. Inhibition of EGFR
kinase activity with erlotinib promoted cNot1 deadenylase activity. Thus, nEGFR
kinase activity acts as a negative regulator of deadenylase activity. This idea
agrees with the observation that nEGFR is a negative regulator of AGO2 in
response to hypoxia. In this context, nEGFR kinase activity is responsible for
the phosphorylation of AGO2 at residue Tyr 393 in response to hypoxic stress,
which suppresses dicer binding that is essential for microRNA maturation and
RISC function.
The present data suggest an inhibitory function of AGO2 phosphorylation in the
AGO2/GW182 complex, which regulates deadenylase activity. On the other hand,
downregulation of EGFR by a specific siRNA was associated with the absence of
the EGFR protein in the nuclear fraction and the subsequent inhibition of cNot1
deadenylase activity. Thus, nEGFR is required for deadenylase activity.
Moreover, the regulatory effect of nEGFR kinase activity on deadenylase activity
was also observed after activation of EGFR kinase by EGF. We detected
deadenylase activity in several tumor cell lines and in normal fibroblasts.
Consequently, general roles for nEGFR kinase activity in regulating mRNA
stability and protein translation are postulated.
After irradiation, the expression of mRNAs associated with VEGF signaling
increased. Interestingly, pretreatment with erlotinib blocked the radiation-
induced increase in mRNA expression. This finding supports the hypothesis that
inhibition of EGFR kinase activity prevents AGO2 phosphorylation/inactivation
and active cNot1-deadenylase degrades mRNAs located in the EGFR complex. The
hypothesis that nEGFR is involved in regulating the expression of proteins
involved in HIF1A/VEGF signaling is supported by previous studies showing that
radiation-induced expression of the HIF-1A protein is reduced in response to
erlotinib-mediated inhibition of nEGFR kinase activity.
The mRNA expression profile obtained from RT-PCR analysis of irradiated cells
favors the increased expression of genes involved in angiogenesis and anaerobe
glycolysis, which are markers of the metastatic tumor cell phenotype in response
to irradiation. In this context, EGFR is reported to be involved in regulating
VEGFA expression in transformed cells and human NSCLCs. Moreover, as shown in
our previous study, radiation induces HIF-1A expression to stimulate a metabolic
shift toward lactate production, which is associated with treatment resistance.
We hypothesize that the mRNA/nEGFR complex may be relevant to radiation-induced
tumor cell resistance.
According to the data presented in, the expression of mRNAs released from the
complex with nEGFR was increased in response to irradiation. This finding may
have resulted from mRNA stabilization and is consistent with the hypothesis that
radiation-induced EGFR translocation and activation of kinase activity blocks
AGO2 activity and deadenylase activity, subsequently resulting in mRNA
stabilization. Importantly, we were able to identify validated miRNAs in complex
with mRNAs and nEGFR. In response to irradiation these miRNAs were removed from
nEGFR complex as AGO2 was phosphorylated and inactivated. These observations
suggest the presence of a miRNA-driven deadenylase activity in complex with
nEGFR. A miRNA-driven model for the regulation of cNot1 activity has been
reported previously. The present study postulates a novel role for nEGFR, which
accumulates in perinuclear and nuclear regions. The nEGFR protein complexes with
mRNA and proteins involved in the RISC and negatively regulates the deadenylase
cNOT1 in a miRNA-directed manner through its kinase activity. Notably, EGFR
kinase activity and nuclear translocation are induced by cellular stress.
Moreover, this novel function of EGFR kinase is also observed in several EGF-
stimulated tumor cell lines and normal skin fibroblasts.
The effect of hsa-mir1180-5p knockdown on NFATC4 protein expression links the
presence of miRNAs in the EGFR/cNot1 complex with the translation of the
corresponding protein. Furthermore, miRNA-regulated NFATC4 protein expression
acts as a negative regulator of the post-radiation cell response and
radiosensitivity. In general, NFATC4 acts as a negative regulator of growth and
its expression is EGFR-dependent. Consequently, as shown here for A549 and FaDu
cells, knockdown of NFATC4 expression results in a radioprotective effect. These
data suggest that NFATC4 controls cell survival in tumor cells after exposure to
stress.
# Conclusions
In summary, the present study is the first time to show that nEGFR kinase-
regulated cNOT1 deadenylase activity enables cells to immediately respond to
cellular stress by interfering with mRNA stability in a miRNA-directed manner.
We postulate a three-step regulatory mechanism. In the first step, mRNA species
are loaded into a complex that contains nEGFR, GW182 and AGO2. The selectivity
of this process has not yet been defined, but we assume that the regulation of
the nuclear translocation of EGFR and constitutive formation of the complex with
HNRN-proteins play roles in selectivity. In the second step, the deadenylase
cNOT1 is loaded onto EGFR-bound mRNAs in a miRNA-guided manner. Third,
deadenylase activity is regulated by EGFR kinase activity and determines mRNA
stability and the translation frequency. Thus, a membrane bound tyrosine kinase
receptor, such as EGFR, can directly increase the stability of mRNAs involved in
regulating survival in a miRNA-dependent manner.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
The Wallacean shortfall, which suggests our understanding of geographical
distribution patterns of the species at large scales is generally poor, can be
minimized by sampling ecosystem gradients at smaller scales and expanding our
knowledge outward. Despite some progress in achieving global strategic goals of
the United Nations and the Aichi Targets, biodiversity has declined,
particularly in developing countries. The lack of information regarding
biodiversity status precludes recognition of impacts of anthropogenic activities
on biodiversity and implementation of conservation strategies and targets.
Therefore, to gain a large-scale understanding of any taxonomic group and to
determine threats and conservation potential within their spatial distribution
in accordance with environmental variability, there must first be fine-scale
(local/regional) baseline data.
Amphibian occurrence and abundance are greatly influenced by localized variation
in geomorphic, geologic, and environmental characteristics. Species Distribution
Modelling (SDM) approaches based on Geographical Information System (GIS) have
been widely used to predict distribution of species of amphibians and other
vertebrate groups such as rodents and passerine birds for conservation purposes.
However, with presence-only records often being the only available data, and
potential for imperfect detection within a study location, one must account for
distribution and absence given contributing environmental covariates.
Amphibians in Pakistan have long been ignored in research, conservation,
management, and policy and legislation. Pakistan’s National Climate Change
Policy (2012), National Biodiversity Strategy and Action Plan (2015) and
Pakistan Wetland Action Plan (2000) proposed guidelines for the conservation of
natural resources, including fauna and flora, and mitigation of threats.
Currently, 21 species of amphibian (all anurans) have been documented in
Pakistan of which nine are believed to be endemic. However, no progress on
integrating anurans in wildlife conservation, and policy development or
legislation has been made. There is no national assessment of conservation
status of anurans of Pakistan which cautions the use of global conservation
status. Only a few published studies report the richness and abundance of
various Pakistani anuran species. For example, the common Skittering Frog
(*Euphlyctis cyanophlyctis*) was reported as abundant in the rice fields of
Gujranwala, Punjab Province, and nine anuran species were recorded from Margalla
Hills National Park, Islamabad. Six anuran species were reported from Rawalpindi
and Islamabad areas including high abundance of Indus Valley Bull Frog
(*Hoplobatrachus tigerinus*) and Skittering Frog from Rawal Lake, Islamabad.
In the current study, we aimed to estimate niche suitability, niche overlap and
model distribution of anuran species within the Rawalpindi District and
Islamabad Capital Territory, which encompasses a unique mosaic of deserts and
xeric shrublands, montane grasslands, temperate broadleaf and mixed forests,
temperate conifer forests, and tropical/subtropical coniferous forests. Our
findings were expected to generate new information on factors affecting niche
suitability, spatial distribution size, and important ecoregions of the studied
species and for anuran diversity to inform future conservation plans in
Pakistan.
# Materials and methods
## Study area
The study was carried out in seven administrative units (Gujar Khan, Kahuta,
Kallar Sayedan, Kotli Sattian, Murree, Rawalpindi, and Taxila) of the Rawalpindi
District and Islamabad Capital Territory, Pakistan. The Rawalpindi District
(33.4620° N, 73.3709° E) is located in the north-west of Punjab Province and
covers an area of 5312 km<sup>2</sup>. Islamabad Capital Territory (ICT)
(33.7205° N, 73.0405° E) is in the north-east of the country and covers an area
of 906.50 km<sup>2</sup>. The elevation ranges from 457–2286 m and 457–610 m in
Rawalpindi District and Islamabad Capital Territory, respectively. The climate
of the study area is humid subtropical (Koppen climate classification). The
summers produce more rain than the winter due to the monsoon season (July-
August). The average rainfall in Islamabad Capital Territory is about 940 mm, in
areas of Rawalpindi, Gujar Khan and Taxila ranges from 970–990 mm and in Murree,
Kotli Sattian, and Kahuta is 1249 mm.
The study area is dominated by tropical and subtropical coniferous forests in
the north; broad-leaf, mixed forest and montane grasslands and shrublands in the
mid and midwest; and arid shrublands in the east and south. The tropical and
subtropical coniferous forests are dominated by *Pinus wallichiana* and *Pinus
roxburgii* and have relatively fewer human settlements. The proximal, central
and southern regions feature urban and semi-urban areas with vegetation species
such as *Acacia modesta*, along with *Olea cuspidata*, and *Dodonea viscosa*.
Of the 9 anuran species that are endemic to Pakistan, and therefore are of
increased conservation interest, our study area encompasses most of the range of
two of these endemics within Pakistan. Hazara Torrent Frog (*Allopaa
hazarensis*) is endemic to the springs and streams of Northern Pakistan. Murree
Hills Frog (*Nanorana vicina*) is endemic to Pakistan and India.
## Data collection
We surveyed 87 randomly selected sites in the seven administrative units of
Rawalpindi District and Islamabad Capital Territory and gathered anuran presence
data from 2016 to 2018 (March–September) on a monthly basis; we visited each
site at least twice from during the study period. The duration and number of
observers varied during the surveys; however, on average each visit consisted of
two days of non-standard field observations from early morning till late night
carried out on a weekly basis, with 4–6 observers. Each visit had at least one
observer familiar with the identification of anurans of the area. Anuran species
richness was recorded using time-constrained visual encounter survey (VES)
technique. Observers actively and thoroughly searched the sampling locations for
a predefined time (\~60–120 min.) in order to record the species. Adult
specimens and tadpoles were collected with dip nets or simply picked up by hand,
were examined, and identified following descriptive identification by Khan and
released back.
## Data analysis and species distribution modelling
All analyses, models, maps and plots were generated using R statistical software
version 3.6.3. A preliminary check on observed environmental values for each
species was conducted by using the extract function in the ‘raster’ package.
Environmental ranges of species were checked before running models in order to
identify potential flaws in the output (e.g. high spatial distribution
probability in montane ranges when the species observations have initial low-
elevation values). Bioclimatic variables (BIO 1–19) were retrieved from the
WorldClim repository via the ‘raster’ package at 0.5 arcmin resolution, and were
tested for collinearity using Variation Inflated Factors (VIF). The following
bioclimatic variables were retained: BIO7: temperature (C°) annual range; BIO8:
mean temperature (C°) of wettest quarter (3 consecutive months); BIO9: mean
temperature (C°) of driest quarter; BIO15: precipitation seasonality; BIO18:
precipitation (mm) of the average warmest quarter; and BIO19: precipitation (mm)
of the average coldest quarter. To map vegetation, we used MODIS (https://develo
pers.google.com/earthengine/datasets/catalog/MODIS_006_MOD13Q1#bands;
https://code.earthengine.google.com/e3a10b1ec6086c3ee7c598cfaca7dd98;
resolution: 250m; scale 0.0001). MODIS has a spatial resolution of 250 m and
provides a Vegetation Index (VI) value at a per pixel basis. We used two primary
vegetation layers: Normalized Difference Vegetation Index (NDVI), which is the
continuity index to the existing National Oceanic and Atmospheric
Administration-Advanced Very High-Resolution Radiometer (NOAA-AVHRR) derived
NDVI; and Enhanced Vegetation Index (EVI), which minimizes canopy background
variations and maintains sensitivity over dense vegetation conditions. In Google
Earth Engine (GEE), both MODIS layers The MODIS NDVI and EVI products were
computed from atmospherically corrected bi-directional surface reflectance that
had been masked for water, clouds, heavy aerosols, and cloud shadows. A 30 m
Digital Elevation Model (DEM) was retrieved from the USGS earth explorer
database (<https://earthexplorer.usgs.gov/>). The DEM was used to generate
slope, aspect, and terrain roughness via the *terrain* function in the ‘raster’
package. Distance to rivers was calculated using a local shape file of all
waterways in Pakistan and the *fasterVectToRastDistance* function of the
‘fasterRaster’ package. River distance values (in meters) were log normalized to
prevent outlier values from overwhelming the model contribution estimates. All
environmental variables were resampled to 0.5 arcmin resolutions thereafter
using the bilinear method, and masked to a shapefile of Pakistan regional
boundaries. The explanatory variables were then stacked and tested for
multicollinearity using the *vifstep* and *vifcor* functions of the ‘usdm’
package with a threshold VIF of 10 and a threshold correlation coefficient with
an absolute value of 0.7; whereby we excluded predictors that were strongly
correlated with ⸺ but considered to be less important for anurans ⸺ than other
predictors.
The Maximum Entropy or “Maxent” modelling method has been shown to perform as
well as, or better than ensemble models when modelling species distributions,
with additional benefits of lower computational power requirements and increased
simplicity of use. Species Distribution Models for this study were created
following guidelines on Maxent parameterization. Species occurrence data was
thinned to one point per raster cell to omit spatial biases. Maxent models were
created using the Maxent software wrapper through the ‘dismo’ package. A kernel
density bias mask was created by querying all available anuran occurrences from
the Global Biodiversity Information Facility (GBIF) database for Pakistan as a
measure of restrictive effort using the ‘MASS’ package using the reference
bandwidth smoothing factor. We used the *occ_search* function in the ‘rgbif’
package in R to access the GBIF database, using the taxon “Anura”, the country
Pakistan (“PK”), and “coordinates = TRUE” as filters to create a text file
containing 345 dataset keys and number of observations per dataset). We
generated 10,000 geographically randomized background points within the bias
mask estimate. Models were tuned using the *ENMeval* function in the ‘ENMeval’
package to identify feature selection variables and regularization multiplier
(beta-multiplier) values selected from the lowest delta Akaike Information
Criterion (AIC<sub>c</sub>) using five random *k*-folds. Maxent datasets were
partitioned into training (3/4) and testing (1/4) data using a 4-way partitioned
*k*-fold. Features selected in the final models were linear and quadratic (lq)
with a beta-multiplier set to between 1–2 for sensitivity testing, and 1 for the
final model. We selected these features for the final Maxent models for each
species based on the statistics we used to validate our model: area-under-the-
curve (AUC) values within receiver-operating characteristic (ROC) curves and the
true skill statistic (TSS \[= TP + TN—1, where TP = proportion of true positive
predictions and TN = proportion of true negative predictions\]). Models were
replicated with replacement using the bootstrap method.
Final models were visualized using the raw model output using the ‘ggplot2’
package and each model prediction was condensed using the highest true skill
statistics (TSS) threshold value as a filter (hereafter called “threshold
model”). The true skill statistics is defined based on the components of the
standard confusion matrix representing matches and mismatches between
observations and predictions. Model outputs were defined as *niche suitability*
on a 0–1 probability scale (1 = highest niche suitability; 0 = lowest niche
suitability), and threshold models were considered to be the niche distribution
of a given species in terms of overlap with others. Each threshold model was
converted to points and extracted from the explanatory variables to derive
summary statistics of environmental values for each species within their niche
distribution. All species threshold predictions were then combined to create
niche overlap raster to determine which eco-regions were most suitable for
anuran species in Pakistan. High-density niche overlap areas were identified by
filtering 50% (4.5 species per raster cell) and 100% (9 species per raster
cell). We then converted the two final niche spaces to a spatial polygon to
identify percentage overlap between each ecoregion and high-density niche
distributions.
# Results
## Niche suitability and overlap
Final predictions for niche suitability of all nine anuran species using Maxent
models indicated good fit in terms of AUC. Environmental variables varied among
species; however, distance to rivers and precipitation in the warmest and
coolest quarters were shown to have significant contribution (% contribution
\>10; higher permutation importance and variable importance in jacknife-testing)
for *Duttaphrynus bengalensis* and *M*. *nilphamariensis;* distance to rivers
and precipitation of the warmest quarter for *Duttaphrynus stomaticus;* distance
to rivers, precipitation of the warmest quarter, and NDVI for *A*. *hazarensis*
and *N*. *vicina*; and precipitation of the warmest quarter and NDVI were most
influential for *H*. *tigerinus*, *Euphlyctis* spp., *Minervarya* spp., and
*Sphaerotheca pashchima*. Habitat suitability for most species was higher in
locations with greater precipitation in the warmest quarter and less
precipitation in the coolest quarter. Habitat suitability for *A*. *hazarensis*
and *N*. *vicina* was higher in locations with greater ecosystem productivity
(higher NDVI) closer to rivers.
## Spatial distribution
Nine anuran species showed a divergent pattern of spatial distribution. We
found that *Euphlyctis* spp., *Minervarya* spp., and *H*. *tigerinus* showed
preference for the lowlands in proximal, central and southern parts of the study
with high urban settlements, little vegetation and higher average temperatures.
The toads (*D*. *bengalensis* and *D*. *stomaticus*) had scattered distributions
throughout the study area with no clear preference for elevation. *S*.
*pashchima* showed a patchy distribution in the midwestern extent of the study
area as well as the foothills to the north. *Microhyla nilphamariensis* also
showed a wide distribution throughout the study area with a preference for both
lowlands and montane terrain. Endemic species such as *N*. *vicina* and *A*.
*hazarensis* were observed in locations with higher elevations, proximal to
streams and lower average temperatures as compared to the other seven species
sampled.
Niche overlap at 50% (4.5 species per area) within the studied ecoregions showed
0.2% overlap in montane grasslands and shrublands, 4.8% in temperate broadleaf
and mixed forests, 23.6% in temperate coniferous forests and 33.9% in tropical
and subtropical coniferous forests. Whereas, the niche overlap (9 species per
area) revealed 0% overlap in montane grasslands and shrublands, 3.2% overlap in
deserts and xeric shrublands, 9.4% overlap in temperate broadleaf and mixed
forests, 17% overlap in temperate coniferous forests, and 70.2% overlap in
tropical and subtropical coniferous forests, providing evidence that tropical
and subtropical coniferous forests support the highest diversity of anuran
species in the region.
# Discussion
Development of analytical habitat distribution models has rapidly increased in
ecology due to the invention of new GIS tools and statistical techniques. Such
models statistically relate the geographical distribution of species or
communities to their present environment. Neither any scientific study on the
distribution patterns nor any species occurrence database of anurans of Pakistan
exists in the country. In forested mountainous regions outside of but similar to
those of Pakistan, amphibians, particularly *Ascaphus truei* and *Dicamptodon
tenebrosus*, were more abundant in stream habitats within older coniferous
forests. Precipitation and soil temperature influences probability of occurrence
for Polish amphibian species while vegetation contributed significantly in the
prediction for salamander species of central Portugal. Our results suggest
similar trends, with precipitation (of the warmest and coldest quarter),
distance to rivers and vegetation being highly deterministic factors of
suitability for anurans in Pakistan.
While Maxent models can accurately predict grid-based habitat suitability and
presence of species when observation data of those species is limited,
predictions from such models could also be used to identify the likeliest
locations of species for further monitoring and obtain actual presence/absence
or abundance data. Actual presence/absence or abundance data at precise
locations enables biologists to use finer-scaled environmental variables that
influence habitat suitability and abundance of species. For anurans and other
aquatic wildlife, the type of wetland habitat or changes in wetland variables
over time at repeatedly monitored sites can increasingly be quantified or
classified at fine spatial scales over large regions away from ground-truthed
locations. Increased availability of wetland data is due to: 1) the accumulation
of decades of remotely sensed environmental data by satellites; 2) the
development of free, open-source, online platforms (e.g., Google Earth Engine)
for processing remotely sensed data and extracting this data to survey
locations; and 3) development and sharing of open-source machine learning
techniques for predicting and classifying wetlands. To improve the chances of
detecting anurans or other vocalising species when they are present at sites,
monitoring could involve obtaining multiple visits per site by using passive
acoustic monitoring with pre-programmed acoustic recorders. Acoustic data could
then be transcribed to obtain detections per visit and hierarchical models can
be used to account for varying detection probability of species among sites when
estimating effects of environmental variables on occupancy or abundance.
The toads (Family Bufonidae) of the study area: *D*. *bengalensis* and *D*.
*stomaticus*, had somewhat scattered distribution throughout the region with no
clear preference for elevations; however, *D*. *stomaticus* showed a clear
preference to the northwestern extent of the study area, mostly concentrating
within and proximal to the lowlands. *D*. *bengalensis* and *D*. *stomaticus*
have been recorded as widespread species found up to 1800 m and 4500 m
elevations, respectively. *D*. *bengalensis* is adapted to various types of
habitats, even degraded ones, and around human habitations. The geographic range
of *D*. *bengalensis* has now been extended due to its introduction and *D*.
*bengalensis* has attained a status of invasive species in various parts of the
world. The two toads are found in the plains, lowlands, sub mountain areas as
well as hilly areas in Pakistan which experience monsoon season (July-August)
during which the species breed. We recorded *D*. *bengalensis* at elevations
higher than previously reported. One reason for their widespread distribution in
our study area is their adaptation to a wide range of habitats. *M*.
*nilphamariensis*, a diminutive frog species (Family Microhylidae), showed a
widespread distribution throughout the study area with a preference for both
lowlands and montane terrain. The species has been recorded from areas up to
2000 m elevation as well as from lowlands, sub mountain areas and foothills in
Pakistan.
The dicroglossid frogs of the study area showed varied distribution patterns.
*S*. *pashchima*, a burrowing frog species, showed a patchy distribution in the
midwestern extent of the study area as well as the foothills to the north. *S*.
*pashchima* has been recorded as a widespread species from lowlands and forested
areas up to 1500 m. This species remains under soft soil for most parts of the
year and emerges during summers to breed during the monsoon and avoids high
altitude areas possibly due to their burrowing habit, since hard substrate makes
it difficult for them to dig. Further, mountains in the north are under less
influence of monsoon, which likely provides less suitable breeding conditions.
Other dicroglossid frogs such as *Euphlyctis* spp. showed scattered distribution
probability through the lowlands. *Minervarya* spp. was shown to prefer
proximal, central, southern, and western lowland areas. *H*. *tigerinus* also
showed variability in preference between lowland and elevated areas, with a
concentrated distribution toward the middle to northern extents of the study
area. *H*. *tigerinus* has previously been recorded from areas up to 2000 m.
These areas are amongst most built up parts of the region in addition to
encompassing other human modified habitats such as croplands. *Euphlyctis* spp.
has been recorded from areas up to 2500 m while *Fejervarya* spp. from areas up
to 2000 m (Dijk 2004), but these dicroglossid frogs are also widespread in
lowlands and forested areas. *Euphlyctis cyanophlyctis* and *Zakerana
syhadrensis* occur along stream banks and water pools between forest edges,
agricultural areas, and residential gardens. Our findings are consistent with
the available information. However, we have thus forth provided empirical data
on the response of these species to the studied environmental factors for the
first time.
The endemic frogs, *A*. *hazarensis* and *N*. *vicina*, showed restricted
occurrence within the northern and north-eastern mountain ranges in areas of
high elevation compared to the proximal lowlands to the southeast. Most of these
areas feature subtropical pine forest (900–1500 m) dominated by *Pinus
roxburghii* trees, while the northernmost areas possess Himalayan moist
temperate forest (1500–3000 m) dominated by *Pinus wallichiana* and *Pinus
roxburghii*. The wetlands throughout this range exist in the form of freshwater
streams. *A*. *hazarensis* is endemic to Pakistan while *N*. *vicina* is known
from Pakistan and India. *A*. *hazarensis* and *N*. *vicina* are known from
streams and pools in forested mountainous areas as high as 1500 m and 3000 m,
respectively. During our study, we found that *A*. *hazarensis* could occur at a
higher elevation (\>1500) than the previously reported range.
# Conservation implications and suggestions
Urban developments have been shown to have negative impacts on amphibians.
Several types of culverts, tunnels and corridors have been developed and their
effectiveness assessed. Many amphibian species in North America and Europe have
used these structures, resulting in reduced mortality from vehicular collisions.
Since anurans in Pakistan enjoy no legal protection, no such consideration is
given during urban planning. With recent urban expansion in Rawalpindi District
and the Islamabad Capital Territory, there has been a noticeable reduction in
forests, open spaces and watersheds. As shown by our results, some of the anuran
study species are tolerant to habitat degradation while others are not. The
creation of human modified habitats may further facilitate the spread of native
species such as *Euphlyctis* spp., while also accommodating invasive species
such as *D*. *bengalensis* and *D*. *stomaticus* which may pose the threat of
resource competition against native species. We recommend studying the
effectiveness of such existing amphibian tunnels and corridors or designing new
ones tailored to the needs of our species. These could then be incorporated in
future urban development programs.
Conservationists put more emphasis on conserving threatened species. Studies,
however, have shown that even common species are subjected to population decline
and local extinction especially if these species are associated with a
particular type of habitat or set of environmental conditions. A 29% decrease in
the population of the Moor Frog (*Rana arvalis*), which is found in heathlands
and moorlands in parts of Europe, was reported between 1950–2006. This decline
was attributed to cultivation of heathlands and moorlands, lowering of ground
water levels and intensification of agricultural practices. In a forest
landscape study, Brown Frogs (*Rana arvalis*, *R*. *temporaria*) bred more in
various wetland habitats (e. g., naturally flooded areas, beaver ponds,
mitigation pools with shallow littoral zones, cleaned ditches) than in ditches
overgrown with forest vegetation.
Climate change during the past two decades has affected several species of
plants and animals in Pakistan. The tadpoles of *A*. *hazarensis* and *N*.
*vicina* responded (under laboratory conditions) to higher temperature
(*\>*26°C) through faster metamorphosis, reduction in the body size, more
frequent developmental complications or deformities such as edema and tail
kinks, lower fitness and higher mortality. Being associated with a particular
set of environmental conditions in the north and northeast of the study area, it
is feared that these two species endemic frogs, which are currently evaluated as
least concern in the IUCN Red List of threatened species, may experience local
extinction in the future. Land use simulators can be used to project changes
over space and time in environmental variables used both in Maxent models (e.g.,
climate) and in models based on surveys whose locations were informed by Maxent
model predictions. Thus, Maxent models can be used to project changes in
distribution of species over time under different climate scenarios, and raster
layers based on these distributions can be used to identify potential refugia
for anurans either under current or future climate conditions. These raster
layers may also be used as inputs in raster overlay-based conservation planning
tools (e.g., Marxan, Zonation) to prioritise locations for protection or
management of threatened species.
# Supporting information
We would like to especially thank Mr. Wajehuddin and Mr. A. Bhutta, Punjab
Forest Department, Government of Punjab, Pakistan for providing assistance
during the field work. We are thankful to Snow Leopard Foundation, Pakistan, for
providing logistics. We are also thankful to Dr. Hussain Ali, Snow Leopard
Foundation, for his help.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
The membrane skeleton is the basis of erythrocyte morphology and deformability.
It is the hexagonal lattice structure formed by 6 spectrin tetramers connecting
to the short actin filaments at the junctional complex. The membrane skeleton is
anchored to the lipid bilayer via ankyrin and band 4.1. The short actin
filaments or protofilaments in the junctional complexes have the constant length
of \~35–37 nm, which plays important roles in keeping the hexagonal structure
and the mechanical property of the membrane skeleton. The short actin filament
consists of 6 pairs of actin monomers and is coated by tropomyosins (TMs), with
erythrocyte tropomodulin (E-Tmod) capping its slow growing end (pointed end).
As a TM-binding protein and the only capping protein at the pointed end in
erythrocytes, E-Tmod plays a critical role in restricting the length of the
short actin filament (F-actin). E-Tmod is a 41 kDa protein (E-Tmod41), which
consists of N-terminal actin binding domain (E-Tmod<sub>1–92</sub>) and
C-terminal actin binding domain with six leucine repeats. E-Tmod41 binds to
TM5/5b (35 nm) at 39–138 residues and the complex functions as a “molecular T
ruler” metering off long actin filaments to short filaments of 37 nm. Recently,
a short E-Tmod isoform of 29 kDa (E-Tmod29) was discovered. It lacks the
N-terminal actin-binding domain but retains the C-terminal actin-binding domain.
It can bind to TM5 or G-actin and is localized in the cytosol of erythrocytes.
The expressions of E-Tmod41 and E-Tmod29 are driven by the alternative promoters
of *E-Tmod* gene. E-Tmod41 null mice display a mild sphero-elliptocytic anemia
with osmotically fragile erythrocytes, due to the misregulation of F-actin
lengths and a disrupted spectrin-actin lattice of membrane skeleton. In addition
to E-Tmod, there are three members in Tmod family, neuronal Tmod (N-Tmod),
ubiquitous Tmod (U-Tmod), and skeletal muscle Tmod (sk-Tmod). E-Tmod is the only
Tmod isoform present in human and mouse mature erythrocytes. But U-Tmod is found
in erythroid progenitors and exists in the erythrocytes of TOT (Tropomodulin
overexpressing transgenic) / E-Tmod<sup>-/-</sup> mice. It may be due to the
wide range of expression and the weak capping activity of U-Tmod.
The topology of membrane skeleton is formed during the development and
maturation of erythrocytes. The synthesis and expression of major cytoskeletal
proteins in erythrocyte membrane occur in an asynchronous manner and the
remodeling of the membrane skeleton begins at a very early stage during
erythrocyte development. In the erythroid differentiation induced by
erythropoietin, interleukin-3 (IL-3) and stem cell factor, etc., the gene
expressions for many membrane skeleton proteins are significantly upregulated.
In addition to chemical factors, the biomechanical force, fluid shear stress,
has been found to contribute to the hematopoiesis in the embryos. In adult, the
reticulocytes have to circulate in the blood vessels before they become mature.
Thus, these indicate that physical environment like fluid shear stress may also
be involved in regulating of gene expressions of membrane skeleton proteins.
microRNAs (miRNAs) are the small non-coding RNA molecules containing about 22
nucleotides and play key roles in the regulation of gene expression. Many
miRNAs, such as miR-451, miR-221/222, and let-7d, etc., are shown to be involved
in the erythroid differentiation. There are mechano-sensitive miRNAs, such as
miR-126, miR-23b, miR-10a, etc., which could be regulated by fluid shear stress
in endothelial cells, vascular smooth muscle cells and macrophages. Therefore,
it is possible that miRNAs may play roles in regulating the gene expressions of
membrane skeleton proteins in the erythroid differentiation induced by fluid
shear stress.
In the present study, we examined the effects of fluid shear stress on the
erythroid differentiation, E-Tmod41 expression, and F-actin cytoskeleton
remodeling. Our data showed that fluid shear stress could induce the erythroid
differentiation and E-Tmod41 expression, thus contributing to the F-actin
cytoskeleton remodeling. Furthermore, shear stress could upregulate E-Tmod41
expression by suppressing E-Tmod41-targeting miR-23b-3p and activating the
alternative promoter upstream of exon 0.
# Materials and Methods
## Cell culture and animals
Mouse erythroleukemia (MEL) cell line derived from Friend virus-infected mice
were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS).
Terminal erythroid differentiation was induced in MEL cells by adding 2%
dimethyl sulphoxide (DMSO) in the culture medium for 24 hours. C57BL mice of 8
weeks old were paired. The vaginal plug was checked on the second day and
counted as embryonic day 0.5 (E0.5). On E10.5, the pregnant mice were sacrificed
by cervical dislocation. Embryonic erythroblasts were isolated from the yolk
sacs embryos and maintained in IMDM medium containing 10% FBS, 10 units
ml<sup>-1</sup> penicillin, 10 μg ml<sup>-1</sup> streptomycin, 0.2 mg
ml<sup>-1</sup> Fe-saturated transferrin, and 0.5 mg ml<sup>-1</sup> ascorbic
acid. The animal protocol was approved by the Ethical Committee of Peking
University Health Science Center.
## Cell exposure to shear stress
MEL cells or erythroblasts were plated in a 6-cm Corning petri dish and the dish
was placed in a cone-plate shearing system (Fig A). When the cone spins, the
cells will be subjected to shear stress. The shear stress, τ, can be calculated
based on the equation, *τ* = *μω*/ *α*, where *μ* is the viscosity of the cell
suspension (2.21 mPa∙s), is the angular velocity, and *α* is the angle of the
cone (0.5°). The shear stress was set to 5 dyn/cm<sup>2</sup>, which is the
shear stress yielded in the dorsal aorta of E10.5 embryos.
## Flow cytometry
Cells were fixed in 4% paraformaldehyde, washed with phosphate buffered saline
(PBS), and incubated with 0.1% Triton X-100 for 5 min. After blocked in 1%
bovine serum albumin (BSA) for 30 min at room temperature, the cells were
incubated with rhodamine phalloidin (Cytoskeleton Inc., USA) for 20 min in the
dark at 37°C. Then the cells were resuspended in 300 μl PBS and analyzed in a BD
FACS Calibur (USA). The mean fluorescent intensity was measured to represent the
F-actin content in the cells.
## Laser scanning confocal microscopy
The cells were stained with rhodamine phalloidin following the procedures
mentioned above. The nuclei were counterstained with
4',6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, China) for 10 min
and then washed with PBS. The samples were resuspended in the mixture of PBS and
glycerol (v/v, 1:1) and placed into a coverglass bottom dish. The cells were
observed with a laser scanning confocal microscope (Leica TCS SP8 MP FLIM,
Germany). The mean fluorescent intensity of the images were calculated by using
ImageJ 1.46r software (National Institutes of Health, USA).
## RNA isolation and Quantitative RT-PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA)
according to the manufacturer’s instructions. Two microgram of total RNA was
reverse transcribed to cDNA using a ReverAid First Strand cDNA Synthesis Kit
(Thermo, USA). Real-time PCR was performed on a Mx3000 Multiplex Quantitative
PCR system (Stratagene, USA) using Brilliant II SYBR Green QPCR Master Mix
(Agilent Technologies, USA). The sequences of the primers used for E-Tmod41 were
as follows: forward: 5’-GAC ACA GCC TCA CAC AAT GT-3’; reverse: 5’-CTT GGT GGT
CTG ATC CTT CT-3’. The sequences of the primers for the markers of erythroid
differentiation, β-major globin (*Hbb-b1*), glycophorin A (*GYPA*), and *GATA1*,
and miR-23b-3p host gene, *2010111I01Rik*, were listed in Table A in. GAPDH
(forward: 5’-ACC ACA GTC CAT GCC ATC AC-3’; reverse: 5’- TCC CCA CCC TGT TGC TGT
A-3’) was used as an internal control. A relative fold change in the gene
expression was calculated using the method of 2<sup>-△△CT</sup>.
## Western blot analysis
Cells were lysed in RIPA buffer and centrifuged at 12000×*g* for 5 min at 4°C.
The supernatant was collected and the protein concentration was quantified by
BCA assay (Applygen Technologies Inc., Beijing, China). Proteins (20 μg) were
separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. The
membranes were incubated with anti-E-Tmod41 antibody (prepared by AbMax
Biotechnology Co., Ltd, Beijing, China), anti-glycophorin A antibody (Beijing
Biosynthesis Biotechnology Co. Ltd, Beijing, China), anti-GAPDH antibody, or
anti-β-tubulin (Santa cruz Biotech., USA), followed by HRP-conjugated goat anti-
rabbit or mouse IgG. The signals were detected by using an Enhanced
Chemiluminescence Detection (ECL) kit (Evergreen, Beijing, China).
## Adenovirus infection
MEL cells (1×10<sup>6</sup> cells) were infected with adenovirus, Ad-E-Tmod41
and Ad-Null (SinoGenoMax, Beijing, China) at 0, 25, 50, 75, and 100 MOI for 48
hours. The cells were either lyzed for protein extraction or fixed in 4%
paraformaldehyde for F-actin content analysis.
## siRNA, mimic, and inhibitor transfection
Small interfering RNA (siRNA) specific to E-Tmod41 was designed using Invitrogen
Block-iT RNAi Designer. The sequence is 5’-GGA AUU UAA GGA CCG AGA A-3’. The
mimic and inhibitor of miR-23b-3p were purchased from RiboBio (Guangzhou,
China). MEL cells were transfected with siRNA, mimic, or inhibitor (50 and 100
nM) by using riboFECT CP transfection kit (RiboBio, Guangzhou, China).
Transfected cells were harvested at 24 hours for mRNA extraction or at 48 hours
for protein isolation.
## MicroRNA array analysis
MEL cells sheared for 12 hours and the unsheared controls (3 pairs) were
harvested and lysed in TRIzol reagent (Life technologies, Carlsbad, CA). After
RNA extraction and quality analysis, miRNAs were labeled and hybridized on the
miRCURY LNA Array (v.18.0, Exiqon, Danmark) at KangChen Bio-tech Inc. (Shanghai,
China). The images were analyzed and data were extracted using GenePix Pro 6.0
software (Axon). The microarray data was validated by using TaqMan Assay (Life
technologies).
## Plasmid construction, site-directed mutagenesis, and dual luciferase assay
P<sub>E0</sub> (\~700 bp) and P<sub>E1</sub> (\~1000 bp) promoter sequences were
amplified from mouse genomic DNA and inserted to pGL3 basic vector (Promega,
USA) at the multiple cloning sites, respectively. The E-Tmod 3’-untranslated
region (3’UTR) was amplified from mouse cDNA and subcloned to pGL3 control
vector at XbaI site. The mutagenesis of potential miR-23b-3p targeting site was
introduced by using site-directed mutagenesis kit (Tiangen Biotech., Beijing,
China). For transfection, 1 μg of plasmid and 100 ng of renilla luciferase
vector were co-transfected into MEL cells by FuGENE 6 (Promega, USA). Cells were
collected after 48 hours for the detection of luciferase activity by the dual-
luciferase reporter assay system (Promega, USA). For miRNA study, pGL3 control
vector containing wild type or mutated E-Tmod 3’UTR were co-transfected into MEL
cells with miR-23b-3p mimics (50 nM) or control mimics. Then the cells were
collected after 24 hours for the dual luciferase assay.
## Statistical analysis
All experiments were performed in duplicate with data averaged from at least
three independent experiments. The data are presented as the mean ± SEM. Direct
comparisons were made using paired or unpaired Student’s *t*-test, and multiple
group comparisons were made using one-way analyses of variance (ANOVA).
Statistical significance was defined as *p*\<0.05, 0.01, or 0.001 (indicated as
\*, \*\*, or \*\*\*, respectively). Prism 5.0 software (GraphPad, Inc., USA) was
used for data analyses.
# Results
## Fluid shear stress induces erythroid differentiation and F-actin cytoskeleton remodeling in MEL cells
We employed MEL cell, a cell model commonly used for the study of erythroid
differentiation, to examine the effects of fluid shear stress on the erythroid
differentiation and cytoskeleton remodeling. A cone-plate shearing device was
developed to generate the laminar shear stress of 5 dyn/cm<sup>2</sup> (Fig A),
mimicking the blood flow in the aortas of embryos at embryonic day 10.5. MEL
cells treated with 2% DMSO served as the positive control for erythroid
differentiation. Wright-Giemsa staining showed that the dark blue color became
reddish in the cytosol of MEL cells treated with shear stress or DMSO and that
the nuclei became condensed. Further analysis showed that their cellular area
significantly decreased as compared to control. Quantitative RT-PCR data showed
that markers for erythroid differentiation, *Hbb-b1*, *GYPA*, and *GATA1*, were
all significantly upregulated by both shear stress and DMSO. These data indicate
that, although it was less competent than DMSO, fluid shear stress indeed
induced the erythroid differentiation in MEL cells.
Since the morphological change is accompanied with F-actin cytoskeleton
remodeling during erythroid differentiation, we examined the F-actin content in
MEL cells treated with DMSO and shear stress. Flow cytometry analysis showed
that the F-actin content increased \~1.5 fold in both DMSO- and shear stress-
treated MEL cells. Confocal images also showed the similar results.
## Fluid shear stress upregulates E-Tmod41 in MEL cells
As the capping protein at the pointed end of F-actin, E-Tmod41 is critical for
F-actin polymerization and stability. It may play a role in the F-actin
cytoskeleton remodeling induced by fluid shear stress. Therefore, we examined
E-Tmod41 expression in shear stress-treated MEL cells. Quantitative RT-PCR
showed that E-Tmod41 mRNA expression was upregulated by 3 h of shearing. As the
shearing time extended, E-Tmod41 mRNA level increased and reached the highest
level at 12 h. As detected by Western Blot, E-Tmod41 protein level did not
change at 12 h (data not shown) but markedly increased 2 fold at 24 h. The data
indicate that fluid shear stress could upregulate E-Tmod41 expression.
## E-Tmod41 regulates F-actin content in MEL cells
The upregulation of E-Tmod41 expression by shear stress may contribute to the
F-actin cytoskeleton remodeling. We next performed the gain or loss of function
experiments to study the effect of E-Tmod41 in F-actin remodeling. E-Tmod41 was
over-expressed by adenovirus infection. Both flow cytometry and confocal
microscopy showed that the F-actin content was significantly increased in MEL
cells infected by 50 MOI Ad-E-Tmod41. On the contrary, when E-Tmod41 was
knocked down by E-Tmod41-specific siRNA, the F-actin content was greatly
reduced. These findings demonstrate that E-Tmod41 affected the F-actin content
in MEL cells, which may mediate the F-actin cytoskeleton remodeling induced by
shear stress. To explore whether the expression of E-Tmod41 would affect the
erythroid differentiation, we detected the expressions of *Hbb-b1*, *GYPA* and
*GATA1* in MEL cells with E-Tmod41 overexpression or suppression. But
quantitative RT-PCR data showed that none of these markers changed (Figs A and
B).
## Fluid shear stress suppresses the expression of an E-Tmod-targeting miRNA, miR-23b-3p
miRNAs play important roles in hematopoiesis and there are many miRNAs that
could be regulated by fluid shear stress. So we collected MEL cells sheared for
12 h and performed miRNA microarray analysis. Data showed that 24 miRNAs were
upregulated and 58 miRNAs were down-regulated \>1.5 fold (with *p* value \<0.05)
by shear stress (Table B). Among the down-regulated miRNAs, miR-23b-3p was
predicted to target E-Tmod mRNA 3'UTR by TargetScan and Pictar. Quantitative RT-
PCR data showed that, in addition to miR-23b-3p, fluid shear stress could also
suppress the expression of miR-23b-3p’s host gene, *2010111I01Rik*. Importantly,
*2010111I01Rik* is not a target of miR-23b-3p (Fig D).
Sequence analysis showed that the targeting site of miR-23b-3p on E-Tmod 3’UTR
is highly conserved in mammals. To test whether miR-23b-3p targets E-Tmod, two
reporter vectors were constructed with a luciferase-coding sequence followed by
wild type or mutant E-Tmod 3’UTR , which were named as pGL3-E-Tmod 3’UTR and
pGL3-E-Tmod 3’UTR mutant, respectively. Dual luciferase assay demonstrated that
the luciferase activity of pGL3-E-Tmod 3’UTR was reduced by miR-23b-3p mimic,
while that of pGL3-E-Tmod 3’UTR mutant did not change. The data suggest that
fluid shear stress downregulated the expression of miR-23b-3p and that
miR-23b-3p was an E-Tmod targeting miRNA.
## miR-23b-3p regulates E-Tmod41 expression and F-actin content in MEL cells
Since miR-23b-3p was shown to be an E-Tmod targeting miRNA, we next examined
whether miR-23b-3p could regulate E-Tmod41 expression and thus affect F-actin
content in MEL cells. miR-23b-3p was overexpressed or knocked down with mimic or
inhibitor (Figs). Quantitative RT-PCR showed that mRNA level of E-Tmod41 was not
changed by miR-23b-3p mimic or inhibitor (Figs). But the E-Tmod41 protein level
was decreased or increased by mimic or inhibitor. F-actin was stained in MEL
cells transfected with miR-23b-3p mimic or inhibitor. Confocal microscope images
were taken (Figs) and the mean fluorescent intensities (representing F-actin
content) were measured. Data showed that miR-23b-3p mimic reduced F-actin
content in MEL cells, while miR-23b-3p inhibitor had the opposite effect.
## Fluid shear stress regulates *E-Tmod* alternative promoters
The expression of *E-Tmod* gene was driven by two alternative promoters. One is
located up-stream of non-coding exon 0 (E0), named as promoter E0
(P<sub>E0</sub>), the other is located up-stream of exon 1 (E1), named as
promoter E1 (P<sub>E1</sub>). Our previous study showed that P<sub>E0</sub>
preferably regulated the transcription of E-Tmod41, while P<sub>E1</sub>
preferably regulated the transcription of E-Tmod29. Therefore, we constructed
two reporter vectors, with the luciferase coding sequence driven by
P<sub>E0</sub> or P<sub>E1.</sub> MEL cells were transfected with two vectors,
respectively, and then subjected to shear stress for 12 hours. Dual luciferase
assay showed that P<sub>E0</sub> activity was augmented over 10 fold by shear
stress , while P<sub>E1</sub> activity was reduced to 1/10 of the control level.
## Fluid shear stress induces erythroid differentiation and E-Tmod41 expression in mouse embryonic erythroblasts
To test the results obtained in MEL cells, mouse erythroblasts were isolated
from mouse E10.5 embryos and subjected to shear stress for 24 hours. Wright-
Giemsa staining showed that un-sheared erythroblasts had the characteristics of
polychromatic erythroblasts. After shearing, the dark blue in the cytosol became
much lighter and the nuclei became condensed. Western blot results demonstrated
that the protein expressions of E-Tmod41 and glycophorin A were greatly
upregulated in sheared erythroblasts. These data suggests that shear stress
could induce erythroid differentiation and E-Tmod41 expression in primary
erythroblasts, which is consistent with the data obtained in MEL cells.
# Discussion
Previous efforts have proved that hemogenic endothelial cells could be induced
to differentiate into hematopoietic cells both *in vivo* and *in vitro* by fluid
shear stress in zebra fish and mouse. Furthermore, hematopoietic stem cells
(HSCs) or multipotent progenitor cells circulate and differentiate in the
embryonic blood vessels. Therefore, erythroid differentiation should also be
regulated by shear stress. Here, by using both MEL cells and embryonic
erythroblasts, we showed that fluid shear stress yielded in aortas of E10.5
embryos, could indeed induce the erythroid differentiation (Figs), which is
consistent with Adamo’s observation. The effect of fluid shear stress on
erythroid differentiation may come from its regulation on the transcriptional
factor Krüppel-like factor 2 (Klf2), which has been proved to play a role in
erythropoiesis. Our preliminary result showed that Klf2 mRNA level was
significantly upregulated in MEL cells sheared for 6 hours (data not shown),
which is consistent with previous studies.
Findings of flow cytometry and confocal microscopy analyses showed that MEL
cells treated with both fluid shear stress and DMSO had increased F-actin
cytoskeleton content as compared to the control cells. This means fluid shear
stress contributes to F-actin cytoskeleton remodeling during erythroid
differentiation. Our previous work showed that DMSO treatment could upregulate
the expressions of E-Tmod41 and its transcription factor GATA1 in MEL cells. In
this study, we found fluid shear stress could upregulate the expression of
E-Tmod41. Further studies with overexpression or knockdown of E-Tmod41 indicate
that E-Tmod41 affects F-actin content (Figs), which may explain the mechanism
for the effect of fluid shear stress on F-actin remodeling. E-Tmod29, an isoform
of E-Tmod that can bind to TM and G-actin in the cytosol, was found
downregulated in MEL cells after shearing (data not shown), suggesting that it
may play a role in F-actin cytoskeleton remodeling. The downregulation of
E-Tmod29 may free more TMs and G-actins, which are available for F-actin
assembly.
As the capping protein at the pointed end of F-actin, E-Tmod and its family
members play important roles in regulating F-actin structures. Recent studies
are mainly focused on E-Tmod and U-Tmod due to their wide expressions. The
abilities of U-Tmod that capping the pointed end of F-actin and binding to TMs
(including α/βTM, TM5b, TM5NM1) are weaker than E-Tmod. Therefore, U-Tmod is
involved in the dynamic change of F-actin structure, and regulates the cell
motility in endothelia and insulin-stimulated GLUT4 exocytosis in adipocytes;
while E-Tmod is widely expressed in the terminally differentiated cells, such as
erythrocytes, cardiomyocytes, and skeletal muscle cells, etc. U-Tmod was found
in the hematopoietic stem and progenitor cells and plays an important role in
terminal differentiation of fetal liver erythroid cells. During the erythroid
differentiation, U-Tmod was downregulated and E-Tmod41 was upregulated, finally,
leaving E-Tmod41 to be the sole protein that caps the pointed end of F-actin.
This information suggests that U-Tmod may be downregulated during the erythroid
differentiation induced by fluid shear stress, which would provide more pointed
ends for E-Tmod41 to cap and accelerate F-actin remodeling. In addition, our
data suggested that E-Tmod41 does not affect the erythroid differentiation (Figs
A and B). This is consistent with previous findings that E-Tmod null mice only
have a mild spherocytic elliptocytosis and seem to have no defect in erythroid
differentiation.
miRNAs are involved in both erythroid differentiation and the mechanical
responses in endothelial cells, vascular smooth muscle cells, and macrophages.
By using microRNA array, we found that many miRNAs are differentially regulated
by fluid shear stress in MEL cells. Among them, mechanosensitive miR-23b-3p,
which was proved to be an E-Tmod targeting miRNA, was found suppressed by shear
stress. The suppression of miR-23b-3p resulted in the upregulation of E-Tmod41
protein expression and contributed to F-actin remodeling in MEL cells (Figs). We
noticed that miR-23b was found upregulated by pulsatile shear stress in human
umbilical endothelial cells and participated in the regulation of cell
proliferation. The contradictory results may come from the different responses
of one miRNA to different flow patterns in various cell types. It was reported
that miR-23b could enhance the connections between breast cancer cells and that
the inhibition of miR-23b enhanced their migration and deformation abilities.
MiR-23b was found to directly target p21-activated kinase 2 (PAK2) and increase
the phosphorylation of myosin II. In addition, miR-23b was found to target many
genes that participate in cell cytoskeleton remodeling. These studies suggest
that miR-23b can regulate cytoskeleton and deformation of cells. In our case, it
contributes to F-actin cytoskeleton remodeling in sheared MEL cells by targeting
E-Tmod41.
Our data showed that fluid shear stress could activate P<sub>E0</sub> promoter
to promote E-Tmod41 expression and suppress P<sub>E1</sub> promoter to inhibit
E-Tmod29 expression. This is consistent with our previous observations that
P<sub>E0</sub> activity is increased and becomes dominant in maturating
reticulocytes, while P<sub>E1</sub> activity is high in undifferentiated
erythroblasts. The differential regulations of E-Tmod41 and E-Tmod29 by fluid
shear stress would be beneficial to F-actin cytoskeleton remodeling and
morphological change in erythroid cells. Besides, it should be pointed out that,
in our study of miRNA, the protein level of E-Tmod29 was not changed by
miR-23b-3p (data not shown), which means E-Tmod41 but not E-Tmod29 is the target
of miR-23b-3p. Therefore, the down-regulation of E-Tmod29 is mainly through the
regulation of alternative promoters by fluid shear stress but not through
miR-23b-3p.
In our study, we used the cone-plate shearing device to shear the cells. Since
MEL cells and embryonic erythroblasts are suspending cells, the shear stress
acting on them in the shearing device may be much more complicated as compared
to adherent cells, e.g., hemogenic endothelial cells. In addition, we used the
shear stress yielded in the aortas of E10.5 embryos, 5 dyn/cm<sup>2</sup>. But
for the erythroblasts in bone marrows and reticulocytes circulating in the
blood, the mechanical forces they sense (including shear stress and hydrostatic
pressure) and their physical environments may not be the same as in the
embryonic aortas. Therefore, it would be useful if we design devices mimicking
the environments of bone marrow or blood vessels by microfluidics and micro-nano
techniques.
In conclusion, by using our experiment setup, our findings suggest that fluid
shear stress induced the differentiation and F-actin cytoskeleton remodeling in
nucleated erythroblasts. Fluid shear stress upregulated E-Tmod41 expression in
MEL cells and embryonic erythroblasts and the change of E-Tmod41 contributed to
F-actin cytoskeleton remodeling. The upregulation of E-Tmod41 by shear stress
could be mediated by two mechanisms: the inhibition of its targeting miRNA,
miR-23b-3p, and the activation of its promoter, P<sub>E0</sub>. Our work would
help us to better understand the factors that regulates erythroid
differentiation, the molecular mechanisms for cytoskeleton remodeling in
erythroid cells, and the mechanisms for the mechanical regulation of E-Tmod.
# Supporting Information
We thank Drs. Jing Zhou and Wei Kong in our department for their useful
suggestions. We thank Dr. Wei Kong for using her microscope to take bright field
images. We thank Ms. Fang Yu for her assists in flow cytometry analysis and Dr.
Qihua He for her assists in confocal microscopy analysis.
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: WY XW LAS. Performed the
experiments: WM. Analyzed the data: WM WY. Contributed
reagents/materials/analysis tools: XZ SZ DS WK. Wrote the paper: MW XW LAS
WY. |
# Introduction
Cloud computing is popular in industry due to its ability to deliver on-demand
resources according to a pay-as-you-go model. Usually, three basic service
models are included in cloud computing: Infrastructure as a Service (IaaS),
Platform as a Service (PaaS) and Software as a Service (SaaS). Namely, SaaS
provides access to complete applications as a service. PaaS provides a platform
for developing other applications on top of it, such as the Google App Engine
(GAE) and Azure. IaaS provides an environment to deploy the managed virtual
machines. Technically, when the users submit the requests, the providers would
provide the resources depending on the users’ demand. As a key technique in
cloud computing, the elasticity has the ability to acquire and release the
resources according to the users’ demand.
Generally, the providers implement an automatic provisioning approach via the
virtualization technique. Virtualization makes it possible to rapidly scale the
resources up or down. The aforementioned approaches present a reactive method,
which is triggered by a certain threshold, such as CPU utilization or memory
utilization. Actually, two or more thresholds should be used as a performance
metric. In addition, it is important to provision the correct amount of the
resources efficiently using a suitable threshold. In fact, the fluctuating
workload would lead to an overprovisioning state or an underprovisioning state.
To avoid these problems, researchers usually use a predictive technique, such as
the proactive method. These feasible predictive approaches, such as machine
learning, Moving Average, and Auto-Regression, would track the dynamic resource
requirement and effectively minimize the energy consumption. This predictive
policy would quantify the requirement in advance in order to flexibly scale the
resources up or down. However, it is a challenging issue to improve the accuracy
of the predictive technique. Additionally, an estimation error would lead to an
overprovisioning or underprovisioning state. When in the sudden workload, this
predictive method is especially inaccurate. Thus, combining this with an
automatic method and a proactive method would be more agile for provisioning the
resources. For example, the Elastic VM architecture provisions the resources
dynamically to reduce the SLA violation. However, the elasticity is necessary to
meet the users’ demand from different perspectives. Some researchers would take
the performance metrics into consideration, such as the SLA and the profit of
the providers. However, more metrics are used in the elasticity to evaluate the
performance. For example, from the purpose of the providers, they might consider
more related elements. That is, they would seek to minimize the renting cost,
the energy consumption and the Service Level Agreement (SLA) violation. In
summary, the elasticity would be implemented for one or two purposes, such as
saving energy or reducing the cost. However, it is difficult to make an
elasticity solution by considering multiple objectives. To solve the mentioned
issues, we propose the ERP approach to provision the resources by the
performance threshold, including the CPU and the memory. According to the
threshold, we would flexibly scale the resources up or down by considering
multiple perspectives. From the perspective of the provider, the goal is aimed
at minimizing the amount of the resources to reduce the energy consumption. From
the perspective of the users, the goal is aimed at rapidly scaling the resources
up or down. In brief, the ERP approach is aimed at maximizing the utilization
and minimizing the SLA violation. Then, the main contributions would be
summarized in the following.
First, this approach solves the suitable threshold to determine the users’
demand. We present the performance threshold by using the GRA method, which
considers such multiobjectives as the CPU utilization and memory utilization.
Meanwhile, it is instructed on the cloud layer model using the MAPE loop.
Usually the MAPE loop includes four phases, such as Monitoring (M), Analysis
(A), Planning (P) and Execution (E).
Second, this approach solves the issue of scaling the resources flexibly.
According to the proposed threshold, we could efficiently scale the resources up
or down. That is, we propose a fine-grained algorithm, which means to scale up
the resources from the PM-level or the VM-level in order to flexibly meet the
users’ demand.
Third, this approach solves the issue of reducing the overheads. When it is
overprovisioned, we would shut down the extra machines to reduce the energy
consumption via a simple predictive technique, such as the weighted moving
average (WMA).
The remainder of the paper is described as listed below. Section 2 analyses the
related literature on the elastic techniques in cloud computing. Section 3
presents the ERP framework based on the layer model. Section 4 provides the
performance threshold via the cloud layer model. Section 5 presents the
effective ERP algorithm, which would scale the resources up or down from
different granularities. Section 6 proves the results by comparing them with the
aforementioned approaches. Finally, section 7 draws conclusions and describes
future development.
# Related work
Usually the elastic solution is implemented by scaling the resources in or out.
By analyzing some related works, we would divide the elastic resource
provisioning approaches into two major aspects, including automatic scaling
methods and elastic mechanisms on the predictive technique.
## Automatic scaling methods
In the automatic policy, the resources would be provisioned and released
automatically according to the demand. Generally, the action is triggered by the
fixed thresholds, such as the utilization. The common techniques are provided by
Amazon and Scalr. However, they provision the resources only based on the
utilization, when in fact more elements have taken effect. Additionally, its
advantage is a kind of coarse-grained provisioning strategy to scale the virtual
machines. When considering the fine-grained provisioning strategy, some
researchers focus on the reactive methods by resizing the resources dynamically
and minimizing the response time and executing cost in cloud computing. However,
they focus more on the fine-grained scaling strategy, and less on multiple
perspectives. Kingfisher proposed an elastic mechanism to reduce the transition
of time and cost. This approach exploits the available resources on the virtual
machines to scale in or out, and uses an integer linear program formulation to
optimize the cost. Leitner et al. proposed the SLA-aware scheduling algorithm,
which would reduce the request execution time. It presents a cost-efficient
method to scale up from the perspective of the providers. In contrast, our
approach considers more factors to formulate the threshold by the cloud layer
model, such as CPU utilization, memory utilization, etc. Additionally, we aim to
scale the resources by minimizing the renting cost and response time. This would
shut down the spare machines from the perspective of saving the consumption. By
analyzing the mentioned works, we determined that most recent elastic strategies
focus on the horizontal elasticity. Therefore, it is important to scale the
resources from different granularities, including horizontal elasticity and
vertical elasticity. By considering the fine-grained elasticity, we present the
ERP algorithm to scale up the resources in the PM-level or VM-level by the
performance threshold. Moreover, when it is in overprovisioning, it would scale
down the resources in the VM-level.
## Elastic mechanisms based on the prediction
In fact, elasticity is essential to meet a fluctuating workload, and it is
necessary to determine the suitable amount of the resources in order to scale
the resources. Actually, the proactive approaches are used to determine the next
demand, such as the Autoregressive moving average model (ARMA) and Holt winter.
These predictive techniques have the advantage of giving an accurate prediction
value in the stable workload. However, these predictive techniques focus more on
the accuracy, but ignore the complexity. Moreover, when a sudden workload
appears it might be in estimation error. To reduce the complexity of the
prediction algorithm, some techniques are used to determine the repetitive
patterns and predict the next values. PRESS is a predictive elasticity system
that analyzes and extracts the workload patterns and provisions the resources
automatically. The advantage of this policy is that improves the prediction
accuracy, and it reduces the resource waste efficiently. However, it only makes
emphasis on the overhead. CloudScale is a system that automates the fine-grained
resources in cloud computing infrastructures, determining the adaptive resources
by the prediction. In addition, it integrates the dynamic CPU voltage scaling to
saving the consumption by migration. This technique puts more emphasis on the
proactive method based on the prediction, which would minimize the energy
consumption and avoid the Service Level Object (SLO) violation. In fact, more
elements should be taken into consideration. Hence, in our approach, we consider
more elements, such as reducing the renting cost, energy consumption and SLA
violation. Additionally, we increase or decrease the resources automatically
from different granularities to meet the demand, including fine-grained scaling
and coarse-grained scaling. Namely, when it is underprovisioning, our approach
scales up the resources from different granularities by the performance
threshold, such as in the PM-level or VM-level. In contrast, we scale down the
VMs by the WMA predictive technique efficiently.
# Proposed approach
In this section, we present our proposed approach for the detailed description.
Our approach is designed on the cloud layer model. That is, this policy is
implemented to determine the performance threshold to flexibly scale the
resources up or down. Additionally, the formulation of the performance threshold
is presented in detail in the next section. Then the ERP framework is explained
in detail in the following.
## Cloud layer model
In this section, our approach describes a cloud layer model to scale the
resources rapidly. The cloud layer model focuses more on the quantitative
analysis, whereas the Delphi method depends more on the subjective assessment.
The ERP approach is implemented on the cloud layer model. The layer model is
composed of three parts: SaaS, PaaS and IaaS. The SaaS determines a series of
requests offered by the users. In the PaaS the broker is responsible for
provisioning the infrastructure resources according to the users’ demandwhich is
presented by the MAPE loop. In IaaS, the datacenter is composed of some PMs and
VMs. The provider would provision the resources according to the requests. As
depicted in, the key components of the MAPE are described in detail as follows.
### Monitor (M)
The monitoring component collects some metrics, such as the CPU utilization,
memory utilization and some available resources. It monitors the information
every five seconds. The key information is collected, aggregated and calculated
by the performance model, which is described in detail in the next section.
### Analyze (A)
The analyzing phase is responsible for analyzing the collected information. The
obtained data is aggregated and calculated by the performance model, and we
achieve the performance value to decide whether the scaling action is triggered.
Moreover, we use the WMA predictive technique to determine the correct number of
the servers and shut down the spare machines.
### Plan (P)
This component is the core of the cloud layer model. According to the users’
demand, it implements the scaling strategy by minimizing the renting cost and
reducing the energy consumption. Additionally, it would increase or decrease the
resources by the performance threshold.
### Execute (E)
In the executing phase, the Nginx load balancing server balances the web
requests by provisioning the servers in the infrastructure. Since the VMs are
hosted in the PMs, the provider would provision the resources according to the
demand by using the proposed plan.
## Proposed framework
In our approach we propose a novel framework to flexibly increase or decrease
the resources aiming at minimizing the renting cost, energy consumption and
response time, as illustrated in. The ERP algorithm is mainly composed of two
phases. In the first phase, the performance model constructs a baseline
threshold, which is aggregated and calculated by the gathered data. From this
the resources would be rapidly scaled up or down. In the second phase, the ERP
algorithm is used to scale the resources by the performance threshold for the
purpose of minimizing the renting cost and saving power consumption.
Then, we explain these two phases in detail. In the first step, the monitoring
component monitors the CPU utilization, memory utilization, CPU clock speed and
some available resources. We aggregate the gathered data to make a performance
evaluation by the proposed cloud layer model. In the second step, we make a
further description on the ERP approach. In the analyzing component, we scale
the resources by the performance threshold. Actually, the planning phase may
lead into two states, including an underprovisioning state or overprovisioning
state. When it is in an underprovisioning state, we execute the action on
increasing the resources at the PM-level. If it continues, we go on scaling up
the resources at the VM-level. The PM-level scaling depends on the available
resources in the same host. The VM-level scaling is based on the VMs hosted on
the PMs. Additionally, the VM could come from the same PM or another PM.
Otherwise, when it is in overprovisioning we scale down the resources by the
prediction. Then the extra spared machines would be shut down by saving the
energy consumption. Moreover, our approach implements the elastic scaling from
different granularities with the consideration of minimizing the cost and the
SLA violation.
# Performance threshold
In this section, we present a performance threshold on multiple elements. From
this we would rapidly scale the resources up or down in cloud computing.
## TOPSIS and GRA policy
This policy presents a multicriteria threshold that takes five related criterion
into account, as shown in. The criteria on the TOPSIS and GRA policy would
include the cost type and benefit type. After the matrix is normalized, the
TOPSIS method evaluates them by the positive ideal solution and negative ideal
solution. Then, the GRA method makes the decision from less information and
explores the system behavior by analyzing the related degree.
Usually the information on the PMs is gathered every 5 seconds to form the
decision matrix, as shown in. The gathered data is described as depicted in.
Then we construct and implement the performance threshold in detail as follows.
<img src="info:doi/10.1371/journal.pone.0216067.e001" id="pone.0216067.e001g" />
R
=
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### Normalization of the decision matrix
In the first step we normalize the decision matrix. Namely, the decision matrix
is normalized by achieving the average value of every column as listed in.
<img src="info:doi/10.1371/journal.pone.0216067.e002" id="pone.0216067.e002g" />
R
=
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### Improved TOPSIS
This is the abbreviation of the Technique for Order Preference by Similarity to
Ideal Solution (TOPSIS). The traditional TOPSIS method depends more on
subjective weights, while the improved TOPSIS solutions depend more on key
factors. In the second step, the ideal solution would be determined by and. That
is, for the cost type the ideal solutions are the smaller ones, and the negative
solutions are the larger ones. It is the opposite situation for the benefit
type. Then, we achieve the positive ideal solution and the negative ideal
solution, respectively.
<img src="info:doi/10.1371/journal.pone.0216067.e003" id="pone.0216067.e003g" />
P
j
\+
=
{
(
max
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<img src="info:doi/10.1371/journal.pone.0216067.e004" id="pone.0216067.e004g" />
P
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### Grey relational analysis
Grey theory is an effective way to solve multiobjective decision problems in the
engineering areas. In the following step, we determine the difference between
the comparative series *r*<sub>*jk*</sub> and the standard series $P_{k}^{+}$ or
$P_{k}^{-}$. Additionally, the distinguish coefficient *ρ* is usually 0.5, and
is generally between \[0, 1\]. Then, the Grey relational coefficients
*ς*<sup>+</sup>and *ς*<sup>−</sup> are constructed by and, respectively.
<img src="info:doi/10.1371/journal.pone.0216067.e007" id="pone.0216067.e007g" />
ς
\+
(
k
)
=
min
j
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<img src="info:doi/10.1371/journal.pone.0216067.e008" id="pone.0216067.e008g" />
ς
−
(
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max
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Actually, the weight coefficients are determined by the analytic hierarchy
process (AHP) method. Then we determine the degree of relation *r* on the weight
coefficients *ω* by multiplying them by Grey relational coefficient *ς*(*k*).
Additionally, the degree of relations *r*<sup>+</sup> and *r*<sup>−</sup> are
formulated by Eqs and, respectively.
<img src="info:doi/10.1371/journal.pone.0216067.e009" id="pone.0216067.e009g" />
r
\+
=
∑
k
=
1
m
ω
k
ς
\+
(
k
)
<img src="info:doi/10.1371/journal.pone.0216067.e010" id="pone.0216067.e010g" />
r
−
=
∑
k
=
1
m
ω
k
ς
−
(
k
)
Then we formulate the relative closeness coefficient *u*<sup>+</sup> to the
ideal solution by, which is implemented on the ideal relational coefficient
*r*<sup>+</sup> divided by the sum of the positive relational coefficient
*r*<sup>+</sup> and negative relational coefficient *r*<sup>−</sup>.
<img src="info:doi/10.1371/journal.pone.0216067.e011" id="pone.0216067.e011g" />
u
\+
=
r
\+
r
\+
\+
r
−
## Performance model
The performance threshold is constructed by the entropy method, which is an
effective method to calculate the deviation degree. The smaller the entropy
value is, the better the performance is. Similarly, the larger entropy value is,
the worse the performance is. Therefore, we determine the performance threshold
by the entropy method, which is listed as in.
<img src="info:doi/10.1371/journal.pone.0216067.e012" id="pone.0216067.e012g" />
Δ
P
=
−
log
2
(
P
2
P
1
)
=
−
log
2
(
u
2
\+
u
max
/
u
1
\+
u
max
)
=
−
log
2
(
u
2
\+
u
1
\+
)
Where Δ*P* is the performance threshold by the entropy method, *P*<sub>1</sub>
is the probability before the demand varies, and *P*<sub>2</sub> is the
probability after the demand varies. Additionally, the probability is
constructed on the ideal relational coefficient *u*<sup>+</sup> divided by the
max relational coefficient *u*<sub>max</sub>.
In the scheduling, a current performance value below 0.1 denotes a better
performance environment. When it is above 3, it denotes a poor performance
environment. In fact, a normal value is between 0.1 and 3, which is described
in. In our experiments, when the performance value is lower than 0.1, we would
scale down the servers. Then, we set 0.1 as the lower threshold
*P*<sub>*d*</sub>. When the value is greater than 0.2, we would scale up the
servers for the purpose of reducing the response time by reserving slightly more
resources. Then, we set 0.2 as the upper threshold *P*<sub>*u*</sub>.
# The ERP algorithm
In this section we describe the ERP algorithm to scale the resources from
different granularities according to the users’ demand.
## ERP algorithm
To provision the resources flexibly, we first discuss some related definitions
on the elasticity, such as the resilience and scalability. Next, we define them
and clarify the difference between them. **Scalability** means to the ability of
the system to deal with an increasing amount of the servers in a capable manner.
However, it focuses more on the increasing ability, and less on the response
time. **Resilience** means to provision the resources rapidly in a flexible way.
Elastic scheduling refers to two core conditions, including the time and speed.
In this paper, we define an elastic scheme *S*, which is represented as *S* = (*
clock*,*U*<sub>*cpu*%</sub>,*U*<sub>*mem*%</sub>,*P*<sub>*u*</sub>,*P*<sub>*d*</
sub>), where *clock* is the CPU cycle, *U*<sub>*cpu*%</sub> and
*U*<sub>*mem*%</sub> are the CPU utilization and the memory utilization,
respectively, which are gathered by monitoring the system, and *P*<sub>*u*</sub>
and *P*<sub>*d*</sub> are the upper and lower thresholds, respectively. In
brief, the main algorithm (refer to Algorithm 1) provisions the resources
rapidly via the MAPE loop. In the monitoring and analyzing components, some key
elements are collected to determine the performance threshold. Then in the
planning and executing components, the elastic scheme would scale the resources
by the performance threshold. To make the ERP algorithm understood for the
further step, lists the main parameters of the ERP algorithm as below.
Next, the ERP algorithm is described in detail. It implements an elastic
resource provisioning approach in the datacenter. This algorithm takes the
performance threshold as the baseline to scale the resources up or down. At
first, the monitoring component would collect and gather the information as
listed in (lines 1–2) every few minutes. In fact, the ERP algorithm would
increase or decrease the resources to meet the users’ demand. When the
performance value *P* is larger than the upper threshold *P*<sub>*u*</sub>, the
algorithm would be triggered to scale up the servers (SUS) (lines 4–5).In
contrast, once the current performance value is below the threshold
*P*<sub>*d*</sub>, the scaling down the servers (SDS) algorithm is triggered
(lines 6–7).
**Algorithm 1**. **ERP (Elastic resource provisioning)**
1: Initialization: Server, P
2: while (the allocation is deploying)
3: monitor the performance value P
4: if P \> *P*<sub>*u*</sub>
5: Scaling up the servers (SUS)
6: else if P \< *P*<sub>*d*</sub>
Scaling down the servers (SDS)
7: End
The proposed ERP algorithm has included two aspects. First, the scaling up the
servers (SUS) algorithm proposes a scaling method that is based on different
granularities. That is, we scale up the VMs in the same available PMs or from
some different PMs. Second, the scaling down the servers (SDS) algorithm
presents the approach to shut down the extra machines.
## The SUS algorithm
The SUS algorithm is intended to scale up the resources in a flexible way,
including from the PM-level or VM-level. The SUS algorithm is described by
Algorithm 2. The monitoring component collects some metrics related to the
resources (lines 1–4). If the performance evaluation reaches the upper threshold
*P*<sub>*u*</sub>, it scales up more available resources on the PM (lines 5–7).
When the updated performance value continues past on the upper threshold
*P*<sub>*u*</sub>, we would provision slightly more resources (lines 8–10).
Additionally, the VMs might come from different PMs.
**Algorithm 2**: **SUS (Scaling up the servers)**
1: Begin
2: Initialization: Server, P
3: while (the allocation is deploying)
4: monitor the performance value P
5: if P \> *P*<sub>*u*</sub>
6: Scaling up the PMs
7: update the performance value P
8: while (P \> *P*<sub>*u*</sub>)
9: Scaling up the VMs
10: update the performance value P
11: End
## The PLI algorithm
The purpose of the PM-Level increasing (PLI) algorithm is to increase the VMs on
the available PMs (refer to Algorithm 3). Then we explore the PLI algorithm in
detail. The monitoring component aggregates the information and calculates the
performance value (lines 1–3). Once the triggered action appears we scale up the
residual resources on the available PMs. Then we would choose the PMs aimed at
minimizing the renting cost (lines 4–7). Additionally, the cost function is
described by Eqs and. Finally, it updates the performance value (line 8).
**Algorithm 3**: **PLI (PM-level increasing)**
1: Begin
2: Initialization: Server, P
3: Calculating the performance value
4: while (P \> *P*<sub>*u*</sub>)
5: if PM is available
6: select the min cost PM to increase
7: update the performance value
8: End
In this phase, is aimed at minimizing the renting cost, where
*u*<sub>*cpu*%</sub> presents the CPU utilization of the VM. The binary variable
*v*<sub>*j*</sub> indicates whether or not the VM is selected, and the binary
variable *p*<sub>*i*</sub> indicates whether or not the PM is selected. The
parameter *m* is responsible for the amount of VMs hosted on the current host,
and *c*(*p*<sub>*i*</sub>) is the expending cost of the current host.
<img src="info:doi/10.1371/journal.pone.0216067.e013" id="pone.0216067.e013g" />
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## The VLI algorithm
In this section, we propose the VM-level increasing (VLI) algorithm (refer to
Algorithm 4) to continue increasing the resources to meet the fluctuating
demand. It consists of three parts: monitoring the component, increasing the
resources and updating the state. Then we describe the algorithm 4 in detail.
First, the monitoring component gathers the information to calculate the
performance value (lines 1–3). Second, we choose the suitable VMs to increase
(lines 4–5), which would determine minimizing the expending cost by. That is, we
calculate the remaining utilization by the CPU and the memory. We implement the
cost in by multiplying the remaining utilization by the single VM renting cost.
The purpose of the function is to achieve the VM with a minimum cost, where the
binary variable *v*<sub>*j*</sub> indicates whether or not the VM is selected
in. Then we make a global search to find a suitable VM to increase. Finally, we
update the state and calculate the performance value (lines 6–7).
**Algorithm 4**: **VLI (VM-level increasing)**
1: Begin
2: Initialization: Server, P
3: Calculating the performance value
4: while (P \> *P*<sub>*u*</sub>)
5: select the min cost VM to increase
6: update the performance value
7: End $$C_{vm} = \arg\min(v_{j}(1 - u_{cpu\%}) \bullet (1 - u_{mem\%}) \bullet
c_{}(v))$$ $$j \in \left\{ 1,2,\ldots,m \right\},v_{j} \in \left\{ 0,1
\right\}$$
## The SDS algorithm
The aforementioned algorithms (refer to Algorithms 2–4) implement increasing the
resources from a different granularity according to the users’ demand. In this
section, the Scaling-down servers (SDS) algorithm is described for the detailed
steps. In the first step we monitor the component and gather some information to
achieve the performance threshold (lines 1–3). Once the SDS algorithm is
triggered we would scale down the resources. Then we select the extra machines
to shut down for the purpose of minimizing the cost (lines 4–5). Hence, we shut
down the machines that occupy the maximum expending cost. is as listed below.
Finally, we update the state and determine the current performance threshold
(lines 6–7).
<img src="info:doi/10.1371/journal.pone.0216067.e017" id="pone.0216067.e017g" />
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**Algorithm 5**: **SDS (Scaling-down servers)**
1\. Begin
2\. Initialization: Server, P
3\. Calculating the performance value
4\. while (P \< *P*<sub>*d*</sub>)
5\. select the max cost VM to decrease
6\. update the performance value
7\. End
# Experiments
In this section, we implement the elastic resource allocation strategy based on
the performance criterion. Meanwhile, the proposed approach proves that it is
appropriate for meeting the demand in different kinds of workloads. In addition,
this approach considers both reducing the renting cost and improving the
utilization.
## Environment setup
We use the CloudStack platform and simulated real-world workloads to evaluate
the ERP approach. We deploy a cluster composed of ten PMs. One installs the
CloudStack platform. The other nine PMs use Xenserver as the management nodes
(2.20GHz Intel(R) Xeon(R) 8 CPU, 8 G memory, running CenOs 6.9). We create 27
VMs (1 VCPU, 1 G memory, running CenOs 6.9) in the cluster. Then, the database
is run off MySQL. When the workload is fluctuating, the Nginx has the function
off balancing the servers. All the configuration information is listed in.
To evaluate the proposed approach, we design two kinds of workloads: synthetic
workloads and real-world workloads. We use the Jmeter to generate the requests
based on the TPC benchmark. First, the synthetic workload would vary from the
users’ demand. The fluctuating process of the workload is described as below.
The load generator would implement 600, 900, 600, 1200, 600, and 1800 users,
which is shown in, which lasts for over 30 minutes. Second, the simulated real-
world workload is extracted from the EPA and NASA traces. The two kinds of real-
world workload traces are generated as shown in. Additionally, the monitoring
service is implemented by the Jmeter plugins, such as monitoring the response
time, CPU utilization or memory utilization. The experiment would last for over
40 minutes.
## Evaluation metric
In the experiments, we consider some performance indicators as the metrics, such
as the renting cost, energy consumption, resource utilization and SLA violation.
### The cost
This metric might be measured by the reserved and on-demand VMs. For example,
the basic unit of the CPU is set at 1 GB in Aliyu. It is charged 0.059 ¥/hour in
the reserved plan and 0.28 ¥/hour for the on-demand plan. The renting cost is
defined in, where *C*<sup>*r*</sup> and *C*<sup>*o*</sup> are responsible for
the renting cost in the reserved or on-demand plan, respectively. Then in the
scheduling the average overhead is described in, where it is calculated by the
sum of the cost divided by the time interval *T*.
<img src="info:doi/10.1371/journal.pone.0216067.e018" id="pone.0216067.e018g" />
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### Energy consumption
This metric might be measured by the average energy consumption, which is
defined as the energy consumption ratio as listed in, where *N* is the total
number of the intervals. Additionally, the energy consumption is expressed in.
<img src="info:doi/10.1371/journal.pone.0216067.e020" id="pone.0216067.e020g" />
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Where the idle power consumption coefficient *k* is equal to 0.7, and the
parameter *P*<sub>max</sub> represents the peak power. Additionally, *u* is
based on the CPU utilization.
<img src="info:doi/10.1371/journal.pone.0216067.e021" id="pone.0216067.e021g" />
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### The utilization
The utilization is one of key indicators to evaluate the performance in the
scheduling. The average utilization is defined as the ratio between the total
CPU utilization and the total number of the intervals, as shown in.
<img src="info:doi/10.1371/journal.pone.0216067.e022" id="pone.0216067.e022g" />
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### SLA violation
The SLA violation can be calculated by the percentage of the difference between
the actual requests and allocated requests divided by the total requests, as
described in. Generally, the SLA violation might be measured by the CPU
utilization, just in. Then the average SLA violation is defined as the ratio
between the total SLA violation and the total number of the intervals, expressed
by. In fact, the SLAV is expressed by the average SLA multiplied by the average
response time, as shown in.
<img src="info:doi/10.1371/journal.pone.0216067.e023" id="pone.0216067.e023g" />
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## Algorithms in comparison
To validate the ERP algorithm, we compare it with other algorithms, such as
lightweight resource scaling (LS) algorithm, the proactive method, and the
reactive method.
### Reactive method
The traditional algorithm is scaled by the CPU utilization, obeying the simple
principle by a rule-condition-action. In the experiments, the threshold is
usually fixed at 0.8 or 0.2. Namely, when the utilization is higher than 0.8,
the VMs would be increased. In contrast, when the utilization is lower than 0.2,
the resources would be decreased.
### Proactive method
The proactive method means that it would scale the servers up or down by the
prediction technique, such as ARMA. That is, it could scale the resources up or
down by the ARMA.
### LS
The LS algorithm focuses more on the response time. When it is higher than the
upper threshold the number of the VMs increases. In contrast, the number of the
VMs would be scaled down. Additionally, the algorithm would shut down the spare
machines by a simple predictive technique.
## Experiment results
Actually, our proposed algorithm is constructed on the performance value, which
is calculated by the GRA and TOPSIS policy. In more experiments we determine
that the performance threshold range is between 0.1 and 0.2. Namely, when it is
greater than 0.2, we would scale up the servers, and when it is lower than 0.1
we would scale down the servers. Moreover, the performance evaluation considers
multiple angles, such as maximizing the utilization, and minimizing the power
consumption and the SLA violation. The results prove the effectiveness of the
ERP approach.
### The number of the servers
In the synthetic load, the reactive algorithm puts a greater emphasis on the
scalability of the servers and reacts quickly at first. The proactive algorithm
would obtain the suitable number of the servers in the regular load test, and
the LS algorithm spends less resources. Our proposed algorithm could
occasionally occupy slightly more resources than the LS algorithm to meet the
multidimension requirement in the simulated experiment at the beginning, as
shown in. In the real-world load, including EPA and NASA, our algorithm would
occupy slightly more resources at first. Next it would outperform other
algorithms in the normal level, as illustrated in Figs and. We determine that
the LS algorithm is unsuitable for various loads. That is, because the LS
algorithm depends more on the response time. When a sudden load appears, it
would increase the overhead. However, our approach has the advantage of
efficiently avoiding a sudden load efficiently by reserving slightly more
resources.
### The renting cost
We measure the renting cost using. As shown in, in the synthetic load the LS
algorithm puts a greater emphasis on the time to scale the resources. Namely, in
the stable workload it gains the smallest average renting cost. We find that the
ERP algorithm spends a slightly higher cost than the LS due to reserving few
resources at first. The proactive algorithm would obtain a better result in the
regular load test by the prediction. Our proposed algorithm obtains a lower cost
than the reactive algorithm. As shown in, in the real-world load we find that
our proposed algorithm obtains a lower cost than the other algorithms, and the
LS algorithm obtains a higher cost depending on the response time. When it
appears in the sudden load, the LS algorithm would scale up the resources more
quickly, which makes the occupied resources greater than in other algorithms.
### Resource utilization
We measure the average resource utilization based on. Figs and show the CPU
utilization during the experiments under different workloads, including the
synthetic load and real-world loads. In these experiments, we determine that the
proposed approach utilizes the resources more fully, which is depicted in Figs
and. In the experiments the ERP method consumes slightly more resources at first
and simultaneously guarantees a lower SLA violation rate. Additionally, it
releases the servers by the WMA prediction by guaranteeing the performance in
the varying workloads. We see that no resource utilization is higher than 100%,
which proves that our approach efficiently reduces the underprovisioning state.
### Response time
The response time is another performance metric that needs to be studied. As
depicted in Figs and, in the synthetic workload, when considering the maximum
response time, we determine that our proposed algorithm obtains a quicker
response than the other algorithms by reserving few resources at first. For the
average response time, these algorithms are in the acceptable level at the
stable workload. As depicted in Figs and, in the real-world loads we find that
our algorithm presents a lower maximum or average response time than others by
reserving slightly more resources at first, while the LS algorithm obtains
slightly higher time due to a longer monitoring time. Additionally, it is unfit
for the sudden load. In the NASA load the variable workload leads to inaccurate
prediction values, so the proactive algorithm obtains a longer average response
time.
### SLA violation
We measured the SLA violation based on. As shown in Figs and, in the workloads
our algorithm presents a lower SLA violation ratio than the other algorithms. In
addition, the error rate is another metric that evaluates the performance. As
listed in, we see that our algorithm produces a slightly lower error ratio and
efficiently avoids the sudden load.
### Average energy consumption
We measure the average energy consumption based on. As shown in, in the
synthetic load our algorithm achieves a lower power than the LS and proactive
algorithms. Since it is in the stable workload, the reactive algorithm obtains a
better result than the other algorithms only by the utilization. As shown in, in
real-world loads the proposed algorithm presents a lower power than the LS and
reactive algorithm. The proactive method consumes less energy consumption than
others, but it cannot meet the demand due to the inaccurate prediction. This is
because that it achieves a higher error rate in.
# Conclusion
Traditional elasticity is often used as a reactive method, which is implemented
by the rule-condition-action. However, it would be a better strategy to combine
this with the prediction. In this paper, we present an elastic strategy that
increases or decreases the resources by the performance threshold in a flexible
manner. To further elaborate, the ERP approach makes the following
contributions. First, we present the performance threshold depending on the CPU
and the memory. By this, we could flexibly scale the resources up or down. This
solves the issue of deciding a suitable threshold on multiple elements. Second,
we propose an SUS algorithm that implements the fine-grained scaling in the PM-
Level or VM-Level to increase the resources flexibly. This solves the issue of
an elastic scaling strategy from different granularities to reduce the SLA
violation and response time. Third, combining this with the WMA prediction we
propose the SDS algorithm to scale down the servers. Then we would shut down the
spare machines to save energy consumption. This solves the issue of effectively
saving the overheads. Finally, we evaluate the proposed ERP approach in the
simulated and real-world workloads. The results show that the ERP method
improves the utilization, minimizes the renting cost, saves the energy
consumption and gives a quicker response time.
In fact, we implement the scaling approach on the premise of regarding the
servers as the available resources. However, no cloud provider offers unlimited
resources, except for Google and Amazon. Thus, a further study should be made on
some aspects. First, it is necessary to find an effective way to minimize the
renting cost by reserving some available resources in advance. However, more
servers would be wasted by reserving too many resources. Therefore, it is
necessary to balance the reserved plan and the on-demand plan. Second, from the
perspective of minimizing the energy consumption, a reasonable dynamical
provisioning approach might efficiently consolidate the available resources by
the migration technique. Then in the future it will be necessary to explore the
dynamical provisioning approach in the complex workloads. Perhaps some typical
types of the workflow would be an interesting extension in the future.
# Supporting information
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Peste des petits ruminants (PPR) is a contagious viral disease that primarily
affects domestic and wild small ruminants. Because of its high morbidity and
mortality, PPR is responsible for heavy economic losses in livestock husbandry
across many developing countries. It is considered a significant threat to the
global goat and sheep industry. PPR is caused by peste des petits ruminants
virus (PPRV), a member of the family *Paramyxoviridae* and genus *Morbillivirus*
along with Distemper, Rinderpest, and Measles viruses. To date, four PPRV
lineages (I-IV) have been identified worldwide. Lineage IV is the dominant
strain in Asia, including the entire Trans-Himalayan region (THR). PPRV has a
tropism for epithelial and lymphoid cells. The virus can exist in different host
body tissues and is discharged from the body through various secretions and
excretions. These secretions and excretions, including the respiratory droplets,
become the source of PPRV, allowing the transmission of the virus through close
contact and aerosols. The clinical symptoms of PPR typically begin with dullness
and fever (\>40 °C). Subsequently, there is the development of oral mucopurulent
discharge, ocular discharge, and eventually, oral lesions, bronchopneumonia, and
diarrhea. The severity of this disease is determined by the strain of the virus,
local environmental features, and the immune status of the infected host. The
morbidity and mortality of PPR can vary between 10%–90% and 50–90%,
respectively. The typical latency period of PPR is 4–6 days, whereas the longest
incubation period reported is 21 days. The major hosts of PPRV are livestock,
such as sheep and goats. Wildlife is also an important target for PPRV. The main
targets are antelope (such as saiga antelope—*Saiga tatarica mongolica*), ibex
(such as Siberian ibex- *Capra sibirica*, Sindh ibex—*Capra aegagrus blythi*),
gazelle (such as goitered gazelle—*Gazella subgutturosa*, Arabian
gazelle—*Gazella arabica*). In Asia, clinical signs and mortality of PPR in
wildlife have been reported essentially as the same as those in livestock, which
provides a basis for interspecific transmission. This situation is different
from the non-clinical infection in Africa, i.e. no viral shedding even if an
antibody is produced. Especially in THR, bharal (*Pseudois nayaur*), markhor
(*Capra falconeri*), blackbuck (*Antilope cervicapra*), and Himalayan goral
(*Naemorhedus goral*) have been found to exhibit obvious clinical signs. Among
them, strains from bharal and markhor were successfully isolated. Phylogenetic
analysis showed that they were closely related to the strains isolated from
livestock, suggesting a potential relationship between them.
PPR was first reported in West Africa in 1942 and spread across Africa and Asia.
Available research indicates that China, India, and Nepal are all PPR epidemic
countries. Two PPR epidemics have been documented in China; the first occurred
in 2007 in the Tibet Autonomous Region of China. The more severe outbreak
occurred between the end of 2013 and the first half of 2014, which was first
identified in the Xinjiang Uygur Autonomous Region and later spread to more than
20 provinces in total. This consequence in more than 30,000 sheep infections, of
which 10,000 animals died.
In response to the heavy losses caused by PPR, the World Organization for Animal
Health (OIE) and the Food and Agriculture Organization (FAO) have set the goal
of eradicating PPR globally by 2030. It has been suggested that the global
eradication of PPR could return benefits of about \$74 billion over 15 years.
However, the continuous epidemiological cycles of PPR worldwide constitute a
great challenge to eradicating the disease. This calls for a deeper
understanding of its temporospatial characteristics. The THR is an important
endemic and high-risk area of PPR where monitoring and prevention measurements
are difficult to implement. This can be majorly attributed to the rugged natural
geographical conditions and the low effective livestock system. The poor
veterinary services further complicate the situation in the region. Thus, a
forecasting system would be a strong aid in defining the key points of
prevention to save time, labor, and products for underdeveloped countries and
regions.
According to reports, the PPR risk exists across the THR, in which livestock
serve as the maintenance hosts. Wildlife possibly plays the role of bridge
hosts, in which virus transmission is not maintained but can persist for a while
and be transmitted back (spillback) to livestock. Although scientists argue for
the direct epidemiological linkage at the interface of livestock and wildlife,
the interspecies transmission of PPRV during grazing and at water sources has
been confirmed. Sharing the use of rangelands by livestock and wildlife can lead
to disease transmission. Abubakar et al. pointed out that an outbreak of PPR in
Sindh ibex was due to the spillover of the virus from a recent outbreak of PPR
in nearby domestic small ruminants. Similar PPR spillovers to wild hosts are
reported in Tibet and the Ngorongoro Conservation Area in northern Tanzania.
Except for trade, free migration of wildlife and nomadism-driven back and forth
movement of livestock enable their meeting in the same space (contaminated or
not) across time. Both animals and humans prefer low-energy-consuming surfaces
during movement, which has become its driving force. If the migration and
grassland sharing of the multiple PPRV hosts last, reliance on the contamination
of habitats within the latent period of the disease is expected, and the
probability of direct contact via contaminated grassland is increased. While it
would be arbitrary to conclude that the infection occurred on the cross-country
paths, the potential communication of risk among the Trans-Himalayan neighboring
countries is worth monitoring.
We assume that interspecies transmission of PPRV occurs on small ruminants in
THR, which forms the basis for the transboundary transmission of PPR. Initially,
we predict the distribution of PPR on both sides using the maximum entropy model
(MaxEnt) and the connectivity of landscapes among different PPR-contaminated
regions using the LCP model, thereby revealing the potential transboundary
communication of PPR.
# Materials and methods
## Research area
Our research area is defined as the THR, which mainly included the Himalayan
mountains, a part of the Tibet plateau, the Ganges plains, a part of the Indus
plains, a part of the Indian Peninsula, and the Arakan Mountains. Altogether,
seven countries are in this region, including China, India, Nepal, Pakistan,
Bhutan, Bangladesh, and Burma, covering approximately 6.89×10<sup>6</sup>
km<sup>2</sup>. The northern, central, and southern parts of the THR differ in
natural geography, ecology, and climatology. The central part, i.e., the
Himalayan mountains, has the highest elevation of approximately 4000–8800 m. It
is a long and narrow mountain range with a length of approximately 3000 km and a
maximum width of only 400 km, covering an area of more than 1×10<sup>6</sup>
km<sup>2</sup>. In the south-facing slope of the mountain, lower elevation
regions were covered by the evergreen broad-leaved forest, and higher elevation
regions were covered by coniferous forests, shrubs, and alpine meadows. The
north-facing slope displayed the alpine climate, dry and cold with little
precipitation. The northern and the southern parts are divided by the Himalayan
mountains. The northern part is constituted by the Tibetan Plateau, with an
elevation of approximately 2500–5000 m. This area is dominated by plateaus and
mountains interspersed with plains and basins. The intense radiation, low
temperature, large daily temperature range, and small annual temperature range
verify a typical plateau climate feature. The elevation in the southern part was
below 1500 m. It mainly consists of flat, fertile plains with a tropical monsoon
climate and a subtropical grassland climate.
There are two different livestock systems in THR. The mixed crop-livestock
farming system is represented by the Ganges plains, the Indus plain, and the
Indian peninsula (low-elevation). The other one consists of the grazing system,
represented by the Tibetan Plateau (high-elevation). The Tibetan Plateau is a
traditional pasture (the number of sheep ≈ 10 million, goats ≈ 5 million). India
(sheep ≈ 65 million, goat ≈ 135 million), Pakistan (sheep ≈ 3.7 million, goat ≈
7.4 million), and Nepal (sheep ≈ 0.8 million, goat ≈ 10.9 million) are also the
core areas of animal husbandry in the world. With respect to free susceptible
hosts, bharal is distributed in the Tibetan frontier, Nepal, and Bhutan, and
population densities in Nepal were found to be 0.9–2.7
individual/km<sup>2</sup>, increasing to a maximum of 10 in the winter, as herds
congregate in the valleys. The number of mature individuals is approximately
47,000–414,000. Himalayan goral occupies the south-facing slope of the Himalayan
mountains, and its population density varies from 2.6–10.5
individuals/km<sup>2</sup>. Blackbuck is widely distributed in the Indian
subcontinent, and the number of mature individuals is around 35,000. Markhor is
mainly distributed in Pakistan. It is also found in small numbers in India
(Jammu Kashmir). The number of mature individuals of this species is
approximately 5,754.
## Research data
There were 1135 recorded PPR outbreak locations collected from the OIE reports
and published studies, including 107 records collected from the latter. The host
datasets were used alongside four fundamental environmental predictor categories
relevant for habitat modeling of terrestrial macro-fauna, i.e., climate,
terrain, vegetation, and human impact to construct the environmental model in
this study. The preprocessing and calculation of all spatial data were conducted
in ArcGIS 10.6 and projected in UTM-WGS-1984 with standard settings or
resampling to 30 arc-seconds.
## PPR spatial distribution model
The MaxEnt model is regarded as one of the best-performing specialty
distribution modeling techniques for analyzing presence-only data. It creates
ecological niche models by combining presence-only data with environmental
variables using a machine-learning approach known as maximum entropy. The
reliability of MaxEnt has been confirmed by its good capacity to predict novel
presence localities for poorly known species/diseases. It has been widely used
in many diseases, including PPR and African swine fever.
The MaxEnt model is applied to the spatial distribution model building to
explore the risk situation of PPR in the THR. In the construction of the model,
the regions with significant differences in elevation are treated separately to
overcome the problem that the model is not robust enough to deal with the DEM
with large differences. The low-elevation model (Model 1) and a high-elevation
model (Model 2) were constructed for regions below and above 1500 m,
respectively, according to the elevation standard of highland climate. The
spatial autocorrelation was minimized by filtering all recorded PPR locations
using the SDM Toolbox v1.1c in ArcGIS 10.6. Filtering was performed by limiting
the minimum distance between each pair of points. In addition, the filtering
program plays the role of systematic sampling. It can delete adjacent records to
reduce spatial aggregation, which is regarded as the most effective method in
correcting sampling bias. Multicollinearity was reduced for both the climate and
non-climate predictors. First, major predictors were selected using principal
component analysis (PCA). The variables with eigenvalues larger than 1.0 and the
scree plot criterion or ‘broken stick’ stopping rule for PCA in item-level
factoring were adopted. Suppression of unnecessary loading and rotation of
factor pattern of variables was used to retain predictors for subsequent
analysis in MaxEnt. Next, variables with low contribution rates were filtered
out using the MaxEnt model. Finally, variance inflation factor (VIF) analysis
was conducted to evaluate the multicollinearity among predictors after the
reduction. A VIF value below 10 indicates low and acceptable multicollinearity.
The filtered PPR locations and predictors were then used as input data to
construct the PPR model using the MaxEnt algorithm. The present models were
developed using occurrence data and 10,000 random background points,
representing the distribution of environmental conditions in the study region.
We divided the selected presence records into 70% training and 30% testing
portions to build and validate the models based on 10 bootstrap replicates. For
the remaining parameters, we kept the default settings in the pilot study.
Predicted PPR risk maps obtained by models 1 and 2 were overlaid using the fuzzy
overlay to construct the final PPR risk map of the THR. For visualization, the
Jenks natural break optimization method was used to classify the model output to
identify high-risk areas. Smoothing was followed for map visualization.
The key component of the model validation procedures is the criterion that
evaluates the model performance. We use threshold-dependent and threshold-
independent criteria. The area under the ROC curve (AUC) is a threshold-
independent criterion based on plotting the true positives against the false-
positive fractions for a range of thresholds in prediction probability.
Currently, the AUC is considered as the best criterion for assessing model
success for presence/absence data. As a threshold-dependent validation measure,
we used confusion matrix-based measures, including the Kappa test and correctly
classified instances (CCI). The Kappa statistic normalizes the overall accuracy
by the accuracy that might have occurred by chance alone. The percentage of CCI
was defined as the rate of correctly classified cells. The thresholds of these
two criteria are determined using the sensitivity-specificity sum maximization
approach.
## LCP model
The least-cost paths (LCPs), the shortest paths between two points with maximum
efficiency for a moving individual, have been advocated as an effective,
operational, and flexible approach to analyzing connectivity in heterogeneous
landscapes. The LCP model allows the integration of multi-dimensional
information, including geographic and behavioral information, to comprehensively
predict the potential transboundary (transregional) path of the animals. LCPs
are employed mainly to determine sites that are potentially used as dispersal
routes for terrestrial animals and have been proven to be applicable in
ruminants. To predict the potential transmission paths of the PPR in the
research area, we created a cost/resistance surface for the migration of small
ruminants using land cover type and elevation as cost variables according to
their movement capability. Two variables were reclassified using the Jenks
natural break method. Cost measurement scale of 1 (lowest cost) to 9 (highest
cost) is determined according to the number of the land cover type (except for
water bodies, which has been assigned as “restricted’ due to its relative
barriers to ruminants). The goat still maintains the same hoof structure as the
wild goat, which is designed for movement and grip in rugged environments.
Similar climbing skills, food and shelter requirements make domestic and wild
goats have similar movement capabilities and habitat preferences. Cost values
were assigned to each classification based on small ruminant habitat preferences
(see and Tables for more details). Land cover and elevation were then combined
using a logical overlay operation. Recorded PPR outbreak locations were then
clustered by K-nearest neighbor cluster analysis, and LCPs between the clusters
were analyzed using the constructed cost surface. After the calculation,
transboundary paths were highlighted by removing the internal paths.
A sensitivity analysis was performed to assess the robustness of the outputs
when they were affected by uncertainty. The main source of uncertainty in
evaluating potential paths for host movements is the cost value used to
constitute the cost surface. The one-at-a-time method was employed in the
sensitivity analysis. This common approach involves changing the input criteria
one at a time to observe the effect it produces on the output. This process was
repeated for each variable. We changed the cost of the different land cover
types/elevation classifications one at a time by adding or subtracting an amount
Δ = 5% or Δ = 10% from the original cost value to build iterative models. Raster
datasets presenting cost surfaces were produced through every iteration. To
measure the outputs, we rely on the Spearman rank correlation coefficient to
compare the ranking of countries obtained with original cost values with those
obtained with different land cover/elevation cost values. The closer the
Spearman rank correlation coefficient is to 1, the more similar the iteration is
to the original model. That is, this classification has less impact on the
model.
# Results
## Results of PPR spatial distribution models
Model 1 (≤1500 m): Altogether, 129 recorded PPR outbreak points at a distance of
at least 10 km away from each other were obtained after filtering. After PCA and
MaxEnt filtering, minimum temperatures of August (Min T Aug.), minimum
temperatures of November (Min T Nov.), human population, land cover, distance to
the river, and slope angle were left. The VIF values among the remaining
predictors were 1.014–1.742, which was in line with the low multicollinearity
standard (\<10). Moreover, AUC = 0.892, SD (standard deviation) = 0.002, Kappa =
0.869, and CCI = 0.869, indicated the robustness of the model. The response
curves of the different predictors are shown in, and the relative contributions
of each predictor are shown in (left).
Model 2 (\>1500 m): A total of 96 recorded PPR outbreak points remained after
filtering for 5 km. After PCA and MaxEnt filtering, the mean temperature of
April (Mean T Apr.), human population, land cover, distance to the river, and
slope angle predictors were left. The robust VIF values among the remaining
predictors were 1.006–1.062. For validation of the model, AUC = 0.934, SD =
0.010, Kappa = 0.880, and CCI = 0.881, indicated the robustness of the model.
The response curves of the predictors are shown in, and the relative
contributions of each predictor are shown in (right).
PPR high-risk areas in the THR were predicted using both models and are shown
in. High-risk areas were distributed along the Himalayas, covering northern
India, Nepal, and central Pakistan. In addition, PPR high-risk areas were
scattered throughout Bangladesh and central India. It is worth noting that in
Tibet, China, high-risk areas show an obvious trend of distribution along
rivers. The results show that the risk of PPR around Pakistan, India, Nepal, and
China borders is extremely high. The possibility of transboundary spread cannot
be ignored, especially since it may be facilitated by wildlife.
## Results of the LCP model
The LCP analysis revealed eight potential transboundary paths in the research
area. The eight identified livestock transboundary paths were: A. Mandi
(India)-Ali region (Tibet, China); B. Almora (India)-Ali region (Tibet, China);
C. Khalanga (Nepal)-Ali region (Tibet, China); D. Chukha (Bhutan)-Lhasa (Tibet,
China); E. Chandpur (Bangladesh) -India-Burma-Dali (Yunnan, China); F. Mahakali
(Nepal)—Uttarakhand/Uttar Pradesh (India); G. Seti (Nepal)—Uttar Pradesh
(India); H. Lumbini (Nepal)—Uttar Pradesh/ Madhya Pradesh (India).
The cost value of each classification is changed by 5% (incremental percent
change) within the range of −10% to +10% (range percent change). Thus, the
sensitivity analysis consisted of 72 model iterations. The results are visually
represented by comparing the iterative models with the original cost value model
through the Spearman rank correlation coefficient, as shown in. indicate that
the cost value of elevation has little effect on ranking. In contrast, the
sensitivity of the cost value of land cover is only slightly higher than the
former (see and Tables for more details). Sensitivity analysis underlined the
significant stability of the rankings with respect to the variation in the cost
value for the land cover and elevation perspectives.
# Discussion
## PPR risk and variable analysis
Human population density is the most important predictive variable in the low-
elevation model, with a contribution of 58.2%. Indeed, the low-elevation areas
in this study are mainly distributed in the Indus plains, which are heavily
populated. Although animal husbandry is the major source of income in this area,
considering the large local population of the herds, both the variables of sheep
and goat density were excluded by the MaxEnt model due to their low contribution
rate (0–0.1). However, this does not indicate that host density has nothing to
do with PPR risk. It may also be due to the correlation between human and host
density that leads to the deletion of collinear variables in the model, which
needs further investigation. In contrast, human population densities were
considered as the third highest prediction variable in the high-elevation model.
Nonetheless, the response curves of the human population density for both models
displayed a similar trend, with a rapid increase in PPR incidence as the
population density increases, followed by a plateau. Therefore, despite the
variations in the contribution of human population density in both models, its
close correlation with the incidence of PPR cannot be overlooked.
The temperature has also been suggested to play an important role in
transmitting and spreading infectious diseases. In this study, the mean
temperature in April had the highest contribution rate in the high-elevation
model. Small ruminants in the Tibetan plateau are mainly raised by transhumance,
and summer pastures in high-elevation areas are commonly used during the warm
season (April to May). Transhumance and migration of wildlife have intensified
the direct and indirect contact between wildlife and livestock, which might
increase the risk of PPRV transmission. In contrast, domestic small ruminants
are kept in farms in low-elevation areas. The effect of seasonal temperatures
was minimized accordingly. However, the response curves remind us that the
appropriate temperature in summer (Min T Aug.: 15–30°C) and winter (Min T Nov.:
0–20°C) can increase the risk of PPR. These alerted us to pay additional
attention to seasonality in preventing PPR, especially the risks of seasonal
pasture transfer in transhumance areas.
Many landform variables related to gathering contributed to the prediction of
the PPR risk in our models. According to the response curves, habitats with
deciduous broad-leaved forests, urban areas, or shrublands had the highest
probability of PPR. Both the deciduous broad-leaved forests and shrublands could
provide food and shelter for small ruminants. In addition, they are mainly
distributed in the temperate zone, which is consistent with the optimum
temperature shown by the climate variables. The insignificant importance of the
slope angle to our models can be explained by the good climbing skill of small
ruminants, i.e., the slope hardly restricted their distribution. For this
reason, slope angles were not included in our LCP model.
The variations in the distances to the river contributed significantly in the
high elevation model (8.9%) than in the low elevation model (1.8%). From the
high elevation areas response curve, it could be understood that a farther
distance from the river decreases the risk. This is different from that in the
low elevation areas. The high-risk areas distributed along the rivers in Tibet
can be explained by the prevalence of a cluster of wildlife around the water
holes, which would increase contact and spread of PPRV. The accessibility of
water resources and the lush vegetation in plain areas dispense the need for
rivers.
Our model shows that the mixed crop-livestock farming system has a very high PPR
risk in areas close to the Himalayas, and human influence (population) is the
main variable in such cases. Most ruminants in mixed crop-livestock farming
systems are found in rural areas and have frequent contact with farmers due to
production demand. Therefore, the risk of PPR being dominated by the human
impact is expected. For grazing systems, high-risk areas are only scattered
around the river valley, and the natural environment (temperature) is the
dominant variable. Transhumance became the link between temperature, ruminants,
and PPRV. The communication of risk between the two livestock systems and two
different landscapes may play a potential role in driving PPR transboundary
transmission.
## The impact of wild and domestic hosts on PPR
The host populations are important for PPR maintenance, bridges, and
transmission. Because of the complex migration of wild susceptible hosts,
obtaining high-quality data for model construction is not easy. However,
population profiles (see the second paragraph of the study area section for more
detail) can still help us analyze its impact on PPR. In , we observe that bharal
is distributed in the Tibetan plateau, providing sufficient bridge hosts.
Himalayan goral occupies the Himalayas with a high population density. In
contrast, the populations of blackbuck and markhor are relatively small.
Moreover, the other seven paths, except path E, are within the territory range
of wild small ruminants, which might become bridge hosts for PPR transboundary
transmission.
## LCP
The LCP analysis returned eight transboundary paths between India, Bhutan,
Bangladesh, Nepal, and China. Next, we describe the two-way communication of PPR
risk from outside China to inner China. One end of Path A connects to Mandi city
in northwest India, which is known to have a prosperous livestock industry with
large populations of small ruminants but poor animal health and veterinary
services. The predicted risk of PPR is extremely high in this area. Path A
further extends southwest to the Himalayas and passes through the middle section
of the Sino-Indian border into China. While the elevation along path A is
generally high with a peak of 5733 m, many wild ruminants (viz. bharal and
Himalayan goral) can cross such rugged terrain. At the other end of path A is
the vast alpine pasture area of the Tibetan Plateau, where nomadic domestic
small ruminants are widely distributed, which provides a sufficient host for
PPRV. Path B is like A, from northwestern India to Tibet, but its length is
shorter, and it might be the fastest path contributing to the spread of PPR
across borders. Path C extends from midwest Nepal, where PPR frequently occurs,
to Tibet. In this path, the porous border and unrestricted animal movement
within the country during festive seasons (August to October) may also aid in
spreading the disease. Path D extends from Chukha (Bhutan) along the river
valley to Lhasa (Tibet), with wild small ruminants distributed along the way. At
one end of path E is Chandpur (Bangladesh) that follows the Jamuna River and the
Brahmaputra rivers to Parshuram Kund (India), and then crosses the Burmese
section of the Arakan Mountains to Dali (Yunnan, China). This path is also
mainly distributed along the river valley, and the bushes on both sides of the
valley make it easier for the animals to cross. Both paths F and G start from
the edge of the Himalayas (within Nepal) and reach the Ganges plain. Path H
crosses the Ganges plain and extends to the Indian plain in central India, and
the upper two paths are in low elevation areas, which do not offer any
obstruction to the movement of small ruminants.
The constructed LCP model involved the main variables that affect the movement
of ruminants. The complicated secondary variables (such as hunting and natural
enemies) were not included because they could not be measured. At the same time,
the merits of the LCP model do exist, especially for a large geographical scale
prediction. The cost value commonly depends on the literature and the
researchers. It is worth noting that identifying the animal corridors is not
easy. LCP is still an effective and universal quantitative method to solve this
problem. Mutual verification between the model and reference confirmed that the
sensitivity of the LCP model is resistant to slight changes in the values of
variables. We put forward a set of methods for countries with data limitations
and regions too vast and/or too difficult to access to provide a quick risk
assessment.
# Supporting information
We appreciated the input from Dr. Hein van Gils that helped shape our early
thinking about the work described here. We would like to thank Tony Wang from
the Garvan Institute of Medical Research for the English editing. The final
version of this manuscript was also improved by the useful comments from Dr.
Simon Clegg, Prof. Richard Kock, and two anonymous reviewers.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Macrophages are a heterogeneous and plastic cell population that respond to
environmental signals in various cholestatic liver diseases. Tissue-resident
macrophages of the liver, also termed Kupffer cells, are self-renewing cells
that are present in the liver at birth and promote tolerance in homeostasis. In
the setting of liver injury, tissue-resident macrophages can adopt a pro-
inflammatory state and additional monocyte-derived macrophages may be recruited
from the peripheral circulation to the liver. This leads to a heterogeneous
population of macrophages that may have distinct functions in disease.
Prior studies have presented conflicting evidence for a role of macrophages in
obstructive cholestasis. Recruited monocytes have been shown to have a
protective role against infection in the setting of murine bile duct ligation.
In contrast, C-C chemokine receptor type 2 (CCR2)-mediated recruitment of
monocyte-derived macrophages in a murine model of primary sclerosing cholangitis
has been implicated in the mechanism of liver injury and fibrosis. Similarly,
macrophages have been associated with the pathogenesis of murine parenteral
nutrition-associated cholestasis via toll-like receptor 4 (TLR4)-mediated
activation and production of interleukin-1 beta (IL-1β). Furthermore, reduced
farnesoid x receptor (FXR) signaling is thought to induce activation of the
macrophage inflammasome in cholestasis and endotoxemia, thereby promoting IL-1β
release and increasing immune susceptibility in cholestasis. However, the
precise subsets of macrophages responsible for cholestatic liver injury and
repair have not been fully characterized.
Macrophages have also been more specifically implicated in biliary atresia (BA),
an obstructive cholangiopathy of infants thought to arise from an aberrant
immune response to a self-antigen. While there are two major forms of BA,
isolated BA (iBA) and syndromic BA (BASM) with associated malformations,
evidence supports a similar antigen-driven immune response in both subtypes.
Evidence supporting a role for macrophages in this mal-adaptive immune response
include the observation that increased numbers of macrophages correlate with
poor prognosis in human BA. Hepatic macrophages are also increased in the
rotavirus-induced murine model of BA. In addition, macrophage depletion in a
murine model of BA improved bile duct obstruction. These studies demonstrate a
central role for macrophages in promoting liver injury in BA but fail to
identify the specific pathogenic versus pro-restorative macrophage subsets.
In our current work, we define human liver macrophage heterogeneity in pediatric
cholestasis by analyzing single-cell RNA-sequencing (scRNA-seq) from patients
with cholestasis from BA or Alagille Syndrome (ALGS, a non-immune etiology of
obstructive cholestasis) and comparing these with non-cholestatic pediatric
liver and previously published normal hepatic macrophages. We identify novel
hepatic macrophage subsets in obstructive cholestasis that are distinct from
non-diseased macrophages by leveraging the ability of scRNA-seq to define cell
sub-populations. We further demonstrate reduced expression of regulatory genes
across all cholestatic macrophage subsets that may contribute to loss of immune
tolerance in cholestasis. Taken together, our results lay the foundation for
future mechanistic studies and development of macrophage-specific immune
modulatory therapies.
# Experimental methods
## Human tissue samples
Formalin-fixed, paraffin-embedded liver tissue sections from non-diseased donor
liver (n = 5), and BA (n = 6), and ALGS (n = 6) patients at the time of liver
transplantation were obtained from the pathology archives of Ann & Robert H.
Lurie Children’s Hospital of Chicago. Fresh liver tissue was obtained from the
explanted liver of 3 patients with cholestatic liver disease (2 with BA and 1
with ALGS) and 1 patient with a hepatic tumor at the time of liver
transplantation. Laboratory data was collected retrospectively from the hospital
admission for liver transplantation. Written informed consent was obtained from
each patient’s legal guardians including in the study. The study protocol
conforms to the ethical guidelines of the Declaration of Helsinki as reflected
in a prior approval by the Institutional Review Board of Lurie Children’s
Hospital of Chicago. All methods were conducted in accordance with the
Institutional Review Board’s guidelines and regulations.
## Macrophage quantification by immunohistochemistry
We performed immunohistochemistry to CD68 (Dako M0876), a cell surface marker on
macrophages in normal, BA, and ALGS patients to determine if the hepatic
macrophage population is expanded in cholestatic liver disease. De-waxing and
antigen retrieval were performed on formalin-fixed, paraffin-embedded tissue
sections following the Leica Bond-Max automated protocols. Image capture was
performed with a 40x objective (400x) on a Nikon 80i (Nikon, Melville, NY, USA)
microscope with a DS-Ri2 color camera. Images were stitched with a Prior Proscan
III (Rockland, MA, USA) 8-slide stage and digital encoder, which allows capture
of the entire tissue biopsy to a single image. The surface area was determined,
and cell quantification was calculated with a thresholding algorithm on RBG
images using NIS-elements AR (version 5.1). The same algorithm was utilized for
all specimens, and the investigator who performed the quantification was blinded
to disease classification. We performed a pairwise comparison between control
and cholestatic groups and determined the level of significance by unpaired
t-test.
## Human liver tissue digestion
We obtained fresh liver samples from explanted liver tissue for iBA, BASM, ALGS,
and non-cholestatic pediatric liver (NC) at the time of transplantation. To
account for variable disease throughout the liver, three 1 cm<sup>3</sup>
samples per patient were obtained from different areas of the explant by the
clinical pathology team. Non-cholestatic liver tissue was taken from a patient
with a hepatic neoplasm distal to the site of tumor. Matched histology slides
from the explant were prepared by random sampling simultaneously from the
explant by the pathology technician. Samples were stored in Tissue Storage
Solution (Miltenyi Biotec) for 6 hours prior to mechanical and chemical
digestion. Each of the three 1 cm<sup>3</sup> samples per patient were split
into thirds and infused with RPMI. We cut each sample into small pieces in a
c-tube and added 2.5 mL digestion buffer per c-tube: 2 mg of DNase I (Sigma),
585 μL of Liberase TL (Sigma), and 9.215 mL of RPMI-1640 (Sigma R8758). Liver
tissue was further digested using a both the Miltenyi Biotec gentleMACS
Dissociator and incubation with shaking at 37°C for 1 hour. We strained the
liver homogenate through a 40 μm filter into a 50 mL conical tube with grinding
and washing to optimize yield. The samples were spun at 300 rcf for 10 min
(4°C), the supernatant was aspirated, and the pellet was resuspended in Pharm
Lyse for 1 minute to lyse remaining red blood cells. The reaction was stopped
with HBSS, the samples were spun again at 300 rcf for 7 min (4°C), and the
supernatant was aspirated. We resuspended the pellet in HBSS and strained twice
over a 40 μm filter into a 15 mL tube. Cell count was performed and the cell
suspension was prepared for fluorescence activated cell sorting (FACS).
## Flow cytometry and scRNA-seq library construction
A total of 1.9 x 10<sup>7</sup> cells were obtained from digestion of ALGS
liver, 2.2 x 10<sup>7</sup> from BASM, 4.92 x 10<sup>7</sup> from iBA, and 1.1 x
10<sup>8</sup> from CL. We stained single cell suspensions from each sample with
antibodies to detect cell viability and expression of the CD45 common leukocyte
antigen. 90–100,000 live CD45+ cells were collected by fluorescence activated
cell sorting with a viability of 94% for ALGS, 84% for BASM, 76% for iBA, and
87% for NC. scRNA-seq libraries were prepared using the Single Cell 3’ v2
Reagent Kit for BASM and ALGS and the v3 Reagent Kit for iBA and NC. Gel Beads
in Emulsion containing single cells were generated by the 10x Genomics Chromium
Controller in the Northwestern Next Generation Sequencing Facility. Barcoded
libraries were sequenced on the Illumina HiSeq 4000 platform. Raw sequence data
was processed using the 10X Genomics Cell Ranger 3.1.0 pipeline for de-
multiplexing, trimming, aligning, and mapping to genes. After filtering of the
scRNA-seq data 5,027 immune cells in ALGS, 2,633 immune cells in BASM, 5,927
immune cells in iBA, and 4,691 immune cells in NC were detected.
## Single-cell RNA-seq analysis
To define the hepatic immune cell heterogeneity, we analyzed each single cell
library using the Seurat version 3.0 R toolkit. Filtering parameters for each
sample were set to include genes expressed in \> 3 cells. Cells were included
with gene counts \>200 and \< 5000, and with \< 20% mitochondrial genome. We
next ran the functions LogNormalize (scale factor 10,000), ScaleData, and RunPCA
on each dataset. Variability in each principal component was visualized by the
ElbowPlot function. Based on this analysis we clustered the cells by the
FindNeighbors function (15 dimensions for ALGS, 17 for BASM, 12 for iBA, and 10
for NC) and FindClusters (resolution of 0.5 for each cholestatic sample and 0.2
for NC). Cell clusters were visualized by Uniform Manifold Approximation and
Projection (UMAP) using the function RunUMAP. Using lineage-specific marker
genes, we annotated each cluster as myeloid (*CD68*, *CEBPB*, *CLEC9A*), T and
natural killer (NK) cells (*CD3D*, *CD8A*, *NKG7*), and B cells (*CD79A* without
*MZB1*) plasma cells (*CD79A* co-expressed with *MZB1*), and dividing cells
(*TOP2A*). To confirm our cell assignments we used SingleR to compare all
clusters from each patient to the reference bulk transcriptome data from Immgen.
We also separately compared our disease-specific myeloid clusters to the Immgen
database to further refine our myeloid subset annotations prior to integrated
analysis. We next performed integrated clustering on the mononuclear phagocyte
cells from each cholestatic patient and ran FindIntegrationAnchors and
IntegrateData on ALGS clusters 5, 8, 10, BASM clusters 0, 6, 7, and iBA 3, 6, 9,
11, and 12. We determined the conserved genes within each integrated myeloid
cluster by the function FindConservedMarkers. To compare our diseased
macrophages to normal macrophages, we imported previously published single-cell
data on non-diseased adult human liver from 5 donors with a median age of 41.0
years (interquartile range 23.5 to 54.5 years). We used the same cell-specific
annotations and assigned this normal data-set as the reference in further
SingleR analysis of our cholestatic macrophages. The degree of similarity
between groups was further assessed visually by UMAP and by correlation analysis
of shared genes (Morpheus, <https://software.broadinstitute.org/morpheus>). To
infer pseudotime values, we used Monocle 3 for trajectory analysis of non-
diseased macrophages. By grouping cells into 5 clusters based on their
pseudotime values, we applied the function FindGeneModules to identify 2 gene
modules upregulated at the beginning (pseudotime 0–5) and end (pseudotime of the
trajectory to best represent the non-inflammatory and inflammatory macrophage
profiles, respectively. Finally, to ascertain if differences in the
transcriptional signatures may be secondary to patient age, we compared
macrophages from the NC liver sample to the adult normal macrophages by
correlation and pseudotime analyses as described above.
## Immunofluorescence and quantification of macrophage subsets
We next characterized protein expression for genes that differentiated the 3
cholestatic macrophage subsets by immunofluorescence using the Vectra
Multispectral Imager in the Northwestern Immunotherapy Assessment Core. Baking
and dewaxing was performed on formalin-fixed, paraffin-embedded tissue sections.
Using the Opal 7-color automation kit (Akoya Biosciences, Marlborough, MA, USA)
slides were stained for CD68 (Abcam ab955), CD69 (Abcam ab233396), C1Q (Abcam
ab268120), and S100A8/9 (Abcam ab22506). Whole slide fluorescent imaging was
performed followed by multispectral imaging of three 2.01 mm x 1.5 mm areas per
slide (Phenochart and Vectra software). We next used inForm software to
phenotype the cells and analyzed the cell data with phenoptrReports 0.2.9
package in R. Based on gene expression data we defined LAM on histology as
CD68<sup>+</sup>C1Q<sup>+</sup>S100A8/9<sup>-</sup>CD69<sup>-</sup>, MLM as
CD68<sup>+</sup>C1Q<sup>-</sup>S100A8/9<sup>+</sup>CD69<sup>-</sup>, and AM as
CD68<sup>+</sup>C1Q<sup>+/-</sup>S100A8/9<sup>-</sup>CD69<sup>+</sup>. Using
these definitions, we compared abundance on histology by disease group.
# Results
## Increased macrophage numbers in obstructive cholestasis as compared to healthy liver controls
We performed immunohistochemistry on histology samples from donor livers, and BA
and ALGS patient livers at the time of liver transplantation to determine
whether the hepatic macrophage population is expanded in cholestatic liver
disease. No histologic abnormality was present among donors with the exception
of one individual liver which exhibited hepatocyte swelling. Mean age for donor
pediatric patients rounded down to the nearest month was 68 months (SD 113, n =
5). No laboratory data was available for donor controls. All BA and ALGS liver
samples had prominent fibrosis or cirrhosis at the time of tissue collection.
Mean age rounded down to the nearest month for BA patients was 7 months (SD 1, n
= 6) and 105 months (SD 78, n = 6) or 8 years and 9 months for ALGS cases.
Difference in age between the 3 groups was not statistically significant by
ANOVA (p = 0.12). Mean direct bilirubin within 24 hours prior to transplant was
not significantly different between disease groups at 9.6 mg/dL (SD 8.0, n = 6)
for BA and 12 mg/dL (SD 7.7, n = 6) for ALGS (p = 0.65 by paired t-test). We
found increased number of CD68<sup>+</sup> macrophages in BA liver as compared
to control with a mean of 1332 cells/mm<sup>2</sup> in BA versus 601
cells/mm<sup>2</sup> in non-diseased pediatric liver tissue (p = 0.04). While
ALGS samples also exhibited greater numbers (1040 cells/mm<sup>2</sup>) of
CD68<sup>+</sup> macrophages, it did not reach significance compared to control.
The pronounced influx of macrophages in cholestatic liver disease suggests they
may play a pathogenic role in cholestatic-induced liver injury.
## Clinical information
We obtained liver tissue at the time of transplantation from three pediatric
patients with cholestatic liver disease. Cholestasis with variable elevation of
liver enzymes was present at the time of tissue collection for the iBA, BASM,
and ALGS patient samples based on laboratory data close to the time of sample
collection. The iBA case was female and presented at nearly 6 months of age with
a diagnosis of BA and did not receive a Kasai Portoenterostomy. She developed
cirrhosis and portal hypertension and was thereby evaluated for primary liver
transplantation. The patient with BASM was also female and received a late
diagnosis of BA without Kasai Portoenterostomy. She developed cirrhosis and had
a pre-transplant course complicated by portal hypertension, liver synthetic
dysfunction, and infection. She received a transplant at 6 months of age. The
patient with ALGS was male and met criteria for liver transplantation due to
refractory fat-soluble vitamin deficiency leading to severe hepatic
osteodystrophy and malnutrition. He had preserved liver synthetic function and
while he did not have clinically evident portal hypertension at the time of
transplant at 22 months of age he had findings of mild to moderate portal
fibrosis with numerous bridges on histology.
## Variable immune cell composition between BASM and ALGS
We next performed scRNA-seq on CD45<sup>+</sup> live cells isolated from each
liver sample to better evaluate immune cell infiltration in obstructive
cholestasis. We classified single-cell clusters into 5 immune cell types and a
population of dividing cells in the cholestatic liver samples using lineage-
specific marker genes. Different clusters of the same cell type were highly
correlated within each sample and between the samples thereby supporting the
lineage annotation. Further, the lineage annotations were confirmed by Single-R,
which compares each cell against a reference dataset of population-level
transcriptional profiles (in this case, the Immgen database). Lastly, one
cluster in each patient expressed high levels of cell cycle genes, which would
indicate dividing cells. T and NK cells were the most abundant immune cell
population in all samples, comprising 73%, 48%, and 54% of total immune cells in
ALGS, BASM, and iBA respectively. Mononuclear phagocytes were the next largest
population in BASM and iBA, but not in ALGS. This discrepancy may reflect the
difference in disease etiology.
## Three distinct macrophage populations in obstructive cholestasis
To better understand macrophage heterogeneity in obstructive cholestasis, we
focused our analysis on the clusters annotated as MNP and other myeloid cells.
Our Single-R results suggested these clusters contained a mixture of
macrophages, dendritic cells (DCs), and neutrophils. For further analysis, we
excluded neutrophils, which were found in BASM (cluster 8) and iBA (cluster 7)
and defined by distinct expression of neutrophil genes, such as *FCGR3B* and
*S100P*, and lack expression of macrophage genes, such as *CD68* and *CTSB*. We
then performed integrated clustering on the remaining cells from all patients to
define 3 macrophage subsets and 3 dendritic cell subsets. Three macrophage
clusters were identified by the lineage-specific markers *CD68*, *CEBPB*,
*CD14*, and *CD69*. The dendritic cells were annotated using markers described
previously to identify a *CD1C* positive subset, *CLEC9A* positive subset, and
plasmacytoid DC (pDC) subset. All macrophage populations were represented in
each patient. Together, these findings suggest common macrophage subsets may
arise from environmental cues in the setting of cholestasis.
We next sought to characterize the cross-disease transcriptional signature of
each inflammatory macrophage subset and defined MΦ1 as lipid-associated
macrophages (LAM), MΦ2 as monocyte-like macrophages (MLM), and MΦ3 as adaptive
macrophages (AM). LAM demonstrated the highest expression of genes associated
with lipid metabolism including *APOC1*, *APOE*, *LGMN*, *FABP5*. There was also
high overlap with genes previously reported in LAM from human adipose tissue
including *TREM2*. MLM were defined by genes previously identified in
monocytes, including *S100A8*, *S100A9*, *VCAN*. Finally, AM were enriched for
genes associated with lymphocyte activation including *CD2*, *CD7*, *CCL5*,
*CCL4*, *CD3D*, *IL7R*. As we have previously defined these immune cells as
macrophages, the increased expression of genes involved in adaptive immunity
suggest these cells may have engulfed lymphocytes or play a role in regulation
of lymphocyte response.
To validate these three populations across cholestatic liver disease, we
performed immunofluorescence on a large cohort of patients. We chose markers for
each population based on their differential gene expression by scRNA-seq. Using
these markers, we demonstrated the presence of all subsets across the fixed BA
and ALGS samples from through overlap with CD68 expression. Since not all
individual cells in a population expressed the relevant marker, we expect this
approach to have lower sensitivity than specificity as supported by differences
between histology and gene expression analyses for the BASM, iBA, and ALGS
samples. Thus, the percent of each population is likely to be an underestimate
and may explain the proportion of CD68+ cells not assigned to any population.
Despite these differences, comparing the number of cells in each population
between 6 BA and 6 ALGS patients shows that LAM tends to account for a greater
proportion of macrophages in BA. In contrast, the AM population is larger on
average in ALGS patients. Further study is required to determine whether this
difference reflects disease pathogenesis.
## Reduced expression of immune-regulatory genes in obstructive cholestasis as compared to non-diseased human liver
We took advantage of single-cell data that was previously published using non-
diseased adult livers to determine how macrophages from cholestatic livers
compared to those from healthy livers. We reproduced the 20 clusters from the
original study of which 2 were labelled as “inflammatory” (IM) and “non-
inflammatory” (NM) macrophages. Although this data included all cell types, not
just CD45+ cells, annotation of immune cell types using lineage-specific markers
led to analogous results. To overcome technical variability between data-sets
limiting the utility of co-clustering, we used Single-R, Correlation analysis,
and single gene and gene set comparisons to evaluate similarities and
differences between macrophage subsets. All 3 populations of cholestatic
macrophages were more similar to the IM than NM; AM was the least correlated
overall (0.84) compared to LAM (0.89) and MLM (0.89).
To determine whether the differences between datasets was due to older age of
the controls, we performed scRNA-seq on a pediatric non-cholestatic (NC)
pediatric liver sample. The NC case was an 11-year-old female whose explanted
liver demonstrated some areas of necrosis consistent with changes after
chemotherapy and chronic inflammation with margins negative for tumor. Through a
comparable scRNA-seq analysis work-flow, we identified two populations of
macrophages, which we label Ped1 and Ped2. Unlike the cholestatic macrophages,
these populations clearly recapitulate the dichotomy of adult NM and IM.
Moreover, while all cholestatic macrophages demonstrated decreased expression of
immunoregulatory genes (*MARCO*, *HMOX1*, and *CD5L*), Ped2 expressed these
genes at comparable levels to NM. The cholestatic populations, LAM and AM,
exhibited distinct transcriptional signatures from both adult and pediatric
macrophages subsets. In contrast, the genes that defined MLM were also increased
in adult IM and Ped2. Interestingly, expression of *NR1H4*, which encodes FXR
and is thought to play a role in macrophage inflammasome activation in
cholestasis, is negligible across all macrophages. Taken together, our findings
support the emergence of disease-specific macrophages in cholestasis that may
mediate inflammation via different pathways than FXR signaling.
Although transcriptionally distinct, macrophages in the diseased liver may be
derived from their healthy counterparts. Using Monocle, we defined a pseudotime
trajectory beginning in NM (pseudotime 0) and ending in IM (pseudotime 25). We
then identified 2 modules associated with high expression at these endpoints:
the non-inflammatory module included genes such as *CD5L*, *MARCO*, and *VCAM1*
whereas the inflammatory module included *LYZ*, *S100A8*, and *VCAN*. In support
of the limited effect of age on healthy macrophage heterogeneity, the former
modules were highest in Ped1, while the latter was highest in Ped2. In contrast,
we found that no cholestatic macrophage subset expressed high levels of the non-
inflammatory module. However, MLM exhibited high expression of the inflammatory
module, possibly indicating a common origin with IM. This analysis demonstrates
the transcriptional variability across cholestatic macrophages beyond the
dichotomy of healthy liver macrophages.
# Discussion
We are the first to perform scRNA-seq on pediatric cholestatic liver to define
the macrophage transcriptional profile in obstructive cholestasis. Hepatic
macrophages play a critical role in maintaining immune tolerance in the setting
of persistent exposure to bacterial antigens from the intestine. Loss of this
tolerogenic phenotype in the setting of inflammation may be of particular
importance in ongoing hepatic injury in obstructive cholestasis. Here, we
identify three populations of pathogenic macrophages independent of underlying
etiology that may contribute to liver injury in obstructive cholestasis. No
cholestatic macrophage subset was characterized by expression of immune
regulatory genes as seen in normal adult NM and a subset of macrophages in non-
cholestatic pediatric liver (Ped1). Our data suggest that tissue-resident
macrophages, such as NM previously reported, may be absent or transcriptionally
altered by the tissue microenvironment in cholestatic liver injury. Instead, all
3 cholestatic macrophage subsets were most similar to IM, which are likely to be
monocyte-derived macrophages. In particular, the population of monocyte-like
macrophages had the greatest upregulation of genes encoding the S100 proteins in
addition to *TREM1*, known to amplify the innate immune response, suggesting
this population may have recently infiltrated. We also defined a subset of
lipid-associated macrophages that had the highest expression of genes involved
in TLR signaling (e.g. *GPNMB*, *MT1G* and *MT1X*). Lastly, we demonstrate the
presence of a novel adaptive macrophage subset with increased *RORA* gene
expression, which has been shown to promote anti-inflammatory polarization of
hepatic macrophages in a murine model of nonalcoholic steatohepatitis and a
human monocyte cell line. The transcriptional profiling of these distinct
subsets may identify macrophage-specific targets to ultimately inhibit monocyte
recruitment, block TLR-mediated macrophage activation, or re-program macrophages
to an anti-inflammatory phenotype.
While macrophages have been implicated in immune-mediated hepatic injury from
cholestasis, the exact mechanism is not well known. Current medical therapies
for cholestatic liver disease include FXR agonists, which in addition to
regulating the bile acid pool may also inhibit macrophage inflammasome
activation based on prior studies. However, we demonstrate an absence of *NR1H4*
encoding FXR in cholestatic macrophages despite evidence of TLR signaling and
inflammasome activation. As macrophages play a role in cholestatic liver injury,
this finding highlights the lack of current cell-specific immune-modulatory
strategies and the need for a deeper understanding of the immune response to
cholestasis.
LAM in our samples had a gene signature that was similar to recently published
data on *TREM2+* lipid-associated macrophages in murine adipose tissue. This
gene signature was also similar to human hepatic macrophages during obesity and
may represent a conserved response to loss of metabolic homeostasis. As
hypercholesterolemia is a common sequela of cholestasis, the *TREM2*+ LAM in our
samples may arise in response to similar metabolic derangements. However, LAM in
our study differed in that they had an overall inflammatory gene signature
despite expression of *TREM2* previously shown to promote anti-inflammatory
macrophage polarization. They also were identifiable by C1Q expression, which
was similarly expressed by NM from the healthy adult dataset. It is possible
that LAM arise from inflammatory activation of healthy tissue-resident
macrophages. Targeting the *TREM2* molecular pathway may be an important
therapeutic target to re-program hepatic macrophages to an immune regulatory
phenotype and reduce the consequences of hypercholesterolemia in cholestasis.
The mechanism of disease pathogenesis in BA is hypothesized to be
multifactorial, including an aberrant immune response to a cognate antigen
whereas ALGS is a genetic disease resulting in bile duct paucity. Thus, while
the aim of the current study was to identify a common cholestatic macrophage
phenotype, there are likely etiology-specific differences in the immune pathways
for macrophage activation that require further investigation in larger studies.
However, despite this limitation, we provide important insight into hepatic
macrophage heterogeneity in cholestatic liver disease compared to healthy
livers. Despite age differences, it is worth noting that macrophages from a non-
cholestatic pediatric patient demonstrated a similar dichotomy as adult hepatic
macrophages. This finding suggests that the distinct transcriptional signature
of cholestatic macrophages is not a result of age-specific differences or
technical differences between datasets such as variation in sample isolation,
processing and digestion protocols, or experimental design. Lastly, we
acknowledge that our findings may not be limited to obstructive cholestasis and
may overlap with other causes of end-stage liver disease characterized by
cirrhosis and portal hypertension. A recent study on adult cirrhotic livers
described a population of scar-associated macrophages in cirrhosis that appear
most similar to cholestatic LAM and express higher levels of *TREM2*, *CD9*,
*LGALS3*, and *SPP1*. Future studies will more clearly define the similarities
and differences in subset-specific macrophage function by patient age, stage of
cholestatic liver disease, and etiology of cirrhosis.
In this study, we have used single-cell transcriptional analysis of pediatric
cholestatic liver samples to identify macrophage subsets at greater resolution
than previously described. With ongoing work, we will strengthen conclusions on
the hepatic macrophage transcriptional signature in different cholestatic liver
diseases and identify common therapeutic targets to reprogram macrophages and
slow disease progression. More specifically, we highlight expression of the
immune regulatory genes *RORA* and *TREM2* within these inflammatory subsets
that may be potential therapeutic targets to ameliorate inflammatory injury in
obstructive cholestasis. Future work to correlate our findings to the immune
cell subsets present earlier in disease will provide important insight into
cell-specific therapeutic strategies to improve prognosis shortly after disease
onset. Identifying molecular targets to reprogram hepatic macrophages in
cholestasis may also have therapeutic implications for other etiologies of liver
diseases and reduce the medical burden of end-stage liver disease.
# Supporting information
The authors acknowledge the support provided by the Northwestern University Flow
Cytometry, Pathology, Immunotherapy Assessment Core, and Sequencing Core
Facilities.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Achilles tendon (AT) is the largest and strongest tendon in humans. Its major
function is to transmit the force produced by calf muscles to heel bone.
Although AT is primarily made up of the type-1 collagen fiber which has high
strength and flexibility, exposure to excessive mechanical loadings during
vigorous exercises including running and jumping may easily result in tendon
injuries such as rupture and tendinopathy. Ultrasound is a useful imaging tool
for the assessment of musculoskeletal structures because of its high image
resolution, non-invasiveness and real-time capability. Shear-wave elastography
(SWE) is a new ultrasound technology which allows quantitative evaluation of
soft tissue stiffness (Young’s Modulus). Its principle is to send acoustic
radiation force impulses through soft tissues with a particular density (ρ) and
to compute the tissue shear elasticity (μ) in kilopascal from the velocity of
shear wave (cT) travelling within the tissue based on the equation: μ =
ρcT<sup>2</sup>. SWE has been used for the assessment of breast and liver
tissues. However, there are only a few studies reported SWE assessment of AT,
and they studied the characterization of normal and abnormal AT, repeatability
of stiffness measurement, AT stiffness augmentation and stretched AT elastic
anisotropy. The feasibility of using SWE in the assessment of AT properties was
supported by these studies due to good agreement in reported results and fair
repeatability in measurements as well as clear morphological delineation.
Static stretching is commonly performed prior to athletic activities to avoid
musculoskeletal injuries by reducing tendon stiffness and enhancing functional
range of movement. It has been reported that static stretching has no
significant immediate effect on cross-sectional area (CSA) and slack length of
AT. However, the immediate effect of static stretching on AT stiffness is
controversial in previous studies. Some studies reported a significant decrease
in AT stiffness after static stretching, whilst Nakamura found a significant
increase in AT stiffness, and other studies showed no alteration in AT stiffness
after static stretching. The inconsistent results of previous studies may be
attributed to the indirect and complex conventional methods of using grey-scale
ultrasound and dynamometer for the measurement of AT stiffness in these studies.
With the advantages of real-time capability and quantitative measurement of soft
tissue stiffness, SWE is an ideal imaging tool for accurate assessment of AT.
However, the value of SWE in assessing the immediate effect of static stretching
on AT has not been reported. Leg dominance arises from predominant loading of
unilateral leg in specific activities. Previous study has suggested the
definition of leg dominance depending on the nature of activities–manipulative
(kicking a soccer ball) or stabilization (standing on one leg). Tendons alter
its structural and biochemical properties to adapt the magnitude and habit of
mechanical loading during activities. The asymmetric loading profiles between
two legs may cause difference in tendon properties, such as higher AT stiffness
in dominant leg. Higher incidence of tendon rupture in left Achilles tendon was
noted in which the left leg was considered as the dominant leg which supported
body stability. The mechanical properties of AT are different in dominant and
non-dominant legs. Static stretching is important for preventing and treating
injuries, and is commonly used as a therapeutic tool in physical rehabilitation
and sports. Understanding the variation of the mechanical properties of AT
between dominant and non-dominant legs in response to static stretching can help
to devise appropriate treatment protocol for dominant and non-dominant legs.
However, the effect of leg dominance on immediate effect of static stretching on
AT properties has not been evaluated in previous studies.
With the use of SWE, the present prospective study aimed to investigate the
immediate effect of static stretching on normal Achilles tendon (AT) properties;
to compare the potential difference of the effect of static stretching on
stiffness of AT between dominant and non-dominant legs, and to examine the
inter- and intra-operator reliability of SWE measurements of AT stiffness. The
primary aims of the study are to provide a new imaging perspective in
determining immediate effect of static stretching on AT properties with the use
of SWE, and support further research on the effectiveness of pre-exercise static
stretching and designation of side-specific stretching programme to optimize
athletes’ performance and prevent AT injuries.
# Methodology
## Subject Recruitment and Experimental Design
A total of 21 Chinese young healthy subjects were recruited on a voluntary basis
via acquaintance. One subject had a previous history of lower leg injury and
Achilles tendon rupture and thus the subject was excluded from the study.
Finally, the remaining 20 subjects were included in the study (13 male and 7
female, mean age = 21.3 ± 1.4 years, mean height = 168.2 ± 8.0 cm, mean weight =
60.8 ± 9.6 kg, body mass index = 21.4 ± 2.5 kg/m<sup>2</sup>). All recruited
subjects were informed on the study aims, examination procedures, rights of
volunteers, and safety of ultrasound by an information sheet, and signed a
written consent before the commencement of the study. The study was approved by
the Human Subject Ethics Subcommittee of the Hong Kong Polytechnic University.
Achilles tendinopathy would affect the morphological and mechanical properties
of AT, and may affect the effect of static stretching on AT stiffness.
Therefore, subjects who were symptomatic of Achilles tendinopathy and have
dysfunction of Achilles tendon were identified. All subjects completed the
Victorian Institute of Sports Assessment–Achilles questionnaire (VISA-A). The
VISA-A evaluates the effect of tendinopathy on function and quantifies the
symptoms and dysfunction of Achilles tendon. According Robinson et al., a
recreational person with Achilles tendinopathy will have a VISA-A score of 70 or
less. In addition, a patient with Achilles tendinopathy who has VISA-A score
reached 70 indicated that he/she was cured. Thus, subjects with VISA-A score
lower than 70 were excluded from the study. Other exclusion criteria were
history of tendon rupture, trauma and surgery in lower leg, experiences of
strength training or flexibility training, history of systemic, metabolic,
endocrine and inflammatory diseases and on hormonal treatment, corticosteroid
drugs and contraceptive pills.
The leg dominance of subjects was determined by asking the subject the preferred
leg to kick a ball. It was suggested that the non-dominant leg refers to the
preferred leg to kick a ball while the dominant leg is defined as the supporting
leg for stability.
## Reliability Tests
To evaluate the inter-operator reliability of SWE measurement of AT stiffness,
another 6 subjects were recruited and scanned by 6 operators. To evaluate the
intra-operator reliability, each operator scanned the subjects three times in
the same scanning session. The operator with the highest intra-rater reliability
was selected to perform the ultrasound scanning in the main study, and performed
the intra-operator reliability test of AT thickness and CSA measurements.
## Ultrasound examination of AT
In the main study, the 21 subjects were reminded to avoid vigorous exercises in
lower limbs for at least 2 hours prior to the examination and were asked to rest
for 30 minutes before pre-stretching ultrasound examination.
All ultrasound examinations were performed by the supersonic shear wave
elastography ultrasound unit (Aixplorer; Supersonic Imagine, Aix-en-Provence,
France) with a SuperSonic SuperLinear<sup>™</sup> 4–15 MHz linear-array
transducer. In the ultrasound examination of AT, the subject laid in a prone
position on the examination couch with the feet hanging freely over the edge of
the couch to avoid tendon stress. A customized ankle fixer was used to
standardize the position of the ankle for ultrasound scanning. A generous amount
of ultrasound gel was applied over the AT to form a gel gap which can ensure
good probe-tissue coupling and prevent tissue distortion due to transducer
compression. B-mode ultrasound and SWE were performed to assess the
morphological and mechanical properties of AT respectively.
In the B-mode ultrasound examination, the AT was scanned transversely until
transverse scans at the level of medial malleolus were obtained. To ensure the
ultrasound beam is perpendicular to the tendon with minimum anisotropy, the
transducer was angled cranially and caudally until a scan plane which showed the
maximum echogenicity of the tendon was obtained, and the thickness and cross-
sectional area of the tendon were measured with the electronic calibers. The
tendon thickness was defined as the maximum anteroposterior diameter of the
tendon, and the tendon CSA was measured by manual outline of the tendon
boundaries.
With the SWE function of the ultrasound unit activated, the stiffness of AT of
the subject was evaluated and the AT was examined with longitudinal scans. To
standardize the region of AT to be assessed, the inferior border of the 1.56 cm
x 1.56 cm acquisition box was placed 2 cm proximal to calcaneal insertion. SWE
scanning was performed until the color display in the acquisition box became
steady and homogenous, and the elastogram was obtained. For each AT, two shear-
wave elastograms were obtained at different longitudinal planes of the tendon.
On each elastogram, the measurement of tendon stiffness was performed using
Q-box measurement tool which was an in-built program for automatic calculation
of mean, maximum, minimum and standard deviation (SD) of stiffness values. A
total of 4 Q-boxes with a diameter of 3 mm were placed along the long axis of
tendon in the image for the measurement (Figs).
## Stretching protocol
Each subject had a pre-stretching ultrasound examination on both legs. The
subject then performed a 5-minute static stretching on the left leg. In the
5-minute static stretching, the subject stepped on a 30° inclined platform with
the body trunk leaned forward until the lower leg was aligned vertically, and
the subject maintained in this posture for 5 minutes. Immediately after the
stretching, a post-stretching ultrasound examination was performed on the left
leg using the same scanning protocol. After the examination of the left leg, the
same stretching and ultrasound protocols were performed on the right leg.
## Data Analysis
All statistical data were expressed as mean ± SD and the effect of static
stretching was defined as the difference in tendon properties after stretching.
For each AT, the thickness, CSA and mean stiffness were obtained and analyzed.
The overall mean stiffness value of each AT was the average of the mean
stiffness measured by the 8 Q-boxes in the two shear-wave elastograms.
Statistical Package of Social Sciences software version 20.0 (SPSS, version 20
for Windows, Chicago, IL) was used to calculate the results of all statistical
tests with a 95% confidence interval. Intra-class correlation (ICC) was used to
evaluate the reliability of AT properties measurements among and within the 6
operators. The normality of the data was analyzed by Shapiro-Wilk test. Data
following or violating normal distribution are analyzed by paired t-test or
Wilcoxon signed rank tests respectively. Paired t-test or Wilcoxon signed rank
test was used to examine change in tendon properties after static stretching and
to compare the difference in tendon properties between dominant and non-dominant
legs.
# Results
In the 20 subjects included in the study, The VISA-A scores were above 70
(Range: 74–98). Seventeen subjects had the dominant leg on the left side whereas
the other 3 subjects had a right dominant leg. The calculated statistical power
for ultrasound stiffness measurement was 0.796 with the 20 subjects (G\*power,
version 3.1.5, Franz Faul, Uni Kiel, Germany).
The mean thickness and CSA of bilateral AT before and after stretching are
summarized in ( and Files). There was no significant difference in the AT
thickness and CSA before and after static stretching (p \> 0.05).
The mean stiffness values of AT before and after stretching exercise are
summarized in. The pre-stretching stiffness of dominant AT (491.3 ± 86.9 kPa)
was significantly higher than that of non-dominant AT (392.6 ± 83.1 kPa) (p \<
0.05) whilst such difference became insignificant after static stretching (p =
0.830). There was a significant increase in the stiffness value in the non-
dominant legs (from 392.6 ± 83.1 kPa to 495.0 ± 96.1 kPa) after performing
stretching exercise (p \< 0.05). Although there was also increase in the
stiffness of AT in the dominant legs but the difference was not significant (p
\> 0.05).
Results showed that the intra-operator reliability \[ICC (3, k) score\] of AT
stiffness measurement ranged from 0.751 to 0.941, whereas the inter-operator
reliability \[ICC (2, k) score\] among the 6 operators was 0.749. The intra-
operator reliability of AT thickness and CSA measurements was 0.991 and 0.962
respectively.
# Discussion
In the present study, the CSA and thickness of AT did not show significant
change immediately after static stretching. These findings were consistent with
previous studies adopting the same stretching protocol with the present study.
Results indicated that static stretching does not have immediate effect on the
morphology of AT.A significant difference in pre-stretched AT stiffness between
dominant leg (i.e. 491.3±86.9 kPa) and non-dominant leg (i.e. 392.6±83.1 kPa)
was found in the present study which is consistent with Bohm’s study, and their
result showed significant higher resting stiffness in dominant leg than non-
dominant leg (p\<0.05). The inherent higher stiffness of dominant AT can be
explained by unequal mechanical loading leading to higher shear stress caused by
micro-tearing and promotion of gene expression for type-I collagen production.
Different mechanical loading of dominant and non-dominant legs during daily
activities may account for the different mechanical properties of AT between
both legs. Therefore, there was difference in AT stiffness before stretching
between dominant and non-dominant legs.Achilles tendon consists of 30% collagen,
2% elastin, and 68% extracellular matrix. Performing the function of stability
and supporting, higher frequency of repeated mechanical loading on dominant AT
had been suggested to contribute to more frequent intra-tendon micro-tearing,
leading to subsequent increased blood supply and extracellular matrix content to
the tendon for structural reconstruction, accounting for a higher hydrostatic
pressure in the tendon region. Stress is defined as function of force per unit
area. Since no significant difference had been found for CSA of bilateral AT,
increased hydrostatic force hence contributes to a higher stiffness of dominant
AT. Increased loading at dominant AT had been suggested to stimulate expression
of insulin-like growth factor-I production (IGF-I) which stimulates synthesis of
type I collagen and cell proliferation, resulting in an increase in cross-
linking along the tendon and therefore increased the stiffness of AT.
The AT stiffness in non-dominant legs was significantly increased after static
stretching whereas there was no significant changes in AT stiffness in dominant
legs after static stretching. The substantial stretching effect in non-dominant
legs may be due to the significant difference in the pre-stretch stiffness of
the tendon between dominant and non-dominant legs. It has been reported that
strain magnitude applied to Achilles tendon must exceed a particular threshold
in order to trigger adaptation effects on the mechanical properties of the
tendon. The strains must be high enough to produce sufficient stimulus that
beyond the strains triggered by the mechanical load applied during daily
activities to trigger adaptation effects on the Achilles tendon. Since the pre-
stretch AT stiffness in dominant legs was higher than that in non-dominant legs,
the strains produced by the 5-minute static stretching on the AT in dominant
legs might not be higher than the mechanical load applied during daily
activities and were not high enough to trigger adaptation effects on the
mechanical properties of the tendon. However, the pre-stretch AT stiffness in
non-dominant legs is lower. Therefore, lower magnitude of strain can produce
sufficient stimulus to trigger adaptation effects on the mechanical properties
of the Achilles tendon in non-dominant legs. The present study showed increased
AT stiffness after static stretching, and this finding is in consistent with the
results of previous studies. Using a machine for material tests to perform
mechanical tensile test of rat’s Achilles tendon, de Almeida et al. found that
Achilles tendons with stretching were stiffer than those without stretching.
Using dynamometer and grey scale ultrasound to measure the passive torque and
the displacement of Achilles tendon after ankle dorsiflexion respectively,
Nakamura et al. found that there was a significant increase in Achilles tendon
stiffness after static stretching.
Compared with the fair inter-operator reliability of SWE measurement of AT
stiffness in Aubry’s study (inter-operator reliability is 0.46), the inter-
operator reliability of the AT stiffness measurement using SWE found in the
present study (inter-operator reliability is 0.749) was higher than the previous
studies. Compared with the highest ICC values in intra-operator reliability test
for SWE measurement of AT stiffness in Peltz’s study (highest intra-operator
reliability is 0.42), the intra-operator reliability of the six operators in the
present study ranged from 0.751 to 0.941, which is higher than Peltz et al.. The
improvement in intra-operator and inter-operator reliability in SWE measurement
of AT stiffness in the present study may be due to the standardization of
scanning protocol and the special equipment used to standardize the ankle
position. Peltz et al. suggested that the measurement repeatability is mainly
influenced by the relative position of transducer and tendon. Angle between
transducer and axis of tendon was suggested to be a factor influencing shear
wave velocity in the tendon and leading to inaccurate calculation of Young’s
modulus. Ankle fixer and standardized measurement site (2 cm proximal to the AT
insertion) were used for the measurement of AT stiffness in the present study
which standardized the position of feet and degree of flexion of ankle
throughout the scanning process. Therefore, higher measurement reliability was
resulted. The significant improvement in the SWE measurement reliability in
present study suggests that standardization of scanning protocol and technique
is crucial for SWE measurement of AT stiffness so that reliable measurements can
be obtained.
The present study demonstrated the asymmetry of stiffness change of dominant and
non-dominant AT where non-dominant AT had significant increase in stiffness
after static stretching. This finding is useful in the design of stretching
protocol in which it should consider the difference in the AT stiffness between
the dominant and non-dominant legs.
There are limitations in the present study. The sample size of the study was
small and only young subjects were recruited. Different static stretching
parameters including loading, dose, magnitude, torque, and number of stretching
cycle and duration of stretching had been reported to have influence on tendon
stiffness. In the present study, only 5 minute continuous and 30 degree
dorsiflexion stretching protocol was adopted. Further SWE investigations to
assess the effect of the number of stretching cycles, stretching angles,
magnitude and duration on AT stiffness are suggested. The present study
investigated the immediate effect of static stretching but the delayed effect
was not studied. Further studies to investigate the delayed effect of static
stretching on AT are suggested.
In conclusion, SWE is a useful and reliable imaging tool for the assessment of
immediate stiffness change of AT upon static stretching. Dominant leg AT has a
higher baseline stiffness than non-dominant leg AT. A 5-minute static stretching
with 30 degree dorsiflexion can lead to significant increase in non-dominant AT
stiffness which may help enhance performance of manipulative activities.
Asymmetry of stiffness change of bilateral AT provides scientific ground for
supporting static stretching as a means to even out the inherent bilateral AT
stiffness difference and provides insight for future studies to manipulate
various stretching parameters in order to optimize athletes’ performance.
# Supporting Information
[^1]: All authors have declared that no conflicts of interest exist for the
data, results and conclusions described in this study.
[^2]: Conceived and designed the experiments: TCRC HCN LWL KWL MHL HFY MY.
Performed the experiments: TCRC HCN LWL KWL MHL HFY. Analyzed the data: TCRC
HCN LWL KWL MHL HFY MY. Wrote the paper: TCRC, MY. |
# Introduction
CD8<sup>+</sup> T cells are critical for control of viral infections and tumors
and their efficient induction requires coordinated signaling through a number of
pathways, including T cell receptor (TCR) ligation with peptide in the context
of major histocompatibility complex class I (MHC I), costimulatory molecules and
cytokines. One of the key effector functions acquired by CD8<sup>+</sup> T cells
upon activation is the ability to produce antiviral and pro-inflammatory
cytokines, including IFNγ and TNF. Typically, cytokine production by antiviral
CD8<sup>+</sup> T cells occurs in an hierarchical fashion, with the majority
producing IFNγ, and a subset of those producing TNF. Such ‘polyfunctionality’
within a T cell response is used to indicate an increased quality of response,
and has been associated with heightened affinity of TCR-pMHCI recognition.
Tumor necrosis factor (TNF) can substantially influence antiviral
CD8<sup>+</sup> T cell responses. TNF can be expressed as a membrane bound
protein (mTNF) or cleaved and released as a soluble protein (sTNF). Following
infection, TNF is expressed by a range of cells, including epithelial cells,
natural killer (NK) cells, macrophages, dendritic cells (DCs), CD4<sup>+</sup>
and CD8<sup>+</sup> T cells. TNF binds to two receptors, ubiquitously expressed
TNFR1, and TNFR2, which is more restricted to haematopoetic tissues and is
upregulated on activated CD8<sup>+</sup> T cells. TNFR1 has a death domain to
drive apoptosis and it also triggers NFκB driven inflammatory pathways. TNFR2
does not have a death domain and only weakly stimulates NFκB, but coordinated
signaling of TNF through TNFR1 and TNFR2 has been shown to have cytotoxic effect
on activated CD8<sup>+</sup> T cells, suggesting that TNF:TNFR2 signaling plays
an immunoregulatory role. It has been shown that global TNF/TNFR2 signaling
inhibits the secondary CD8<sup>+</sup> T cell response to influenza in the
lungs. Studies investigating the role of TNF in anti-influenza immune responses,
viral clearance and immunopathology have indicated that TNF is not required for
viral clearance in the lungs, but is essential in controlling lung damage.
Others reported that sTNF is responsible for limiting the extent of lung injury
and this interaction was mediated via TNFR1. Moreover, the latter study
demonstrated that TNF expression is required early during infection to regulate
the magnitude of CD8<sup>+</sup> T cell responses. However, studies with TNF
knockout (*Tnf-/-*) mice are limited as this genotype causes defects in the
follicular DC network as well as in B cell follicle and germinal centre
formation. Consequently, *Tnf-/-* mice have a profound defect in their immune
architecture and cellular composition. Therefore, studies using global *Tnf-/-*
mice do not allow us to investigate the role of intrinsic TNF produced by
CD8<sup>+</sup> T cells and its role in the infection. Recently, Wortzman *et
al*. used an adoptive transfer model of TNF- or TNFR2-deficient transgenic
CD8<sup>+</sup> T cells, and demonstrated that TNF produced intrinsically by
CD8<sup>+</sup> T cells enhanced effector functions and regulated contraction of
those cells via TNFR2 signaling.
In our study, we show that the rapid and robust production of TNF after *in
vitro* stimulation is dependent on co-stimulation and is associated with changes
in histone post-translational modification (PTM) deposition at the *Tnf* gene
locus. We also demonstrate that, following intranasal infection with influenza A
virus (IAV), global TNF deficiency increased the magnitude of IAV-specific
CD8<sup>+</sup> T cell responses, as measured in the periphery, but did not
significantly affect the recruitment of IAV-specific CD8<sup>+</sup> T cells to
the lungs. Moreover, this TNF-mediated attenuation of the IAV-specific
CD8<sup>+</sup> T cell response was found to be largely dependent on extrinsic
TNF production, with only a moderate contribution by intrinsic CD8<sup>+</sup> T
cell-derived TNF. These data clearly indicate an immunoregulatory role for TNF
during IAV infection, which occurs at the global, rather than local, level and
is mediated predominantly by extrinsic TNF production.
# Materials and methods
## Mice
Female 6–12 week old C57BL/6J (WT), ovalbumin transgenic-I (OT-1), *Tnf -/-* and
*Tnf -/-/*OT-I mice were bred and housed in specific pathogen-free conditions at
the Biomedical Research Facility, Department of Microbiology and Immunology, The
University of Melbourne (Parkville, VIC, Aust.). *Tnf -/-* mice were obtained
from the Heath Laboratory (University of Melbourne) with permission from the
Centenary Institute (Sydney, Australia). Within experiments, mice were age-
matched to within 1 week. Mice were killed by CO<sub>2</sub> asphyxiation using
a slow fill rate of 20% per volume per minute. All animal experimentation was
conducted following the Australian National Health and Medical Research Council
Code of Practice for the Care and Use of Animals for Scientific Purposes
guidelines for housing and care of laboratory animals and performed in
accordance with Institutional regulations after pertinent review and approval by
the University of Melbourne Animal Ethics Committee.
## In vitro stimulation and intracellular cytokine staining
Sorted CD8<sup>+</sup>CD44<sup>lo</sup> T cells stimulated at 2 x 10<sup>4</sup>
cells per well in mAb-coated 24-well plates for up to 72 hours (h) were
incubated in the presence of 10 U/mL recombinant human IL-2 (Roche Diagnostics,
Mannheim, Germany). The mAb were coated onto Nunc plates overnight in PBS at the
following concentrations: anti-CD3ε (clone 145-2C11) at 10 μg/mL; anti-CD8α
(clone 53–6.7) at 10 μg/mL; anti-CD11a (clone I21/7.7) at 5 μg/mL; anti-CD28
(clone 37.51) at 10 μg/mL. To permit intracellular cytokine staining, GolgiPlug
at a 1:1000 dilution (BD Biosciences, San Diego, CA, USA) was added for the last
5 h of the incubation. After incubation, cells were surface-stained using anti-
CD8α-PE (BD Pharmingen; clone 53–6.7), fixed and permeabilised using
Cytofix/Cytoperm buffer and 1 × Perm/Wash buffer (BD Biosciences) according to
manufacturer’s instructions and stained with anti-IFNγ-FITC (BD Pharmingen;
clone XMG1.2) and anti-TNF-APC (BD Pharmingen; clone MP6- XT22). Cells were then
acquired using FACSCalibur flow cytometer (BD Biosciences) and data were
analyzed by using FlowJo software (Versions 9&10) (FlowJo LLC, Ashland, Oregon).
## Chromatin immunoprecipitation (ChIP) for histone modifications and RNA polymerase II
Sort purified lymphocytes from LNs and spleen (\~5 x 10<sup>6</sup> cells total)
were fixed in 1% formaldehyde, resuspended in ChIP lysis buffer (1% v/v SDS,
10mM EDTA, 50 mM Tris-HCl) and sonicated to generate 200–1000 base pair
fragments. Samples were then precleared using Protein A-agarose/salmon sperm DNA
(Millipore, 16–157), split into 5 and incubated overnight with either 5 ug
anti-H3K27me3, 3 ug anti-H3K4me3 or 4 ug anti-RNA polymerase II (all
Invitrogen). A no antibody control and a total input positive control were also
included. After washing, all samples (except the ‘total input’) were incubated
with Protein A-agarose/salmon sperm DNA with rotation for 1 hour followed by a
series of washes in low salt, high salt, lithium chloride, and TE buffers. DNA
was eluted before crosslink reversal with 0.2M NaCl at 66°C overnight, followed
by protein digestion with proteinase K (Promega). Immunoprecipitated DNA was
extracted by phenol: chloroform:isoamyl (25:24:1) extraction and resuspended in
HPLC water. For analysis, real-time PCR was used to measure the levels of ChIP-
DNA, such that resulting cycle threshold (Ct) values were converted to copy
number (#copies = 10<sup>5</sup>/2<sup>Ct-17</sup>) and samples were normalised
to their corresponding total inputs with background subtraction (no-antibody
control).
## Influenza A virus infection and determination of viral titer
Mice were anesthetized by isofluorane inhalation and infected intranasally with
1x10<sup>4</sup> PFU of the HKx31 (H3N2) IAV strain in 30 μL of PBS. Weights
were monitored daily and mice typically lost \<20% of original starting body
weight. Viral titres were determined at the indicated timepoints using a plaque
assay on monolayers of Madin-Darby Canine Kidney (MDCK) cells. Briefly, lungs
were homogenised in 2 mL of incomplete RPMI, the cell suspension was centrifuged
and a titration of supernatant was applied to the MDCK monolayers, overlaid,
incubated for up to 3 days before plaques were counted.
## T cell adoptive transfer
CD8<sup>+</sup>CD44<sup>lo</sup> T cells (OT-I or *Tnf -/-/*OT-I) were sort
purified and 1x10<sup>4</sup> cells were transferred via intravenous injection
into WT or *Tnf -/-* recipient mice. Recipient mice were infected the following
day with IAV as described above and spleen and bronchoalveolar lavage (BAL) were
taken for tetramer analysis at day 10 after infection.
## Tetramer staining
Spleen or BAL from IAV-infected mice at day 10 were processed to single cell
suspensions, red blood cells were lysed and the remaining leukocytes stained. We
used PE-labeled MHCI tetramers (D<sup>b</sup>NP<sub>366</sub> or
K<sup>b</sup>PB1<sub>703</sub>) and APC-labeled MHCI tetramers
(D<sup>b</sup>PA<sub>224</sub> or D<sup>b</sup>PB1-F2<sub>62</sub>) (University
of Melbourne Tetramer Facility), stained with Fixable Live/Dead AquaBlue
viability dye (Life Technologies), blocked with anti- CD16/32 mAb (clone 2.4G2),
and stained with anti-CD3ε-PerCPCy5.5 (BD Pharmingen; clone 145-2C11), anti-
CD8α-PacBlue (BD Pharmingen; clone 53–6.7) and anti-CD4-AF700 (BD Pharmingen;
clone RMA4-5). Cells were acquired on a FACS Canto II flow cytometer (BD
Biosciences), and data were analyzed by using FlowJo software (Treestar).
## Statistical analyses
All experiments contained a minimum of 4 mice (4–10) and were repeated at least
twice, with similar results. Each symbol represents either data from an
individual sample or the mean of multiple samples. Error bars represent standard
error of the mean (SEM). Mann-Whitney test with Bonferroni correction for
multiple comparisons was used to compare multiple samples and statistically
significant differences between groups are indicated as follows: ns = not
significant (p\>0.05), \*p ≤ 0.05, or \*\*p ≤ 0.01.
# Results
## TNF expression kinetics correlate with epigenetic regulation upon TCR stimulation
We first assessed the kinetics of TNF expression following TCR engagement with
various levels of co-stimulation, in a population CD8<sup>+</sup> T cells
isolated from pooled lymph nodes of naïve WT mice. Cells were stimulated with
anti-CD3 monoclonal antibody (mAb) in combination with co-stimulation from anti-
CD28 mAb alone, anti-CD8/CD11a mAbs or anti-CD8/CD11a/CD28 mAbs. At 0, 5, 24, 48
and 72 h after stimulation, cells were harvested and the percentage of
CD8<sup>+</sup> T cells that produced TNF or IFNγ was assessed by flow
cytometry. Total cell counts for a distinct but representative experiment were
also determined. There was extremely rapid production of TNF by naïve
CD8<sup>+</sup> T cells after stimulation, with TNF detectable under all
stimulation conditions within 5 h. In the presence of anti-CD8/CD11a/CD28 mAb,
TNF production appeared biphasic, with an immediate peak at 5 h, followed by a
drop at 24 h and then a steady sustained increase that continued out to at least
72 h. The immediate production of TNF appeared dependent on CD8/CD11a co-
stimulation, since the only condition that didn’t induce robust immediate TNF
production lacked anti-CD11a, while the later sustained production correlated
with CD28 co-stimulation, since the condition lacking anti-CD28 costimulation
showed relatively poor TNF production at the later timepoints. In contrast,
IFNγ<sup>+</sup> cells, although detectable at 5 h, steadily increased in
proportion over the course of the assay under all conditions.
The extremely rapid and robust production of TNF after CD8<sup>+</sup> T cell
stimulation suggested that naïve CD8<sup>+</sup> T cells are poised for TNF
production, as noted previously and supported by the observation that naïve
CD8<sup>+</sup> T cells contain substantial levels of pre-existing TNF mRNA. To
determine whether differential kinetics of TNF and IFNγ expression were
associated with distinct epigenetic signatures, we stimulated sort purified
naïve CD8<sup>+</sup> T cells as before and performed chromatin
immunoprecipitation (ChIP) for histone modifications followed by quantitative
PCR targeting the proximal promoter regions (PPRs) of *Ifng* and *Tnf* loci. We
targeted histone 3 lysine 4 tri-methyl (H3K4me3) and H3K27me3 as these are
associated with permissive or repressive chromatin structure and thereby
transcriptional activity or inactivity, respectively. The patterns of histone
modification largely correlated with the frequency of cytokine-producing cells,
with rapid deposition of the activating H3K4me3 mark at the *Tnf* PPR at 5 h
post-stimulation, corresponding to the peak frequency of TNF<sup>+</sup>
CD8<sup>+</sup> T cells, and a subsequent removal (at 48 h) of the repressive
H3K27me3 mark at this site. In contrast, the *Ifng* locus retained enrichment of
the repressive H3K27me3 mark at 5 h but showed an increase in the relative
enrichment of H3K4me3 at 48 h, corresponding with a substantial increase in the
frequency of IFNγ<sup>+</sup> cells at this time point. Binding of RNA
polymerase II (pol II) is a further indication of transcriptional potential.
Prior to stimulation the PPRs of *Ifng* and *Tnf* loci appeared poised for
transcription, with both showing enrichment of pol II. Upon stimulation pol II
binding was only retained at the *Tnf* PPR at 5 h, and was reduced but still
present at 48 h. In contrast, pol II was lost from the *Ifng* PPR at 5 h, only
becoming detectable again at 48 h, broadly corresponding to the relative
kinetics of production of the two cytokines.
Thus, these data suggest that the differential kinetics of TNF and IFNγ
production by CD8<sup>+</sup> T cells after stimulation is, at least in part,
controlled by the accessibility of local chromatin structure determined by
deposition of key histone modifications at the PPR.
## TNF attenuates virus-specific CD8<sup>+</sup> T cell responses
To investigate the role of global TNF on antiviral CD8<sup>+</sup> T cell
responses, we intranasally infected WT and TNF knockout mice (*Tnf*-/-) with the
HKx31 (H3N2) strain of IAV. Global loss of TNF in influenza-infected mice led to
an increased level of protein in cell-free bronchoalveolar lavage fluid (BAL) on
d7 post-infection, typically used as an indication of loss of alveolar barrier
integrity and accumulation of protein-rich fluid in the alveolar space but may
also reflect increased levels of inflammatory mediators. Despite differences in
local lung injury, there was no difference in overall disease severity, as
indicated by weight loss, or in viral clearance between WT and *Tnf*-/- mice
during primary influenza infection. We then analysed the endogenous IAV-specific
CD8<sup>+</sup> T cell response in the spleens and BAL of WT and *Tnf*-/- mice
at day 10 (d 10) post-infection, which represents the peak of the acute
response. To measure CD8<sup>+</sup> T cell response magnitude, we used MHC
class I tetramers, D<sup>b</sup>NP<sub>366</sub>, D<sup>b</sup>PA<sub>224</sub>,
K<sup>b</sup>PB1<sub>703</sub> and D<sup>b</sup>PB1-F2<sub>62</sub>, to identify
four influenza-specific populations. This analysis revealed significantly higher
proportions and a consistent trend toward increased absolute numbers of IAV-
specific CD8<sup>+</sup> T cells in the spleen following infection of *Tnf*-/-
mice. However, we found no noticeable difference in the proportions or absolute
numbers of IAV-specific CD8<sup>+</sup> T cells in the BAL of infected mice at d
10. Total splenic or BAL-derived CD8<sup>+</sup> T cell numbers were not
different between WT and *Tnf-/-* mice. This suggests that TNF-mediated
attenuation of splenic CD8<sup>+</sup> T cell responses does not impact the
efficiency of IAV-specific CD8<sup>+</sup> T cell recruitment into the lung.
## Both intrinsic and extrinsic TNF can attenuate IAV-specific CD8<sup>+</sup> T cell responses
A global absence of TNF affects lymphoid secondary structure and has a profound
effect on a range of cell types and their function, especially given that the
predominant sources of TNF are macrophages and dendritic cells. Thus, to
understand the relative impact of intrinsic versus extrinsic TNF on the
antiviral CD8<sup>+</sup> T cell response, we used an adoptive transfer model in
which WT or *Tnf*-/- TCR transgenic CD8<sup>+</sup> T cells expressing a
K<sup>b</sup>-restricted ovalbumin (OVA<sub>257-264</sub>)-specific TCR (OT-I)
were transferred into either WT or *Tnf*-/- mice, followed by IAV infection
(gating strategy included as). As controls, we included intact WT and *Tnf*-/-
mice that did not receive a transfer (No Tx) and we included WT OT-I→WT and
*Tnf*-/- OT-I→*Tnf*-/- transfers to control for the effect of cell transfer.
Both of these sets of controls were able to recapitulate our observations from,
with higher proportions and numbers of OVA-specific CD8<sup>+</sup> T cells in
the global absence of TNF in the spleen at d10 post-infection. In WT
OT-I→*Tnf*-/- transfers, only responding CD8<sup>+</sup> T cells can produce TNF
and the OTI CD8<sup>+</sup> T cell response was significantly higher than that
observed when intrinsic and extrinsic TNF were present in WT OTI→WT transfers.
This suggests that CD8<sup>+</sup> T cell-derived TNF plays a modest role in
attenuating the IAV-specific response. In *Tnf*-/- OT-I→WT transfers,
CD8<sup>+</sup> T cells selectively lack the capacity to produce TNF but the
CD8<sup>+</sup> T cell response magnitude was comparable to that observed in the
complete absence of TNF in the *Tnf*-/- OT-I→*Tnf*-/- transfers. Finally, the
loss of global, extrinsic or intrinsic TNF had no impact of the proportion of
CD8<sup>+</sup> T cells that were OVA-specific in the BAL. Again, this finding
is consistent with and further reinforces that CD8<sup>+</sup> T cell- or
extrinsically-derived TNF has no impact on local antigen-specific
CD8<sup>+</sup> T cell recruitment. Collectively, these data indicate that
extrinsically-derived TNF has the dominant impact on antiviral CD8<sup>+</sup> T
cell responses by attenuating the magnitude in the secondary lymphoid organs,
but CD8<sup>+</sup> T cell-derived TNF can mediate a modest attenuation.
# Discussion
TNF is one of the major mediators of inflammation and its role during influenza
infections has been described in several studies. While our work and previous
studies in *Tnf-/-* mice demonstrate that TNF is not needed for efficient
clearance of virus from the lungs as it is not required for the antiviral
activity of CD8<sup>+</sup> T cells, TNF is important for other aspects of
antiviral immunity as it displays functional duality, being both pro-
inflammatory and immunoregulatory. With regard to its proinflammatory role,
TNF has been linked to increased morbidity upon influenza infection, possibly
through exacerbation of lung inflammation by promoting recruitment of immune
cells. The source of this TNF may be stromal cells, macrophages or dendritic
cells but TNF derived from virus-specific CD8<sup>+</sup> T cells has also been
shown to contribute to lung damage. With regard to its immunoregulatory role,
TNF may simultaneously limit lung immunopathology by attenuating CD8<sup>+</sup>
T cell responses. In the global absence of TNF, infected mice displayed
numerically higher and prolonged IAV-specific CD8<sup>+</sup> T cell responses
in the lungs, and this accumulation was specifically linked to the action of
sTNF. In addition, it has been shown that TNF produced by activated
CD8<sup>+</sup> T cells can act in an autocrine fashion via TNFR2, to regulate
the peak magnitude of the CD8<sup>+</sup> T cell response and promote
contraction. The latter study used the adoptive transfer model of OT-I
transgenic cells thus eliminating all other factors that can affect the immune
response in global *Tnf -/-* mice.
In this study, we showed, in accordance with previous studies, that TNF is
produced by CD8<sup>+</sup> T cells in two waves; namely an immediate burst of
TNF that peaks at \~5 h, followed by a more rapid accumulation of TNF production
that continues to increase out to 72 h. Early TNF upregulation is consistent
with an observation that TNF expression is necessary during priming of T cells
in order to attenuate the subsequent effector response. Moreover, early
inflammatory events have been shown to control contraction of CD8<sup>+</sup> T
cell responses and TNF has been implicated as a critical factor, sensitizing
activated T cells for subsequent apoptosis. Intriguingly, the two waves of TNF
production were associated with different patterns of histone modifications,
with early TNF production associated with rapid enrichment of the permissive
H3K4me3 mark, and later TNF production associated with a loss of the repressive
H3K27me3 mark. This is in line with a previous global analysis of epigenetic
control of CD8<sup>+</sup> T cell function. In that study, the gain of H3K4me3
and the loss of H3K27me3 represented two distinct epigenetic mechanisms
associated with transcriptional activation, with gain of H3K4me3 correlating
with more rapid gene transcription.
Apart from rapid epigenetic changes, we have also previously demonstrated that
naïve OTI CD8<sup>+</sup> T cells contain substantial levels of TNF, but not
IFNγ, mRNA transcript, which is also likely to contribute to the very early (5h)
production of TNF by CD8<sup>+</sup> T cells after activation. It is difficult
to compare these very early epigenetic changes and cytokine production
characteristics immediately after *in vitro* stimulation with the events that
occur *in vivo*. This is largely because *in vivo* cell division is initiated in
very rare, antigen-specific T cell population 2–3 days after infection, which
means cells cannot be readily detected *in vivo* until well beyond the
timepoints analysed here. However, we have previously shown that OTI cells
either activated *in vivo* by infection with HKx31-OVA or *in vitro* by a 5h
peptide stimulation, produce TNF protein prior to IFNγ, which supports the
current findings. Characterization of H3K4me3 and H3K27me3 deposition on the
*Tnf* and *Ifng* loci in naïve, effector (10 d) and memory (\> 70 d) OTI cells
has also been assessed. Although these *in vivo* activated populations are not
directly comparable to our analyses immediately after stimulation, naïve OTI
cells exhibited elevated H3K27me3 deposition at the *Ifng* compared to *Tnf*
locus, as well as detectable levels of H3K4me3 only at the *Tnf*, but not the
*Ifng*, locus, consistent with our data and with rapid TNF production after
stimulation.
Our analysis of the impact of global loss of TNF on IAV-specific CD8<sup>+</sup>
T cell responses was consistent with previous studies in several respects.
Firstly, we found that TNF was not required for efficient clearance of virus
from the lung, but did reduce lung injury following intranasal infection.
Additionally, we found that the absence of TNF resulted in an increased acute
IAV-specific CD8<sup>+</sup> T cell response. Collectively, these data support
the idea that TNF plays an immunoregulatory role following IAV infection at a
number of points: early after infection (by d 7) to mitigate lung damage, later
(d10) to attenuate peak CD8<sup>+</sup> T cell response magnitude, and
thereafter to drive the contraction of the IAV-specific CD8<sup>+</sup> T cell
response.
Our study also contrasted with previous studies that had demonstrated that the
loss of TNF increased the severity of influenza infection, as assessed by weight
loss, and increased CD8<sup>+</sup> T cell response magnitude in the lung. We
found that an absence of TNF had no impact on disease severity and caused an
increase in the proportion and number of IAV-specific CD8<sup>+</sup> T cells in
the spleen, but not in the lung. This suggests that TNF modulates the overall
magnitude of the CD8<sup>+</sup> T cell response but does not affect the
recruitment of CD8<sup>+</sup> T cells to the site of infection. This second
discrepancy may be explained by the fact that previous studies investigated the
role of TNF in response to relatively virulent strains of IAV, associated with
high levels of virus replication and pulmonary inflammation. The HKx31 strain,
in comparison, is known to induce a relatively mild infection when delivered
intranasally. The requirement for TNF by the CD8<sup>+</sup> T cell response has
previously been shown to differ depending on the inflammatory context, with T
cell responses to weak tumor antigens showing greater TNF dependence than a
strong anti-LCMV response.
Our dissection of the relative impact of CD8<sup>+</sup> T cell extrinsic versus
intrinsic TNF during IAV infection demonstrates that CD8<sup>+</sup> T cell-
derived TNF mediates a modest attenuation of CD8<sup>+</sup> T cell responses.
This is supported by a previous study which concluded that intrinsic TNF
potentiated the contraction of the CD8<sup>+</sup> T cell response after viral
clearance. Our study extends these observations by demonstrating that
CD8<sup>+</sup> T cell-derived TNF is not sufficient for full contraction of
virus-specific CD8<sup>+</sup> T cells and that extrinsically-derived TNF was
largely responsible for this attenuation in the periphery. Thus, although
intrinsically-derived TNF does not appear to play a critical role in this
context of mild IAV infection, the fact that its role varies in a context-
dependent manner means that an understanding of the kinetics and epigenetic
control of TNF expression in CD8<sup>+</sup> T cells is essential.
In conclusion, TNF is essential for attenuating the peak CD8<sup>+</sup> T cell
response following acute IAV infection, with extrinsically-derived TNF having
the greatest impact on response magnitude and quality. This study also found
that the effects of TNF were most apparent in the global peripheral response but
had little effect on recruitment of CD8<sup>+</sup> T cells to the site of
infection.
# Supporting information
This work was supported by Australian National Health and Medical Research
Council (NHMRC) funding (APP1071916). and a Sylvia and Charles Viertel Senior
Medical Research Fellowship awarded to N.L.L.G.
[^1]: The authors have declared that no competing interests exist.
[^2]: Current address: The Walter and Eliza Hall Institute, Parkville,
Victoria, Australia
[^3]: Current address: Broad Institute of MIT and Harvard, Cambridge, MA,
United States of America
[^4]: Current address: Babraham Institute, Babraham Research Campus,
Cambridge, United Kingdom |
# Introduction
Breast cancer is the leading cause of cancer in women with an estimated
1,383,500 new cases and 458,400 deaths worldwide. Despite improvements in
treatment strategies recurrence rates are still high among breast cancer
patients. This may be attributed to heterogeneous nature of breast cancers
representing varied morphologic and biological features, behavior, and response
to therapy. Even among breast tumors of similar histologic type and grade,
prognosis varies. The clinical decisions for management of breast cancer
patients rely on the availability of robust well validated clinical and
pathologic prognostic factors to support treatment related decision making.
Routine physical examinations along with imaging, histopathological analysis and
clinical parameters (tumor size, lymph node status, stage and grade) largely
impact the management of breast cancer patients. Currently, breast cancer
prognosis assessment methods have limited accuracy, are expensive, and in 20–30%
of cases lead to over-treatment with adverse effects. None of the currently
known prognostic factors has the ability to predict accurately which breast
cancer patients are at high risk of recurrence. Thus, there is an increasing
need for identification and validation of prognostic markers for assessment of
risk for disease recurrence in breast cancer patients.
Tumors are characterized by alterations in the epithelial and stromal
components, which both contribute to disease progression. Recent reports
demonstrate synergy between stromal and epithelial interactions, even at the
initial stages of breast carcinogenesis, appears necessary for the acquisition
of malignancy and provides novel insights into where, when, and how the tumor
stroma develops, allowing development of new molecular markers and therapeutic
targets. It is now well recognized that stromal cells within and surrounding
pathologic lesions also actively contribute to malignant phenotypes through
elevated expression of cytokines and growth factors. They exert their effects
through increased deposition and remodelling of the extracellular matrix (ECM).
The clinical impact of changes in ECM on tumor aggressiveness and disease
outcome needs in depth investigation.
Transglutaminase 2 (TG2), a member of multifunctional enzyme family, modifies
glutamine residues by cross-linking proteins, demonstrates protein disulphide
isomerase and kinase activities, mediates transmembrane signal transduction and
interacts with cell surface and extracellular matrix proteins. TG2
overexpression has been reported in cytoplasm, nucleus, membrane or ECM in tumor
cells. Increased expression of cytoplasmic TG2 is associated with increased cell
survival, anchorage-independent growth, loss of cell polarity, increased
invasion and resistance to chemotherapy in mammary epithelial cells. TG2
promotes tumor progression by initiating a comprehensive program of de-
differentiation by inducing epithelial mesenchymal transition (EMT) and cancer
stem cell like phenotype,. The resulting tumors remain dependent on
TG2-regulated pathways for their growth and survival. Increased TG2 induces
expression of transcription repressors including Snail1, Twist, Zeb1, and Zeb2,
the key regulators in development of EMT phenotype in cancers,. TG2
overexpression results in constitutive activation of NFKB, the inflammatory
transcription factor known to regulate various genes involved in cancer
initiation and progression. Nuclear TG2 in association with pRb, p53 and
histones regulates cellular functions. Cell surface TG2 in association with
β-integrins serves as a co-receptor for integrin-mediated binding to fibronectin
(Fn), thereby regulating cellular adhesion, spreading, motility and survival.
Extra-cellular matrix TG2 regulates cell–matrix interactions. TG2 serves as a
signalling molecule transmitting signals from outside the cell through Alpha1B
adrenergic receptors to a downstream cytoplasmic target, phospholipase C,
through hydrolysis of GTP. These findings suggest differential localization of
TG2 in cancer cells impacts tumor development, growth, survival or invasion by
different cellular mechanisms.
Till date, most investigations on determining clinical relevance of TG2
overexpression in epithelial malignancies including breast cancer are limited to
its expression in cytoplasm of tumor cells. However, studies demonstrating an
association of TG2 overexpression in ECM with disease recurrence
(locoregional/metastasis) are lacking. In this study, we focussed on evaluating
the prognostic significance of TG2 overexpression in ECM in breast cancer
patients. Further, to evaluate the crosslinking i.e. transamidating activity of
TG2 (stroma/cytoplasm), we determined the expression of N-epsilon gamma-glutamyl
lysine amino residues (to detect any potential TG2-mediated protein crosslinking
events) in the same cohort of the breast cancers using immunohistochemistry. In
addition, we stained representative tissue sections (where TG2 is expressed in
the stroma) with anti-phospho-FAK or anti-phospho-ERK antibodies to evaluate the
effect of stromal TG2 on activation of integrin dependent downstream signaling
in breast cancer tissues.
# Materials and Methods
## Patients, Clinicopathological Data Collection and Tumor Specimens
The study was approved by Mount Sinai Hospital Research Ethics Board, Toronto,
Canada. Written informed consent was obtained for the acquisition and use of
patient tissue samples and anonymized clinical data. The breast cancer database
maintained in the Department of Pathology and Laboratory Medicine (PLM), Mount
Sinai Hospital (MSH), Toronto, Canada was reviewed for the last 12 years to
select breast cancer cases wherein complete clinical, pathological and follow up
data were available. Tissue specimens were retrieved from the archived blocks of
253 breast cancer patients (mean age: 59 years; range: 29 to 89 years)
undergoing curative cancer surgery during the period 2000–2002. Comprehensive
clinicopathologic data were available in digital databases for each of these
cases including demography, clinical tumor staging (American Joint Committee on
Cancer staging guidelines), surgical; histological grade; recurrence including
local, regional, locoregional or distant; treatment, subsequent management and
disease status at last clinical review. The hematoxylin and eosin (H & E)
stained slides of these cases were reviewed and tumor tissues confirmed by the
pathologist (MC). These 253 breast cancer cases were classified as ductal
carcinoma in situ (DCIS, n = 60), invasive ductal carcinomas (IDC, n = 168),
invasive lobular carcinoma (ILC, n = 16) and invasive mucinous carcinoma (IMC,
n = 9). In addition, archived blocks of normal breast tissues (n = 40) obtained
from patients undergoing breast reduction surgery were retrieved from MSH tissue
bank.
## Treatment and Follow-up
Breast cancer patients (n = 253) were treated with a primary surgery i.e. either
breast conserving surgery (BCT), or a mastectomy, as per the hospital protocol.
Breast cancer patients who were ER<sup>+</sup>/PR<sup>+</sup> were given
hormonal treatment. Pre-menopausal women were given tamoxifen as their primary
treatment option. Post-menopausal patients were given an option of using
tamoxifen followed by aromatase inhibitors, which included anastrozole,
letrozole, and exmestane. Patients were given tamoxifen for 5 years and then an
aromatase inhibitor for 5 years for risk reduction. Patients who received BCT
were treated with radiation therapy (RT). Radiation therapy was given from 40 Gy
to 50 Gy in fractions of 1.8 to 2.0 Gy. Patients receiving adjuvant chemotherapy
(CT) were defined as patients, who were ER<sup>−</sup>/PR<sup>−</sup> with a
tumor size of \<0.5 cm, patients who were node negative with a tumor size \>2
cm, and patients who had a positive nodal status. These patients were given CT
regimens regardless of histology grade, and tumor size. Patients with rapidly
progressive disease or visceral crisis received combination chemotherapy (CT)
including AC (doxorubicin, cyclophosphamide)/CEF (cyclophosphamide, epirubicin,
5-flourouracil)/CMF (cyclophosphamide, methotrexate, 5-fluorouracil)/FAC
(5-fluorouracil, doxorubicin, cyclophosphamide). Patients with metastatic
disease were treated with single agents (doxorubicin, docetaxel or paclitaxel).
Follow-up data were available for all 253 breast cancer patients. Survival
status, loco-regional relapse or distant metastasis of the breast cancer
patients was verified and updated from the records of the Tumor Registry, Mount
Sinai Hospital (MSH), Toronto, Canada as of August, 2012. Breast cancer patients
were monitored for a maximum period of 143 months (range: 4–143 months; mean
83.9 months and median 93 months). The patients were reassessed on a regular
basis and the time to recurrence was recorded. If a patient died, the medical
history, clinical examination, and radiological evaluation were used to
determine whether the death had resulted from recurrent cancer (relapsing
patients) or from an unrelated cause. Disease-free survivors were defined as
patients free from clinical and radiological evidence of local, regional, or
distant relapse at the time of the last follow-up. Disease-free survival (DFS)
was evaluated in the present study for statistical analysis. Disease-free
survival was expressed as the number of months from the date of surgery to loco-
regional relapse or till date distant metastasis was diagnosed.
## Immunohistochemistry (IHC)
Serial paraffin embedded tissue sections (4 µm thickness) were deparaffinized in
xylene, hydrated through graded alcohol series, pre-treated in a microwave oven
for 15 min in Tris-EDTA (0.1 M, pH = 9.0) containing Tween 20 (0.05% v/v) for
antigen retrieval. Slides were washed with Tris-buffered saline (TBS, 0.1 M,
pH = 7.2) containing Triton X-100 (0.1%) followed by treatment with 0.3%
H<sub>2</sub>O<sub>2</sub> at room temperature for 10 minutes to block the
endogenous peroxidase activity. Thereafter, sections were incubated with normal
horse serum (10%) prepared in 5% bovine serum albumin (BSA) to preclude any non-
specific binding. The sections were incubated with either TG2 antibody (mouse
mAb cat \# MS-300-PABX, 1∶4,000 dilution, Lab Vision Corporation, Fremont,
CA)/N-epsilon gamma-glutamyl lysine amino residues antibody (mouse mAb cat \#
ab424, Abcam, Cambridge) for 60 minutes/anti-FAK (phospho Y397) antibody (rabbit
pAb cat \# ab4803, Abcam, Cambridge)/anti-ERK1+ERK2 (phospho T202+ T185+ Y187)
antibody (rabbit mAb cat \# ab32538, Abcam, Cambridge). Slides were washed with
Tris-buffered saline (TBS, 0.1 M, pH = 7.2) containing Triton X-100 (0.1%)
followed by incubation with biotinylated secondary antibodies for 20 minutes.
The sections were finally incubated with VECTASTAIN Elite ABC Reagent (Vector
labs, Burlingame, CA) and diaminobenzidine was used as the chromogen. All
procedures were carried out at room temperature unless otherwise specified.
Slides were washed with Tris-buffered saline (TBS, 0.1 M, pH = 7.4), 3–5 times
after every step. Finally, the sections were counterstained with Mayer’s
hematoxylin and mounted with D.P.X mountant. In negative control tissue
sections, the primary antibody was replaced by isotype-specific non-immune mouse
IgG. The sections were evaluated by light microscopic examination.
## Evaluation of Immunohistochemical Staining
IHC scoring was performed under supervision of the pathologist (MC).
Immunopositive staining was evaluated in five pathological areas of the tissue
sections as described earlier. Immunostaining for all the proteins in this study
was evaluated independently in tumor cell cytoplasm, nucleus and stroma by the
intensity and percentage of positive staining. Sections were scored as positive
if TG2/N-epsilon gamma-glutamyl lysine amino residues/anti-FAK (phospho
Y397)/anti-ERK1+ERK2 (phospho T202+ T185+ Y187) immunostaining was observed in
the tumor cell cytoplasm or in the stroma when observed by two evaluators (JA &
GS) who were blinded to the clinical outcome. These sections were scored as
follows: 0, \<10% cells; 1, 10–30% cells; 2, 31–50% cells; 3, 51–70% cells; and
4, \>71% cells showed immunoreactivity. Sections were also scored semi-
quantitatively on the basis of intensity as follows: 0, none; 1, mild; 2,
moderate; and 3, intense. Finally, a total score (ranging from 0 to 7) was
obtained by adding the scores of percentage positivity and intensity for each of
the breast cancer tissue sections. This integrated scoring has proven to work
well in our previous investigations.
## Statistical Analysis
The IHC data was subjected to statistical analysis using SPSS 20.0 software
(SPSS, Chicago, IL) and GraphPad Prism 5.0 software (GraphPad Software, La
Jolla, CA). Scatter plots were used to determine the distribution of total score
of cytoplasmic or stromal TG2 expression in all tissues examined. The p-value
\<0.05 was considered significant for statistical analysis. The cut-off of IHC
score ≥3.0 for cytoplasmic/stroma TG2 immunostaining was considered as
overexpression for further analysis. For N-epsilon gamma-glutamyl lysine amino
residues immunostaining, the cut-off of IHC score ≥2.0 for cytoplasmic/stroma
was considered as overexpression for further analysis. Expression data thus
generated was analyzed to determine significant correlations between TG2
overexpression, clinical parameters and prognosis of breast cancer patients. The
correlation of TG2 expression with patient survival (i.e. disease free survival)
was evaluated using life tables constructed from survival data with Kaplan-Meier
plots as described earlier. Multivariate analysis was carried out using Cox
regression models to determine the performance of TG2 overexpression as a marker
in comparison to other clinical and pathological prognostic parameters including
age, histological grade, tumor size, stage, grade and nodal status of breast
cancer patients.
# Results
## Immunohistochemical Analysis of TG2 Expression in Breast Cancer
To determine the clinical significance of TG2 overexpression in
cytoplasm/stroma, immunohistochemistry was performed in breast normal (n = 40)
and cancer tissues (n = 253). Scatter plot analysis shown in depicts the
distribution of IHC scores for TG2 immunostaining in breast normal and cancer
tissues. Of the 40 breast normal tissues, 14 cases (35%) showed weak to moderate
immunostaining for TG2 in cytoplasm of epithelial cells. However, no TG2
immunostaining was observed in stroma of the breast normal tissues used in this
study. Immunohistochemical analysis of 253 breast cancers revealed 199 cases
(78.6%) showing strong TG2 immunostaining either in cytoplasm (33.6%) or stroma
(45.0%). Among DCIS, 22 of 60 (36.7%) showed cytoplasmic TG2, while majority of
the cases (50/60; 83.3%) showed no detectable TG2 expression in stroma. Fifty
four of 168 (32.1%) IDCs showed cytoplasmic TG2, while 97 cases (57.7%) showed
TG2 expression in stroma. Of 16 invasive lobular carcinomas, 6 (37.5%) showed
cytoplasmic TG2, while only 4 cases (25.0%) showed TG2 overexpression in stroma
(,). Among IMCs analyzed in this study, 3 of 9 (33.3%) showed cytoplasmic TG2
while only 3 cases (33.3%) showed TG2 expression in stroma. Negative control
sections, wherein primary antibody was replaced by isotype IgG, no
immunostaining was observed in cytoplasm/stroma of breast cancer tissue sections
(data not shown).
Box plot analysis revealed significant increase in stromal TG2 with advancing
stage (p = 0.020), tumor size (p\<0.001), lymph node metastasis (p\<0.001) and
recurrence (loco-regional recurrence/distant metastasis) (p\<0.001) (
respectively;).
## Potential of TG2 as a Marker for Breast Cancer Recurrence and Distant Metastasis
Follow up data of 253 breast cancer patients for up to 12 years was used to
assess the prognostic relevance of TG2 for predicting Disease free survival
(DFS) including both loco-regional recurrence and distant metastasis. Recurrence
(loco-regional/distant metastasis) was observed in 57 breast cancer patients
including DCIS (n = 15), IDC (n = 37) and (ILC = 5) over a time period of 4–143
months. Kaplan-Meier survival analysis showed significantly reduced DFS of
breast cancer patients showing TG2 accumulation in stroma (mean DFS = 112
months, p = 0.002) in comparison with patients showing lower expression (mean
DFS = 127 months). Among the clinicopathological parameters, T-stage (p\<0.001),
nodal status (p = 0.001), and histology grade (p = 0.002) correlated with
reduced DFS in breast cancers. Among IDC patients, increased TG2 expression in
tumor stroma correlated significantly with reduced DFS (mean DFS = 110 months)
in comparison with patients showing lower stromal TG2 (mean DFS = 130 months,
p\<0.001). All the 5 ILCs showing reduced DFS showed increased TG2 accumulation
in tumor stroma. However, Kaplan Meier analysis could not be performed for ILC
due to the small sample size. No significant association of cytoplasmic TG2
overexpression with recurrence was observed in all breast cancers analyzed as
well as in DCIS and IDCs.
In multivariate Cox regression analysis using TG2 overexpression (cytoplasm or
stroma), age, ER/PR status, tumor stage, grade and nodal status as variables in
the model, TG2 accumulation in tumor stroma (p = 0.014, Hazard’s ratio,
H.R. = 2.7, 95% C.I. = 1.2–5.9), T-stage (p = 0.001) and grade (p = 0.034)
emerged as independent factors associated with poor prognosis of breast cancer
patients. In IDC, TG2 stromal accumulation was associated with poor prognosis in
a Cox multivariate analysis (p = 0.006, H.R. = 3.79, 95% C.I. = 1.4–9.8) and
tumor stage (p\<0.001, HR = 5.26, 95% C.I. = 2.1–13.7).
## Evaluation of TG2 Transamidating Activity in Invasive Ductal Carcinomas (IDCs)
To evaluate the transamidating activity of TG2 overexpression in
stroma/cytoplasm of IDCs, we determined the expression of N-epsilon gamma-
glutamyl lysine amino residues in representative tissue sections of IDCs showing
either low or high scores of stromal TG2 immunostaining. Of the 40 IDCs of the
breast demonstrating high immunostaining scores of stromal TG2, 34 cases (85%)
showed stromal staining of N-epsilon gamma-glutamyl lysine amino residues.
Interestingly, all IDCs (n = 35) showing low stromal/cytoplasmic TG2 staining
also showed weak immunostaining for N-epsilon gamma-glutamyl lysine amino
residues in cytoplasm and stroma of IDCs. No immunostaining was observed either
in cytoplasm or stroma of breast cancer tissues used as negative controls. A
correlation (R = 0.671, p = 0.016) was observed for co-localization of TG2 and
N-epsilon gamma-glutamyl lysine amino residues immunostaining in stroma in
tissue sections of IDCs.
## Evaluation of Phospho-FAK and Phospho-ERK1+ ERK2 in IDCs Showing Overexpression of Stromal TG2
To determine the effect of TG2 on activation of integrin dependent downstream
cell signaling in IDCs showing overexpression of stromal TG2, we performed
immunohistochemical analysis of phospho-FAK (Y397) and phospho-ERK1+ ERK2
(phospho T202+ T185+ Y187) in representative tissue sections. Our results of
phospho-FAK (Y397) showed strong nuclear staining in all IDC sections analyzed,
whereas no immunostaining in nucleus/cytoplasm was observed in IDC tissue
sections used as negative controls. No significant difference in the nuclear
expression for phospho-FAK (Y397) was observed among these IDC sections.
Immunohistochemistry of phospho-ERK1+ERK2 (phospho T202+ T185+ Y187) showed no
detectable immunostaining in IDCs showing overexpression of stromal TG2.
However, thyroid cancer tissue sections used as a positive controls showed
strong nuclear expression of phospho-ERK1+ERK2 (phospho T202+ T185+ Y187).
Negative control tissue sections showed no immunostaining in cancer cells.
# Discussion
The development of metastasis poses a major clinical problem in treatment of
breast cancer patients leading to poor cancer free survival. Identification of
molecular markers which can predict recurrence is imperative for developing
effective therapies for more effective disease management. Our study provides
clinical evidence that TG2 expression is up-regulated in the primary tumor of
patients likely to develop distant metastasis. The majority of IDC patients
showed TG2 accumulation mainly in tumor stroma. Notably, significant association
of stromal TG2 in IDC was observed with clinicopathologic features including
increased tumor size, grade, stage and nodal metastasis which contribute to
aggressive phenotype in these patients. Further, our results clearly
demonstrated accumulation of TG2 in tumor stroma associated with loco-regional
recurrence, distant metastasis, and hence decreased DFS of IDC patients. Our
findings are supported by the report of Girgoriev *et al*., which showed stromal
TG2 expression in 50% of the human breast carcinomas, while 15% cases stained
positive for cytoplasmic TG2; however the correlation of TG2 with disease
outcome was not assessed in this study. Mehta *et al*., reported TG2 expression
was significantly higher in lymph nodes than in primary breast tumors, but their
study was limited by a small size of 30 cases only and no follow up data were
provided. Further in another independent study by same group, Mangala *et al*.,
compared stromal TG2 expression in 189 early stage breast cancer cases and
demonstrated significant association of higher stromal TG2 expression with
negative lymph nodes (p\<0.001). Although, authors reported median follow-up of
4 years for these patients with negative lymph nodes, statistical analysis
including multivariate Cox regression for evaluating prognosis in breast cancer
patients was not provided. Thus, our study assumes importance as the first
report demonstrating association of TG2 accumulation in tumor stroma with poor
disease outcome and its significant association with aggressive features such as
increasing tumor size, grade, stage, lymph node and distal metastasis in a large
cohort of breast cancers with special emphasis on invasive ductal carcinomas.
TG2 overexpression in metastatic breast cancer promotes apoptosis-resistance
phenotype, cell migration and invasion by initiating integrin-mediated cell
attachment and cell survival signalling pathways,. A review of TG2 expression in
other human cancers revealed overexpression of TG2 in pancreatic tumor cells
associated with nodal metastasis, lymphovascular invasion and poor overall
patient survival. Further, TG2 overexpression in ovarian cancer patients was
associated with poor overall survival. Increased expression of TG2 in ovarian
cancer cells enhanced their adhesion to fibronectin and promoted directional
cell migration, whereas knockdown of TG2 showed diminished tumor dissemination
on the peritoneal surface and in mesentery in an intraperitoneal ovarian
xenograft mouse model.
Extra-cellular TG2 along with β1 and β3 integrins serves as a co-receptor for
fibronectin. Interestingly, this integrin mediated interaction of TG2 and
fibronectin promotes adhesion, migration, and spreading of cells on fibronectin-
coated surfaces and is independent of the TG2 enzymatic activity. TG2 in ECM
associates with integrins inducing activation of anti-apoptotic protein Bcl-2,
focal adhesion kinase (FAK) dependent signal transduction pathways including
PI3K/Akt, and Ras/Erk, pathways which contribute to cancer aggressiveness.
Moreover, TG2 overexpression in ECM leads to increased accumulation of matrix
bound transforming growth factor beta 1(TGF-β1), both in vitro and in vivo. TG2
expression signals the onset of EMT in epithelial cells and contributes to their
increased survival and metastatic potential. The association of stromal TG2 with
lymph nodal metastasis in breast cancer patients in our study provides clinical
evidence in support of its utility as a marker of metastatic potential in these
patients. The mechanistic basis of aberrant stromal TG2 expression contribution
to EMT and metastatic capabilities of breast cancers warrants investigation in
future studies.
Our results also demonstrated overexpression of N-epsilon gamma-glutamyl lysine
amino residues in cytoplasm and stroma of IDCs demonstrating presence of active
TG2 in these breast cancers. Notably, most of these breast cancer cases had poor
prognosis indicating a plausible role of active TG2 in stroma in recurrence
among breast cancer patients. However, lack of significant difference of
phospho-FAK expression and absence of phospho-ERK in IDCs showing overexpression
of stromal TG2 suggests that stromal TG2 may not activate integrins. Taken
together our results suggest the crosslinking function of stromal TG2 might be
important in IDCs of breast.
# Conclusions
Our study clearly demonstrates the clinical significance of stromal TG2
overexpression in breast IDCs and may serve as an independent risk factor for
identifying patients with high risk of recurrence and metastasis. These patients
can be followed more closely and managed appropriately by selecting other
treatment modalities and thereby potentially reducing the morbidity due to
recurrence. Further, it may also help avoid overtreatment of patients at low
risk of disease recurrence reducing harmful side effects of therapy and reduce
the economic burden on health care providers as well.
# Supporting Information
[^1]: The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: RR PGW AM. Performed the
experiments: JA GS. Analyzed the data: JA GS AM. Contributed
reagents/materials/analysis tools: PGW RR MC. Wrote the paper: JA AM.
Histopathology reporting: MC. Chart review (clinical and followup): JA. |
# 1. Introduction
Canadian boreal forests represent 24% of the world’s boreal forest. In these
forests, anthropogenic disturbances pose serious threats for boreal flora. This
is particularly true for sensitive plant species such as bryophytes, which have
been recognized as reliable indicators of environmental changes. Bryophytes are
key constituents of biodiversity in Canadian boreal forests, promoting species
richness and supporting important ecosystem functions.
Forest management pressure is however affecting bryophyte diversity and
community composition in the boreal biome, either through direct species removal
or by altering habitat conditions originally suitable for bryophytes. Forestry
practices are also reducing the ecological continuity of forests, jeopardizing
the recolonization processes after disturbance events. Highly habitat-specific
and/or dispersal-limited bryophyte species harbored by old-growth boreal forests
may therefore be at risk. Despite their ecological importance and sensitivity to
disturbances, bryophytes are part of the vast unseen biodiversity that is
currently ignored in most conservation plans.
Less known and represented in natural history collections than other groups such
as birds, mammals or flowering plants, the large contribution of inconspicuous
taxonomic groups to diversity is difficult to assess, and thus commonly
operationalized using diversity measures of these other groups as surrogates.
However, these better-known taxonomic groups are poor surrogates for highly
diverse but less showy or studied taxa. Including inconspicuous species groups,
such as bryophytes, representativeness in systematic conservation planning
assessments would lead to more robust conservation measures.
From a conservation perspective, rare species deserve priority attention as
they are at a high risk of extinction. However, because of their own nature,
many rare species of unseen biodiversity groups suffer from a lack of
information on environmental requirements or their distribution. Species
Distribution Models (SDMs), which allow to quantify the statistical
relationships between species observations and environmental conditions from
known locations, can provide useful tools for assessing ecological preferences
of rare species or predicting their distributions. More precisely, SDM-based
predictions are achieved by using the relevant environmental conditions as
proxies of species occurrence. However, the ability of traditional SDMs to
predict rare species has been strongly limited by the number of occurrences
available, with increases in prediction accuracy with increased sample size.
Furthermore, modeling species with low prevalence often results in a high
predictors/occurrences ratio, which can lead to model overfitting and reduced
applicability to new data. Fortunately, recent advances in modeling techniques
and approaches such as Ensembles of Small Models (ESMs) have been shown to
provide robust predictions for rare plants. ESMs are ensembles of bivariate
models generated from all pairwise predictor combinations from a larger set of
predictors. ESMs can produce more accurate predictions than traditional SDMs and
reduce model overfitting for rare species. In parallel, remote sensing (RS)
offers a powerful tool to derive and integrate environmental information into
SDMs and generate predictions on species distribution over large areas. Although
a considerable number of studies have successfully integrated RS predictors into
SDMs, no study has generated ESMs using only RS predictors, nor has used this
approach to generate SDMs of inconspicuous organisms such as bryophytes, much
less of their rare species.
In this paper we use RS-derived predictors in an ESMs framework to produce
predictive models of rare bryophyte species in Eastern Canadian boreal forests.
Bryophyte rare species were selected based on their prevalence in the study area
(\<30 occurrences;). This rare species selection approach was chosen because of
the lack of knowledge on bryophytes related to their distribution, ecological
preferences and abundance in the region, which make it difficult to apply more
informative approaches such as multicriteria rare species classification
methods. In fact, the most complete rare bryophyte species list published to
date for the region used species’ prevalence as the only criterion for rare
species classification. It should be noted that rare bryophytes from were not
targeted here as their low prevalence (≤5 occurrences) greatly restricts the
development of SDMs. We hypothesize that high ESMs-based prediction accuracy can
be achieved for rare bryophyte species despite their low number of occurrences.
Our specific objectives are to assess i) if there is a relationship between the
number of occurrences and the predictive performance of ESMs, ii) if the
predictive performance of models varies by the modeled bryophyte guild (mosses,
liverworts and sphagna), and iii) if there is a spatial relationship between the
richness patterns of rare bryophyte species and overall bryophyte species both
for bryophytes as a whole and at the guild level. A total of 52 rare bryophyte
species were targeted in the present study, including 33 mosses, 14 liverworts
and 5 sphagna.
# 2. Materials and methods
## 2.1 Bryophyte field data set
We used a 389-plot database of presences-only including the field data from
three studies previously conducted in our study area, which integrated young,
mature and old-growth forests and both recent fires and cut-blocks. The study
area of 72,292 km<sup>2</sup> is located in the southwest of the Nord-du-Québec
administrative region of western Quebec (48° 51’ to 50° 42’N and 74° 31’ to 79°
26’W;), within the Black spruce–feathermoss forest bioclimatic domain. Natural
dynamics of these forests are primarily driven by stand-replacing fires, whose
cycle has been estimated at 398 years after 1920. The region is characterized by
a flat topography, dominance of poorly drained clay soils and a moderately humid
and cold climate (927.8 mm annual precipitation and 1.0°C annual mean
temperature). These conditions favor the accumulation of organic layer between
fires, which is known as the paludification process.
Bryophytes were collected following a “floristic habitat sampling” method, which
consists in collecting all bryophytes found in all microhabitats within 5 x 10 m
plots. Rare bryophyte species were selected based on their prevalence within the
study area (\<30 occurrences). From an initial set of 214 species, 142 rare
species were pre-selected, and among them, only those with a minimum of 5
occurrences were retained for modeling, since meaningful predictions can be
achieved at this sample size. A total of 52 rare bryophyte species (33 mosses,
14 liverworts and 5 sphagna;) were finally selected for modeling (species
occurrence coordinates are shown).
## 2.2 Remote sensing environmental predictors
The selection of RS-derived predictors was carried out based on their
sensitivity to environmental factors known to influence bryophyte distribution,
namely topography, canopy cover and structure, and vegetation and soil moisture.
Climatic variables were not included due to their coarse spatial resolution (≥ 1
km) and low spatial variability across the study area (annual mean temperature
and total precipitation with an approximate variability range of 1°C and 150 mm
respectively), which could lead us to overestimate the distribution of rare
species. In addition, the climatic variability that could be integrated into the
individual models of our rare species would be even more limited by the low
number of available occurrences. It should be noted that climate variables also
present lower reliability compared to RS variables at the scale of our study.
This is because climatic variables are based on interpolation methods with high
uncertainty, especially in northern latitudes where weather stations are scarce,
while RS information is spatially continuous by nature. Therefore, we selected
RS variables showing higher variability across the study area and capable of
detecting changes in local conditions more closely related to bryophyte
occurrence.
RS-derived environmental data were acquired using Google Earth Engine (GEE). The
initial set of 6 predictors included topographic position index (TPI), 2-band
enhanced vegetation index (EVI2), normalized difference water index (NDWI1),
vegetation continuous fields (VCF), PALSAR HV/HH polarization index
(PALSAR_HVHH), and bare soil index (BSI; see for predictor descriptions). TPI
was derived from the Shuttle Radar Topography Mission (SRTM) digital elevation
model in ArcGIS v.10.5 using an annulus neighborhood with inner and outer radius
of 15 and 20 pixels, respectively. EVI2, NDWI1, and BSI predictors were derived
from Sentinel-2 spectral bands. For each band, a mosaic was built from the
images available for the summer season (July 1-August 31) between 2015–2019 to
ensure homogeneity in the reflectance values. Cloudy pixels were masked in all
selected images using the Sentinel-2 QA60 band, which allows to identify pixels
with opaque clouds and cirrus clouds. Mosaics were performed by applying the
median of the overlapping pixel values. We chose EVI2 instead of EVI since EVI2
does not require the blue band, which is sensitive to the presence of residual
clouds and aerosols. VCF represents percent tree cover at 30 m resolution, after
rescaling the 250 m MODIS VCF Tree Cover layer using circa-2010 and 2015 Landsat
images and incorporating the MODIS Cropland Layer to improve accuracy in
agricultural areas (<https://catalog.data.gov/dataset/global-forest-cover-
change-tree-cover-multi-year-global-30m-v003>). The VCF predictor presented
pixels (0.1% of the total) with missing values in the study area. PALSAR_HVHH
was calculated as the ratio of HV-polarized to HH-polarized L-bands from the
Advanced Land Observing Satellite (ALOS) Phased Arrayed L-band Synthetic
Aperture Radar (SAR). HV-polarized and HH-polarized L-bands were averaged from
yearly mosaics between 2015 and 2017. All predictors were generated and
standardized at a 30 m spatial resolution (see for original spatial
resolutions). Pearson correlation coefficient was used to identify pairs of
highly correlated predictors (\|r\|)\> 0.7) from a set of 10,000 random
background points. Only the NDWI1-BSI predictor pair showed a high correlation
(r = -0.87). We retained NDWI1 which is sensitive to vegetation and soil
moisture, since bryophytes are poikilohydric organisms whose distribution is
highly dependent on available moisture. This resulted in a final set of 5
uncorrelated predictors to run the models.
## 2.3 Modeling approach: Ensembles of small models
ESMs based on bivariate models were developed to spatially predict 52 rare
bryophyte species (5–29 occurrences) using two modeling machine-learning
techniques: Maxent and Random Forest (RF). Both Maxent and RF techniques can
provide robust predictions when few occurrences are available. Maxent estimates
the probability distribution for a given species by finding the probability
distribution of maximum entropy according to a set of constraints representing
the input known locations. RF uses a bootstrap aggregation technique to provide
mean predictions from a multitude of independent decision trees built from
randomly selected subsamples from the training dataset. A random subset of
candidate predictors is assessed to split each node of each individual tree,
selecting the predictor that provides the most information in each case.
ESMs were generated in R v.3.6.3 using the *biomod2* package v.3.4.6. As we used
presence-only data, 10,000 background points were randomly generated within the
study area and used as pseudo-absences for all species. Presences and pseudo-
absences were weighted equally for training the ESMs. The pairwise combinations
of our 5 final predictors resulted in 10 candidate bivariate models per modeling
technique (Maxent and RF) for each species. We used default settings of the
*biomod2* package for computing Maxent and RF models. Predictive performance of
each bivariate model was assessed via 10-fold cross-validation procedure, using
80% of the data to train the model and 20% for its validation. While we
acknowledge that validation would be optimal using an external dataset, this is
hardly available when dealing with rare species. The Somers’ D metric was used
to identify and select bivariate models better than random (Somers’ D score \>
0, i.e. AUC \> 0.5). Maxent-ESMs and RF-ESMs were then performed using a
weighted mean of predicted probabilities from their corresponding retained
bivariate models based on their Somers’ D scores. The contribution of each
bivariate model was thus proportional to its predictive accuracy. The final ESMs
selected for each species was generated by weighted averaging predictions from
Maxent-ESMs and RF-ESMs. Predictive performance of final ESMs was evaluated
using the area under the receiver operating characteristic curve (AUC), and the
true skills statistic (TSS). AUC is not dependent on a threshold and ranges from
0.5 for an uninformative model to 1 for a perfect fit model, while TSS ranges
from -1 to 1 and was chosen instead of kappa because it is not affected by
prevalence. Since AUC and TSS values were highly correlated (Pearson r \> 0.95),
the results and discussion on models’ overall predictive performance will be
based on the AUC statistics, following and. The statistic sensitivity was also
calculated, which allows the assessment of the proportion of actual presences
correctly predicted. We computed sensitivity for those species whose final ESMs
were better than random (AUC \> 0.5). Besides of the continuous models (values
0–1000), we generate binary models (presence/absence) using the maximum training
sensitivity plus specificity threshold, or TSS optimum (predictive mapping of
the distribution of the target species is available in). Finally, we mapped the
richness patterns (species number) for total rare bryophyte species, as well as
for rare species by guild, by stacking their binary predictions
(presence/absence). Missing values associated with the predictions of the three
species that included the VCF predictor in their final models were classified as
absences before richness computation. We then compared the spatial richness
patterns obtained here for rare species with those obtained recently for overall
bryophyte species in a smaller region (28,436 km<sup>2</sup>) but fully included
in our study area at the same spatial resolution (30 m). The comparison was
performed for bryophytes as a whole (i.e. rare bryophyte richness versus overall
bryophyte richness), and between homologous bryophyte guild pairs. This spatial
correspondence analysis was carried out using Lee’s L statistic through the
*lee* function from the *spdep* package v.1.1–5.. Lee’s L statistic, in contrast
to non-spatial bivariate association measures such as Pearson’s correlation
coefficient, integrates and corrects for the spatial autocorrelation of each
variable when computing the pixel-to-pixel spatial correlation. Due to the high
computational requirements to carry out this analysis, the 30 m pixels were
previously averaged into 300 m pixels through the *aggregate* function of the
*raster* package v.3.4–5. Outputs of *lee* function were centered at 0 and re-
scaled to -1 and 1 to facilitate the interpretation of the results by
subtracting the overall mean and dividing by the maximum value. We then
calculated, for each pixel, the quantile associated with its Lee’s L value using
a Monte Carlo test with 999 simulations in order to identify significant
positive (quantile \>0.975) or negative (quantile \<0.025) spatial associations.
## 2.4 Species traits characterization
Species traits can influence the accuracy and therefore the ability of SDMs to
predict their occurrence. We evaluated the relationship between ESMs’ model
performance, as measured by AUC, and rare species traits, namely substrate
preference (six categories), reproduction mode (three categories), and spore
size (maximum and minimum;), as well as their interactions. This assessment was
performed using a multiple linear regression through the *lm* function from the
*stats* package v.3.6.3. Relationships were considered significant at α = 0.05.
# 3. Results
## 3.1 ESMs’ predictive performance versus number of occurrences and bryophyte guilds
RS-based ESMs provided poor to excellent predictive accuracy for 38 of the 52
modeled rare species, with AUC values ranging from 0.551 to 0.979 and a mean AUC
(mAUC) of 0.795 ± 0.132. Of these 38 species, 19 species were predicted with AUC
values greater than 0.8, confirming our hypothesis that high ESMs-based
prediction accuracy can be achieved for rare bryophyte species despite their low
number of occurrences (\<30). Sensitivity for these 38 species ranged from 0.8
to 1 with an average of 0.959 ± 0.063, indicating that actual presences were
usually accurately predicted. Only predictions for 14 species were not better
than random (AUC ≤ 0.5). Regarding our first specific objective, a negative
correlation (Pearson r = -0.34) was found between the number of occurrences of
the 52 target species and the predictive accuracy as measured by AUC. This
negative correlation was also observed at the guild level.
To accomplish our second specific objective, we grouped the 52 modeled species
by guild and found that predictive accuracy was similar for mosses (mAUC = 0.
715 ± 0.167) and liverworts (mAUC = 0.735 ± 0.185), and lower for sphagna (0.663
± 0.208). No significant relationships were found between ESMs’ performance and
rare species traits (or their interactions).
## 3.2 Richness patterns of rare bryophyte species
Predictive mapping of richness patterns of total rare bryophyte species and rare
species at the guild level (mosses, liverworts and sphagna) are presented in.
Predicted richness values ranged from 0 to 30, 21, 9, and 3 species,
respectively. The richness pattern of total rare bryophytes was largely
structured by the similar richness patterns observed for rare mosses and
liverworts, with high richness values mostly found towards the center and
southwest of the study area. Conversely, rare sphagna species were concentrated
in very specific areas mainly towards the north of the study area with two
additional spots towards the southeast.
Regarding our third specific objective, the Lee’s L statistic identified areas
of significant positive and negative spatial association between rare and
overall species richness for the four homologous bryophyte group pairs. Large
areas in which the spatial association between the two types of richness was not
significant were also consistently observed across pairs.
# 4. Discussion
Boreal regions are large areas lacking sharp environmental contrasts, as shown
by the low variability of our predictors, and thus a habitat where obtaining
high-performance SDMs can be challenging. Despite this, our ESMs provided
reasonably accurate predictions for rare bryophytes using only 5 uncorrelated RS
predictors. Specifically, RS-based ESMs provided poor to excellent predictive
accuracy for 73% of the target species despite their very low number of
occurrences. Indeed, 16 species with less than 10 occurrences showed an AUC \>
0.7. In addition, the computation of the metric sensitivity allowed us to
independently show the ability of our ESMs to accurately predict known
presences, with high values for the 38 species modeled better than random.
Therefore, the combination of RS data at 30 m spatial resolution and ESMs proved
to be a powerful approach to predict the distribution of rare bryophyte species
in Eastern Canadian boreal forests.
The negative relationship found between models’ predictive performance and the
number of occurrences of all bryophytes, as well as at the guild level,
illustrated the suitability of ESMs for predicting the distribution of very rare
bryophyte species regardless of guild. This result agrees with those obtained
in, who showed a higher predictive performance of ESMs for the rarest vascular
plants. Regarding bryophyte species by guild, we consider that the lower overall
predictive performance obtained for sphagna species compared to that of mosses
and liverworts may be an artifact resulting from the low number of rare sphagna
species modeled (n = 5). In fact, the occurrences of two of these five sphagna
species were successfully predicted (AUC values of 0.76 and 0.97). However, we
do not exclude the possibility that some ecologically meaningful variables that
describe the habitat of these species, such as drainage class, were missing from
our models.
In general, our results show that the development of SDMs from RS data allows
not only to make predictions of rare species distribution at spatial scales
relevant to ecological planning, but also to do so at a level of detail (30 m
resolution) that can not be achieved using the traditionally used climatic
variables at coarse resolutions (≥ 1 km). This is particularly important for
inconspicuous species such as bryophytes, which interact with their environment
at more local scales and for which the use of coarse resolutions can result in a
critical lose of information. Likewise, SDMs developed at coarse resolutions can
overestimate species distribution and greatly limits the practical utility of
derived predictions to subsequently detect species in the field. On the other
hand, the wide variety of potentially relevant predictors for rare plants that
can be derived from RS (related to vegetation, humidity, forest structure,
topography, etc.), can allow a more realistic approach to the environment-
species relationship, which can be particularly useful for species with complex
ecological niches. Thus, our methodology can play an important role in filling
existing knowledge gaps on bryophyte distribution ranges, as well as their
ecological preferences, in largely unexplored regions such as boreal forests.
The Identification of diversity hotspots has been one of the most used criteria
in biodiversity conservation planning in order to locate areas of biological and
ecological interest that should be prioritized by decision makers. Conservation
measures targeting these areas will be more effective if multiple components of
biodiversity are spatially concentrated. Specifically, both species richness and
the presence of rare species have frequently been cited as the main criteria to
select areas for conservation, while many rare species might not be represented
in species-rich areas. Our study however revealed a spatial concordance between
the richness of overall bryophyte species and that of their rare taxa in
different regions of the study area. While more bryophyte biodiversity
components could be subsequently evaluated, this result have important
implications for Canadian conservation planning. We consider that the
identification of areas harboring high level of both overall and rare bryophyte
species diversity, as well as the development of informative tools that serve
these purposes, is a significant and necessary step to promote the systematic
integration of these species into conservation plans and programs. Likewise,
conservation planning targeting bryophytes and other inconspicuous taxa could
further benefit from individual SDMs-based predictions as a basis for assessing
their representation in nature reserve networks, to quantify the impact of land
use changes on their distribution ranges, to inform assessments of their
conservation status, and to identify suitable areas for their recovery or
reintroduction.
# 5. Conclusions
Our work demonstrates the ability for RS data to characterize the habitat of
rare bryophyte species and predict their distribution patterns across the
landscape. This study also reaffirms the effectiveness of ESMs in estimating
rare plant distributions, and highlights, for the first time, the suitability of
this modeling approach for making predictions of inconspicuous rare species. We
consider that our methods and results provide an important advance in the
application of techniques focused on the study of bryophytes, with potential
valuable applications for their management and conservation. In fact, although
our study focuses on a particular taxonomic group, the combined use of ESMs and
RS would lend useful results for other overlooked inconspicuous taxa lacking
information on distribution, which would facilitate their integration in
systematic conservation planning.
# Supporting information
We thank Marion Barbé, Chafi Chaieb and Joëlle Castonguay for sharing their
bryophyte field data, and Julie Arseneault for her help in identifying samples.
We thank Rubén G. Mateo for his suggestions and assistance in the preliminary
analyzes of the data. We also thank the anonymous reviewers for the constructive
comments and suggestions provided on our manuscript. This research was funded by
Environmental Damages Fund, Environment and Climate Change Canada.
[^1]: The authors have declared that no competing interests exist. |
# Introduction
Multiple system atrophy (MSA) denotes an adult-onset neurodegenerative disorder
of relentless progression and unknown aetiology that is clinically characterized
by the variable combination of autonomic failure, levodopa-unresponsive
parkinsonism, cerebellar ataxia, and pyramidal signs. MSA affects men and woman
equally, usually starting in the sixth decade and progresses rapidly with death
occurring after an average of nine years. Pathological features cover selective
neuronal cell loss and gliosis in the basal ganglia, cerebellum, pontine and
inferior olivary nuclei, pyramidal tract, intermediolateral cell column and
Onuf's nucleus.
Morphologically, MSA is considered a primary oligodendrogliopathy based on the
cellular hallmark, the glial cytoplasmic inclusions (GCIs). GCIs contain
primarily α-synuclein (αSYN) and hence link MSA with other α-synucleinopathies,
such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). Still the
underlying mechanism of αSYN aggregates, which appear to play a fundamental role
in disease pathogenesis, remains to be determined. However, several molecular
and cellular changes, including oxidative stress, mitochondrial dysfunction and
apoptotic processes might be involved in neuronal degeneration.
Microglial activation has been reported to parallel the neuronal multisystem
degeneration in MSA, suggesting neuroinflammation as a key pathogenic mechanism
comparable to findings in PD. During the last years, studies analysing
polymorphism of genes involved in inflammatory processes, such as
interleukin-1alpha (IL-α), interleukin-1beta (IL-1β), interleukin-8,
intercellular adhesion molecule-1 and tumor necrosis factor showed elevated MSA
risk. These studies point towards a possible role of neuroinflammation in MSA
pathogenesis.
At present, MSA therapy is only symptomatic and mainly targets parkinsonism and
autonomic failure as there is no drug treatment that provides MSA patients with
consistent long-term benefits. Neuroprotective or regenerative strategies,
including neurotransplantation, appear to be an alternative therapeutic approach
for managing MSA patients. Experimentally, different cell types for neural
restoration in MSA have been tried. E13 whole ganglionic eminence grafts survive
and exert functional benefit in toxin-based MSA models. Moreover, survival,
integration and functional benefit of E13 ventral mesencephalic (VM) grafts in
toxin-based MSA models has been demonstrated.
A particular type of stem cells which is considered clinically more attractive,
ethically less problematic and exhibiting immunological properties that make
them superior over other cell types are mesenchymal stem cells (MSCs). First
described by Friedenstein and colleagues, as a population of bone marrow (BM)
cells, also known as fibroblast-colony-forming cells, which adhere to cell
culture plastic surfaces, these cells were shown to differentiate into many
mesodermal derivatives, such as adipocytes, osteocytes and chondrocytes, *in
vitro* and *in vivo* when exposed to appropriate stimuli.
The MSCs' ability to differentiate into neural-like and glial-like cells could
be shown, albeit *in vitro* only. Based on this and similar results, subsequent
studies have been initiated and designed in order to prove these cells'
potential to support neuroregeneration and also to provoke their
immunomodulatory properties in regions, which are actually void of MSC
(reviewed). This particular body of literature is vastly growing, yet lacking
strong *in vivo* evidence which demonstrates that MSCs, unless they are
ectopically placed there or infused in large numbers, are indeed capable of
bringing forth neuro-ectodermal derivatives. In light of this, many findings and
interpretations remain elusive.
Long-term clinical and radiological effects of MSCs in patients with MSA have
been described by Lee and co-workers in 2008. In an open-label study design, the
neurological deficits in 11 patients with the cerebellar type of MSA (MSA-C),
who received consecutively intra-arterial and three repeated intravenous
injections for three months, were compared with non-treated MSA patients,
demonstrating a delay in progression of neurological deficits after MSC therapy.
A recent study by the same group investigated successful neuroprotective and
immunomodulatory effects of human MSCs in a double-toxin induced animal model of
MSA-P. However this double-toxin induced model solely represents striatonigral-
like pathology, without reproducing oligodendroglial inclusion pathology,
mediating secondary neuronal multisystem degeneration.
At present to our knowledge, there is no experimental evidence for the
neuroregenerative potential of MSCs in transgenic mice, overexpressing
oligodendroglial αSYN, mimicking important aspects of MSA, such as neuronal loss
linked to MSA-like progressive autonomic failure, cerebellar ataxia and
parkinsonism, GCI pathology, astrogliosis and microglial activation. For this
reason we applied murine MSCs intravenously in aged (PLP)-αSYN transgenic mice
and analysed possible neuroprotective effects and the capacity of modulating
neuroinflammation.
# Methods
## Animals
In the present study homozygous (PLP)-α-SYN mice at the age of 18 months were
used. The animal study was designed compliant with the Austrian guidelines for
the care and use of laboratory animals and all experiments were approved by the
Federal Ministry for Education, Science and Research of Austria with the
reference number do. ZI. 5004. Animals were housed at the Animal Facility of the
Innsbruck Medical University under a 12-hour light/dark cycle with food and
water available *ad libitum*.
## Isolation of GFP MSCs
MSCs were obtained from C57BL/6-Tg(UBC-GFP)30Scha/J mice (Charles River,
Germany), 6–8 weeks old, expressing the enhanced green fluorescent protein (GFP)
gene under the human ubiquitin C promoter. Primary GFP mMSC cultures were
isolated according to established protocols.
Briefly, tibia and femur were treated with collagenase (Sigma, St. Louis, MO,
USA) for 2 h, 37°C, 20% O<sub>2</sub>, 5% CO<sub>2</sub>. Thereafter, fragmented
bones were centrifuged and cell fractions were loaded on a Ficoll-Paque Plus
gradient (Amersham Biosciences, Piscataway, NJ, USA) to harvest cells from the
interphase, followed by a washing step. The isolated bone marrow cells were
seeded for expansion in complete isolation medium constituted of RPMI-1640
(Gibco, Invitrogen, Carlsbad, Ca, USA) supplemented with 20% fetal bovine serum
(Invitrogen), 100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen).
After 24 hours non-adhering cells were removed by extensive washing with
Dulbecco's phosphate buffered saline (DPBS Invitrogen). The attached cells were
cultured until confluent and subsequently subcultured at low density (50
cells/cm<sup>2</sup>) with complete expansion medium consistent of Iscove's
Modified Dulbecco's Medium (IMDM, Invitrogen) supplemented with 20% fetal bovine
serum (Invitrogen), 100 units/ml penicillin and 100 µg/ml streptomycin
(Invitrogen). Medium exchange was performed twice weekly.
## Characterization of GFP MSCs by flow cytometry
GFP MSCs were washed with DPBS, harvested with 0.25% trypsin and 1 mM EDTA
(Invitrogen) for five minutes at 37°C, divided into round-bottom polystyrene
tubes and incubated with pooled mouse IgG (Sigma Aldrich, St. Louis, MO, USA)
for 15 minutes at room temperature (RT). Subsequently, cells were labelled with
phycoerythrin (PE)- or peridinin-chlorophyll protein complex (PerCP)-conjugated
monoclonal antibodies (mABs) specific for CD29, CD11b, CD105, CD34, CD117
(c-kit), CD44, Ly6A/E (Sca-1) (all Biolegend, San Diego, CA, USA) as wells as
SSEA-4 (R&D Systems, Mineapolis, MN, USA), MHC Class II (I-A/I-E), MHC Class I
(H2D) and CD45 (all Becton Dickinson Biosciences, San Jose, CA, USA) for 30
minutes at 4°C in the dark. PE- as well as PerCP-conjugated isotype-matched mABs
were used as negative controls. To asses cell viability Via**-**Probe™ Cell
Viability solution (Becton Dickinson Biosciences, San Jose, CA, USA) was added
shortly before flow cytometric analysis. Two additional washes were performed
and cell surface antigen expression was analyzed on a FACScan using
CellQuest<sup>TM</sup> software (both BD Biosciences, San Jose, CA, USA) with
10,000 events recorded for each sample.
## In vitro differentiation of GFP MSCs
Assessing the potential of isolated cells to differentiate into osteogenic and
adipogenic lineages was performed as previously described. Briefly osteogenic
differentiation was induced culturing MSCs in 6-well culture plates (TPP,
Trasadingen, Switzerland) in IMDM medium containing 10% FBS, 100 units/ml
penicillin and 100 µg/ml streptomycin and supplemented with 50 µM ascorbate
2-phosphate, 10 mM β-glycerol phosphate and 100 nM dexamethasone (all from Sigma
Aldrich, St. Louis, MO, USA). Medium was changed twice a week for a period of
2–3 weeks. To observe calcium deposition, cultures were washed with PBS, fixed
with 4% paraformaldehyde (PFA, Sigma Aldrich, St. Louis, MO, USA) for ten
minutes and stained with Alizarin Red, pH 4.1, for ten minutes on a rotating
platform. Cultures were rinsed two or three times with PBS to reduce non-
specific staining.
Adipogenic differentiation was induced after growing MSCs as a monolayer and
allowing them to become confluent. Complete medium was exchanged to adipogenic
induction medium consisting of IMDM medium containing 10% FBS, 100 units/ml
penicillin and 100 µg/ml streptomycin, 1 µM dexamethasone and 0.5 mM methyl-
isobutylxanthine, 10 µg/ml insulin and 100 µM indomethacin (all from Sigma
Aldrich, St. Louis, MO, USA). Cells were incubated in this medium 48–72 hours
and then adipogenic maintenance medium containing 10 µg/ml insulin and 10% FBS
in IMDM was applied for 24 hours. Cells were then again treated for 48–72 hours
with adipogenic induction medium followed by a period of 24 hours in maintenance
medium and a third treatment with induction medium. Finally, cultures were kept
for one week in adipogenic maintenance medium. Cells were fixed in 4% PFA and
lipid droplet staining was performed using Oil Red O (Sigma Aldrich, St. Louis,
MO, USA).
## Cell transplantation
For MSC transplantation, two groups of (PLP)-α-SYN mice were included in the
study, one group termed (PLP)-α-SYN+MSC (n = 12) receiving 500,000 cells in 150
µl of saline through the tail vein, and the control group termed (PLP)-α-SYN
(n = 6) sham injected with an equal amount of saline only. Survival in the
(PLP)-α-SYN+MSC group was around 60%.
## Behaviour
To determine the efficacy of intravenously transplanted MSCs with respect to a
potential amelioration of motor dysfunction in the (PLP)-α-SYN mouse modelling
MSA, the following motor function assessment was carried out: beam walking test
and stride length analysis with DigiGait.
### Beam walking test
Fine motor coordination and balance capabilities of mice were assessed by the
beam walking test. The beams consisted of long stripes of wood (each measuring
70 cm) with square cross sections of 0.9 cm and 1.6 cm, horizontally placed 50
cm above the bench surface. The mice were encouraged to walk a distance of 50
cm. For training, three daily sessions of three trials (9 crossings) were
performed using the 1.6 cm square large beam. Mice were then tested 1 week, 2
weeks and 4 weeks post transplantation (p.t.) using the 0.9 cm square beam. Mice
were allowed up to 60 seconds to traverse the beam. The latency to traverse the
beam and the number of times the hind feet slipped off, over the given distance
of 50 cm, were recorded for three consecutive runs. Analysis of each session was
based on the mean score of the three trials.
### Stride length analysis with DigiGait
The DigiGait System (DigiGait Imaging System, Mouse Specifics, Boston, MA, USA)
is a non-invasive method for quantitatively compare gait dynamics. Each mouse
was placed on a transparent belt of a treadmill enclosed by a plastic scaffold.
The speed of the treadmill was set to 20 cm/s for all experimental groups. The
ventral side of the mice as they walk was imaged by a high-speed camera, which
captured the dynamics of the paws and corresponding limbs as they approach and
move away from the belt. A special software, DigiGait Imaging System, Mouse
Specifics, Boston, MA, USA) automatically calculated stride length and other
spatial and temporal gait indices for each limb. DigiGait analysis was performed
at 1 week, 2 and 4 weeks p.t.
## Tissue processing
Four weeks after MSC treatment, animals were transcardially perfused with PBS
under deep thiopental anaesthesia. Brains were removed and cut to separate the
hemispheres. One hemisphere was put into 4% PFA overnight and cryoprotected with
20% sucrose. Brains were slowly frozen and kept at −80°C for further processing.
The other hemisphere was cut to obtain midbrain-brainstem tissue, put into a
cryovial (Nunc, Rochester, New York, USA) and frozen in liquid nitrogen and
stored at −80°C until use. A modified RIPA buffer was used to homogenize brain
tissue of each preparation. Brain homogenates were centrifuged at 16,000 g for
ten minutes at 4°C and supernatants were stored at −80°C until further
processing.
## Immunohistochemistry
Six series of 40 µm sections throughout the whole hemisphere were cut on a
cryostat (Leica, Nussloch, Germany). One series was directly mounted on
gelatine-coated slides and used for cresyl violet (Nissl) staining.
Immunolabelling was performed on free floating sections using the following
antibodies: rat anti-mouse CD11b (1∶150; AbD Serotec, Oxford, UK), polyclonal
rabbit anti-green fluorescence protein (1∶1000; GFP, Abcam, Cambridge, UK),
polyclonal rabbit CD3 (1∶7500, Abcam, Cambrige, UK) and monoclonal mouse anti-
tyrosine hydroxilase (TH, 1∶1000, Sigma, St. Louis, MO, USA). Secondary
antibodies were biotinylated anti-rat IgG, biotinylated anti-rabbit IgG, anti-
mouse IgG (1∶200, all Vector Laboratories, Burlingame, CA), Alexa-fluor
488-conjugated goat anti-rabbit or Alexa-fluor 594-conjugated goat anti-rat
(both 1∶500, Molecular Probes, Leiden), respectively.
Endogenous peroxidase activity was quenched in H<sub>2</sub>O<sub>2.</sub> After
normal serum blocking, sections were incubated with the primary antibody
overnight at 4°C, followed by incubation in biotinylated secondary antibody.
After incubation in Vectastain ABC reagent (Vectastain ABC kit, Vector
Laboratories, Burlingame, CA), the immunohistochemical reaction was developed
with 3,3′-diaminobenzidine (DAB) and sections were mounted onto gelatine-coated
slides, counterstained with cresyl violet or Mayer's haematoxylin solution,
dehydrated and coverslipped with Entellan. Immunofluorescence staining performed
for tracing GFP positive MSCs was carried out by normal serum blocking and
overnight incubation with the anti-GFP primary antibody followed by incubation
with the respective secondary antibody, and counterstaining of the nucleus by
4′, 6-Diamidin-2′-phenylindoldihydrochlorid (DAPI, Sigma Aldrich, St. Louis, MO,
USA). For GFP staining, brain sections from C57BL/6-Tg(UBC-GFP)30Scha/J mice
were used as positive controls and tissue sections from (PLP)-αSYN transgenic
animals as negative controls. Double-immunofluorescence staining for GFP and
CD11b was performed as described above; dilution of the commercial rat anti-
mouse CD11b antibody was 1∶50.
## Quantification of α-synuclein concentration in brain lysates
Brain lysates were analysed for αSYN concentration using an α-synuclein
immunoassay kit (Invitrogen, Carlsbad, CA, USA), following manufacturer's
instructions. ELISA plates were analysed with a multi-well plate reader at 450
nm (Beckman Coulter, Brea, CA, USA).
## Quantification of cytokine concentrations in brain lysates
Brain lysates were analysed using the mouse Th1/Th2 10-plex kit, MCP-1 and
TGF-β1 (Flow Cytomix, Bender MedSystems, Vienna, Austria) according to the
manufacturer's instructions. Data were acquired using a FACScan (BD Biosciences,
San Jose, CA, USA) with 1500 events recorded for each sample and further
analysed by the Flow Cytomix Software version 2.3 (Bender MedSystems, Vienna,
Austria).
## Microscopy and image analysis technique
Cell culture microscopy was performed using a Leica DMI 4000B microscope and
Application Suite V3.1 (Leica, Wetzlar, Germany). Fluorescent histological
sections were analysed with the aid of an ApoTome® microscope and AxioVision
Software (both Carl Zeiss Microimaging GmbH, Jena, Germany).
All morphometric analysis was done in a blinded way applying a computer-assisted
image analysis system (Nikon E-800 microscope, CCD video camera, Optronics
MicroFire, Goleta, USA; Stereo Investigator Software, MicroBrightField Europe
e.K., Magdeburg, Germany). The optical fractionator method, was used to estimate
the total number of neurons and microglia in the substantia nigra pars compacta
(SNc).
## Statistics
All data are given as means ± standard error of the mean (SEM). Behavioural data
were compared by two-way analysis of variance (ANOVA) for time and treatment
effects followed by a *post hoc* Bonferroni test (corrected for multiple
comparisons). Data from cytokine measurements were subjected to two-tailed
unpaired Student's *t-test* with regard to treatment. Data obtained from image
analysis technique were analysed by unpaired Student's *t-test*. Correlations
between cytokines and the number of TH<sup>+</sup> neurons were performed with
the Pearson correlation analysis. All statistical analyses were performed with
GraphPad Prism 5 Software (GraphPad Software Inc., San Diego, CA, USA). A
p-value of p\<0.05 was considered significant.
# Results
## Isolation and characterization of murine GFP MSCs
MSCs of GFP transgenic mice were isolated from tibia and femur and kept in
culture for several passages. For characterization, differentiation and
transplantation, cells at passage 8 were used (**, A**). Before intravenous
application the cells were characterised, flow cytometry analysis confirmed that
the cells at the stage of transplantation were positive for GFP (96.65%), CD29
(98.72%), CD44 (99.51%), CD105 (99.43%), MHC Class I (H2D, 48.23%), Sca-1
(99.48%), SSEA-4 (95.91%) and had a low number of CD11b (13.28%), CD34 (13.10%),
CD45 (1.72%), CD117 (c-kit, 15.76%) and MHC Class II (I-A/I-E, 11.84%). At the
stage of transplantation 99.92% of all cells were viable, as revealed by Via
Probe staining.
Multilineage potential was demonstrated by differentiation into adipocytes,
indicated by Oil-Red O staining (**, B**) as well as calcium deposits indicating
osteogenic lineage differentiation when stained with Alizarin Red (**, C**).
## Cell transplantation and tracing of GFP MSCs
Two groups of aged (PLP)-α-SYN mice were included in the study, one group
receiving 500,000 cells/150 µl of saline through the tail vein designated
(PLP)-αSYN+MSC, and one group serving as controls with an equal amount of saline
only into the tail vein termed (PLP)-αSYN. Immunofluorescence with a polyclonal
rabbit anti-GFP antibody was performed on PFA fixed MSCs in culture to ensure
tracing of GFP MSCs. Single engrafted donor cells were detected four weeks post
MSC injection in the (PLP)-αSYN+MSC group whereas sections from PLP-αSYN mice
served as negative controls. To control the efficiency of the antibody in fixed
tissue, brain sections from (PLP)-αSYN control group served as negative control
while brain sections from C57BL/6-Tg(UBC-GFP)30Scha/J served as positive
control. In face of potential contaminations of myeloid cells (CD11b) in the MSC
culture, double staining with GFP and CD11b **(**) was performed in sections of
the (PLP)-αSYN+MSC group to prove the phenotype of GFP positive cells being MSCs
and not myeloid cells.
## Behaviour
The efficacy of intravenously transplanted MSCs to restore motor function in
aged (PLP)-αSYN mice versus controls, was measured with the beam walking test,
and stride length was analysed with the DigiGait system. The beam walking test
determines fine motor coordination and balance capabilities. Traversing the beam
was performed 1 week, 2 and 4 weeks p.t. Over the period of 4 weeks, no
significant improvement in the time traversing the beam (p\>0.05) or in the
number of sideslips (p\>0.05) was detected.
We analysed the stride length on both hindlimbs (left, right) with the DigiGait
System in the transplant and control group. Previous results from our research
group have demonstrated that (PLP)-αSYN transgenic mice show shortening of
hindlimb stride length associated with TH<sup>+</sup> cell loss in the SNc. In
the current study we analysed the stride length of the left and right hindlimbs
in the transplant and control group at 1 week, 2 week and 4 weeks p.t. Stride
length in animals treated with i.v. MSCs was not significantly altered from
stride length in the control group (for both hindlimbs p\>0.05).
Both tests demonstrate that i.v. MSC treatment in aged (PLP)-αSYN mice has not
induced changes in motor behaviour.
## Neuroprotective effect on TH<sup>+</sup> neurons in the SNc after MSC treatment
Reduction of TH-immunoreactive neurons was previously reported in the SNc of
(PLP)-αSYN transgenic mice suggesting that the presence of αSYN in
oligodendrocytes induces dopaminergic neuron loss. We performed TH staining and
stereological counting in the SNc in the MSC treatment and control group ****
demonstrating a significant recovery of the total number of TH<sup>+</sup>
neurons in the MSC treated group ((PLP)-αSYN+MSC 4747±356.8 vs. (PLP)-αSYN
3510±368.8; p = 0.036) 4 weeks after transplantation.
## αSYN concentration in midbrain-brainstem lysates
In previous studies, research on the (PLP)-α-SYN mouse model has demonstrated
that pathological αSYN accumulation promotes degeneration of neurons in the SNc,
locus coeruleus, nucleus ambiguous, laterodorsal tegmental nucleus,
pedunculopontine nucleus and Onuf's nucleus, similar to findings in MSA patients
(reviewed). With a αSYN immunoassay we investigate whether MSC treatment had an
effect on αSYN concentration in midbrain-brainstem lysates. We chose midbrain-
brainstem samples since the dissected area includes the affected nuclei.
Statistical analysis by unpaired Student's *t-test* did not show differences on
αSYN concentration in MSC treated (n = 7) versus control animals (n = 6)
((PLP)-α-SYN+MSC 1.899±0.1032 vs. (PLP)-α-SYN 1.692±0.1322, p = 0.237).
## MSCs influence cytokine levels in midbrain-brainstem lysates
IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, GM-CSF, INFγ, MCP-1, TGF-β1 and
TNFα were assessed in midbrain-brainstem lysates of (PLP)-αSYN+MSC versus
(PLP)-αSYN animals. Four weeks after intravenous MSC application, we observed a
significant downregulation of IL-1α (p = 0.0014), IL-2 (p = 0.019), IL-10
(p = 0.046), IL-17 (p = 0.056), GM-CSF (p = 0.029), TGF-β1 (p = 0.001) and TNFα
(p = 0.001) in midbrain-brainstem lysates of MSC treated animals versus control
group, whereas no significant difference was found for IL-4 (p = 0.16), MCP-1
(p = 0.18), IL-5 (0.072), IL-6 (p = 0.18) and INFγ (p = 0.31).
Due to a significant reduction of the lymphocytic signalling molecules IL-2 and
IL-17, we performed a staining for the T-cell marker CD3 in (PLP)-αSYN+MSC and
(PLP)-αSYN animals. We could detect single CD3<sup>+</sup> cells throughout
different brain regions.
Moreover, we analysed microglia as an additional source of cytokines. Previous
work of our group has shown that microglial activation in the (PLP)-αSYN mouse
is present in the SNc and mediates neurodegeneration. Hence counting
CD11b-immunoreactive cells in the SNc as a marker for microglia was performed
(**, B–C**). Statistical analysis with two-tailed unpaired Student's *t-test*,
revealed no significant difference in the number of CD11b<sup>+</sup> cells in
transplanted animals (n = 7) versus the control group (n = 6) ((PLP)-αSYN+MSC
6588±242 vs. (PLP)-αSYN 6880±529.3; p = 0.6075) (**, A**).
Finally, in order to evaluate whether the cytokines in midbrain-brainstem
lysates (IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, GM-CSF, INFγ, MCP-1,
TGF-β1 and TNFα) contribute to TH<sup>+</sup> neuronal rescue we established a
Pearson correlation between the cytokines and TH<sup>+</sup> neurons. We could
demonstrate significant inverse correlations for IL-1α (Pearson r = −0.697,
p = 0.0081), IL-2 (Pearson r = −0.823, p = 0.0005), TGF-β1 (Pearson r = −0.647,
p = 0.0169) and TNFα (Pearson r = −0.0575, p = 0.0397).
# Discussion
In the present study we aimed to determine whether intravenous application of
murine MSCs in an aged (PLP)-αSYN MSA mouse model ameliorates behavioural
deficits and exerts neuroprotective and immunomodulatory properties. To date, to
our knowledge there is no experimental evidence of a MSC therapeutic approach in
transgenic MSA models. Recently, human MSCs have been demonstrated to protect
against loss of neurons in SN and striatum in an animal model of double toxin-
induced MSA-P. A few years ago, in an open-label study design, disease modifying
effects of intra-arterial and intravenous injected MSCs in eleven patients with
MSA-C, have been reported. Together with *in vitro* and *in vivo* findings in PD
and other neurodegenerative disorders, MSCs seem to be an attractive and
feasible therapeutic intervention,.
As a matter of fact, experimental results are often difficult to compare, in
lieu of standards for MSC isolation, cultivation and in vivo application.
Furthermore there is no unique marker to identify MSCs. In general, MSCs are
characterised upon expression of a group of surface receptors and upon their
multilineage potential. Prior to transplantation we have characterized GFP MSCs,
based on a set of criteria proposed by the International Society for Cellular
Thearpy, which includes expression and lack of surface markers and
differentiation potential into adipocytes and osteocytes.
In the (PLP)-αSYN+MSC transplant group the survival rate after intravenous MSC
infusion was low, probably due to the high age of the animals. Further we
presume that due to their size MSCs got trapped within the pulmonary
capillaries, causing pulmonary and hemodynamic alterations. On the other hand
numerous animal studies and clinical trials have reported favourable outcomes
following systemic infusion of MSCs.
Since we have only encountered a few single donor engrafted GFP<sup>+</sup> MSCs
four weeks after transplantation, the use of GFP as experimental tool to examine
the survival and fate in future studies is in question, since there are a lot of
inconsistent results in the literature, on tracking and determining cell fate of
MSCs using GFP as a reporter in transplantation studies. In addition, we cannot
exclude a detrimental effect of the GFP protein on MSC survival and therefore
preventing proper integration into sites of neurodegeneration. On the other
hand, a number of reports state that MSCs exert effects on tissue repair despite
exhibiting low and/or transient levels of engraftment. This foreshadows a novel
concept of tissue repair relying on secretion of trophic factors and/or
crosstalk with the microenvironment rather than MSC transdifferentation.
The (PLP)-αSYN transgenic mouse model has been widely characterized in terms of
effects of αSYN overexpression on neurodegeneration and motor activity.
Furthermore, this animal model has been successfully applied in neuroprotective
studies as a preclinical rationale for phase II clinical trials. We performed
the beamwalking test to define fine motor coordination and balance capabilities
as well as stride length with a digital system, since shortening of the stride
length has been reported in the (PLP)-αSYN transgenic mouse. In a recent study,
intravenous human MSCs have ameliorated behavioural deficits in a double-toxin
induced mouse model of MSA-P. Our experimental data show no significant effects
of intravenously applied MSCs to alleviate behavioural failure. On the other
hand, the study with double-toxin induced striatonigral degeneration has been
performed in mice lacking αSYN expression in oligodendroglia and thus
replicating solely striatonigral-like pathology without reproducing other
cardinal features of MSA. It remains elusive whether oligodendroglial αSYN
exerts a deleterious effect on transplanted MSCs, however in a recent study
addressing the fate of embryonic striatal grafts in presence of oligodendroglial
αSYN inclusions, disturbed dopaminergic re-innervation and reduced p-zone volume
of the grafts in the MSA mouse model has been attributed to effects of host αSYN
pathology.
For our pilot study, we have chosen transgenic animals at the age of 18 months,
overexpressing αSYN under control of the PLP promoter, since GCIs are the
hallmark of MSA and MSA is a late onset disease. Previous studies have clearly
demonstrated that in this MSA animal model αSYN overexpression leads to
neurodegeneration, resembling human neuropathology. At the age of 18 months,
neurodegeneration due to αSYN overexpression is at a much more progressed stage,
impairing motor activity drastically as shown in MSA mouse models overexpressing
αSYN under control of oligodendroglial promoters. Currently we are examining
this issue more closely in the (PLP)-αSYN mouse. Nevertheless, the absent
behavioural improvement after MSC treatment, leads to the conclusion, that MSC
treatment at later disease stages does not induce the desired effect of
ameliorating behavioural deficits.
Since one pathological feature in MSA patients covers selective neuronal loss in
the SNc, and the (PLP)-αSYN mouse model replicates this feature, we evaluated
whether MSC treatment had an effect on number of TH<sup>+</sup> neurons. There
was a subtle but significant recovery of numbers of dopaminergic neurons in the
MSC transplant group compared to transgenic controls.
We further investigated putative factors that may contribute to this “rescue” of
dopaminergic neurons after MSC treatment. We analysed whether TH recovery is
caused by the decrease of αSYN concentration in midbrain-brainstem lysates. αSYN
is known to be a key factor involved in oligodendroglial and neuronal loss in
MSA patients and in the (PLP)-αSYN transgenic animal model. Recently genetic
variants in the αSYN gene *SNCA* have been associated with an increased risk in
developing MSA. However, αSYN concentration in the midbrain-brainstem region was
not significantly altered between (PLP)-αSYN+MSC treatment compared to
(PLP)-αSYN control group. This finding highlights the concept of tissue repair
of MSCs by releasing anti-inflammatory and trophic molecules.
Neuroinflammation has been widely regarded as a possible key player in
progressing disease pathogenesis in various neurodegenerative diseases. In PD
patients as well as PD animal models, neuroinflammation in terms of microglial
activation has been observed. Lately emerging evidence for the presence of
T-lymphocytes in the midbrain of PD patients suggests that a potential role of
infiltrated peripheral cells is related to PD pathogenesis. In a recent study
infiltration of T-cells into the brain actively participated in dopaminergic
neuron degeneration in the SNc. Additionally, overexpression of human αSYN in
mouse SN neurons, induced by an adeno-associated viral vector, has led to
activation of microglia, production of inflammatory cytokines and stimulated the
adaptive immune response. In our experiment we analysed twelve cytokines, IL-1α,
IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, GM-CSF, INFγ, MCP-1, TGF-β1 and TNFα in
midbrain-brainstem lysates and found significant downregulation of IL-1α, IL-2,
IL-10, IL-17, GM-CSF, TGF-β1 and TNFα four weeks after intravenous MSC
application in the treatment group. Additionally we encountered CD3<sup>+</sup>
T-cells throughout the brain in (PLP)-αSYN and (PLP)-αSYN+MSC treated animals.
After MSC treatment, however, the T-cell specific cytokines IL-2 and IL-17 were
significantly downregulated. Furthermore, TH neuronal “rescue” was inversely
correlated with IL-2, indicating that MSC treatment influenced pathogenic T-cell
response in (PLP)-αSYN mice. Similar effects have been widely investigated for
multiple sclerosis and experimental autoimmune encephalitis and support the role
of MSC treatment by modulation of T-cell response.
Finally we analyzed the influence of MSC treatment on microglial activation,
since microglia have been reported to parallel the neuronal multisystem
degeneration in MSA and mediates dopaminergic neuronal loss related to
oligodendroglial α-synucleinopathy in the (PLP)-αSYN mouse. We quantified the
number CD11b<sup>+</sup> microglial cells in the SNc, yet could not demonstrate
a significant difference in microglial cell number after MSC treatment. However,
this finding cannot exclude a modulatory effect on microglial activation status.
Furthermore, the major proinflammatory cytokine TNFα and IL-1α, also released by
activated microglia and astroglia was significantly decreased in brain lysates
of the MSC treatment group and inversely correlate with the number of TH neurons
indicating that suppression of microglial activation and astrogliosis may
contribute to dopaminergic neuronal survival. Similar results, demonstrating
decreased activation of astrocytes and microglia by human MSCs in a mouse model
of MSA-P have been recently reported and are in good concordance with our
findings that MSCs exert modulatory effects on neuroinflammation and promote
survival of dopaminergic neurons in the SNc.
In summary, our study describes the first experimental attempt using MSCs as a
therapeutic intervention in an aged transgenic mouse model of MSA featuring
oligodendroglial α-synucleinopathy. We have demonstrated that intravenous
application of MSCs leads to a rescue of dopaminergic neurons in the SNc.
Furthermore we could demonstrate a profound immunomodulatory effect after MSC
treatment, resulting in downregulation of various proinflammatory cytokines,
which are linked to microglial activation, astrogliosis and mediation of
adaptive immunity. However, MSC treatment did not alter behavioural deficits in
aged transgenic MSA mice.
Our data have potential implications for MSCs as a future stem cell source in
MSA therapies. Nevertheless, further experimental studies on the efficacy of
MSCs as disease modifying candidates in MSA as well as different routes of
application have to be performed. Prior to embarking on further human trials,
preclinical studies are necessary since they will reveal if all that glitters
experimentally is truly clinical gold.
# Supporting Information
The authors are grateful for technical assistance by Monika Hainzer. Sincere
thanks to Maria Auer for assistance with the ApoTome.
[^1]: Conceived and designed the experiments: SS MR. Performed the
experiments: SS AJ. Analyzed the data: SS NS MR. Wrote the paper: SS.
Revised the manuscript for important intellectual content: NS GL MR GKW.
[^2]: The authors have declared that no competing interests exist. |