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INTRODUCTION {#s1}
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The activation of a neuroendocrine axis leading to the production of steroid hormones is a conserved trigger of maturation onset in animals ([@DEV188110C42]; [@DEV188110C47]; [@DEV188110C50]). In vertebrates, neuronal gonadotropin-releasing hormone (GnRH) secretion awakens the hypothalamic-pituitary-gonadal (HPG) axis leading to the production of sex steroids at puberty. Similarly, the insect neuropeptide Prothoracicotropic hormone (PTTH) induces steroidogenesis that drives the juvenile-to-adult transition. Multiple signals converge on these neuroendocrine axes to couple the timing of maturation with external and internal stimuli including nutritional and metabolic states ([@DEV188110C25]; [@DEV188110C32]). In humans, the prevalence of childhood obesity is believed to be a key factor in the growing incidence of early puberty, which affects growth and final body size ([@DEV188110C2]; [@DEV188110C19]). In *Drosophila* and other insects, the onset of maturation is governed by a nutritional checkpoint associated with the attainment of critical body size or weight, after which maturation will occur irrespective of nutritional intake ([@DEV188110C11]; [@DEV188110C25]). Thus, maturation is not initiated until the juvenile has accumulated sufficient energy and material to ensure successful maturation and adult fitness.
PTTH is produced by a pair of neurosecretory cells (PTTHn) in each brain hemisphere ([@DEV188110C24]). The axons of these neurons terminate on the steroid-producing cells of the prothoracic gland (PG) ([@DEV188110C24]), forming a neuroendocrine circuit functionally similar to the mammalian HPG axis. PTTH released here binds to Torso, its cognate receptor tyrosine kinase, initiating a mitogen-activated protein kinase (MAPK) cascade that leads to the production of the steroid ecdysone ([@DEV188110C41]). Ecdysone is then released from the PG cells through a regulated vesicle-mediated process ([@DEV188110C55]) and, in peripheral tissues, is converted into its more potent form, 20-hydroxyecdysone (20E) ([@DEV188110C42]). Actions of 20E are mediated by its binding to the nuclear Ecdysone Receptor (EcR), which, in a heterodimeric complex with Ultraspiracle (Usp), induces time- and tissue-specific transcriptional responses that initiate metamorphosis, a transition from the juvenile larval stage to the adult similar to mammalian puberty ([@DEV188110C21]).
Uncovering the signals that affect the production and release of PTTH is key to understanding how maturation timing is controlled. *Drosophila* Insulin-like Peptide 8 (DILP8; Ilp8 -- FlyBase) secreted by adult precursor tissues developing within the larva prevents maturation onset until these tissues are sufficiently developed, through regulation of neurons that inhibit the PTTHn ([@DEV188110C7], [@DEV188110C6]; [@DEV188110C14], [@DEV188110C15]; [@DEV188110C51]). The neuropeptide Pigment-Dispersing Factor (PDF) ([@DEV188110C24]), a signal associated with the circadian clock, also regulates PTTH, suggesting that the fly maturation pathway, like the mammalian HPG axis, is under photoperiodic control ([@DEV188110C52]). In mammals, the peptide kisspeptin (KISS1) and its receptor GPR54 (KISS1R) are key triggers of puberty onset that regulate GnRH secretion. Similarly, the *Drosophila* KISS1 ortholog, Allatostatin A (AstA), and its receptor regulate maturation onset via control of PTTH signaling ([@DEV188110C9]; [@DEV188110C35]). Furthermore, the HPG axis is under feedback control, whereby gonadal steroids act on the upstream neuroendocrine components to regulate signaling through this axis ([@DEV188110C1]). In *Drosophila*, ecdysone provides positive and negative feedback on the PG in a classical feedback loop to shape the pulses of ecdysone necessary to drive the genetic programs underlying the juvenile-to-adult transition ([@DEV188110C30]). However, whether feedback also regulates the neurocircuitry that triggers these steroid pulses in animals, and thus the overall timing of the juvenile-to-adult transition, is presently unknown.
We report here the identification of *EcR* and *usp* in an RNAi-based screen for regulators of PTTH production or release. We find that ecdysone-mediated feedback via EcR in the PTTHn drives the pupariation-triggering PTTH surge, thereby determining the timing of maturation. Under rising ecdysone levels, EcR mediates *Ptth* transcriptional upregulation, leading to the steep rise in PTTH prior to metamorphosis. This generates a PTTH surge that induces a high-level ecdysone peak that initiates maturation; high ecdysone levels subsequently feed back negatively to suppress PTTH production. Mammalian gonadal steroids act in positive and negative feedback loops to modulate the HPG axis ([@DEV188110C1]). Our results show that the developmental transition to adulthood in *Drosophila* is similarly controlled by positive and negative feedback mechanisms that modulate the PTTHn, suggesting that neuroendocrine feedback control of developmental maturation is evolutionarily ancient.
RESULTS {#s2}
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EcR induces maturation onset through positive regulation of the PTTHn {#s2a}
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PTTH release is thought to be the main trigger of the juvenile-adult transition in insects ([@DEV188110C42]). Loss of PTTH extends the larval period of feeding and growth, leading to increased pupal and adult size. To identify signals controlling maturation onset, upstream of PTTH, we performed an RNAi-based pupal-size screen targeting 608 membrane-associated proteins (known or potential receptors) and transcription factors in the PTTHn ([Fig. 1](#DEV188110F1){ref-type="fig"}A, [Table S1](Table S1)). In this screen, expression of RNAi constructs (along with the RNA-processing enzyme Dicer-2) was driven in the PTTHn by *NP423-GAL4* (*NP423\>*) ([@DEV188110C9]; [@DEV188110C16]; [@DEV188110C54]). Among the strongest hits was *EcR*; knockdown of this gene, or of *usp*, which encodes a transcriptional co-factor of EcR, in the PTTHn led to larval overgrowth and thus to increased pupal size ([Fig. 1](#DEV188110F1){ref-type="fig"}B). To verify these findings, we reduced *EcR* expression in the larval PTTHn and measured developmental timing and final body size. Knockdown using two independent RNAi lines (\#1 and \#2) confirmed that loss of *EcR* in the PTTHn causes developmental delay and overgrowth, like reduced expression of *Ptth* itself ([Fig. 1](#DEV188110F1){ref-type="fig"}C-E). This suggested that EcR might act in the PTTHn as a positive regulator of PTTH production and release. Fig. 1.**Screening for regulators of PTTH identifies the Ecdysone Receptor (EcR) complex.** (A) Outline of the PTTHn screen for factors regulating growth. Gene expression was knocked down in the PTTHn using the strong *NP423-GAL4* (*NP432\>*) driver, and pupal size was measured. In *Drosophila*, growth is restricted to the larval stage, and pupal size thus determines final adult size. (B) Pupal-size distribution from the screen, presented as a Z score (standard deviations from the mean of all RNAi lines). RNAi against *EcR* or its partner *ultraspiracle* (*usp*) led to increased pupal size (+3.1 and +1.7 s.d.). (C) Images of representative pupae of animals with NP423-driven overexpression of *UAS-Ptth* (*Ptth* ↑), *UAS-usp-RNAi* (*usp* ↓), *UAS-EcR-RNAi* (*EcR* ↓) or *UAS-Ptth-RNAi* (*Ptth* ↓). (D) *EcR* knockdown using *NP423\>* with two independent RNAi lines led to increased pupal size, similar to RNAi against *Ptth*. (E) RNAi knockdown of *EcR* or *Ptth* delays pupariation, prolonging the feeding stage of development. Top: curve showing the fraction of pupated animals over time; bottom: the corresponding 50%-pupariated 'P50' times. Statistics: one-way ANOVA with Dunnett\'s multiple comparison test; \**P*\<0.05; \*\**P*\<0.01; \*\*\**P*\<0.001.
Next, we used the *Ptth-GAL4* (*Ptth\>*) driver, a weaker but PTTHn-specific line, to confirm that these effects were caused by knockdown of *EcR* in the PTTHn. Because of *Ptth-GAL4*'s relative weakness, we recombined *EcR-RNAi* constructs \#1 and \#2 to increase the strength of RNAi-mediated knockdown. Animals expressing *Ptth\>EcR-RNAi-1+2* exhibited delayed pupariation and increased pupal size comparable to knockdown of *Ptth* itself, and we re-confirmed these effects using a third independent *EcR-RNAi* line, \#3 ([Fig. 2](#DEV188110F2){ref-type="fig"}A,B). All further *EcR-RNAi* experiments used RNAi lines \#1 and \#2 combined. To further attribute these defects to PTTHn-specific EcR deficiency, we disrupted the *EcR* gene in only these cells using tissue-specific somatic CRISPR/Cas9 gene editing. We generated a transgenic UAS-regulated construct that expresses a pair of guide RNAs (gRNAs) targeting exon 3 of the *EcR* gene, which encodes the DNA-binding domain of EcR and is shared between all its isoforms. By expressing this construct along with *UAS-Cas9* under *Ptth\>* control (at 29°C to boost the activity of the GAL4 transcription factor and the Cas9 enzyme), we disrupted the *EcR* locus specifically in the PTTHn. To assess the efficacy of this setup, we immunostained brains from larvae at 96 h AEL (after egg laying) using antibodies against the PTTH neuropeptide, EcR, and the ecdysone-biosynthetic enzyme Phantom (Phm) to label the PG ([@DEV188110C40]; [@DEV188110C53]). No anti-EcR signal was visible in the PTTHn of knockout animals, whereas clear anti-EcR signal was present in the PTTHn nuclei of controls, and in neighboring cells in both controls and *EcR* knockouts ([Fig. 2](#DEV188110F2){ref-type="fig"}C,D). Although the CRISPR/Cas9 system has induced cytotoxicity in some reports, we did not observe any morphological abnormalities in the PTTHn or their projections to the PG. Next, to investigate how this manipulation affected PTTHn activity, we measured developmental timing and final body size. PTTHn-specific *EcR* disruption significantly delayed pupariation and resulted in larger pupae, compared with controls ([Fig. 2](#DEV188110F2){ref-type="fig"}E,F). Furthermore, PTTH levels (anti-PTTH staining intensity in the PTTHn cell body) were significantly reduced in *EcR* knockouts ([Fig. 2](#DEV188110F2){ref-type="fig"}G). Together, these data support a role for EcR as an inducer of PTTH expression. We therefore next overexpressed *EcR* in the PTTHn to analyze whether this might lead to a precocious juvenile-adult transition. Indeed, overexpressing *EcR.A* or *EcR.B1* variants resulted in advanced pupariation onset, thereby shortening the larval growth period and reducing pupal size ([Fig. 2](#DEV188110F2){ref-type="fig"}B,H), consistent with a positive effect of EcR on PTTHn activity. Fig. 2.**EcR activity in the PTTHn controls the onset of maturation.** (A) RNAi-induced knockdown of *EcR* with RNAi lines \#1 and \#2 combined or a third (single) RNAi line (\#3) against *EcR* using the specific *Ptth-GAL4* (*Ptth*\>) driver delayed pupariation to a similar extent as knockdown of *Ptth*, recapitulating the results seen with the strong *NP423\>* driver. Top: pupariation over time; bottom: P50 values. (B) PTTHn-specific manipulations of *EcR* expression alter pupal size. Overexpression of the A isoform of EcR led to reduced pupal size, whereas RNAi against *EcR* or *Ptth* increased pupal size. Bimodal distributions reflect sexual size dimorphism. (C,D) At 96 h AEL at 29°C, EcR immunostaining is present in the nuclei of PTTHn and of neighboring neurons in the control genotype (*Ptth-GAL4, UAS-Cas9*,+; *Ptth\>Cas9*,+) for PTTHn-specific CRISPR/Cas9-induced *EcR* deletion (C). EcR immunostaining is eliminated specifically in the PTTHn in animals expressing both Cas9 and the *EcR* guide-RNA construct in these cells, illustrating the cell-specificity of the deletion (D). Scale bars: 50 μm (main panels); 5 μm (insets). (E,F) CRISPR/Cas9-mediated disruption of the *EcR* locus in the PTTHn leads to (E) developmental delay and (F) a corresponding pupal size increase (at 30°C for stronger GAL4 and Cas9 activity; note the temperature-induced growth acceleration of the control animals, compared with the data shown in A). (G) *EcR* disruption in the PTTHn leads to reduced PTTH immunostaining in these cells. The number (*n*) of individual neurons measured is indicated on the bars. Intensity values are normalized against the average value of controls. (H) Overexpression of EcR A or B1 isoforms in the PTTHn leads to accelerated development and premature metamorphosis. Statistics: one-way ANOVA with Dunnett\'s multiple comparisons or an unpaired two-tailed *t*-test for pairwise comparison; \**P*\<0.05; \*\**P*\<0.01; \*\*\**P*\<0.001.
EcR induces the PTTH surge prior to the juvenile-adult transition {#s2b}
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Because EcR induces pupariation through its actions in the PTTHn, we investigated EcR levels in these cells during the juvenile-adult transition. We found that EcR is present in PTTHn nuclei from early L3 (80 h AEL) throughout the L3 stage, increasing in abundance at the onset of the wandering stage (112 h AEL), when ecdysone levels rise prior to pupariation ([Fig. 3](#DEV188110F3){ref-type="fig"}A). Knockdown of *EcR* in the PTTHn attenuated this EcR rise, confirming that *EcR* expression is reduced by the RNAi, albeit not eliminated as with CRISPR-induced deletion ([Fig. 2](#DEV188110F2){ref-type="fig"}C,D). Fig. 3.**EcR is required in the PTTHn to induce PTTH toward the end of larval development.** (A) Late-third-instar upregulation of EcR in nuclei of PTTHn is attenuated by *EcR* knockdown. PTTHn were identified by PTTH immunostaining (the same cells are shown in B) and are circled in the representative image pairs below. (B) *EcR* knockdown in the PTTHn prevents the late-third-instar increase in PTTH expression. PTTH immunostaining intensity increases in control animals at 112 h AEL, whereas this increase does not take place in *EcR*-knockdown animals. Top: quantification of PTTH immunostaining intensity; bottom: representative images. In A and B, the number (*n*) of individual neurons measured at each time point is indicated in bars. All intensity values are normalized against the average value of controls at 80 h for each channel. (C) *EcR* knockdown in the PTTHn reduces *Ptth* expression at all time points and prevents or diminishes the upregulation of its expression toward the end of larval development (a 128-h time point is included for knockdown animals to illustrate the lack of further increase). (D) The late-third-instar peak in 20-hydroxyecdysone levels is reduced or delayed by *EcR* knockdown in the PTTHn. (E) The corresponding expression increase in the ecdysone-responsive proxy gene *E75B* is delayed in *EcR*-knockdown animals; again, a 128-h measurement is included for the developmentally delayed knockdown animals, illustrating that the increase in *E75B* expression eventually appears, although it is delayed by 8-12 h. (F,G) Rescue of developmental timing and growth phenotypes induced by CRISPR/Cas9-mediated *EcR* knockout in the PTTHn by *Ptth* overexpression. Statistics: unpaired two-tailed *t*-test for pairwise comparison; \**P*\<0.05; \*\**P*\<0.01; \*\*\**P*\<0.001.
As *Ptth* is transcriptionally upregulated to induce the ecdysone pulse that triggers maturation ([@DEV188110C24]), we analyzed whether EcR is required in the PTTHn for this upregulation. Anti-PTTH immunostaining intensity in cell bodies of the PTTHn displayed a dynamic profile in control animals, with a strong increase at 112 h AEL that coincided with the increase in EcR abundance ([Fig. 3](#DEV188110F3){ref-type="fig"}B), consistent with EcR being responsible for PTTH upregulation at this time. PTTHn-specific *EcR* knockdown markedly changed this pattern, eliminating the increase in PTTH intensity at 112 and 116 h AEL observed in control animals. Although a reduction in PTTH staining could potentially be explained by increased secretion, this would be inconsistent with these animals\' delayed development. Thus, this altered profile suggests that EcR positively regulates PTTH production, consistent with the results showing that EcR expression in the PTTHn correlates with pupariation advance. Because EcR is a transcriptional regulator, we asked whether EcR is required for transcriptional upregulation of *Ptth* during the late L3 stage by performing a temporal gene-expression analysis of larvae expressing *EcR-RNAi* in the PTTHn throughout the second half of L3. Consistent with previous findings ([@DEV188110C24]), in control animals, *Ptth* expression underwent a dramatic upregulation from about 12 h prior to pupariation (112-124 h AEL) until the onset of metamorphosis ([Fig. 3](#DEV188110F3){ref-type="fig"}C). *EcR* knockdown significantly reduced *Ptth* expression across the time series and prevented its pre-wandering enhancement, indicating that EcR is required for the late-larval transcriptional upregulation of *Ptth*.
Loss of *EcR* in the PTTHn impairs the steroid increase that triggers maturation {#s2c}
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PTTH signaling stimulates the production of ecdysone in the PG by inducing transcriptional upregulation of the ecdysone-biosynthetic Halloween genes ([@DEV188110C24]; [@DEV188110C45]). To examine the effects of EcR in the PTTHn on ecdysone production, we first analyzed the expression of these genes in the PG. Expression of *spookier*, *phantom*, *disembodied* and *shadow* are all upregulated in the PG in response to PTTH/Torso signaling. The enzyme Shade mediates the conversion of ecdysone to 20E in peripheral tissues, and its expression is therefore not regulated by PTTH signaling ([@DEV188110C24]; [@DEV188110C34]). Consistent with the requirement of EcR for inducing PTTH production, we found that the ecdysone-synthesis genes were not properly upregulated in *Ptth\>EcR-RNAi* animals, whereas levels of *shade* were unaffected ([Fig. S1A-E](Fig. S1A-E)). This is consistent with a model in which the delay observed in *EcR*-knockdown animals is caused by a failure to upregulate PTTH towards the end of larval development, thus impairing the ecdysone biosynthetic pathway. To determine whether this is the case, we analyzed ecdysone levels directly by enzyme-linked immunosorbent assay (ELISA) and found reduced levels of ecdysone in animals with *EcR* knockdown in the PTTHn towards the end of larval development ([Fig. 3](#DEV188110F3){ref-type="fig"}D), indicating that they do not produce a proper maturation-promoting ecdysone pulse. As a result, expression of the ecdysone-inducible genes *E75A* and *E75B*, which serve as proxies for the ecdysone titer, show a significantly reduced and delayed rise in *Ptth\>EcR-RNAi* animals ([Fig. 3](#DEV188110F3){ref-type="fig"}E, [Fig. S2](Fig. S2)). These data suggest that EcR stimulates *Ptth* transcription to generate the PTTH surge that initiates metamorphosis. We therefore examined whether the developmental-delay phenotype caused by knockout of *EcR* in the PTTHn could be rescued by simultaneous overexpression of *Ptth*. Indeed, *Ptth* overexpression in the PTTHn of animals with CRISPR/Cas9-mediated knockout of *EcR* in these same neurons partially rescued the delayed pupariation and completely rescued their overgrowth, showing that the phenotype is caused by lack of PTTH ([Fig. 3](#DEV188110F3){ref-type="fig"}F,G). Together, our results suggest that EcR acts as a positive regulator of *Ptth* expression, just as steroids modulate the mammalian HPG axis, and that feedback through the steroid ecdysone is a key trigger of the neuroendocrine cascade that drives maturation onset in *Drosophila*.
Initiation of maturation is triggered by feedback that activates PTTH {#s2d}
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EcR and 20E sit atop a large network of transcriptional regulators ([@DEV188110C21]). Our results show that EcR is required in the PTTHn for *Ptth* upregulation, suggesting a model in which PTTH or other factors induce a small ecdysone rise that acts via EcR in the PTTHn to increase PTTH production, leading to the activation of the metamorphosis-initiating neuroendocrine cascade. In this model, *in vivo* ecdysone manipulations should feed back to affect PTTH levels. Thus, we reduced ecdysone synthesis by silencing *torso*, which encodes the PTTH receptor, in the PG \[PG-specific *phm-GAL4* (*phm*\>) driving *UAS-torso-RNAi* (*torso-RNAi*): *phm\>torso-RNAi*\]. As ecdysone synthesis is downregulated in these animals, their larval feeding stage was prolonged, resulting in overgrowth and increased pupal size ([Fig. S3A](Fig. S3A)). Next, we measured *Ptth* levels during L3 prior to the onset of pupariation. In controls, *Ptth* expression increased as expected from 96 h AEL to 120 h AEL ([Fig. 4](#DEV188110F4){ref-type="fig"}A). By contrast, when ecdysone synthesis was reduced via *phm\>torso-RNAi*, *Ptth* expression remained uninduced, even when these animals eventually pupariated (at 189 h AEL). Expression of the PG-specific ecdysone-biosynthetic gene *disembodied* and the ecdysone-induced gene *E75B* remained low in *phm*\>*torso-RNAi* animals, confirming reduced ecdysone synthesis ([Fig. S3B,C](Fig. S3B,C)). Thus, blocking ecdysone synthesis in the PG prevents the pre-metamorphic upregulation of *Ptth*. Fig. 4.***Ptth* is upregulated by ecdysone-mediated feedback at the onset of maturation.** (A) The larval peak of *Ptth* expression observed in the control genotype does not appear in *phm*\>*torso-RNAi* animals, indicating feedback from ecdysone produced by the PG to the PTTHn. (B,C) Expression of (B) *Ptth* in *ex-vivo* brains is increased by low levels of 20-hydroxyecdysone (20E, the more active form of ecdysone), but inhibited by larger concentrations, whereas expression of (C) the EcR-regulated proxy gene *E75B* increases in *ex vivo* cultured brains with increasing concentration of 20E in the medium, indicating biphasicity of ecdysone response at the *Ptth* locus. (D) RNAi-induced knockdown of *EcR* beginning 10 h before pupariation leads to increased *Ptth* transcription 3 h post-pupariation, consistent with a lack of EcR-mediated inhibition. (E) Levels of the ecdysone-induced transcript *E75A* are higher at the time of pupariation in temperature-induced *EcR*-knockdown animals than in controls, but after 3 h, *E75A* levels have fallen to a lower level in these animals than in controls, suggesting increased or prolonged ecdysone levels, consistent with loss of EcR-mediated *Ptth* inhibition. Colors in E are the same as in D. Statistics: one-way ANOVA with Dunnett\'s multiple comparisons or an unpaired two-tailed *t*-test for pairwise comparison; \**P*\<0.05; \*\**P*\<0.01; \*\*\**P*\<0.001. (F) Graphical summary of the model presented here. A small rise in ecdysone production by the PG feeds back in an EcR-dependent manner in the PTTHn to drive the metamorphosis-inducing surge of PTTH release and ecdysone production; high ecdysone levels at the peak of the surge in turn inhibit further PTTH expression.
We next dissected larval brains, preserving PTTHn projections to the ring gland, which contains the PG, and cultured them *ex vivo* for 6 h in media containing 20E. Consistent with our model, 20E at 5 ng/ml (in the low physiological concentration range occurring during L3, prior to the large pre-metamorphosis pulse) increased *Ptth* expression ([Fig. 4](#DEV188110F4){ref-type="fig"}B). This suggests that pre-pulse ecdysone levels suffice to awaken the neuroendocrine system through *Ptth* upregulation, which then induces the steroid pulse initiating maturation. Interestingly, higher concentrations of 20E inhibited *Ptth* transcription, inconsistent with a solely positive role for 20E/EcR. *Ptth-*inhibitory 20E concentrations (500 ng/ml) correspond to high physiological levels that occur during the large pupariation-associated ecdysone pulse. Consistent with this, these concentrations induced *E75B* upregulation ([Fig. 4](#DEV188110F4){ref-type="fig"}C), which occurs during this maturation-inducing pulse. We further tested the potential of negative feedback downregulation of PTTH following pupariation when the ecdysone titer falls rapidly. To do this, we induced RNAi-mediated knockdown of *EcR* 10 h before pupariation, at a time when *Ptth* expression has already been upregulated, to prevent interference with the positive effect of EcR on the pupariation-triggering rise in *Ptth* expression. This manipulation led to increased *Ptth* expression 3 h following pupariation ([Fig. 4](#DEV188110F4){ref-type="fig"}D), suggesting that EcR is required following pupariation to downregulate *Ptth* transcription to suppress ecdysone production. Consistent with this, these animals exhibited ecdysone levels similar to (or perhaps lower than) those of controls at pupariation, as reflected by *E75A* transcription, but they displayed increased *E75A* expression 3 h later, potentially because of a failure to downregulate ecdysone production ([Fig. 4](#DEV188110F4){ref-type="fig"}E; note that in [Fig. S2](Fig. S2), *E75A* expression was delayed when *EcR* was knocked down constitutively, suggesting that at pupariation in the temperature-shift experiment, *EcR-RNAi* had not had time to affect EcR levels). These data are consistent with a model in which an initial small 20E rise during L3 triggers a positive feedback circuit that generates the metamorphic PTTH/ecdysone surge. Surge-level 20E then suppresses PTTH production following pupariation ([Fig. 4](#DEV188110F4){ref-type="fig"}F).
DISCUSSION {#s3}
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EcR-mediated feedback induces developmental maturation by triggering PTTH neuronal activity {#s3a}
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The activation of a neuroendocrine signaling cascade triggers maturation onset in most animals. This activation is associated with body-size gating to ensure the fitness of the reproductive adult. In insects, attainment of 'critical weight' during the last larval instar is the main such checkpoint gating the transition to adulthood ([@DEV188110C25]). After this checkpoint, a larva becomes committed to maturing on a fixed schedule irrespective of further nutrition. Thus, critical weight likely reflects energy stores sufficient to survive the non-feeding maturation process (metamorphosis) and obtain a final adult body size that maximizes fitness ([@DEV188110C42]). Nutritional status is likewise a main factor permitting the entry into maturation in mammals ([@DEV188110C25]; [@DEV188110C32]). In humans, body weight correlates with the timing of menarche, which led to the use of the term 'critical weight' for the onset of reproductive cycles in humans ([@DEV188110C12]). Obese children enter puberty earlier than height-matched non-obese children, and malnutrition and lack of body fat can lead to delayed puberty ([@DEV188110C19]; [@DEV188110C48]). These observations suggest that the maturation gate reflects not body size per se but rather the amount of body fat, and thus that the neuroendocrine system controlling the timing of this process somehow assesses nutritional and energetic stores. Interestingly, the adipokine leptin regulates pubertal maturation in mammals ([@DEV188110C44]). Circulating leptin levels correlate with adiposity, and leptin-deficient humans and mice fail to undergo puberty. Leptin may therefore communicate body-fat levels to the neuroendocrine system controlling puberty, which could explain the link between childhood obesity and early onset of puberty. In insect larvae, the fat body is the main nutrient-storage and -sensing organ, releasing numerous nutrient-dependent insulin-regulating hormones ([@DEV188110C3]). Insulin is a stimulator of ecdysone production, thus coupling adipose-tissue nutrient sensing to the neuroendocrine maturation axis in *Drosophila* ([@DEV188110C5]; [@DEV188110C26]). Among the insulinotropic adipokines is Unpaired 2 (Upd2), which is structurally and functionally similar to human leptin. Upd2 acts through the JAK/STAT receptor Domeless (Dome) in GABAergic neurons that regulate insulin secretion from the insulin-producing cells (IPCs) in the brain, which are the primary source of circulating insulin ([@DEV188110C39]). Thus, related adiposity hormones that signal nutrition and energy storage influence the neuroendocrine events that lead to the onset of maturation in divergent systems.
Mammalian GnRH-producing neurons regulate the timing of puberty onset, and these cells are activated by the neuropeptide KISS1. The PTTHn, activated by the KISS1 ortholog AstA and its receptor AstA-R1 ([@DEV188110C9]), serve this function in *Drosophila*. This suggests conservation of the overall neuroendocrine architecture of the maturation-initiation system. AstA is regulated by nutritional intake, providing another potential link between energy status and maturation onset ([@DEV188110C17]). Furthermore, PTTHn-specific knockdown of *Insulin receptor* (*InR*) or *dome*, encoding the Upd2 receptor, produced size phenotypes in our screen ([Table S1](Table S1)), suggesting that the PTTHn integrate systemic nutrition-regulated signals and may also receive input via insulin from the IPCs themselves. Because PTTH controls developmental timing, and insulin is the main growth-regulatory factor, these results suggest that Upd2 may link growth and maturation by coordinating the activity of both the IPCs and the PTTHn. Knockdown of the amino-acid transporters Polyphemus and Minidiscs also induced strong growth effects in our screen, suggesting that the PTTHn may also sense nutrient status autonomously; in the IPCs, Minidiscs is required for inducing insulin secretion after intake of the amino acid leucine ([@DEV188110C23]).
This raises the key question of how these nutritional cues lead to the surge mode of GnRH/PTTH release that initiates maturation. Our findings suggest that ecdysone feedback, via EcR in the PTTHn, is the mechanism that induces the PTTH and ecdysone surge towards the end of larval development. This is further reinforced by EcR-mediated positive feedback on ecdysone production in the PG ([@DEV188110C30]). We propose that the triggering event that begins the feedback cycle is a small nutrient-dependent ecdysone peak early in the L3 stage. Nutritional signaling via insulin acts directly on the PG and is required for ecdysone production pre-critical weight but not post-critical weight ([@DEV188110C22]; [@DEV188110C46]). Furthermore, PTTH secretion is also controlled by nutrition and is required for normal attainment of critical weight ([@DEV188110C13]; [@DEV188110C45]), suggesting that PTTH acts together with insulin before attainment of critical weight to generate a small nutrient-dependent rise in ecdysone production at the beginning of L3. This small ecdysone peak upregulates *Ptth* via EcR and, under this scenario, corresponds to critical weight, which occurs ∼10 h after the L2-L3 transition. Thus, when ecdysone reaches the threshold corresponding to critical-weight attainment, it generates an irreversible, self-sustaining feedback activation of the neuroendocrine system by promoting the PTTH surge that triggers the maturation-inducing ecdysone pulse towards the end of L3 ([Fig. 4](#DEV188110F4){ref-type="fig"}F). This model is supported by findings showing that a small nutrient-sensitive ecdysone peak early in L3 does indeed signal critical weight ([@DEV188110C22]).
The main feature of this model is ecdysone feedback onto the PTTHn via a mechanism requiring EcR in these cells. EcR/Usp may regulate *Ptth* expression by direct binding to the *Ptth* enhancer or through downstream target transcription factors regulated by this complex. Many transcription factors are known to be targets of EcR ([@DEV188110C21]), and EcR may indirectly regulate *Ptth* expression by altering the expression of one or more of these. Indeed, RNAi against certain known EcR-induced transcription factors, such as *Hr39*, *Hr3* and *ftz-f1*, produced phenotypes in our screen, consistent with a possible role in *Ptth* regulation. Hr3 and Ftz-F1 are also known to participate in ecdysone regulation in the PG ([@DEV188110C36], [@DEV188110C37]), as is another nuclear receptor, Knirps ([@DEV188110C8]), which was also identified in our screen as a potential regulator of PTTH. Clarifying the precise mechanism by which EcR controls *Ptth* expression will be an interesting topic for future investigation.
Conserved neuroendocrine circuitry triggers maturation onset {#s3b}
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Early maturation is associated with smaller adult size in both flies and humans, as this event limits the juvenile growth period ([@DEV188110C4]; [@DEV188110C41]). The prevalence of precocious puberty has been linked with the increasing rates of childhood obesity; however, the mechanisms that gate GnRH secretion at the time of puberty are poorly understood ([@DEV188110C49]). The mammalian HPG axis controlling the onset of puberty is regulated by feedback control in which steroid hormones act to regulate the GnRH-expressing neurons, but whether these neurons themselves are direct steroid targets is still debated ([@DEV188110C20]). However, the KISS1-expressing neuronal population has also emerged as a possible link between sex steroids and the GnRH neurons ([@DEV188110C10]).
Many studies in *Drosophila* and other insects have explored the neuroendocrine PTTH-PG-ecdysone axis. The existence of feedback control between ecdysone and PTTH has been hypothesized for decades and is supported by studies of PTTH in other insects, especially in lepidopterans ([@DEV188110C18]; [@DEV188110C43]), in which hemolymph titers of PTTH and ecdysone are clearly correlated during the last larval instar ([@DEV188110C28], [@DEV188110C27]). A PTTH surge immediately precedes a rise in ecdysone levels, and gradual increases in ecdysone levels appear to reinforce the peak levels of circulating PTTH, suggesting that ecdysone might positively influence PTTH release. This is supported by findings that injection of ecdysone before an endogenous PTTH peak induces a premature rise in PTTH, whereas injection of ecdysteroid-22-oxidase, a potent enzymatic inactivator of ecdysteroids, inhibits this rise ([@DEV188110C29]). We demonstrate here an EcR-dependent positive-feedback mechanism, operating specifically within the PTTHn, that regulates the transcription of *Ptth*. Our findings show a mechanism by which steroid-mediated feedback signaling triggers the PTTH surge at the onset of metamorphosis, suggesting that feedback control is an evolutionarily conserved regulator of the neuroendocrine signaling that initiates the onset of maturation.
MATERIALS AND METHODS {#s4}
=====================
Fly husbandry {#s4a}
-------------
All animals were reared on a standard cornmeal diet (Nutri-Fly Bloomington formulation) at 25°C under 12-h light/dark cycle conditions, with 60% relative humidity, unless otherwise stated. Larvae of mixed sex were used in all experiments. The following fly lines were used: *UAS-EcR.gRNA* was generated in this study; *Ptth*-*GAL4, UAS-Dicer-2* and *NP423-GAL4, UAS-Dicer-2* were generous gifts from Pierre Léopold (Institut Curie, Paris, France); *Ptth-GAL4* and *UAS-Ptth::HA* ([@DEV188110C24]) and *phm-GAL4* ([@DEV188110C33]) were kindly provided by Michael O\'Connor (University of Minnesota, Minneapolis, MN, USA); *w^1118^* (\#60000), *UAS-EcR-RNAi* \#1 (\#37058), *UAS-EcR-RNAi* \#2 (\#37059), *UAS-Ptth-RNAi* (\#102043) and *UAS-torso-RNAi* (\#101154) were obtained from the Vienna *Drosophila* Resource Center (VDRC); *UAS-EcR.B1* (\#6469), *UAS-EcR.A* (\#6470), *UAS-EcR-RNAi* \#3 (\#50712) and *UAS-Cas9.P* (\#54594) were obtained from the Bloomington *Drosophila* Stock Center (BDSC).
Fly genetics {#s4b}
------------
To achieve CRISPR/Cas9-mediated disruption of *EcR* under GAL4/UAS control, we generated a UAS construct expressing two *EcR*-targeted gRNAs (below) in the backbone of vector pCFD6 ([@DEV188110C38]), which was obtained from AddGene (\#73915). The gRNA sequences were designed and checked for specificity and efficiency using online tools at <http://www.flyrnai.org/crispr/> and <http://targetfinder.flycrispr.neuro.brown.edu/>. Two sequences with high predicted efficiency and no off-target binding sites were chosen 232 base pairs apart within exon 3 of *EcR*, an exon shared between all *EcR* transcripts that encodes the protein\'s DNA-binding domain. Efficient induction of double-strand breaks should thus delete the DNA-binding domain and likely introduce frame-shift mutations as well, rendering the locus nonfunctional. Oligonucleotides containing the gRNA sequences were synthesized and inserted into *pCFD6*. The correct *pCFD6-UAS-EcR.gRNA* product was verified by sequencing, and transgenic animals were generated in-house and by Bestgene (Chino Hills, CA, USA). Fly stocks were constructed using standard techniques. gRNA sequence \#1: TTCATCGCACATTGGTTCTC; gRNA sequence \#2: GCAAGAAGGGACCTGCGCCA.
Synchronization of development {#s4c}
------------------------------
To synchronize development for timed experiments, parental flies were allowed to lay eggs for 2-4 h on an apple-juice agar plate coated with a thin layer of yeast paste; hours AEL was measured from the midpoint of this time. After 24 h, newly hatched L1 larvae were collected and transferred to vials containing standard food at a density of 30 larvae per vial.
*Ex vivo* incubation with 20E {#s4d}
-----------------------------
Four biological replicates of ten brains with an intact ring gland were dissected from synchronized L3 larvae at 110 h AEL in Schneider\'s insect medium (Sigma-Aldrich, S0146). The tissue was transferred to Schneider\'s medium containing 20E (Sigma-Aldrich, H5142) at 0, 5, 50, 500 or 5000 ng/ml and incubated for 6 h at room temperature (roughly 24°C). RNA was then extracted from tissue as described below.
Real-time quantitative PCR (qPCR) analysis {#s4e}
------------------------------------------
Four to six biological replicates of five whole larvae or ten dissected brains of each genotype were collected at the indicated times after egg laying (AEL) or puparium formation. Samples were flash-frozen in dry ice and stored at −80°C. The samples were thoroughly homogenized in 350 µl ice-cold lysis buffer containing 1% β-mercaptoethanol, and RNA was extracted using RNeasy mini kit (Qiagen) with DNase treatment according to the manufacturer\'s instructions. RNA concentrations from whole-larval samples were measured using a NanoDrop spectrophotometer (Thermo Fisher) and adjusted to 300 ng/µl. Synthesis of cDNA from RNA was carried out with a 10 µl RNA sample using a High-Capacity cDNA Transcription Kit (Applied Biosystems). qPCR reactions were performed using the QuantiTect SYBR-Green PCR Kit (Qiagen) on an Mx3005p qPCR system, and transcript levels were normalized against *RpL23*, which is stably expressed across tissues and larval stages ([@DEV188110C8]). Primers used for this study are listed in [Table S2](Table S2).
Immunostaining and confocal microscopy {#s4f}
--------------------------------------
For each genotype, 15 larvae were collected at the indicated times AEL. Each larva was rinsed in water and dissected in cold PBS, and tissues were fixed in 4% formaldehyde in PBS for 30 min, washed in PBS+0.1% Triton X-100 (PBST; one quick rinse followed by three 15-min washes with slow rocking motion), and blocked in PBST+5% normal goat serum (NGS) for at least 1 h. Blocking buffer was exchanged with PBT+5% NGS containing primary antibodies, and tissues were incubated overnight at 4°C. Samples were washed as before and incubated at 4°C overnight with secondary antibodies in PBST. Samples were washed again as above, washed in PBS to remove Triton X-100, and incubated in PBS at 4°C. Brains were mounted on poly-lysine-coated glass slides in ProLong Gold anti-fade reagent (Invitrogen). Fluorescence images were captured using a Zeiss LSM 800 confocal laser scanning microscope coupled with AiryScan technology and were then analyzed using the Fiji software package (<https://imagej.net/Fiji>). All samples for time-course data were imaged with identical settings. Quantifications of fluorescence intensity were performed by creating summed projections of each individual PTTHn followed by measurements of the anti-PTTH signal in the cell body and the anti-EcR signal in the nuclei using the following formula: integrated density−(area×mean background fluorescence). Mean fluorescence of brain tissue without positive PTTH and EcR signal in each individual projection was subtracted as background for each channel. Guinea pig anti-PTTH was purified using the Melon Gel IgG Spin Purification Kit (Thermo Scientific) from anti-PTTH serum generously provided by Pierre Léopold ([@DEV188110C54]); the purified IgG was used at 1:500. Rabbit anti-Phm (1:400) ([@DEV188110C33]) was a generous gift of Michael O\'Connor. Mouse monoclonal anti-EcR (clone Ag10.2) was obtained from Developmental Studies Hybridoma Bank and was used at 1 µg/ml. Secondary antibodies used were Alexa Fluor 555-conjugated goat anti-mouse, Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 647 goat anti-guinea-pig (Thermo Fisher, A21422, A1108 and A21450), all used at 1:200.
Ecdysteroid measurements by ELISA {#s4g}
---------------------------------
Ecdysteroid levels were measured using a competitive 20-hydroxyecdysone ELISA kit (Bertin Bioreagent Cayman, 501390). Four biological replicates of ten larvae from each genotype were collected at the indicated times AEL. Larvae were washed in water, dried on a Kimwipe, and weighed in groups of ten before they were transferred into empty Eppendorf tubes, flash-frozen on dry ice, and stored at −80°C. Extraction of ecdysone was performed by thoroughly homogenizing the frozen samples in 300 µl ice-cold methanol with a plastic pestle. Samples were centrifuged at 17,000 ***g*** for 10 min, and the supernatant was transferred and split into two Eppendorf tubes, each containing approximately 150 µl supernatant. Methanol from both tubes was evaporated in a vacuum centrifuge for 60 min, and pellets were re-dissolved by adding 200 µl supplied EIA buffer to one of the two Eppendorf tubes. This was vortexed, and the same 200 µl of EIA buffer was transferred to the second tube followed by further vortexing. The ELISA was performed using rabbit anti-20E-coated wells, acetylcholinesterase-conjugated 20E, and serial dilutions of 20E as a standard. In brief, mouse anti-rabbit-coated wells were washed five times with 200 µl EIA buffer. EIA buffer, rabbit anti-20E, acetylcholinesterase-conjugated 20E, and standards/samples were then added to their respective wells, and the plate was covered and incubated at 4°C in darkness overnight. The next day, wells were washed six times with 200 µl EIA buffer, and the activity of the remaining acetylcholinesterase enzyme was quantified by adding Ellman\'s reagent and reading absorbance at 405 nm every 15 min with an ELx800 plate reader (BioTek).
Measurement of developmental timing and pupal size {#s4h}
--------------------------------------------------
Synchronized larvae were assayed for pupariation timing by noting newly pupated animals at regular time intervals. The time at which 50% of animals had pupariated, P50, was determined by linear extrapolation between scores before and after reaching 50%. To measure pupal size, pupae were mounted on a glass slide, and images were captured with a Chameleon3 camera (FLIR Systems). Images were processed using a custom script ([@DEV188110C31]) in the MATLAB environment (MathWorks). The MATLAB script for quantification of pupal sizes has been published previously ([@DEV188110C31]).
Statistics {#s4i}
----------
Statistical analysis was performed in Prism software (GraphPad). Statistical differences between a control group and several other groups were analyzed by one-way ANOVA followed by Dunnett\'s multiple-comparisons tests; the difference between one control group and one other group was analyzed by an unpaired two-tailed Student\'s *t*-test. Bar graphs show mean±s.e.m. *P*-values are indicated as: \**P*\<0.05, \*\**P*\<0.01, \*\*\**P*\<0.001.
Supplementary Material
======================
###### Supplementary information
###### Reviewer comments
We thank Michael B. O\'Connor and Pierre Léopold for kind gifts of fly stocks and antibodies. We also thank the Bloomington *Drosophila* Stock Center, the Vienna *Drosophila* Resource Center, the University of Iowa Developmental Studies Hybridoma Bank, and AddGene for making other fly lines, antibodies and plasmids available.
**Competing interests**
The authors declare no competing or financial interests.
**Author contributions**
Conceptualization: C.F.C., T.K., E.T.D., K.R.; Methodology: C.F.C., T.K., S.N., E.T.D., K.R.; Software: S.N.; Investigation: C.F.C., T.K., S.N., E.T.D.; Resources: T.K.; Writing - original draft: M.J.T., K.R.; Writing - review & editing: C.F.C., T.K., M.J.T., K.A.H., K.R.; Visualization: C.F.C.; Supervision: K.A.H., K.R.; Funding acquisition: K.R.
**Funding**
This work was supported by a Novo Nordisk Fonden grant (16OC0021270 to K.R.) and Danish Council for Independent Research Natural Sciences (Natur og Univers, Det Frie Forskningsråd) grants (4181-00270 and 8021-00055B to K.R.). T.K. and K.A.H. were supported by a Villum Fonden grant (15365 to K.A.H.). Deposited in PMC for immediate release.
**Supplementary information**
Supplementary information available online at <https://dev.biologists.org/lookup/doi/10.1242/dev.188110.supplemental>
Peer review history {#s5}
===================
The peer review history is available online at <https://dev.biologists.org/lookup/doi/10.1242/dev.188110.reviewer-comments.pdf>
[^1]: Handling Editor: [Cassandra Extavour](https://dev.biologists.org/content/editor-bios/#extavour)
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
In a recent paper \[[@b1-v114.n03.a04]\] the reaction of slow neutrons with gasous ^3^He was studied experimentally and it was estimated that that in the experiment 46 Lyman alpha photons were produced for each slow neutron (wavelength 0.496 nm) absorbed in a chamber containing ^3^He at a pressure of 93 kPa. It was also noted that the most likely mechanism for the radiation produced was emission produced during the slowing down and charge exchange of the protons and tritons produced in the initial absorption of the neutrons via the reaction $$\left. {}^{3}\text{He} + n\rightarrow{}^{3}H^{+} + {}^{1}H^{+} + 764\ \text{keV} \right.$$and in principle it was possible to calculate the ratios of the various processes which could produce Lyman alpha radiation; i.e., collisions of ^3^H^+^ (tritons), ^1^H^+^ (protons), ^3^H (tritium) and ^1^H (hydrogen atoms) with the background ^3^He gas in the chamber as well as the spatial distribution of the radiation produced. This has now been done and the detailed description of the steps necessary to perform these calculations as well as some results of the calculations will be given here.
The paper is divided into the following parts: The first is a general analysis of the slowing down of the products produced by the neutron absorption described by [Eq. (1)](#fd1-v114.n03.a04){ref-type="disp-formula"}. The second is concerned with the detailed modelling of the slowing down process specific to the apparatus used in Ref. \[[@b1-v114.n03.a04]\]. The third gives results of calculations and comparison with the experiment of Ref. \[[@b1-v114.n03.a04]\]. The final section will discuss how the modelling may be improved or be used to design other experiments.
2. The Slowing Down Model
=========================
The reaction of [Eq. (1)](#fd1-v114.n03.a04){ref-type="disp-formula"} for a slow neutron produces a proton and a triton moving in opposite directions with the same momentum since practically no momentum is transferred from the neutron. Ignoring the electrons this means that the energies of each proton and triton initially will be 573 keV and 191 keV respectively. Although protons and tritons corresponding to each neutron absorption move in opposite directions, each proton or triton has no particular direction. Furthermore, at these energies the particles will tend to move in straight lines as they lose energy until their energy becomes low enough for elastic collisions to cause them to deviate from straight line paths.
With these considerations in mind, it is useful to investigate a model in which the protons or tritons move in straight lines in a gas (either ^3^He or ^3^He in combination with some other buffer gas) but may change charge as they move along that path and lose energy. Experimentally the charge exchange process has been studied in detail for protons or hydrogen atoms moving in beams for a number of gases \[[@b2-v114.n03.a04],[@b3-v114.n03.a04]\]. For atoms or ions moving in helium gas (^3^He or ^4^He) both the cross sections for charge loss (*σ*~10~) or charge gain (*σ*~01~) have been measured as a function of incident energy. The mean free path for a proton with given energy which picks up a charge is simply Δr = 1/*σ*~01~N where N is the number of atoms per unit volume which is proportional to the pressure. Similarly the mean free path for charge loss for the neutral atom will be the same with *σ*~01~ replaced by *σ*~10~. In addition to the cross sections for charge exchange, the energy loss of a particle per unit path length for protons in helium dE/dr (known as the stopping power) is also known as a function of proton energy \[[@b4-v114.n03.a04]--[@b6-v114.n03.a04]\]. Thus the energy loss that occurs in the time between each charge changing collision will be Δr dE/dr. To the extent that the particles move in straight lines the effective range of the particles in slowing down from energy E~0~ to E~f~ is simply the integral of 1/(dE/dr) from E~0~ to E~f~. In our model we assume that protons or tritons initially move a distance given by the mean free path calculated from the pressure and charge transfer cross section as indicated above. During it's flight in addition to charge transfer it may make a number of elastic or inelastic collisions with the gas which do not result in charge transfer but cause energy loss. The total energy loss can be calculated from the distance travelled and the stopping power. The neutral particle is then assumed to move a distance calculated using the pressure and the charge loss cross section *σ*~01~ and as before, the energy loss for moving that distance is calculated from the stopping power. The two collisions constitute a charge changing cycle resulting in an energy loss which is the sum of the losses in each collision and a change in position which is the sum of the mean free paths. The process is than repeated recording the distance travelled in each charge changing cycle and the energy lost until either the energy of the particle has reached a lower limit or the distance travelled has exceeded a predetermined value. By computing each charge changing collision separately we effectively integrate over the reciprocal of the stopping power by summing over the individual path lengths between collisions and the energy loss for each collision and the total distance travelled corresponds to the effective range. The total number of charge changing cycles which occur during the slowing down process is also obtained. The model described above differs from the way slowing down of tritons and protons following slow neutron absorption via [Eq. (1)](#fd1-v114.n03.a04){ref-type="disp-formula"} is usually viewed. The normal procedure \[[@b5-v114.n03.a04]\] is to describe the collisions as heavy particle elastic collisions ignoring the fact that at high energies most of the energy loss is due to the interaction of the fast particle with the atomic electrons of the background gas. This is taken into account by assigning an energy loss to the distance the particle has travelled using the known stopping power. The main object is to determine where the energy loss of the moving particle occurs and the number of electrons produced during the slowing down process. Here we are interested in where during the slowing down process Lyman alpha radiation is produced. We ignore the elastic collisions and hence ignore any deviation from straight line trajectories (straggling), but correctly calculate the energy loss and effective range by using the measured stopping power.
The next step is to estimate the probability of producing Lyman alpha radiation during each charge changing cycle. When the particle is charged, radiation can be produced by direct charge transfer, i.e., $$\left. p + \text{He}\rightarrow H(2p) + \text{He}^{+}\rightarrow H(1s) + \text{Lyman}\ \text{alpha} \right.$$
When the particle is neutral radiation can be produced by direct excitation, i.e. $$\left. H + \text{He}\rightarrow H(2p) + \text{He}\rightarrow H(1s) + \text{Lyman}\ \text{alpha} \right.$$
[Equations (2)](#fd2-v114.n03.a04){ref-type="disp-formula"} and [(3)](#fd3-v114.n03.a04){ref-type="disp-formula"} are written only for protons. We assume that the same processes occur for tritons and tritium atoms and that the cross sections for Lyman alpha production as well as those for charge exchange are the same as those for protons or H atoms at equal energies \[[@b7-v114.n03.a04]\]. The cross sections for all of these processes have been measured as a function of energy and from these cross sections the probability of producing Lyman alpha radiation as neutral or charged particles move a distance Δr can be calculated.
Calculations of the formation of Lyman alpha for protons slowing down in helium is a straightforward procedure, but for the tritons and tritium additional modifications must be made. The stopping power for tritons or tritium used was that for protons moving in helium at one third of the energy. At energies above 10 keV this is reasonable since stopping power has been measured for protons, deuterons and tritons and the results are the same for equal velocities \[[@b7-v114.n03.a04]\]. At energies below 10 keV the data on stopping power may not be reliable \[8)\].
3. The Monte Carlo Model
========================
In order to explain how the experiment is modelled it is useful to review the way the results reported in Ref. \[[@b1-v114.n03.a04]\] were obtained. A diagram of the apparatus used in Ref. \[[@b1-v114.n03.a04]\] is reproduced here in [Fig. 1](#f1-v114.n03.a04){ref-type="fig"}. The neutrons enter the 25 mm cylindrical reaction chamber where protons and tritons are produced in a beam of less than 4 mm diameter. Lyman alpha photons are detected which pass through the MgF~2~ window at the top end of the chamber. However, before entering the cylinder the neutrons must first pass through a "dead space" of length Ldead which contains helium at the same pressure and are attenuated there but no protons or tritons produced enter the cylinder and consequently no Lyman alpha radiation produced in the dead space will reach the detector.
The incident neutron flux and its wavelength were measured. From the measured wave length and previous experimental results assuming that the absorption cross section in ^3^He follows a 1/v dependence the cross section at this wavelength is determined. Knowing the neutron cross section and the pressure, the number of neutrons/sec absorbed in the 25 mm path length within the reaction chamber was determined as follows. Let I~0~ be the incident neutron flux. Then $$I = I_{0}e^{- \text{Ldead}\ \sigma N}(1 - e^{- d\sigma N})$$will be the flux absorbed in the reaction cell where *σ* is the neutron cross section, N the density of helium atoms in the chamber and Idead and d are the lengths of the dead space and the diameter of the reaction chamber (25 mm) respectively. The first term represents the flux transmitted through the dead space and the second term the flux absorbed within the chamber. Note that I is the absorption produced in the total path length within the reaction chamber.
It was assumed that all photons observed were produced within the reaction chamber and have no preferred direction. If this is true the number of photons observed originating at a particular point will depend on the ratio of the solid angle subtended by the window of the detector at the top of the chamber from that point within the cylinder to 4π. This ratio on average will be the same as that for a point at the center of the cylinder. The solid angle subtended by the detector window for this point is simply 2π(1 − cosΘ) where Θ is the angle whose tangent is the ratio of the window radius (2.7 mm) to the distance of the window from the center of the chamber.
Finally, the attenuation of the window and the efficiency of the detector was accounted for. Measurements were made to estimate these quantities.
We take as a coordinate system *x*, *y* and *z* coordinates with the origin at the center of the 4 mm cylinder describing the neutron path and midway between the walls of the 25 mm cylindrical reaction chamber where radiation is produced with the *z* direction along the axis of the 4 mm cylinder and *x* along the axis of the 25 mm cylinder. *x*~up~ and *x*~down~ are the lengths from the origin to the detector window at the top and to the bottom of the reaction chamber respectively. In our model we assume that there are T point sources of protons or tritons distributed along the *z* axis. The basic idea is to choose the starting points for these point sources randomly and to follow the path of each proton or triton within the chamber as it undergoes charge changing collisions until it either reaches a preset minimum energy or collides with the walls or ends of the reaction chamber. Since the particles are assumed to be emitted isotropically their initial directions are also chosen randomly and they are assumed to move in the same direction until their trajectories are terminated either by falling below a minimum energy or hitting the walls or ends of the 25 mm diameter cylinder containing ^3^He. As they are slowed down within the reaction chamber the probability of emitting Lyman alpha radiation at that point is calculated from the slowing down model and the probability of it's reaching the detector is estimated.
Two simplifying approximations which can be improved have been made it setting up this model. First, we have ignored the finite dimension of the neutron beam and treated it as a line source. Second, our method of calculating the solid angle for radiation emitted at a point within the chamber is only approximate. There have been a number of detailed calculations of this for various geometries \[[@b9-v114.n03.a04],[@b10-v114.n03.a04]\] and they show that a moderately good approximation to make here is to assume that the effective solid angle for a point source of radiation within the chamber depends only on the distance of the source point from the detector window and we used this approximation to estimate the number of photons entering the detector. A more detailed calculation is possible.
The setting up of the Monte Carlo calculation is straightforward. Depending upon whether protons or tritons are being tracked the incident energy is set to 573 keV or 191 keV and the particle mass to either 1 or 3 u (unified atomic mass unit). Next three random numbers are generated. The first is used to define the starting point of the track on the *z* axis. Since the total path of the neutron beam within the cylinder is d (25 mm) and we have chosen the center of the cylinder as the zero of our coordinate system we choose the initial starting point randomly between − d/2 and d/2. The second and third random numbers define a random direction by choosing a value of cos θ between − 1 and 1 and ϕ between 0 and π. The direction of the particle is then specified after the first charge changing collision by: $$x = \Delta x = \Delta r\ \sin\theta\ \cos\ \phi$$ $$y = \Delta y = \Delta r\ \sin\theta\ \sin\phi$$ $$z = \Delta z = \Delta r\ \cos\theta$$where Δr is the distance travelled before the first collision and the direction is assumed to remain the same for all subsequent collisions. The path of the particle is then followed as charge changing collisions occur and the energy loss in each collision, the estimated total amount of Lyman alpha radiation formed during the path length Δr, and the fraction of that radiation reaching the detector are calculated and recorded. As in the slowing down model charge changing cycles are then followed until the particle either strikes the walls of the reaction chamber or slows down below a preset minimum energy. The criteria for striking the walls or top or bottom of the cylindrical chamber are simply: $$z^{2} + y^{2} \geq d^{2}/4$$or $$x \geq x_{\text{up}}$$or $$- x \geq x_{\text{down}}$$
Once a given track is terminated, either by hitting the walls of the reaction chamber or by the energy falling below the cutoff energy, three more random numbers are generated and a new track begins.
One other correction is made to the procedure described above. Since each track starts at some distance along the *z* axis from its entrance into the 25 mm cylinder (at *z* = − d/2) there are more protons and tritons to be produced at *z* = − d/2 than at *z* = d / 2. We correct for this by computing the flux reduction for a track starting at a given value of *z* and weighting the Lyman alpha production produced by a given track by this reduction.
As will be shown in the next section, typically about 1500 charge changing cycles occur for protons and 2000 for tritons for a cut off energies of 1 keV and 3 keV respectively. For the Monte Carlo calculations 2000 tracks were enough to get a good estimate of the Lyman alpha radiation produced at a given pressure and the calculations typically required about 10 to 20 seconds.
4. Results for the Slowing Down Model
=====================================
The cross sections for charge exchange and for Lyman alpha production via [Eq. (2)](#fd2-v114.n03.a04){ref-type="disp-formula"} and [Eq. (3)](#fd3-v114.n03.a04){ref-type="disp-formula"}. were obtained from the ORNL 'Redbook' compilation \[[@b11-v114.n03.a04]\] which is available on line. The cross sections have been fitted to Chebyshev polynomials so that only 11 numbers are needed to obtain a cross section at any energy within the range of the tabulated values. The cross sections for charge exchange were available for the entire energy range from from 600 keV to 1 keV but the cross sections for Lyman alpha production via [Eq. (2)](#fd2-v114.n03.a04){ref-type="disp-formula"} and [Eq. (3)](#fd3-v114.n03.a04){ref-type="disp-formula"} were only for energies below 90 keV for production by atoms and below 300 keV for production by ions. No attempt was made to estimate Lyman alpha production outside of these ranges.
The stopping power used here for protons in helium came from the NIST compilation of ranges \[[@b4-v114.n03.a04]\] and stopping powers which is also available on line. The tabulation only extends to a lower energy of 1 keV so no results were estimated for final energies below 1 keV for protons and below 3 keV for tritons.
In order to verify the validity of our slowing down model the ranges in helium for protons at 573 keV and tritons at 191 keV were estimated from the NIST compilation for a helium pressure of 101 kPa. The results were 50 mm for ^4^He, 67 mm for ^3^He for protons and 19 mm for tritons in ^3^He.[1](#fn1-v114.n03.a04){ref-type="fn"} Calculating directly the distance traveled for protons slowing down to 1 keV and tritons to 3 keV in ^3^He we obtained distances of 67 mm for protons and 18 mm for tritons in good agreement with the compilation values. It is important to note that the effective ranges for ^4^He and ^3^He are different.
For protons although our model predicts that there will be about 1500 charge changing cycles in slowing down from 573 keV to 1 keV, charge exchange is relatively unimportant as an energy loss mechanism at the higher energies. This is due to the fact that at higher energies the cross section for losing charge is much larger than that for gaining charge \[[@b2-v114.n03.a04]\]. It is only after slowing down to an energy where the two cross sections are equal (\~ 100 keV) that energy loss via charge exchange becomes important. This is shown in [Fig. 2](#f2-v114.n03.a04){ref-type="fig"} where the energy loss for protons in ^3^He at atmospheric pressure (101 kPa) is shown as a function of the distance travelled. The distance travelled before charge exchange is large, i.e., greater than 50 mm at atmospheric pressure. The situation for tritons is completely different as is shown in [Fig. 3](#f3-v114.n03.a04){ref-type="fig"}. There the number of charge changing cycles increases gradually with the energy loss and, as expected, the distance travelled during the slowing down process is much less.
The model predictions for Lyman alpha production (number of photons produced for each neutron absorbed as a function of distance travelled) by atoms or ions is shown for protons and for tritons both at atmospheric pressure in [Figs. 4](#f4-v114.n03.a04){ref-type="fig"} and [5](#f5-v114.n03.a04){ref-type="fig"}. What is plotted is the total number of photons produced after the particle has travelled a given distance. The important thing to note here is that for protons, practically no Lyman alpha radiation is produced until the protons have travelled a distance of about 60 mm whereas for tritons there is appreciable production when the particle has travelled a much shorter distance. Within our model, the distance travelled by a particle in losing a given amount of energy will be inversely proportional to the pressure. At lower pressures the particles must travel further before they produce Lyman alpha radiation. In an infinite media the total number of photons produced will be independent of pressure. Nevertheless, the total amount of energy for Lyman alpha production is a small fraction of the total energy loss. These results are summarized in [Table 1](#t1-v114.n03.a04){ref-type="table"} which gives the total number of charge changing cycles, the number of Lyman alpha photons produced by neutrals and ions and the percent of the total energy loss due to Lyman alpha production.
5. Results for the Monte Carlo Model
====================================
There are two ways that we can compare the results of the Monte Carlo model with the experiment described in Ref. \[[@b1-v114.n03.a04]\]. We could, given an incident neutron flux and cross section, use the model to estimate the count rate of photons entering the window of the photon detector. Alternatively we could use the model to predict the total number of photons produced for each absorbed neutron and compare this with the results of Ref. \[[@b1-v114.n03.a04]\]. We have chosen the second alternative since the key results of Ref. \[[@b1-v114.n03.a04]\] are the observations that the reaction yield of Lyman alpha photons per neutron absorption is pressure dependent and much greater than one.
At the pressures used in Ref. \[[@b1-v114.n03.a04]\], the model predicts that there will be no Lyman alpha produced by protons. This is understandable since one can see from [Fig. 2](#f2-v114.n03.a04){ref-type="fig"} that at atmospheric pressure a proton must travel at least a distance of approximately 55 mm before any Lyman alpha is produced whereas the largest distance that a proton can travel within the reaction chamber is 51 mm. This will also be true at lower pressures, but at higher pressures some signal is to be expected from the protons. We find that at 200 kPa the model predicts for that for 2000 proton trajectories 10 photons would be produced per neutron absorbed even though 1844 of the protons will have hit the walls of the reaction chamber. For slowing down in ^4^He 16 would be produced per neutron absorbed and 1670 would have hit the walls owing to the smaller effective range in ^4^He.
For tritons at atmospheric pressure (101 kPa) the model predicts that 60 photons will be produced for each neutron absorbed which is less than the infinite medium prediction shown in [Table 1](#t1-v114.n03.a04){ref-type="table"} of 154. The reason is that for 2000 trajectories 1443 hit the walls of the chamber before they reached the cutoff energy of 3 keV.
The pressure dependence of the model calculation is compared with that of the experiment of Ref. \[[@b1-v114.n03.a04]\] in [Table 2](#t2-v114.n03.a04){ref-type="table"} and in [Fig. 6](#f6-v114.n03.a04){ref-type="fig"}. Also shown in the table are the number of triton trajectories that terminate before reaching the cutoff energy of 3 keV. The model results are in moderate agreement with the experiment. At the higher pressures the model predicts approximately the same number of photons/neutrons as the experiment even though over half of the trajectories are terminated. The main difference is that the model predicts a more rapid falloff of the number of photons produced at lower pressures than is observed. Note that the model predicts that all of the tritons will have hit the walls at 40 kPa but that 6 photons/neutron are produced.
At higher pressures one might expect the number of photons observed to approach the infinite medium prediction of 154 photons/neutron since all of the tritons will be slowed down to 3 keV at higher pressure. While this is true, with increasing pressure neutron absorption is much more likely to occur in that part of the chamber nearest the source and thus close to the walls of the cylinder. At high pressure we would expect half of the tritons to hit the wall and the other half to produce photons as if they were moving in an infinite medium. The results shown in [Table 2](#t2-v114.n03.a04){ref-type="table"} at the higher pressures show this. The number of photons/neutron produced is roughly one-half of the infinite media results and the number hitting the wall is larger than at lower pressures and approximately one-half of the total number of trajectories.
6. Summary and Suggestions for Future Work
==========================================
The results described in the previous sections are an initial attempt to understand the production of Lyman alpha radiation observed in Ref. \[[@b1-v114.n03.a04]\].
The relatively good agreement of the model calculations at higher pressures with the experimental results is rather gratifying in view of the number of approximations that have been made both in analysing the experiment and in setting up the model calculations. The fact that the calculations underestimate the production at lower pressures seems to indicate that there is another source of Lyman alpha radiation that is not accounted for by the model. One possibility is that no production of Lyman alpha by neutrals was assumed to occur at energies above 90 keV.
Two unanticipated results of the calculations are the prediction that with the present apparatus at pressures below one atmosphere all of the signal is produced by tritons and that different results would be obtained for slowing down in ^4^He due to the change in range. These predictions can be checked using the present apparatus by working at higher pressures and by using a mixture of ^4^He and ^3^He. Experiments using such mixtures are currently being done and will be reported in a future publication.
From the standpoint of designing a cold neutron detector using Lyman alpha radiation, the model described here will be useful. Provided the cross sections and stopping powers for any mixture of gases are known or can be estimated the slowing down model can be used to estimate the total Lyman alpha production and the Monte Carlo model modified to provide predictions of the signal strength at any given detector and neutron source configuration.
Dr. Alan K. Thompson made a number of useful suggestions during the course of this work and substantially improved the quality of the presentation. J.W.C. acknowledges support from OPM under contract No. CSA-3115486.
The ranges given in Ref. \[[@b4-v114.n03.a04]\] are in units of gm/cm^2^. Therefore the range in ^4^He will be 3/4ths the range in ^3^He.
**About the authors:** John W. Cooper is a Faculty Research Assistant at the Institute for Physical Science and Technology, University of Maryland and a former Physics Editor of the NIST Journal of Research. Michael A. Coplan, a Professor at Maryland, is Associate Director of the Institute for Physical Science and Technology and Director of the University's Chemical Physics Graduate Program. Patrick Hughes is a post-doctoral researcher in the Chemical Physics Graduate Program at Maryland and holds a post doctoral fellowship at NIST's National Center for Neutron Research. The National Institute of Standards and Technology is an agency of the U.S. Department of Commerce.
![Drawing of the reaction cell used in Ref. \[[@b1-v114.n03.a04]\].](v114.n03.a04f1){#f1-v114.n03.a04}
{#f2-v114.n03.a04}
{#f3-v114.n03.a04}
{#f4-v114.n03.a04}
{#f5-v114.n03.a04}
![A comparison of the pressure dependence of number of L*α* photons produced per neutron absorbed from the experiment of Ref. \[[@b1-v114.n03.a04]\] and from the model calculation.](v114.n03.a04f6){#f6-v114.n03.a04}
######
Lyman alpha photons/neutron produced by tritons and protons and the percent of the total energy loss due to Lyman alpha production at a pressure of 101 kPa
Charge changing Cycles Photons produced by Energy loss %
------------------------ --------------------- --------------- ---- ----- -------
Tritons 1841 128 26 154 0.082
Protons 1411 85 18 103 0.018
######
Comparison of the pressure dependence of the number of Lyman alpha photons produced in the experiment of Ref. \[[@b1-v114.n03.a04]\] and the model calculation. Results are based on 2000 trajectories of tritium at each pressure. The last column gives the number of trajectories which hit the walls before reaching the cutoff energy of 3 keV
Pressure (kPa) Photons/Neutron (Model) Photons/Neutron (Ref. \[[@b1-v114.n03.a04]\]) Wall hits
---------------- ------------------------- ----------------------------------------------- -----------
10 0 0 2000
20 0 0 2000
30 2 26 2000
40 6 31 2000
50 18 35 1930
60 26 38 1819
70 36 40 1719
80 45 42 1620
90 52 45 1528
100 60 46 1443
500 78 -- 862
660 80 -- 842
| {
"pile_set_name": "PubMed Central"
} |
All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Global temperatures are increasing due to greenhouse-gas emissions from fossil-fuel combustion and from deforestation, and this is an important threat to aquatic systems \[[@pone.0188791.ref001]\]. The Intergovernmental Panel on Climate Change (IPCC) estimates under its most-likely scenario without mitigation (RCP8.5) that by the year 2100 average mean temperature is "likely" (66% probability) to increase by 4.8°C over the 1996--2005 average, and that carbon dioxide (CO~2~) levels will reach \~1000 parts per million by volume (ppmv) \[[@pone.0188791.ref001]\]. Because global mean temperature is mainly affected by temperature over the oceans, the expected increases over continents are higher than these values. In Amazonia, year 2100 temperatures in the June-August period would be 6-8°C over the 1996--2005 average under the same scenario (\[[@pone.0188791.ref001]\], p. 1343). Global warming can also alter rainy events in some regions \[[@pone.0188791.ref002]\]. In the Amazon Basin, effects of precipitation changes may vary between regions; for example, an increase in rainfall with subsequent flooding is expected in Western Amazonia, and an increased frequency of droughts is expected in the Central and Lower Amazon areas \[[@pone.0188791.ref002]\]. Therefore, climate change may also alter flood-pulse dynamics and cause changes in the composition of riparian vegetation \[[@pone.0188791.ref003]\], with consequent alteration of aquatic communities and detritus input in low-order streams \[[@pone.0188791.ref004]--[@pone.0188791.ref006]\]. Additionally, in freshwater environments flooding results in an increase of water velocity, depth, and hydrological connectivity and may positively affect aquatic fauna and the leaf-breakdown process \[[@pone.0188791.ref007]--[@pone.0188791.ref008]\].
As a result of global warming, modifications of leaf-morphological traits, structural and secondary compounds (e.g., tannins) in leaves are expected to increase \[[@pone.0188791.ref009]--[@pone.0188791.ref011]\], reducing nutrient concentrations and decreasing litter palatability for decomposer communities \[[@pone.0188791.ref012]\]. Thus, climate and litter changes can also modify metabolic activities of decomposer communities, and, consequently, trophic interactions in streams \[[@pone.0188791.ref013]--[@pone.0188791.ref017]\]. In low-order streams, the availabilities of carbon and nutrients (e.g., nitrogen and phosphorus) for organisms are driven by leaf-litter breakdown \[[@pone.0188791.ref018]\]. Therefore, global warming can have important effects on aquatic biogeochemical cycles \[[@pone.0188791.ref004], [@pone.0188791.ref011], [@pone.0188791.ref019]\]. These facts increase the importance of studies that predict the effects of climate change on communities and on ecological processes (e.g., leaf-litter breakdown) in aquatic ecosystems \[[@pone.0188791.ref020]\].
In general, increases in abundance, biomass, and activity of invertebrate shredders and microorganisms (mainly fungi and bacteria) accelerate leaf-litter breakdown \[[@pone.0188791.ref021]--[@pone.0188791.ref022]\]. Leaf-litter trends determine palatability, where lower leaf hardness (resulting from decreases of fiber, lignin and cellulose concentration) and secondary compounds (e.g., polyphenols and tannins), and higher nutrient concentrations, can increase leaf-breakdown rates \[[@pone.0188791.ref021], [@pone.0188791.ref023]\]. Due to high plant diversity in tropical systems, studies of the interactions of shredders, microorganisms and litter quality are fundamental for understanding the leaf-breakdown process (e.g., \[[@pone.0188791.ref024]--[@pone.0188791.ref026]\]). Microorganism colonization of leaves, which increases nutritional quality, also increases leaf-litter colonization and consumption by invertebrate shredders. However, little information is available with respect to synergistic effects of increased temperature and CO~2~ on decomposers (shredders and microorganisms), especially in tropical ecosystems \[[@pone.0188791.ref027]\].
Ecological aspects of shredder invertebrates in tropical ecosystems have been tested in experimental studies, mainly using *Phylloicus* caddisflies (e.g., \[[@pone.0188791.ref025], [@pone.0188791.ref028]--[@pone.0188791.ref029]\]). This genus is present in South and Central American countries \[[@pone.0188791.ref030]\]. Specifically, *Phylloicus elektoros* the species used in our study, has been recorded in Brazil, Peru and Venezuela \[[@pone.0188791.ref030]--[@pone.0188791.ref031]\]. Larvae of *Phylloicus* feed on leaf litter and build cases using leaf discs, thus increasing leaf-breakdown rates through leaf fragmentation \[[@pone.0188791.ref032]\]. However, leaf consumption by *Phylloicus* may be negatively influenced by low litter palatability \[[@pone.0188791.ref033]\], temperatures beyond tolerance level \[[@pone.0188791.ref014], [@pone.0188791.ref027]\], intra-specific competition and predation \[[@pone.0188791.ref025]\].
Microorganisms are also influenced by climate change and litter quality \[[@pone.0188791.ref034]\]. In general, fungi and bacteria have higher activity with increased temperature \[[@pone.0188791.ref035]\], but litter quality effects (mainly from increased atmospheric CO~2~) are not clear. Some studies show a decrease of microorganism biomass and activity resulting from increased leaf structural compounds \[[@pone.0188791.ref034], [@pone.0188791.ref036]\]. However, no difference has been observed in leaves growing under different CO~2~ conditions, and water temperature has been found to be the major factor affecting the colonization process \[[@pone.0188791.ref037]\]. In a previous study using the same conditions as the present study (experimental chambers; see [methodology](#sec002){ref-type="sec"}), but with leaves not grown in simulated conditions of temperature and CO~2,~ Martins et al. \[[@pone.0188791.ref027]\] reported that fungal biomass was positively affected by litter quality and negatively affected by increases in temperature and CO~2~.
We evaluated the effects of different scenarios combining increased temperature and CO~2~ on leaf detritus and decomposers. We analyzed leaf consumption of *Hevea spruceana* (Benth) Müll. grown under different air temperatures, CO~2~ concentrations and water availabilities, by shredders (*Phylloicus elektoros*) and microorganisms in experimental chambers simulating climate change. In addition, we also assessed shredder survival and fungal biomass (as ergosterol concentration) under simulated conditions. In general, leaf-litter consumption by shredders and microorganisms increases at high temperatures, but after passing the temperature-tolerance level, consumption decreases due to high metabolic cost. Thus, our hypotheses are that simulated climate change: i) decreases leaf-litter quality, ii) decreases survival and leaf consumption by shredders, and iii) increases microbial decomposition and fungal biomass.
Methodology {#sec002}
===========
Ethics statement {#sec003}
----------------
Fieldwork was carried out with authorization from the Divisão de Suporte às Estações e Reservas (DSER) of the Instituto Nacional de Pesquisas da Amazônia (INPA). Shredders were collected with authorization and approval of the Brazilian Biodiversity Authorization and Information System (SISBIO; Permit 43934--1). Fieldwork did not involve endangered or protected species.
Microcosms {#sec004}
----------
We performed the experiment in four microcosm chambers (4.05 m × 2.94 m) that simulated air temperature and CO~2~ changes in relation to a real-time control following current conditions in Manaus, Amazonas, Brazil ([S1 Fig](#pone.0188791.s002){ref-type="supplementary-material"}). In accord with estimates of air temperature and CO~2~ for the year 2100 \[[@pone.0188791.ref038]\], the chambers were named: i) Control: real-time current conditions; ii) Light: increases of \~1.5°C and \~220 ppmv in relation to the control condition; iii) Intermediate: increases of \~3.0°C in and \~420 ppmv in relation to the control condition; iv) Extreme: increase of \~4.5°C and \~870 ppmv in relation to the control condition (for temperature and CO~2~ concentration, respectively). Note that these dominations, which have been adopted for all work in these chambers, are overly cautious, since the "Extreme" condition (\~4.5°C) is based on the most likely scenario for global mean temperature in the absence of mitigation, and, as noted earlier, the temperature in Manaus would increase by 6--8°C in the hottest part of the year under this scenario \[[@pone.0188791.ref001]\]. For more information regarding the chambers see \[[@pone.0188791.ref027], [@pone.0188791.ref039]--[@pone.0188791.ref040]\]. Due to the high cost of building and maintaining chambers with real-time variations in air temperature and CO~2~, our design was not completely orthogonal (see more details in \[[@pone.0188791.ref041]\]). Accordingly, a potential malfunction or any other problem affecting a single chamber would affect all arenas and their growing plants \[[@pone.0188791.ref027]\]. However, to avoid this potential problem, values of temperature and CO~2~ were recorded every 2 min in all chambers and have high precision \[[@pone.0188791.ref027]\]. The microcosm was constructed by BIOTEC following instructions for a vivarium cleanroom system with adaptations to increases of temperature and CO2.
Leaf-litter {#sec005}
-----------
In this experiment (see [S1 Protocol](#pone.0188791.s001){ref-type="supplementary-material"} and [S1](#pone.0188791.s002){ref-type="supplementary-material"}--[S4](#pone.0188791.s005){ref-type="supplementary-material"} Figs), we used leaves of *Hevea spruceana* that were grown under different scenarios combining increased temperature and CO~2~. In the four experimental chambers, plants were subjected to field-capacity soil (non-flooded soil) and flooded soil, the latter with seedlings being flooded up to 5 cm above the soil level. *Hevea spruceana* is a medium-sized rubber tree (up to \~ 27-m height) found on floodplains in the Amazon region \[[@pone.0188791.ref042]\]. Seeds of *H*. *spruceana* are important food resources for adult fish, among others, including the commercially important "tambaqui", *Colossoma macropomum*. Moreover, *H*. *spruceana* can be used for rubber production, although it is of relatively poor-quality owing to high proportions of resins \[[@pone.0188791.ref042]\].
Seedlings (six months) of *H*. *spruceana* were collected in riparian vegetation along the Tarumã-Mirim stream, a tributary of the Rio Negro (03°00'27.47" S, 60°12'14.97 W). Thereafter, plants were acclimatized for a month in a greenhouse and subsequently distributed among the four climatic chambers (Control, Light, Intermediate, and Extreme). Twenty plants were placed in each chamber, which were divided into two soil conditions (flooded and non-flooded). Thus, we had eight initial treatments: 4 chambers × 2 soil conditions. Seedlings were subjected to treatments over a 115-day period, during which fallen leaves were collected. Leaves in all treatments were separately air-dried and stored in an air-conditioned room (20°C). All leaves from each treatment (n = 8) were mixed. We adopted this strategy because plants in each treatment were closely spaced. However, the plants growing on different soil but in the same chamber were spatially isolated. We determined leaf chemical composition using leaves that had not been incubated in the stream (non-leached and non-conditioned). Organic carbon (%) was estimated from the ash-free dry mass. Nitrogen concentration (N; %) was obtained from dry combustion and gas chromatography mass spectrometry analysis \[[@pone.0188791.ref043]\]. Phosphorus concentration (P; %) was measured by nitric-perchloric digestion and subsequently, determined by spectrophotometry \[[@pone.0188791.ref043]\]. Cellulose and lignin were measured gravimetrically using acetone and sulfuric acid \[[@pone.0188791.ref044]\].
Before starting the leaf-breakdown experiment, air-dried leaves of *H*. *spruceana* were incubated for 14 days in the Barro Branco stream (Reserva Ducke---02°55\' to 03º01\'S; 59º53\' to 59º59\'W) using litter bags with fine mesh (0.5 mm) in order to leach soluble compounds and allow conditioning by microorganisms \[[@pone.0188791.ref018]\]. We incubated leaves in the stream to allow natural conditioning before the leaves were broken down by shredders and microorganisms. The stream has dense riparian forest, acidic water (pH = 4.6 ± 0.1), high dissolved oxygen concentration (6.6 ± 0.1 mg l^-1^), low electrical conductivity (10.7 ± 0.4 αS cm^-1^) and mean temperature of 24.5 ± 0.5°C \[[@pone.0188791.ref029]\]. The leaves were then cut into discs (14 mm in diameter), avoiding the midrib.
Shredders {#sec006}
---------
We performed the experiment with *P*. *elektoros* larvae. This species is an important shredder in Amazon streams \[[@pone.0188791.ref029]\]. The larvae are usually abundant in pool mesohabitats \[[@pone.0188791.ref030]\], favoring their use in laboratory experiments. Larvae of *P*. *elektoros* were also collected from the Barro Branco stream. We collected 120 individuals manually, avoiding last-instar individuals. Subsequently, individuals, were acclimatized for 48 h in plastic containers containing calcined sand (burned at 450°C for 4 h), bottled water (ÁguaCrim®), and partially decomposed leaves from the stream \[[@pone.0188791.ref027]\].
Experiment {#sec007}
----------
The experiment lasted seven days, and we used arenas (plastic bottles 11.95-cm height × 9.80-cm diameter; 700-ml volume) with bottled water (500 ml), calcined sand (\~ 1 cm height) and constant aeration. We had 15 replicates for each treatment, totaling 120 arenas (4 chambers × 2 soil conditions × 15 replicates). In each chamber, we used only leaves that had grown under the temperature and CO~2~ conditions of the chamber. For example, in the Control chamber, we had 30 arenas with leaf discs from plants grown under control conditions (flooded soil = 15; non-flooded soil = 15; see Appendix). Each arena had disks for only one treatment.
We included five disks in a single treatment in each arena. These disks were used to obtain the total leaf-breakdown rate (by shredders and microorganisms) and were placed on the sands with pins. Six arenas for each treatment had litter bags attached (10 cm × 10 cm; 0.5-mm mesh) with five disks to estimate the leaf-breakdown rate by microorganisms, totaling 12 litter bags per chamber. The disks from the litter bags were used to estimate fungal biomass by ergosterol, filtering with methanol and potassium hydroxide, subsequently analyzed by high-performance liquid chromatography (HPLC), in accord with \[[@pone.0188791.ref045]\]. Only one *P*. *elektoros* individual was placed in each arena to avoid intraspecific interactions (e.g., cannibalism; \[[@pone.0188791.ref025]\]). Arenas were inspected daily to verify shredder survival. Arenas where the shredder pupated or died were not considered in the estimates of shredder survival (pupae) or of leaf-breakdown rate.
The leaf-breakdown rate was estimated by mass loss between the initial and final mass \[[@pone.0188791.ref027]\]. Leaf disks were freeze dried until constant mass (\~ 24 hours) and weighed on a precision balance (accuracy = 10 αg). Total leaf-breakdown rate was calculated by dividing the ingested leaf mass (initial dry mass---final dry mass) by the exposure time. Microbial leaf-breakdown rate was obtained by dividing the difference between the initial and final masses of the leaves from the litter bags (not accessible to shredders) by the exposure time. Shredder leaf-breakdown rate was calculated by the difference between total and microbial leaf breakdown. All leaf-breakdown rates were expressed in mg day-1.
We assessed the values of dissolved oxygen (mg l^-1^; oximetry TSI, model 55), pH (WTW, model PH90) and electrical conductivity (μS cm^-1^; WTW, model LF90) in the water in three arenas for each treatment, totaling 24 records per day. In addition, water temperature was recorded each hour using data loggers.
Statistical analysis {#sec008}
--------------------
All data were tested for normality and homogeneity using Kolmogorov-Smirnov and Levene's tests, respectively, and, when necessary, data were log transformed. Paired t-tests were used to test the chemical composition of leaf litter between different soil conditions (flooded and non-flooded). We did not find differences in chemical composition between soils conditions (see [Results](#sec009){ref-type="sec"}). Thus, we used data from flooded and non-flooded soils together to test differences of leaf breakdown rates, fungal biomass and P. *elektoros* survival.
We used a linear regression to evaluate the relationship between chemical composition (carbon, nitrogen and phosphorus concentrations) and climate change (Principal Component Analysis--Axis 1 using temperature and CO~2~ data). We used a Repeated Measures Analysis of Variance (RM-ANOVA) to test differences in abiotic variables among simulated climatic conditions (control, light, intermediate, and extreme). One-Way ANOVA was used to test differences in breakdown rates (total, microbial and shredders) and fungal biomass among simulated climatic conditions. We calculated the median time to death (TTD) for each treatment using the Kaplan--Meier product-limit method with the log-rank test \[[@pone.0188791.ref046]\]. Additionally, survival curves were evaluated using a Chi-square test \[[@pone.0188791.ref046]\]. All statistical analyses were performed in R software.
Results {#sec009}
=======
Abiotic variables {#sec010}
-----------------
Mean air CO~2~ concentration during the experiment ranged from 537.86 ± 18.36 ppmv in the Control to 1363.78 ± 17.99 ppmv in the Extreme chamber ([Table 1](#pone.0188791.t001){ref-type="table"}). Mean air temperature ranged from 26.96 ± 0.98ºC in the Control to 31.75 ± 0.50ºC in the Extreme chamber. The mean electrical conductivity was similar in all chambers (RM-ANOVA: F~3,47~ = 1.50; p = 0.227). Mean dissolved oxygen (RM-ANOVA: F~3,47~ = 4.92; p = 0.005) and pH (RM-ANOVA: F~3,47~ = 47.36; p \< 0.001) were higher in the control chamber and decreased with increased air temperature and CO~2~ concentration. The mean water temperature was lower in the Control chamber (RM-ANOVA: F~3,47~ = 44.10; p \< 0.001) and ranged from 25.32 ± 0.41°C in the Control to 29.62 ± 0.37°C in the Extreme treatment. Water abiotic variables were similar between arenas with discs from plants growing in flooded and non-flooded soils ([Table 1](#pone.0188791.t001){ref-type="table"}).
10.1371/journal.pone.0188791.t001
###### Mean values (standard error) and results of abiotic variables under simulated climate conditions during seven days of the experiment.
{#pone.0188791.t001g}
Abiotic variables Simulated climate conditions RM-ANOVA
-------------- ------------------- ------------------------------ ----------------- ----------------- ------------------ ------------ ------------- -------------
**Air** **CO**~**2**~ 537.86±18.36^a^ 750.98±16.07^b^ 953.13±24.73^c^ 1363.78±17.99^d^ **3,18** **3335.00** **\<0.001**
**T** 26.96±0.98^a^ 28.23±0.96^b^ 29.27±0.92^c^ 31.75±0.50^d^ **3,18** **475.90** **\<0.001**
**Water** **EC** 121.13±14.46^a^ 140.69±10.86^a^ 149.41±38.30^a^ 142.16±57.05^a^ **3,47** 1.50 0.227
**O**~**2**~ 6.70±0.54^a^ 6.36±0.64^ab^ 6.28±0.32^b^ 6.03±0.42^b^ **3,47** **4.92** **0.005**
**pH** 7.47±0.34^a^ 7.25±0.28^ab^ 6.90±0.16^b^ 6.85±0.24^b^ **3,47** **47.36** **\<0.001**
**T** 25.32±0.41^a^ 26.55±0.35^ab^ 27.36±0.56^b^ 29.62±0.37^c^ **3,47** **44.10** **\<0.001**
In bold, p values \< 0.05. AV = Abiotic variables; CO~2~ = Carbon dioxide (ppmv); T = Temperature (^o^C); EC = Electrical conductivity (μS cm^-1^); O~2~ = Oxygen (mg l^-1^). Con = Control; Lig = Light; Int = Intermediate; Ext = Extreme.
Different letters (a-d) indicate significant differences between groups.
Leaf-litter chemical concentration {#sec011}
----------------------------------
We did not find significant differences in leaf percentages of carbon, nitrogen, phosphorus, cellulose and lignin (p \> 0.223; [Table 2](#pone.0188791.t002){ref-type="table"}) between plants growing on flooded and non-flooded soils. The phosphorus concentration in the leaf litter decreases with increases in temperature and CO~2~ (F~1,6~ = 14.73; R^2^ = 0.66; p = 0.009) and ranged from 0.43 ± 0.08% in the control to 0.20 ± 0.04 in the extreme chamber. Carbon (F~1,6~ = 3.01; R^2^ = 0.22; p = 0.133), nitrogen (F~1,6~ = 0.16; R^2^ = 0.14; p = 0.705), cellulose (F~1,6~ = 0.25; R^2^ = 0.12; p = 0.635), and lignin (F~1,6~ = 3.73; R^2^ = 0.28; p = 0.102) concentrations were not related to the simulated climate change.
10.1371/journal.pone.0188791.t002
###### Chemical characteristics of *Hevea spruceana* leaves growing under different conditions of temperature, CO~2~ (Carbon dioxide) and humidity availability.
We included paired t-tests for leaf characteristics of plants grown on flooded and non-flooded soils.
{#pone.0188791.t002g}
Flooded Non-flooded Paired t-test
---------------- --------- ------------- --------------- ------- ------- ------- ------- ------- --- ------- -------
Carbon (%) 54.56 51.29 51.23 52.34 53.12 52.46 51.76 51.29 3 -0.54 0.626
Nitrogen (%) 1.67 3.85 3.76 2.54 1.99 2.14 2.27 2.26 3 1.18 0.323
Phosphorus (%) 0.37 0.48 0.39 0.17 0.49 0.35 0.28 0.23 3 0.79 0.487
Cellulose (%) 26.95 22.07 24.5 26.78 22.85 26.25 24.04 24.44 3 -0.40 0.716
Lignin (%) 28.13 33.31 28.21 25.83 31.14 27.3 29.89 26.24 3 1.53 0.223
Con = Control; Lig = Light; Int = Intermediate; Ext = Extreme.
Fungal biomass {#sec012}
--------------
The mean value of fungal biomass (ergosterol) ranged from 23.06 ± 4.75 μg g^-1^ in the Light to 42.85 ± 18.03 μg g^-1^ in the Intermediate condition ([Fig 1](#pone.0188791.g001){ref-type="fig"}). We found higher fungal biomass in the Intermediate conditions as compared to the other conditions (ANOVA: F~3,45~ = 4.74; p = 0.006).
{#pone.0188791.g001}
Survival {#sec013}
--------
We did not find any *P*. *elektoros* death in the control and light chambers. Only three (2.5%) *P*. *elektoros* out of the 120 initial individuals died during the experiment ([Fig 2](#pone.0188791.g002){ref-type="fig"}). Survival rates were similar in all treatments (Log-rank, test statistic = 3.7, df = 3, p = 0.290), and were not affected by increased temperature and CO~2~. In addition, pupae were recorded in all treatments (n = 9; [Fig 2](#pone.0188791.g002){ref-type="fig"}), and their number was not found to be affected by climate conditions (Log-rank, test statistic = 2.4, df = 3, p = 0.492).
{#pone.0188791.g002}
Leaf-breakdown rate {#sec014}
-------------------
Under all climate conditions, leaf breakdown was mainly by shredders (Control = 95.15%; Light = 99.11%; Intermediate = 83.46%; Extreme = 93.36%; [Fig 3](#pone.0188791.g003){ref-type="fig"}). However, under Intermediate conditions, microorganisms (22.31%) had a key role in leaf breakdown rate. Microbial leaf-breakdown (ANOVA: F~3,113~ = 5.55; p = 0.001) and total leaf-breakdown (ANOVA: F~3,102~ = 2.79; p = 0.044) rates were higher under the Intermediate condition, as compared to the other conditions. On the other hand, leaf breakdown by shredders was similar under all climate conditions (ANOVA: F~3,102~ = 1.48; p = 0.224; [Fig 3](#pone.0188791.g003){ref-type="fig"}).
{#pone.0188791.g003}
Discussion {#sec015}
==========
Leaf-litter chemical concentration {#sec016}
----------------------------------
We did not find any difference in the chemical composition of *H*. *spruceana* grown on flooded versus non-flooded soil. The effects of flooding on plants may vary according to plant species, flood tolerance, flood time and climatic conditions \[[@pone.0188791.ref008], [@pone.0188791.ref011], [@pone.0188791.ref047]\]. The magnitude of the flooding may also play a role. Under natural conditions, seedlings may remain totally flooded for periods of months, according to their position in the flooding gradient \[[@pone.0188791.ref048]\]. In general, plants in flooded environments absorb less oxygen and nutrients, resulting in lower concentrations of N and P in the leaves and lower assimilation of CO~2~ \[[@pone.0188791.ref049]--[@pone.0188791.ref051]\]. However, a non-significant effect of floods on the concentration of N in *Salix sericea* Marshall was reported by Lower et al. \[[@pone.0188791.ref052]\], probably because their experiment was on the partial flooding of plants. The results may also change if the seedlings remain for a longer period in the experimental chambers.
We found a significant difference in the phosphorus (P) concentration in the leaf litter. P showed a negative correlation with increase in temperature and CO~2~ concentration. This fact is an important sign because P may be a limiting nutrient in addition to nitrogen (N), and may play a key role in global carbon storage \[[@pone.0188791.ref053]--[@pone.0188791.ref054]\]. Several studies have demonstrated that higher CO~2~ concentrations in the air increase wheat root phosphatase activity (organic phosphate mineralization) and increase the inorganic P supply for plant utilization \[[@pone.0188791.ref054]\]. This creates a positive feedback, increasing biomass production on P-limited soils \[[@pone.0188791.ref053]\]. However, this depends in part on plant stoichiometric flexibility \[[@pone.0188791.ref055]\], and we did not find negative feedback (probably due to the increased use of P with the increased rate of metabolism).
Another aspect is the decrease of P concentration by effects of elevated CO~2~. One would expect the same pattern in biomass-C and N \[[@pone.0188791.ref054]\], but this was not observed. This may indicate negative effects of CO~2~ increase depending on the species studied, and P may play a more pronounced role than N in regulating riparian vegetation growth \[[@pone.0188791.ref055]\]. Moreover, interaction with other abiotic factors, such as temperature and flooding can significantly alter the response of plants to CO~2~. Thus, the effect of CO~2~ enrichment may not be manifest if stressors are activated that affect plant growth.
Fungal biomass {#sec017}
--------------
Temperature increase may result in either positive \[[@pone.0188791.ref056]--[@pone.0188791.ref057]\] or negative \[[@pone.0188791.ref058]\] effects in leaf-litter breakdown under constant conditions of CO~2~. In our study, fungal biomass showed no linear response to increase in temperature and CO~2~, showing lower values in the Light condition and higher in the Intermediate condition. Soil microorganisms showed increases in most C degradation genes under conditions with high CO~2~ concentration \[[@pone.0188791.ref059]--[@pone.0188791.ref060]\]. Moreover, in our study higher fungal biomass in the Intermediate condition may be associated with optimal temperature in this chamber, resulting in elevation of metabolic activity, reproductive rates, and fungal biomass \[[@pone.0188791.ref061]--[@pone.0188791.ref062]\]. However, this stimulus to fungi in warmer water (27.36 ± 0.56°C) may be associated with duration of the experiment (7 days), since some fungal species can tolerate higher temperatures for a brief period \[[@pone.0188791.ref061]--[@pone.0188791.ref063]\]. For example, a previous study in the same microcosm with a duration of 30 days found different responses of fungal biomass for two leaf species \[[@pone.0188791.ref027]\]. In leaves of *E*. *glabriflora* there was no variation in fungal biomass among the climatic conditions, whereas in *G*. *glabra* there was a decrease of these microorganisms with increased temperature and CO~2~.
Another important aspect for higher fungal biomass in the Intermediate climate condition is the dominant presence of species adapted to warm water in fungal communities in the tropical region, in contrast to temperate ecosystems (\~21 to 27°C) \[[@pone.0188791.ref034], [@pone.0188791.ref064]\]. An example is the dominance of *Anguillospora filiformis*, which is normally associated with warm water \[[@pone.0188791.ref064]\], confirming the positive effect of temperature \[[@pone.0188791.ref034], [@pone.0188791.ref062]\]. This effect indicates that ecological processes in tropical streams may show less change, for example, in microbial species composition, as compared to temperate streams.
Survival / pupae {#sec018}
----------------
We also observed low mortality of *P*. *elektoros* during the experiment (2.5% of initial individuals). Mortality was not affected by increase in temperature \[[@pone.0188791.ref014]\] and CO~2~, contrary to the finding of Martins et al. \[[@pone.0188791.ref027]\]. Survival rates of shredders in laboratory experiments are generally associated with leaf quality, with high mortality on low-quality plant species \[[@pone.0188791.ref058]\]. However, due to similar chemical composition of the litter, the survival rates between plant species did not change. Increase in temperature and CO~2~ also increase metabolic cost (higher respiratory and excretion rates) and decrease digestive enzyme efficiency \[[@pone.0188791.ref027], [@pone.0188791.ref065]\]. These authors found low survival and higher leaf consumption by *P*. *elektoros*, but this was not observed in our study. Moreover, the short duration of our experiment may have favored the low mortality we observed.
Leaf breakdown rate {#sec019}
-------------------
Leaf breakdown was mainly by *P*. *elektoros*, but was similar under all climate conditions. This result shows an important participation of shredders in the leaf-litter breakdown process, despite the low density of shredders in many tropical streams \[[@pone.0188791.ref024], [@pone.0188791.ref056], [@pone.0188791.ref066]\]. *Phylloicus* larvae are commonly found on submerged leaves, where they obtain their food and the raw materials for the construction of their cases \[[@pone.0188791.ref021], [@pone.0188791.ref032]\]. Another aspect is the large body size of these larvae and high biomass compared to other shredders in tropical streams \[[@pone.0188791.ref066]\], which increases the individual importance of *Phylloicus* larvae for leaf-litter breakdown \[[@pone.0188791.ref014], [@pone.0188791.ref025]\]. In the larval phase, *Phylloicus* is exclusively a shredder and helps to convert coarse particulate organic matter into fine particulates and dissolved organic matter \[[@pone.0188791.ref032]--[@pone.0188791.ref033]\]. The similar leaf-litter breakdown rates under all climate conditions by *Phylloicus* larvae (associated with higher importance) show that the occurrence of these organisms in tropical stream ecosystems may increase the ecological resistance in global-warming scenarios \[[@pone.0188791.ref014]--[@pone.0188791.ref015], [@pone.0188791.ref056]\]. On the other hand, Martins et al. \[[@pone.0188791.ref027]\] recorded a negative effect of climate change on total and shredder leaf breakdown. Thus, the short duration of our study means that our results regarding the survival and importance of shredders in climate change scenarios should be treated with caution.
Microorganisms are the major decomposers in tropical streams due to the low density of shredders \[[@pone.0188791.ref056], [@pone.0188791.ref067]\]. However, microorganisms show higher sensitivity to extrinsic environmental factors (CO~2~ and temperature) in comparison to shredders \[[@pone.0188791.ref034], [@pone.0188791.ref018]\]. These results were contrary to those observed in the same ecosystem when distinct species are compared (*Goupia glabra* and *Eperua glabriflora*) \[[@pone.0188791.ref027]\], indicating a: i) a species-specific relationship between decomposer community and leaf-litter quality and ii) the necessity of additional studies to clarify the real effects of climate change on the decomposer community (tri-trophic interaction among leaf-litter, fungi and invertebrates) in litter breakdown. The increase of microbial leaf-breakdown and total leaf-breakdown rates under intermediate conditions can indicate an increase of riparian metabolism in this condition \[[@pone.0188791.ref019], [@pone.0188791.ref057]\]. This highlights the risk (e.g. decreased productivity) of global warming for these ecosystems. Extreme factor values (positive or negative) are within a well-defined range (inside or outside) in the physiological tolerance of organisms \[[@pone.0188791.ref068]\]. Therefore, advanced stages of global warming could lead to a decrease in decomposing activity, compromising nutrient cycling by microorganisms in aquatic ecosystems.
Conclusions {#sec020}
===========
Plants in riparian vegetation under different environmental conditions (e.g., soil nutrient and water availability) can produce leaf-litter of differing quality \[[@pone.0188791.ref004]\]. Different leaf-litter qualities can change their use (food and building-case use) by shredders \[[@pone.0188791.ref014], [@pone.0188791.ref024]\]. However, we did not record significant differences in leaf quality between the leaf litter from plants growing on flooded versus non-flooded soil in the same microcosm (differences were only observed among microcosms). On the other hand, we can also infer that breakdown rates have been changed by global warming \[[@pone.0188791.ref056], [@pone.0188791.ref058]\], independent leaf-litter quality (soil conditions or biome). However, the question remains open as to whether leaf-quality changes are due to the effects of climate change or nutrient concentrations between the leaf-litters sources. Since our case could be due to experimental design or to adaption of this species, further studies are needed.
In conclusion, leaf breakdown was mainly by *Phylloicus* but was similar under all climate conditions. However, the finding that there was low mortality and similar shredder leaf consumption under all climate conditions must be carefully interpreted due to the short duration of our experiment and consequently low lethal effect. We need to investigate chronic effects (over a longer time). We found an increase of microorganism biomass and leaf-breakdown rate under Intermediate conditions, showing higher sensitivity to extrinsic environmental factors. These results also indicate an increase in riparian metabolism under intermediate conditions of temperature and CO~2~, highlighting the risk of global warming for tropical streams.
Supporting information {#sec021}
======================
###### Experimental protocol to study leaf breakdown in four climate conditions.
(DOCX)
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Click here for additional data file.
###### Architectural plan of microcosm chambers.
(TIF)
######
Click here for additional data file.
###### Representation of arena used to perform experiments.
This arena was used to obtain total and shredders leaf breakdown rates.
(TIF)
######
Click here for additional data file.
###### Sample design of shredder and microbial leaf-breakdown rates in treatments with leaf disks of *Hevea spruceana* under four climate conditions during the experiment in the Control, Light, Intermediate and Extreme treatments.
(TIF)
######
Click here for additional data file.
###### Representation of arena with litter bags.
This arena was used to obtain microbial, total and shredders leaf breakdown rates.
(TIF)
######
Click here for additional data file.
We thank Dr. Adalberto L. Val for microcosm use, Ph.D. Phillip Fearnside for English review and text suggestions, Dra. Ana M.O. Pes for *Phylloicus elektoros* identification, Dr. Sérgio Nunomura for lyophilizer use, Fernanda Dragan and Jéssica Oliveira for help during the experiment, and Celso Rabelo Costa and Elizabeth Rebouças for field and laboratory assistance. RTM and AL received a fellowships from Programa de Apoio à Fixação de Doutores no Amazonas--FIXAM/AM (FAPEAM). RTM received a fellowship from Programa Nacional de Pós-Doutorado---PNPD/CAPES Programa de Pós-graduação em Ecologia e Evolução--PPGEE/UFG. RSR received a Post-Doctoral Scholarship from the National Institute for Research in Amazonia (INPA) through CNPq grant number 151375/2014-3. JFG, MTFP and NH received research grants (302957/2014-6, 310547/2016-4 and 307849/2014-7, respectively) from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). CT-Hidro/Climatic Changes/Water Resources/CNPq (Proc. 403949/2013-0), INCT/ADAPTA-II (CNPq/ FAPEAM), PELD MAUA (CNPq, FAPEAM) and INPA supported the invertebrate samples, laboratory analyses and microcosm experiment.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Current address: Programa de Pós-Graduação em Ecologia e Evolução, Universidade Federal de Goiás, Goiânia, Goiás, Brazil
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Since randomized controlled trials (RCTs) assessing the effects of influenza vaccination on clinical outcomes are scarce, evidence on influenza vaccine effectiveness (VE) mainly derives from observational studies \[[@CR1]\]. However, these studies are prone to bias and have been suspected to systematically overestimate VE, particularly against unspecific outcomes such as all-cause mortality and among the elderly \[[@CR2]\]. Although it has been accepted that observational studies are susceptible to bias, there is an ongoing controversy whether and to what extend confounding by indication and healthy vaccinee bias affect influenza VE estimates \[[@CR3]--[@CR9]\]. Both forms of bias/confounding have been described in such studies, but it is important to note that their presence has opposing consequences for the VE estimates: "confounding by indication" is likely to be present if patients with underlying chronic diseases are more likely to be vaccinated than healthy study participant. If no adequate statistical adjustment (e.g., for comorbidities) is made, this leads to an underestimation of VE since the less healthy population is at higher risk of adverse health outcomes. The alternative scenario is called "healthy vaccinee bias" and refers to a situation when patients, who are in better health conditions, are more likely to adhere to the annually recommended influenza vaccination \[[@CR10]\]. If not corrected for (e.g., by adjustment for comorbidities or indicators of health seeking behavior), healthy vaccinee bias leads to an overestimation of VE.
To test whether residual confounding by healthy vaccinee effects is still present in the adjusted data, it has been suggested by some authors that investigators should obtain "off-season" estimates. Off-season estimates are calculated for time periods outside influenza seasons when the virus is (virtually) not circulating and therefore no vaccine effect should be present \[[@CR10], [@CR11]\]. Any VE obtained during this control period would be attributable to unmeasured confounding, whereas successful adjustment would have removed the effect.
A systematic analysis of these two forms of bias/confounding and their consequences for influenza VE studies has not been published so far. We therefore addressed this issue by a systematic review.
Methods {#Sec2}
=======
Question framing {#Sec3}
----------------
This study addressed the following questions: (i) How often do observational studies on influenza VE show indication of confounding by indication and/or healthy vaccinee bias? (ii) What is the impact on VE point estimates? And (iii) how many of these studies show indication of unmeasured (residual) confounding in the adjusted analyses? To define the conceptual framework of the study, we identified five indicators from the literature, which allow conclusions on the presence of the two forms of bias/confounding in the included studies (Table [1](#Tab1){ref-type="table"}).Table 1Conceptual framework: Indicators and conclusions for presence of confounding by indication and healthy vaccinee bias in influenza vaccine effectiveness studiesIndicatorConclusionReferencesVaccinated study participants have a higher proportion of comorbidities than unvaccinated study participants, as indicated by baseline characteristicsHigh risk of confounding by indication in the unadjusted data set\[[@CR6], [@CR38]\]Vaccinated study participants have a lower proportion of comorbidities than unvaccinated study participants, as indicated by baseline characteristicsHigh risk of healthy vaccinee bias in the unadjusted data set\[[@CR35], [@CR36]\]Inclusion of comorbidities in the regression model increases vaccine effectivenessConfounding by indication has led to underestimation of vaccine effectiveness in the unadjusted data set\[[@CR7]\]Inclusion of comorbidities in the regression model decreases vaccine effectivenessHealthy vaccinee bias has led to overestimation of vaccine effectiveness in the unadjusted data set\[[@CR7]\]Significant effects of influenza vaccination appear outside the influenza season ("off-season estimates"), despite adjustment for comorbiditiesResidual confounding by healthy vaccinee bias\[[@CR3], [@CR11], [@CR36]\]
Study protocol {#Sec4}
--------------
We performed the systematic review according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement \[[@CR12]\]. The respective protocol for this review is shown in Additional file 1.
Eligibility criteria {#Sec5}
--------------------
Studies were included if they fulfilled the following criteria defined a priori: (i) observational (non-randomized) study; (ii) calculated influenza VE by comparing vaccinated and unvaccinated participants; (iii) reported baseline characteristics of vaccinated and unvaccinated participants; (iv) reported data on at least one clinical outcome; (v) reported crude and confounder-adjusted VE estimates from at least one influenza season; (vi) reported confounder-adjusted VE estimates from at least one "control" period outside the influenza season (off-season estimate).
Literature search {#Sec6}
-----------------
Two reviewers (CR and TH) searched MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials (date of last search: 25.05.2014) and independently screened each citation and subsequent full text articles. The complete search strategy is shown in Additional file 2. Electronic searches were complemented by manually searching the reference lists of all identified studies and reviews for additional studies. No restrictions were made regarding publication language and publication status (published/unpublished).
Data extraction {#Sec7}
---------------
From each included study, two investigators (CR and TH) independently extracted the following information: country, study design, age, sex, characteristics of study population (e.g., patients with underlying comorbidities), source of patient data, identification of clinical outcomes and vaccination status, definition of influenza season and off-season, and population size. In addition, we extracted data on crude and adjusted VE point estimates for all reported outcomes during influenza seasons, adjusted off-season point estimates, and which confounder were considered. Extraction forms were pilot tested with the first two identified studies.
Assessment of risk of bias {#Sec8}
--------------------------
Two investigators (CR and TH) independently assessed risk of bias. In case of disagreements, a final decision was made by consensus or resolved by a third reviewer (OW). We used the predefined criteria derived from the above mentioned methodological framework (see Table [1](#Tab1){ref-type="table"}) to assess the risk of healthy vaccinee bias and confounding by indication in the included studies: A study was judged to be at high risk of healthy vaccinee bias if vaccinated participants had significantly fewer comorbidities (or respective indicators such as medical visits) than unvaccinated participants, as indicated by baseline characteristics. A study was judged to be at high risk of confounding by indication if vaccinated participants had significantly more comorbidities (or respective indicators) than unvaccinated participants, as indicated by baseline characteristics. For case--control studies, vaccinated and unvaccinated participants of the control groups were compared. The results of these assessments were expressed as a considered judgment, using the categories "high risk of bias", "low risk of bias" and "unclear risk of bias".
Two approaches, a descriptive and a meta-analytical, were used to assess whether the included studies successfully corrected for bias/confounding. First, we compared crude VE estimates to confounder-adjusted in-season estimates from the same studies. If the studies reported more than one confounder-adjusted estimate, we used the fully adjusted model. If adjustment increased the estimated VE, we concluded that data were at least in part corrected for confounding by indication. If adjustment decreased the estimated VE, we concluded that data were at least in part corrected for healthy vaccinee bias (see Table [1](#Tab1){ref-type="table"}).
Second, we used the approach suggested by Hrobjartsson et al. \[[@CR13], [@CR14]\] to quantify the extent by which adjustment for confounders increased the in-season estimate compared to the crude estimate: For each outcome for which more than one study reported data, we calculated the ratio of odds ratios (crude/adjusted VE during influenza season) per study. A ratio of \>1 indicates that adjustment led to a stronger effect of vaccination, i.e., an increased VE. For calculation of 95 % CI, we used the formula provided by Hrobjartsson et al. \[[@CR13]\]. To quantify the impact of adjustment for confounders, we then meta-analysed the individual study ratios of odds ratios for each outcome separately, using random-effects models with inverse-variance methods. For this analysis, we excluded two studies \[[@CR15], [@CR16]\] which did not report 95 % CI for the respective point estimates.
To evaluate the presence and magnitude of off-season VE estimates, being proposed as indicators of healthy vaccinee bias, we contrasted confounder-adjusted in-season estimates to "pseudo-effectiveness" estimates measured during off-seasons.
All statistical analyses were performed using STATA 12 (StataCorp LP, Texas, USA).
Results {#Sec9}
=======
By systematic literature search we identified 3385 publications, of which 23 were finally included in our analysis (Fig. [1](#Fig1){ref-type="fig"}) \[[@CR3], [@CR5], [@CR7], [@CR15]--[@CR33]\]. Details on the excluded studies are reported in Additional file 3. Baseline characteristics of the 23 included studies are shown in Table [2](#Tab2){ref-type="table"}. Of these, 20 were cohort studies, while the remaining three had a case--control design. The studies were conducted in North America (*n* = 14), Europe (*n* = 6), Taiwan (*n* = 2) or in multiple continents (*n* = 1) and mainly used disease classification codes (e.g., ICD-9) or civil register data for the identification of outcomes. In three studies interviews were conducted or self-administered questionnaires were applied to collect primary data on relevant outcomes or vaccination status \[[@CR24], [@CR25], [@CR27]\]. Except of four studies, which were either performed in students (*n* = 1), in adults aged 40+ years (*n* = 1), or in women who recently experienced live birth (*n* = 2), all studies were conducted in elderly persons. Seven studies covered populations with underlying comorbidities, namely with (chronic) heart disease (CHD), \[[@CR21], [@CR22]\] end-stage renal disease (ESRD), \[[@CR17], [@CR23]\] chronic obstructive pulmonary disease (COPD), \[[@CR28], [@CR29]\] or patients with diabetes or vascular disease \[[@CR33]\].Fig. 1Flow chart for the systematic reviewTable 2Baseline characteristics of included studiesAuthor, yearCountryStudy designInfluenza season(s)Age-group (yrs) or risk groupAge (yrs), range or mean (± SD)% maleData sourcesIdentification of outcomesStudy size (n)Bond et al., 2012 \[[@CR17]\]USCohort2005/06Patients with ESRDV, 60.6 (15.2)V, 52.53 ESRD Networks, records of the US Renal Database (USRDS)All-cause death through ESRD death notification form20,220 (without pneumococcal vaccine)UV, 57.9 (15.9)UV, 50.8Campitelli et al., 2010 \[[@CR7]\]CanadaCohort8 seasons between 1996 and 2007Elderly ≥ 65V, 75.3 (6.6)V, 40.8National health surveys data linked to Ontario Health Insurance (OHIP) and Discharge Abstract (CIHI) databasesRegistered persons database and ICD-9/-10 admission codesV, 14,512UV, 74.2 (6.7)UV, 40.7UV, 11,410Foster et al., 1992 \[[@CR18]\]USCase--control (matched)1989/90Elderly ≥ 65V, 65-94+V, 50.8Databases of participating hospitalsHospital discharge ICD-9 codesCases, 721UV, 65-94+UV, 47.3Controls, 1786France et al., 2006 \[[@CR34]\]USCohort1995/96-2000/01Women and their newbornsV, 30.8 (5.5)NAHealth maintenance organization (Kaiser Permanante and Group Health Cooperative)ICD-9 codes for medically attended acute respiratory illnesses in infantsV, 3160UV, 29.7 (5.5)UV, 37,969Groenwold et al., 2009 \[[@CR19]\]NetherlandsCohort1995/96-2002/2003Elderly ≥ 65V, 75 (median)V, 39.4GPRD of University Medical Center UtrechtICPC coding systemV, 37,501UV, 74 (median)UV, 35.2UV, 13,405Hottes et al., 2011 \[[@CR20]\]CanadaCohort2000/01-2005/06Elderly ≥ 65V, 75 (median)V, 43Manitoba Immunization Monitoring System (MIMS) and Manitoba health policy databaseAll-cause mortality or hospital admission ICD-9/-10 codes139,185 (00/01) to 140,735 (05/06)UV, 73 (median)UV, 44Jackson et al., 2006 \[[@CR35]\]USCohort1995/96-2002/3Elderly ≥ 65V, 51 % \>74V, 42.7Health maintenance organization (Group Health Cooperative)All-cause mortality or hospital ICD-9 discharge codes72,527UV, 46 % \>74UV, 41.9Jackson et al., 2002 \[[@CR21]\]USCohort1992-1996Patients with nonfatal MIAll subjects, 64 (median)among ≥ 65:Health maintenance organization (Group Health Cooperative)Hospital discharge ICD-9 codes, confirmed by chart reviewV, 1016UV, 362V, 47UV, 59Jackson et al., 2008 \[[@CR5]\]USCase--control (matched)2000/1-2001/2Elderly 65-94Cases, 62 % \>74cases, 51Health maintenance organization (Group Health Cooperative)ICD-9 code for CA pneumonia and validation using hospital recordsCases, 1173 Controls, 2346 (1838 V, 508 UV)Johnstone et al., 2012 \[[@CR33]\]40 countriesCohort2003/04-2006/7Elderly ≥ 65 with VD or diabetesMean (4 seasons)Range (4 seasons)Clinical databases from two RCTs (ONTARGET- and TRANSCEND-trial)Outcomes adjudicated by independent committee using clinical data31,546V, 67-68V, 72-73UV, 65-66UV, 67-70Liu et al., 2012 \[[@CR22]\]TaiwanCohort2002-2006Elderly \> 65 with heart diseaseV, 74.8 (6.3)V, 58.3National Health Research Institute-released cohort datasetICD-9 codes for heart diseasesV, 2760UV, 75.7 (7.0)UV, 51.8UV, 2288Mangtani et al., 2004 \[[@CR16]\]UKCohort1989/90-1998/99Elderly ≥ 65Not reportedNot reportedGeneral practice research database (GPRD)ICD-9 codes for acute respiratory illnesses; all respiratory-related deathsPerson-years: influenza season, 419,748 summer, 692,415McGrath et al., 2012 \[[@CR23]\]USCohort1997-1999 and 2001Patients with ESRDMean (4 seasons)Range (4 seasons)Medicare claims from the US Renal Data System (USRDS)All-cause death through ICD-9 codes; Medicare claims from the USRDS107,465 (1997) to 126,699 (2001)V, 62.3-63.9V, 52.2-53UV, 60.3-61.7UV, 50.4-51.6Nicol et al., 2008 \[[@CR24]\]USCohort2002/03-2005/06Adults (students)V, 25.2 (7.9)V, 25.5Internet-based surveySelf-reported occurrences of ILI and health care use12,795UV, 23.3 (6.3)UV, 29.4Nicol et al., 2009 \[[@CR25]\]USCohort2006/07Adults, 50-64Not reportedV, 24Internet-based surveySelf-reported occurrences of ILI and health care use479UV, 16Ohmit et al., 1995 \[[@CR26]\]USCase--control (matched)1990/91-1991/92Elderly ≥ 65not reportednot reportedAdmission and discharge data of 21 participating hospitalsICD-9 hospitalization code for pneumonia/influenzaCases, 1557 Controls, 3401Omer et al., 2011 \[[@CR27]\]USCohort2004/05-2005/06Women and their newbornsV, 18.3 % \< 19NAGeorgia Pregnancy Risk Assessment Monitoring System (PRAMS)PRAMS database (questionnaire or interview)V, 578UV, 3590UV, 14.5 % \< 19Örtqvist et al., 2007 \[[@CR15]\]SwedenCohort1998/99-2000/01Elderly ≥ 65V, 51 % \>74V, 41.3Population register (via national identification number)ICD-9/-10 codes and cause of death register260,155UV, 51 % \>74UV, 39.0Schembri et al., 2009 \[[@CR28]\]UKCohort1998-2006Adults \> 40 with COPDV, 27 % \> 69V: 42.8The Health Improvement Network database (THIN), covering data of general practicesDiesease classification codes of THIN databases ("Read codes")V, 9679UV, 31,062UV, 12 % \> 69UV: 42.8Sung et al., 2014 \[[@CR29]\]TaiwanCohort2000-2007Elderly ≥ 55 with COPDV, 20 % \>74V: 58.7Reimbursement claims from National Health Insurance Research Database (NHIRD)ICD-9 codes for acute MI or angina pectoris with invasive therapyV, 3027UV, 17 % \>74UV: 60.8UV, 4695Tessmer et al., 2011 \[[@CR30]\]GermanyCohort2002-2006Adults with pneumonia (CAP)V, 67.6 (14.5) \*1V: 57.2National Community Acquired Pneumonia Competence Network (CAPNET)CAPNET database entriesV, 1721UV, 3279UV, 55.7 (19.0)UV: 52.5Vila-Córcoles et al., 2007 \[[@CR31]\]SpainCohort2002-2005Elderly ≥ 65V, 51 % \> 74V, 44.3Databases of Primary Health Care Centres (PHCC)ICD-9 codes of PHCC and Civil Registry OfficesV, 6051UV, 5189UV, 38 % \> 74UV, 43.6Wong et al., 2012 \[[@CR32]\]CanadaCohort2000/01-2008/09Elderly ≥ 65V, 75.5 (6.6)V, 43.8Ontario Health administrative databasesICD-9/ -10 codes of databases and registered persons database1,297,051 (00/01) to 1,527,364 (08/09)UV, 74.5 (6.8)UV, 43.6*SD* standard deviation, *V* vaccinated, *UV* unvaccinated, *ESRD* end-stage renal disease, *ICD* international classification of disease, *CHD* chronic heart disease, *GPRD* general practice research database, *ICPC* international classification of primary care, *MI* myocardial infarction, *CA(P)* community acquired (pneumonia), *VD* vascular disease, *ONTARGET-trial* Ongoing Telmisartan Alone and in Combination With Ramipril Global EndPoint Trial, *TRANSCEND-trial* Telmisartan Randomized Assessment Study in ACE Intolerant Subjects with Cardiovascular Disease, *ILI* influenza-like illness, *COPD* chronic obstructive pulmonary disease
Reported outcomes {#Sec10}
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The included studies reported VE estimates (crude and adjusted in-season plus adjusted off-season estimates) related to 11 different clinical outcomes: all-cause mortality (*n* = 12 studies), death due to respiratory event (*n* = 2), major adverse vascular event (*n* = 1), hospitalization due to influenza and/or pneumonia (*n* = 7), hospitalization for acute coronary syndrome (*n* = 1), influenza-like illness (*n* = 3), cardiac death (*n* = 1), hospitalization due to cardiovascular disease (*n* = 1), prematurity (*n* = 1), small for gestational age (*n* = 1), and medically attended respiratory infections in infants (*n* = 1). None of the clinical outcomes was required to be confirmed by laboratory testing for influenza viruses.
Risk of healthy vaccinee bias and confounding by indication {#Sec11}
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Of the included 23 studies, 19 (83 %) showed a high risk of bias (either healthy vaccine bias, confounding by indication, or both). Two studies we judged to be at high risk of healthy vaccinee bias but not confounding by indication (Table [3](#Tab3){ref-type="table"}). One of these studies was performed in patients with end-stage renal disease, with vaccinated participants having more favorable prognostic markers than unvaccinated participants; \[[@CR23]\] the other study covered patients suffering from COPD and indicated that vaccinated patients had less (severe) comorbidities as indicated e.g., by the Charlson comorbidity index, when compared to unvaccinated patients.Table 3Risk of healthy vaccinee bias and confounding by indication in the included studies, as judged from the baseline characteristics of vaccinated and unvaccinated participantsStudyHealthy vaccinee bias^a^Confounding by indication^b^Indicated byBond et al. (2012) \[[@CR17]\]{#d30e1694}{#d30e1700}Vaccinated participants have more comorbidities; unvaccinated have worse laboratory valuesCampitelli et al. (2011) \[[@CR7]\]{#d30e1716}{#d30e1722}Vaccinated participants have more comorbidities; unvaccinated patients have worse functional statusFoster et al. (1992) \[[@CR18]\]{#d30e1738}{#d30e1744}More comorbidities in vaccinated participantsFrance et al. (2006) \[[@CR34]\]{#d30e1760}{#d30e1766}More comorbidities in vaccinated participantsGroenwold et al. (2009) \[[@CR19]\]{#d30e1782}{#d30e1788}More comorbidities, medications and medical visits in vaccinated participantsHottes et al. (2011) \[[@CR20]\]{#d30e1804}{#d30e1810}More medical visits in vaccinated participantsJackson et al. (2002) \[[@CR21]\]{#d30e1826}{#d30e1832}More comorbidities in vaccinated participantsJackson et al. (2006) \[[@CR3]\]{#d30e1848}{#d30e1854}More comorbidities in vaccinated participantsJackson et al. (2008) \[[@CR5]\]{#d30e1870}{#d30e1876}No major differences in baseline characteristics between groupsJohnstone et al. (2012) \[[@CR33]\]{#d30e1892}{#d30e1898}Vaccinated participants have more CAD; unvaccinated have more diabetes and hypertensionLiu et al. (2012) \[[@CR22]\]{#d30e1914}{#d30e1920}More comorbidities in vaccinated participantsMangtani et al. (2004) \[[@CR16]\]{#d30e1937}{#d30e1943}More comorbidities and medications in vaccinated participantsMcGrath et al. (2012) \[[@CR23]\]{#d30e1959}{#d30e1965}Better adherence to dialysis and fewer years with end-stage renal disease in vaccinated participantsNichol et al. (2008) \[[@CR24]\]{#d30e1981}{#d30e1987}More comorbidities in vaccinated participantsNichol et al. (2009) \[[@CR25]\]{#d30e2003}{#d30e2009}No major differences in baseline characteristics between groupsOhmit et al. (1995) \[[@CR26]\]{#d30e2025}{#d30e2031}Unclear data and descriptionOmer et al. (2011) \[[@CR27]\]{#d30e2047}{#d30e2053}Some comorbidities different (diabetes) between groups, other not (hypertension)Örtqvist et al. (2007) \[[@CR15]\]{#d30e2069}{#d30e2075}More comorbidities in vaccinated participantsSchembri et al. (2009) \[[@CR28]\]{#d30e2091}{#d30e2097}More comorbidities in vaccinated participantsSung et al. (2014) \[[@CR29]\]{#d30e2113}{#d30e2119}More comorbidities in unvaccinated participantsTessmer et al. (2011) \[[@CR30]\]{#d30e2135}{#d30e2141}More comorbidities in vaccinated participantsVilla-Corcoles et al. (2007) \[[@CR31]\]{#d30e2157}{#d30e2163}More comorbidities in vaccinated participantsWong et al. (2012) \[[@CR32]\]{#d30e2180}{#d30e2186}More comorbidities and medications in vaccinated participantsgreen circle/+: low risk of bias; red circle/-: high risk of bias; yellow circle/?: unclear risk of bias; CAD, coronary artery disease^a^indicated by: vaccinated participants were healthier (fewer comorbidities) than unvaccinated participants at study entry (cohort studies) or vaccinated controls were healthier (fewer comorbidities) than unvaccinated controls (case--control studies)^b^indicated by: vaccinated participants were sicker (more comorbidities) than unvaccinated participants at study entry (cohort studies) or vaccinated controls were sicker (more comorbidities) than unvaccinated controls (case--control studies)
Fourteen studies showed a high risk of confounding by indication, but not of healthy vaccinee bias. In 13 of them, \[[@CR3], [@CR15], [@CR16], [@CR18], [@CR19], [@CR21], [@CR22], [@CR24], [@CR28], [@CR30]--[@CR32], [@CR34]\] this was indicated by a significantly higher proportion of vaccinated patients with comorbidities (compared to unvaccinated participants), whereas in one study \[[@CR20]\] medical visits served as indicator. In three studies, we found indication for both types of bias/confounding occurring simultaneously \[[@CR7], [@CR17], [@CR33]\]. In these studies, the group of vaccinated participants had a higher proportion of comorbidities, while at the same time unvaccinated participants showed a higher proportion of functional impairments or other relevant comorbidities. In a further three studies, \[[@CR5], [@CR25], [@CR27]\] no major differences in baseline characteristics between vaccinated and unvaccinated study participants were found. In the remaining one study, risk of bias was unclear due to unclear data and reporting (Table [3](#Tab3){ref-type="table"}) \[[@CR26]\].
Adjustment for confounders and impact on point estimates {#Sec12}
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In ten of 12 studies reporting on all-cause mortality, adjustment for confounders increased the estimate of VE. The same effect of adjustment was observed in all studies reporting on hospitalization, major adverse vascular events, influenza-like illness and cardiac death. For the remaining outcomes, the effect was either very small or adjustment decreased the VE estimate. All studies adjusted at least for age and comorbidities, although definitions of the latter differed between individual studies (Table [4](#Tab4){ref-type="table"}).Table 4Crude and confounder-adjusted estimates of vaccine effectiveness during the influenza season in the included studiesOutcome by studyCrude OR (95 % CI)Adjusted OR (95 % CI)Confounders considered in the adjusted analysisAll-cause mortalityBond et al. (2012) \[[@CR17]\]0.79 (0.72--0.87)0.73 (0.67--0.81)Age, race, sex, time on dialysis, diagnostic mode, diabetes, comorbidities, laboratory parametersCampitelli et al. (2011) \[[@CR7]\]0.65 (0.51--0.84)^a^0.61 (0.47--0.79)Demographics, comorbidities, health care utilization, functional status indicatorsGroenwold et al. (2009) \[[@CR19]\]0.86 (0.69--1.06)0.56 (0.45--0.69)Age, sex, prior healthcare use (GP visits), comorbidities, medication useHottes et al. (2011) \[[@CR20]\]0.87 (0.80--0.94)0.70 (0.64--0.77)Age, sex, SES, residency, prior influenza/pneumococcal vaccination, medical visits, Elixhauser indexJackson et al. (2006) \[[@CR35]\]0.56 (0.52--0.61)^b^0.51 (0.47--0.55)Age, sex, comorbidities, previous pneumonia hospitalization, number of outpatient visitsLiu et al. (2012) \[[@CR22]\]0.40 (0.34--0.47)0.42 (0.35--0.49)Age, comorbiditiesMcGrath et al. (2012) \[[@CR23]\]0.77 (0.76--0.78)^c^0.71 (0.70--0.72)^c^Age, race, sex, cause of ESRD, vintage, adherence, hospital days, mobility aids, comorbidities, oxygenÖrtqvist et al. (2007) \[[@CR15]\]0.50 (−)0.56 (0.52--0.60)^d^Age and sex, socioeconomic status, marital status, comorbiditiesSchembri et al. (2009) \[[@CR28]\]0.70 (0.58--0.86)^e^0.59 (0.57--0.61)Age, sex, year and serious comorbiditiesTessmer et al. (2011) \[[@CR30]\]0.85 (0.61--1.17)0.63 (0.45--0.89)Age, sex, pneumococcal vaccination status, body mass index, nursing home residency, smoking, previous antibiotic therapy, long-term oxygen therapy, number of comorbiditiesVilla-Corcoles et al. (2007) \[[@CR31]\]0.77 (0.65--0.89)0.63 (0.54--0.74)Age, sex, chronic lung disease, chronic heart disease, diabetes, hypertension, immunocompromised, immunocompromised x ageWong et al. (2012) \[[@CR32]\]0.72 (0.67--0.77)0.67 (0.62--0.72)Demographics, comorbidities, use of health care service, medication use, special medical proceduresDeath due to respiratory eventSchembri et al. (2009) \[[@CR28]\]0.3 (0.0--7.4)^e^0.63 (0.55--0.77)Age, sex, year and serious comorbiditiesMangtani et al. (2004) \[[@CR16]\]1.32 (−)0.88 (0.84--0.92)Risk, age, repeat prescription statusMajor adverse vascular event (cardiovascular death or nonfatal myocardial infarction or nonfatal stroke)Johnstone et al. (2012) \[[@CR33]\]0.77 (0.61--0.97)^f^0.65 (0.58--0.74)Propensity score (body mass index, age, sex, ethnicity, education, vitamin use, smoking history, alcohol use, history of pneumococcal vaccination), history of coronary artery disease, diabetes, hypertension, stroke, admission to nursing home, use of aspirin, ß-blocker, lipid-lowering drug, angiotensin-converting enzyme inhibitor, angiotensin II inhibitorHospitalization due to influenza and/or pneumoniaFoster et al. (1992) \[[@CR18]\]0.78 (−)^g^0.55 (0.36--0.86)Sex, race, age, information source, hospital type, region, survival, months, duration of recallHottes et al. (2011) \[[@CR20]\]1.09 (0.98--1.21)0.94 (0.82--1.07)Age, sex, SES, residency, prior influenza/pneumococcal vaccination, medical visits, Elixhauser indexJackson et al. (2006) \[[@CR35]\]0.82 (0.75--0.89)^b^0.71 (0.65--0.78)Age, sex, comorbidities, previous pneumonia hospitalization, number of outpatient visitsJackson et al. (2008) \[[@CR5]\]1.04 (0.88--1.22)^b^0.92 (0.77--1.10)Age, sex, asthma, smoking, antibiotics, FEV1, oxygen, previous pneumonia, steroids, other drugsMangtani et al. (2004) \[[@CR16]\]1.18 (−)^g^0.79 (0.74--0.83)Risk, age, repeat prescription statusMcGrath et al. (2012) \[[@CR23]\]0.90 (0.87--0.92)^c^0.84 (0.82--0.86)^c^Age, race, sex, cause of ESRD, vintage, adherence, hospital days, mobility aids, comorbidities, oxygenOhmit et al. (1995) \[[@CR26]\]1.0 (0.82--1.22)^h^0.68 (0.54--0.86)^h^Sex, age, smoking, information source, region, survival, hospital typeHospitalization for acute coronary syndromeSung et al. (2014) \[[@CR29]\]0.52 (0.41--0.66)0.45 (0.35--0.57)Age, gender, comorbidity condition, hypertension, diabetes, dyslipidemia, arrhythmia, anemia, pneumonia, monthly income, level of urbanization, geographic regionInfluenza-like illnessMcGrath et al. (2012) \[[@CR23]\]0.93 (0.91--0.95)^c^0.88 (0.86--0.89)^c^Age, race, sex, cause of ESRD, vintage, adherence, hospital days, mobility aids, comorbidities, oxygenNichol et al. (2008) \[[@CR24]\]0.77 (−)^g^0.70 (0.56--0.89)Age, sex, high-risk status, smoking, general health, undergraduate status, medical visits, virus matchNichol et al. (2009) \[[@CR25]\]0.55 (−)^g^0.48 (0.27--0.86)Sex, smoking, general health, high-risk status, functionality, activity limits, previous vaccinationCardiac death^i^Jackson et al. (2002) \[[@CR21]\]1.24 (0.84--1.84)^j^1.06 (0.63--1.78)Age, gender, severe heart failure during hospitalization, smoking status, comorbidities, medicationCVD hospitalizationLiu et al. (2012) \[[@CR22]\]0.85 (0.76--0.94)0.84 (0.76--0.93)Age, comorbiditiesPrematurityOmer et al. (2011) \[[@CR27]\]0.56 (0.33--0.96)^k^0.40 (0.24--0.68)^k^Gestational age, maternal age, multiple births, maternal risk factors and comorbidities, labor/delivery complications, birth defects, insurance, smoking, alcohol, race, education, marital status, weightSmall for gestational ageOmer et al. (2011) \[[@CR27]\]0.73 (0.40--1.33)^k^0.68 (0.32--1.46)^k^Gestational age, maternal age, multiple births, maternal risk factors and comorbidities, labor/delivery complications, birth defects, insurance, smoking, alcohol, race, education, marital status, weightMedically attended respiratory illness in infantsFrance et al. (2006) \[[@CR34]\]0.90 (0.80--1.02)^l^0.96 (0.87--1.07)^l^Infant gestational age at birth, infant sex, maternal age, Medicaid coverage, maternal history of prior influenza vaccination, and maternal high-risk status^a^adjusted for season and demographics; ^b^adjusted for age and sex; ^c^data were pooled first from 4 seasons; ^d^adjusted point estimates from season 98/99; ^e^unadjusted odds ratios and 95 % CI calculated from death rates for all seasons (1988--2006); ^f^data were pooled first from 4 seasons; ^g^95 % CI not reported; ^h^data from season 1990/91 were used; ^i^defined as death due to myocardial infarction, ischemic heart disease, congestive heart failure, hypertensive heart disease, cardiac arrest, and atrial fibrillation; ^j^adjusted for age; ^k^point estimates reported here were calculated for local influenza activity and included periods of regional and widespread influenza activity; ^l^matched by study site and birth week
We pooled the data for the outcomes all-cause mortality, hospitalization due to influenza or pneumonia, and ILI since more than one study reported on these outcomes. For all-cause mortality, this ratio of odds-ratio analysis indicated that adjustment for confounders increased the effect of vaccination by 12 % (95 % CI: 7--17 %) (Fig. [2a](#Fig2){ref-type="fig"}). For hospitalization due to influenza or pneumonia, effect size increased by 9 % (95 % CI: 4--14 %) after adjustment for confounders (Fig. [2b](#Fig2){ref-type="fig"}). For the outcome ILI, adjustment for confounders increased VE estimate by 7 % (95 % CI: 4--10 %).Fig 2Impact of adjustment for confounders, expressed as ratio of odds ratios (crude/adjusted): (**a**) All-cause mortality, (**b**) Hospitalization due to influenza or pneumonia
Off-season estimates {#Sec13}
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The included 23 studies reported a total 31 off-season estimates. Three of the studies reported pre-season as well as post-season estimates \[[@CR7], [@CR20], [@CR35]\]. Two studies reported only pre-season estimates, \[[@CR5], [@CR23]\] while five studies provided data on post-seasons "effectiveness" only \[[@CR15], [@CR16], [@CR18], [@CR19], [@CR32]\]. The remaining studies reported off-season estimates either for the whole period outside the influenza season or for single months before and after the seasons. Most studies defined beginning and end of influenza periods according to national influenza surveillance data. If more than one off-season estimate was provided, we decided to use the post-influenza season estimate for analysis (for a detailed description of the definition of "off-season" in the studies, see Additional file 4).
Analyzing the 31 adjusted off-season estimates that were reported by the 23 included studies, we found statistically significant effects of influenza vaccination outside the influenza season in 13 studies (Figs. [3](#Fig3){ref-type="fig"} and [4](#Fig4){ref-type="fig"}). Nine (39 %) of the 23 included studies reported at least one statistically significant VE estimate outside the influenza season (Figs. [3](#Fig3){ref-type="fig"} and [4](#Fig4){ref-type="fig"}). These off-season effects were not restricted to the outcome all-cause mortality, but were also reported for four other outcomes (major adverse vascular events, hospitalization due to influenza/pneumonia, acute coronary syndrome, ILI). However, significant off-season estimates were more likely to occur when all-cause mortality was used as an outcome (8/13; 67 %) compared to other outcomes (5/19; 26 %; *p* = 0.03 by chi^2^ test). We then evaluated whether the occurrence of significant off-season estimates was related to the risk of healthy vaccinee bias, as judged from the baseline data of the respective study populations. We found that 46 % (6/13) of the significant off-season estimates were associated with high risk of healthy vaccinee bias at baseline. In contrast, only 6 % (1/18) of non-significant off-season estimates were associated with high risk of healthy vaccinee bias (*p* = 0.01 by chi^2^ test). Studies covering non-elderly populations did not report statistically significant off-season estimates for neither outcome.Fig. 3Odds ratios (95 % CIs) of influenza vaccine effectiveness during influenza seasons (black square), during pre-influenza seasons (striped circle) and post-influenza seasons (white circle) against all-cause mortality (**a**), death due to respiratory event (**b**), death due to cardiac event (**c**), and major adverse vascular event (**d**)Fig. 4Odds ratios (95 % CIs) of influenza vaccine effectiveness during influenza seasons (black square), during pre-influenza seasons (striped circle) and post-influenza seasons (white circle) against hospitalization due to influenza or pneumonia (**a**), hospitalization for acute coronary syndrome (**b**), hospitalization due to cardiovascular diseases (**c**), influenza-like illness (**d**), prematurity (**e**), small for gestational age (**f**), and medically attended respiratory illness in infants (**g**)
Discussion {#Sec14}
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In this review, we systematically assessed the frequency and impact of two major forms of bias/confounding commonly found in observational studies assessing influenza vaccine effectiveness. Our analysis revealed that the majority of included studies showed evidence for confounding by indication, as judged from the baseline characteristics of vaccinated and unvaccinated study participants. Analysis of crude and adjusted estimates showed that statistical adjustment for confounders corrected for this form of bias, at least partially. However, despite adjustment, nearly half of the studies still showed significant estimates of vaccine effectiveness outside the influenza season, which indicates the presence of unmeasured confounding due to healthy vaccinee bias. Remarkably, significant off-season estimates were not only observed in studies on all-cause mortality, but also regarding other outcomes. However, all outcomes that were used in the included studies were only based on clinical criteria, none of the studies used outcomes with laboratory confirmation of the virus.
At population level, implausibly high mortality benefits of influenza vaccination have been observed particularly in elderly persons. Observational studies found a reduction of mortality of about 50 %, while it was estimated that influenza-related mortality attributed to less than 10 % in this age-group \[[@CR2]\]. These and other observations led to the hypothesis of healthy vaccinee bias \[[@CR36]\]. In healthy vaccinee bias, healthy persons are preferentially vaccinated against influenza, while persons with comorbidities have a lower likelihood to get vaccinated. A small subset of unvaccinated frail and terminally ill patients are suggested to explain the large/implausible results regarding mortality mentioned above. Adjustment for conventional comorbidities as confounders has been suggested to insufficiently capture the functional status of this subgroup \[[@CR35]\]. In fact, in the majority of included studies comorbidities were identified through ICD-codes in administrative databases, which have been shown to fail to control adequately for confounding \[[@CR37]\].
On the contrary, other authors have suggested the opposite form of bias/confounding to be present in observational studies on influenza vaccination. They concluded that patients with comorbidities are preferentially vaccinated against influenza, which reflects current recommendations by the World Health Organization (WHO) and several National Immunization Technical Advisory Groups (NITAGs), but might result in confounding by indication \[[@CR6], [@CR38]\]. Looking at the baseline characteristics of the included studies, we found that the majority of studies showed indication for this type of confounding rather than for healthy vaccinee bias. Remarkably, our meta-analytic approach showed that adjustment for comorbidities had only a small impact on the point estimate of VE. Although this procedure increased the VE estimates in the majority of studies, which is consistent with removal of confounding by indication, the effect size changed on average by only 7 to 12 %. However, since nearly all studies adjusted for comorbidities and other confounders such as sex and age simultaneously in one single step, it is unclear whether and to what extent this effect can be attributed to removal of confounding by comorbidities.
Interestingly, the analyses performed in the study by Campitelli et al. \[[@CR7]\] showed that it is possible to adjust, at least in part, for both forms of bias/confounding, given that enough information have been collected regarding comorbidities and functional status of study participants. Those authors demonstrated that the addition of comorbidities as confounders to the regression model shifted the effect estimate away from 1.0, which indicates correction for confounding by indication. They then added indicators of functional status to the model and observed a shift of the estimate towards 1.0, indicating correction for healthy vaccinee bias. However, additional analyses performed in this study demonstrated that residual confounding was likely to be still present in those data since adjustment for comorbidities and frailty indicators could not eliminate significant off-season estimates.
Our systematic review shows that these findings can be generalized to the body of literature on this issue. In nearly half of the studies identified here, significant off-season estimates were observed despite adjustment for confounders. Although significant off-season estimates were more likely to occur in studies which showed high risk of healthy vaccinee bias at baseline, they were also observed in studies that did not find indication of healthy vaccinee bias by comparing the characteristics of vaccinated and unvaccinated study participants. Interestingly, in studies covering non-elderly participants' significant off-season estimates were not identified. However, there were only five of these studies and it is unclear whether this could be attributed to a lower prevalence of comorbidities or frailty indicators in these age groups or whether this is a chance finding.
The significance of healthy vaccinee bias as well as the suitability of off-season estimates as indicators for its presence has been debated in several publications. Nichol et al. discussed that influenza vaccination is common in patients with functional impairments and frailty, \[[@CR6]\] speaking against the assumption that a terminally ill and frail subgroup of patients is responsible for the observation of off-season estimates. Hak et al. suggested that circulation of influenza in the few months before and after the influenza season might account for "off-season" estimates, as well as a prolonged impact of influenza on mortality which extends several months beyond illness \[[@CR4]\]. On the contrary, the publication by Wong et al. \[[@CR32]\] provided additional evidence that off-season estimates result from healthy vaccinee bias for which the conventional analysis failed to adjust for. Those authors used the same data base that was primarily analyzed as a cohort study to apply instrumental variable technique. Using this study design, they were able to show that quasi-randomization eliminates off-season effects of influenza vaccination, supporting the interpretation that study design and data analysis are crucial here.
The recent debate on bias in influenza VE studies mainly focusses on the outcome all-cause mortality \[[@CR2], [@CR6], [@CR10], [@CR35]\]. Our systematic review demonstrates that significant off-season estimates were also observed in the context of three other clinical outcomes, although significantly less frequent than in mortality studies. All of these outcomes have in common that they are based on unspecific case definitions without laboratory confirmation of influenza infection, which is likely to lead to outcome misclassification. It should be evaluated in future studies whether significant off-season VE estimates are still present when influenza-specific outcomes with laboratory confirmation are assessed.
Our study has several strengths. It is the first systematic review which focused on this issue and examined all published studies with relevant data for the assessment of these two types of bias. In addition, we quantified the extent to which adjustment could correct for confounding by indication regarding different clinical outcomes. However, some limitations of our study have to be addressed although they are mainly caused by limitations of the included studies: First, a number of studies could not be included since they did not provide enough information to assess risk of bias. Some of them included also more specific endpoints with laboratory confirmation. For this reason, the proportion of studies with such biases might be an overestimation. Second, as it is often the case in administrative database-related studies, multiple groups of authors used the same data base and potential overlap between study populations cannot be completely excluded. We detected for example potential overlap between the studies by Campitelli et al. \[[@CR7]\] and Wong et al. \[[@CR32]\]. Third, since studies used different covariates for confounder-adjusted VE estimates and different definition of influenza and off-season periods, direct comparison of the results have to be taken with caution. Furthermore, in nearly all studies statistical adjustment was made in multivariate analysis for a variety of confounder simultaneously. Those sets of confounders did not only include comorbidities, but also age, sex and demographic characteristics. Therefore, the adjusted odds ratios used for our analysis do not accurately reflect confounding by indication. Finally, other types of bias, such as errors in diagnosis or vaccination status, could also have influenced study findings but were not in the focus of our analysis.
Conclusions {#Sec15}
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To conclude, this systematic review supports the hypothesis that confounding by indication and healthy vaccine bias operate simultaneously in observational studies on influenza vaccination using unspecific outcomes. Consequently, it seems impossible to infer whether the adjusted vaccine effectiveness estimates under- or overestimate the true effect of the vaccine. Cohort study designs using administrative data bases with unspecific outcomes such as all-cause mortality should no longer be used to measure the effects of influenza vaccination. Instead, other study designs, including test-negative design studies \[[@CR39]\] and quasi-randomized studies using influenza-specific laboratory-confirmed outcomes, are needed to obtain more reliable estimates of influenza vaccine effectiveness. However, one should be aware that in these study types other forms of bias might operate. This should be assessed in further methodological studies.
Additional files {#Sec16}
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Additional file 1:**Protocol for systematic review: Frequency and impact of selection bias in observational studies assessing influenza vaccine effectiveness: A systematic review.** (DOCX 24 kb)Additional file 2:**Search strategy for the systematic review on frequency and impact of selection bias in observational studies on influenza vaccine effectiveness.** (DOCX 21 kb)Additional file 3:**List of excluded studies.** (DOCX 147 kb)Additional file 4:**Definitions of influenza season periods and control periods (off-season) in the included studies.** (DOCX 28 kb)
**Competing interests**
The authors declare that they have no competing interests.
**Authors' contributions**
CR and TH were responsible for the study design. CR and TH performed the literature search, data extraction, statistical analysis and interpretation of the data and drafted the manuscript. OW interpreted the data, gave important intellectual content and revised the manuscript critically. All authors read and approved the final manuscript.
Funding {#FPar1}
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No funding was received for this study or for manuscript preparation.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
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The chicken gut microbiota influences nutrient utilization ([@B1], [@B2]), immune development ([@B3]), endocrine activity ([@B4]), development of gastrointestinal tract ([@B5]), and detoxification, thus contributing to the improved performance of the birds. The chicken gastrointestinal tract harbors complex communities of bacteria ([@B6], [@B7]). The highest species diversity in the chicken gastrointestinal tract is observed in the cecum, which contains up to 10^11^/g organisms ([@B8][@B9][@B10]) and therefore has been widely studied. In addition to commensal bacteria, cecum also could harbor enteric pathogens that pose both avian and zoonotic health risks ([@B11]). The commensals could prevent the colonization of pathogens by competitive exclusion ([@B12]) and through the production of bacteriocins ([@B13], [@B14]).
Several experiments were conducted previously to study the microbial dynamics in broiler chicken intestinal tract ([@B7], [@B15][@B16][@B17]). Furthermore, studies were performed to determine the effect of gut microbes on feed utilization and conversion ([@B1], [@B18]). However, to determine the microbiome community composition, these experiments used 16S rRNA amplicon-based sequencing or culture-based techniques. 16S rRNA amplicon sequencing is inherently limited due to bias introduced during PCRs. Also, the data have lower resolution and are less efficient in predicting the functional properties of the microbiome. The accuracy of culture-based enumeration of the bacterial population is negatively affected by the inability to grow all the bacteria under culture conditions. In this respect, shotgun metagenomics provides a comprehensive representation of both taxonomical and functional properties of the microbiome. The two studies that used shotgun metagenomics for analyzing chicken microbiome were limited by the number of birds used in those experiments ([@B6], [@B19]).
Feral chickens are derived from domestic chickens that are released to the wild and that survive many generations. Living in the wilderness induces differences in the feeding habits and social behavioral patterns. Previous research in wild fowls and turkeys showed that the microbial communities in these birds differ considerably from those in the domesticated counterparts ([@B20][@B21][@B23]). Therefore, we hypothesized that the feral-adult microbiome could be substantially diverse from the microbial population of the commercial poultry. Use of feral-chicken microbiome as probiotic in commercial poultry practices could increase the diversity of the microbial populations and thereby possibly provide colonization resistance against enteric pathogens.
The objective of this study was to analyze the gut microbial colonization dynamics in gnotobiotic chicken (Gallus gallus) model under conditions of inoculation with the microbiome of adult feral chickens using shotgun metagenomics. Our findings suggested that the feral-chicken microbiome could colonize successfully in the young-chicken gut without causing detrimental health effects on the host.
RESULTS {#s2}
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Determination of *Salmonella* absence in the feral-chicken microbiota inoculum and germfree status of the hatchlings. {#s2.1}
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Gut samples were collected from the viscera of six feral chickens following slaughter. These samples were transferred to an anaerobic chamber, and the pooled cecum and colon contents of 6 feral chickens and the absence of Salmonella enterica in the samples were determined following the protocol described in the Bacteriological Analytical Manual (BAM), United States Food and Drug Administration. Briefly, samples were subjected to enrichment in tetrathionate broth followed by plating in xylose lysine deoxycholate (XLD) for agar plates. Absence of black colonies after 24 h of incubation at 37°C for 24 h was considered to represent a *Salmonella*-negative result. The sterility of the isolator and that of the hatchlings were determined by culturing fecal droppings and drag swabs from gnotobiotic isolators. All samples were found to be negative for bacterial colonies, indicating the germfree status of the chicks.
Phylogenetic distribution of microbiome in gnotobiotic chicken gut compared to inoculum. {#s2.2}
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**(i) Microbiome composition in the inoculum and cecal contents at phylum level.** We sequenced each sample using paired-end 250-base or 300-base chemistry and an Illumina sequencing platform. The sequences generated per sample were on average 3.31 ± 0.43 Gb (mean ± standard error of the mean \[SEM\]). The details of the statistical data are provided in [Table S1](#tabS1){ref-type="supplementary-material"}. A taxonomical abundance table based on reads after host read removal and quality filtering with phylum-level distribution was generated in MG-RAST server version 4.0.3 using the RefSeq database.
10.1128/mSphere.00035-19.1
The overall statistics of data generation for each chicken sample in the current study. Download Table S1, XLSX file, 0.01 MB.
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Thomas et al.
This content is distributed under the terms of the
Creative Commons Attribution 4.0 International license
.
The five major phyla in all the samples were *Bacteroidetes*, *Firmicutes*, *Proteobacteria*, *Actinobacteria*, and *Spirochaetes* ([Fig. 1](#fig1){ref-type="fig"}; see also [Table S2](#tabS2){ref-type="supplementary-material"}). The proportion of *Bacteroidetes* in the feral-chicken inoculum was 66.44% but was lower in the samples from day 9 (50.54% ± 4.77%) and day 18 (43.03% ± 3.19%). There was no significant difference between the day 9 and day 18 samples with respect to the levels of *Bacteroidetes* (*P* = 0.22). The abundance of *Firmicutes* in the inoculum was 20.3% and increased to 29.92% ± 4.77% in the day 9 samples and to 38.51% ± 2.67% in the day 18 samples. The *Firmicutes* abundance in the chicken gut did not increase significantly by day 18 compared to day 9 (*P* = 0.15). In addition, *Proteobacteria* levels increased at day 9 (9.34% ± 1.64%) compared to inoculum (7.63%) but decreased by day 18 (6.38% ± 0.7%) (*P* = 0.13), suggesting the role of facultative anaerobes in initial gut colonization ([@B24]). Similarly to that of *Firmicutes*, the abundance of *Actinobacteria* also increased temporally. In the inoculum, the fraction of *Actinobacteria* was 1.96%, whereas, in the day 9 and day 18 samples, the abundance increased to 5.18% ± 1.57% and 6.77% ± 0.7%, respectively (*P* = 0.38). The percentages of *Spirochetes* remained similar in the inoculum and gnotobiotic chicken samples at day 9 and day 18 (2.09%, 2.45% ± 0.92%, and 2.71% ± 0.55%).
{#fig1}
10.1128/mSphere.00035-19.2
Taxonomically assigned reads at phylum level in different samples. Download Table S2, XLSX file, 0.01 MB.
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2019
Thomas et al.
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Creative Commons Attribution 4.0 International license
.
**(ii) Microbiome composition in the inoculum and cecal contents at genus level.** At the genus level, the inoculum and the day 9 and day 18 samples were composed predominantly of *Bacteroides* ([Fig. 2](#fig2){ref-type="fig"}). However, the abundance was higher in the inoculum (47.78%) than in the day 9 samples (36.98% ± 2.87%) and day 18 samples (30.6% ± 2.12%). *Clostridium* levels increased in the day 9 (7.49% ± 1.15%) and day 18 (9.96% ± 0.76%) cecal contents compared to the inoculum (5.23%). The next-most-abundant genus was *Prevotella*, with the inoculum and gnotobiotic chicken samples showing similar percentages. *Eubacterium* levels increased from and abundance of 1.3% in inoculum to 2.4% ± 0.45% in day 9 samples and 2.99% ± 0.23% in day 18 samples. The abundance of *Ruminococcus* was higher in the day 9 samples (4.24% ± 1.11%) than in the inoculum (1.61%) and then decreased by day 18 (3.33% ± 0.4%). The level of *Parabacteroides*, which represented 3.68% in the inoculum, also decreased, similarly to *Bacteroides*, to 2.06% ± 0.29% and 1.98% ± 0.14% in the day 9 samples and the day 18 samples, respectively. The other three major genera whose levels increased in the cecal contents of gnotobiotic chicken compared to inoculum were *Lactobacillus*, *Collinsella*, and *Blautia* ([Table S3](#tabS3){ref-type="supplementary-material"}).
{#fig2}
10.1128/mSphere.00035-19.3
The percentage abundance of different genera within the inoculum and day 9 and day 18 samples. Download Table S3, XLSX file, 0.01 MB.
Copyright © 2019 Thomas et al.
2019
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.
**(iii) Principal-coordinate analysis (PCoA) and β-diversity.** The principal-coordinate analysis (PCoA) data were calculated using Euclidean distance as the similarity metric for clustering the metagenomes ([Fig. 3](#fig3){ref-type="fig"}). While the day 9 communities were randomly distributed across space, the day 18 communities formed comparatively close clusters, which indicated that the microbial communities had evolved and matured temporally and had attained similar community profiles. These findings are similar to those revealing the microbial shift occurring in a previously uninhabited environment, such as infant gut, where the microbial community attains maturity and stability in the initial years of life ([@B25]).
{#fig3}
Shotgun metagenomics was used to study the dynamics of the microbial community structure in the cecum of gnotobiotic chicken and the inoculum. The β-diversity represents the diversity between the samples with respect to the compositional units. The Morisita-Horn index values ranged from 0 to 1, where 1 indicates similar communities and 0 indicates dissimilar communities, and are given in [Fig. 4A](#fig4){ref-type="fig"}. All the values ranged between 0.7 and 1.0, indicating that the communities were more similar than dissimilar. However, individual variations in the colonization pattern were evident. For example, the communities in the samples from from birds 1C, 1D, and 1F on day 9 and birds 2K, 2L, 2M, 2N, and 2O on day 18 were dissimilar from the inoculum communities. However, the similarity between the samples with respect to the functional characteristics of the communities was higher ([Fig. 4B](#fig4){ref-type="fig"}). The Morisita-Horn index values ranged between 0.98 and 1, suggesting that the functional properties of the inoculum and the cecal samples collected at day 9 and day 18 from gnotobiotic birds were similar.
{#fig4}
**(iv) Functional analysis of the cecal microbiome in the gnotobiotic chicken.** Analysis of functional categorization of the bacterial metagenome provides an understanding of the metabolic profile of the community. The metagenomic reads for feral-chicken inoculum and cecal samples of gnotobiotic chicken collected on day 9 and day 18 were assembled into contigs and then annotated against the MG-RAST server at different levels of hierarchy ([Table S4](#tabS4){ref-type="supplementary-material"}).
10.1128/mSphere.00035-19.4
Overall functional annotation of the samples at the subsystem level using mgRAST server version 4.0.3. Download Table S4, XLSX file, 0.4 MB.
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The overall distribution patterns analyzed at the subsystem level showed similar patterns for inoculum and gnotobiotic chicken microbiome collected on both day 9 and day 18 except for sample 2O due to the higher abundance of reads associated with genus *Brachyspira* ([Fig. 5](#fig5){ref-type="fig"}). The metagenome was enriched for enzymes involved in carbohydrate and protein metabolism. ([Table S4](#tabS4){ref-type="supplementary-material"}). Other predominant functions belonged to categories such as DNA metabolism, RNA metabolism, cofactors, vitamins, and prosthetic groups ([Fig. 5](#fig5){ref-type="fig"}). These findings resembled the β-diversity determined for the functional characteristics, where all the communities exhibited similar profiles.
{#fig5}
DISCUSSION {#s3}
==========
The major objective of this experiment was to develop a gnotobiotic model to investigate the gut microbial colonization dynamics in the cecum of gnotobiotic chickens. Various methods of rearing gnotobiotic chicken have been described previously ([@B17], [@B26][@B27][@B29]). Gnotobiotic chickens have been reared using a custom-designed Gustafsson germfree apparatus ([@B27]). However, simpler methods were developed which made use of various disinfectants to reduce the bacterial load on eggs and sterile isolators ([@B17], [@B26], [@B28], [@B29]). Generally, the disinfectants used were mercuric chloride, quaternary ammonium, iodoform, and sodium hypochlorite solutions and commercially available chlorine dioxide solutions. In this study, Sporicidin was highly efficient in achieving disinfection without damaging the eggshells. Bacterial growth was not observed from samples collected from bird droppings and eggshells 2 days after hatching.
The conventionalization of gnotobiotic chickens using cecal microbial populations derived from adult chickens has been previously conducted ([@B17], [@B30]). The major shortcoming of those studies was that the microbial community was identified using culture-based technique and only a few organisms could be identified ([@B17], [@B30]). In this study, we used shotgun metagenomics to compare the microbiomes of the donor material derived from apparently healthy feral chickens and the gnotobiotic chickens. By enriching for the microbial genomic DNA, shotgun metagenomics could be successfully performed using a MiSeq Illumina platform ([@B31]). The findings from this study indicated that gnotobiotic chicken model, paired with next-generation sequencing techniques, could be an excellent tool to study the dynamics of gastrointestinal microbes in the chicken and could also be utilized in future experiments for studying the pathogenesis of enteric pathogens such as *Salmonella*.
The microbial population for inoculating gnotobiotic chickens in this study was collected from feral chickens that were *Salmonella* negative. The feral chickens originated from domesticated birds that had been released to the wild and had adapted to the wilderness through multiple generations. The process of feralization involves changes in social behavioral patterns, sexual selection, foraging requirements, and adaptation to predation in the wild. The expression of the genes that control these phenotypes also changes in the wild ([@B32]). Along with the host genetic changes, the microbiome could also diverge from that of the domesticated fowls. A study comparing the microbiome of wild and domesticated turkeys indicated that although the levels of diversity and richness of the microbial population were similar, only 30% of the operation taxonomic units (OTUs) were shared between them ([@B23]). In general, these results suggest that the gut microbiota composition of wild birds is more diverse and complex than that of the domesticated ones. This raises the possibility that introduction of these new species to the domesticated poultry could possibly alter the microbial community in a beneficial way in the fight against enteric pathogens. In this study, we found that the feral microbiome could successfully colonize in the young-chicken gut without causing problems such as reduced feed intake, diarrhea, or sepsis.
Metagenomics analysis of the cecal samples revealed that at the genus level, *Bacteroides* (47.78%) represented the most abundant organisms in the feral-chicken microbiome. The next-most-abundant organisms were *Clostridium* (5.23%) and *Prevotella* (7.12%), while *Ruminococcus* (1.61%) and *Lactobacillus* (0.34%) formed lower proportions. This contrasts with reports from studies of broiler chicken cecal microbiome where *Ruminococcus* and *Lactobacilli* were found to be the predominant genera. *Ruminococcus* species formed 15.6% of the total sequences in 3-day-old chicken cecum ([@B16]), and the proportion was 6% in 5-week-old chicken cecum ([@B33]). Similarly, *Lactobacillus* species were detected at 7% to 8% in broiler chicken cecum in those studies. A stable proportion of 16% to 23% *Ruminococcus* species in the total cecal microbiome, which did not alter with age, was observed by Ranjitkar et al. ([@B7]).
The gnotobiotic chicken microbiomes from this study showed that the proportions of *Bacteroidetes*, *Firmicutes*, and *Actinobacteria* in the samples collected at both day 9 and day 18 differed from the proportions in the microbiome of the feral-chicken inoculum. The abundance of *Bacteroidetes* was lower in conventionalized chicken than in the feral-chicken microbiome whereas that of both *Firmicutes* and *Actinobacteria* was higher. Furthermore, in the inoculated germfree chickens used in this study, the proportion of phylum *Bacteroidetes* was initially high and later decreased by day 18 with an increase in *Firmicutes*. Furthermore, the abundance of *Firmicutes* and *Actinobacteria* increased with the increasing age of chicks. The differences in feed and in the ages of the birds could possibly explain these variations in the colonization profile. Dietary intervention is a primary driving force that causes alterations in the microbiome ([@B34], [@B35]). Feral chickens forage in the wild on a variety of feed, including insects, berries, and worms, while the gnotobiotic chicken was fed on poultry starter diet. Another reason for the discrepancy between the feral and gnotobiotic chicken microbiome profiles could be that the inoculum was derived from pooled colon and cecal contents of feral chicken, while the analysis of gnotobiotic chicken microbiome was performed using solely the samples that were collected from the cecum.
Another finding was that although the proportion of *Proteobacteria* was higher at 9 days of age, it decreased with age and reached a level lower than that in the inoculum by day 18. This shift is analogous to the microbial dynamics in human infant gut, where initial colonization is by *Enterococcus* and *Escherichia* followed by *Bifidobacterium* and further by obligate anaerobes belonging to *Firmicutes* and *Bacteroidetes* ([@B36][@B37][@B38]). Similarly, a higher proportion of *Escherichia* was reported in young chicken which was later replaced by obligate anaerobes ([@B7], [@B39]). However, the initial colonization by *Proteobacteria* in broiler chicken can represent a public health risk, especially in the context of infection by enteric pathogens such as *Salmonella* and *Campylobacter*. An early bloom in such pathogenic *Proteobacteria* populations in broiler chicken may not be sufficiently countered by the late colonizers, thus resulting in the risk of infection even at market age ([@B17]). In this study, a decrease in the abundance of *Proteobacteria* was correlated with an increase in the population of *Firmicutes* and *Actinobacteria* on day 18. Our findings suggest that early administration of adult-feral-chicken microbiome could effectively prevent prolonged colonization of facultative anaerobes in chickens.
The microbial profile given in [Fig. 2](#fig2){ref-type="fig"} shows the interindividual variation. The differences between individual birds were more pronounced at day 9 as indicated by the PCoA plot data ([Fig. 3A](#fig3){ref-type="fig"}). Similar variations in microbial composition between the experimental birds have been reported previously ([@B1], [@B2]). Similarly to our findings where the samples from 18-day-old chickens clustered much more closely, the microbial communities from older broiler chicken were previously reported to cluster with less variation than the communities from younger birds ([@B39]). In that experiment, the inoculum served as the sole source of microbes and successful colonization of the microbiome happened by 9 days of age.
In contrast, the functional properties of the microbial communities were more stable at day 9 and day 18 and were similar to those seen in the feral-chicken inoculum even while the microbial compositions were different. There were no significant differences between the inoculum and the day 9 and day 18 samples with respect to functional properties even at level 2 in the hierarchy determined using the SEED Subsystems database. Similar results for functional properties of chicken cecal microbiomes were observed previously ([@B6], [@B19], [@B39]).The variability between individuals for the taxonomic profile occurring during the microbial dynamics was not reflected in the functional profiles in these studies. It has been found that microbes occupying equivalent niches share similar functional properties even in diverse hosts ([@B40]). The microbial assemblage characteristics in a previously uninhabited habitat could be driven by equivalence in functional aspects rather than by the stochastic nature of microbial colonization. In this study, with the host niches being similar, the evenness in functional properties of the communities despite taxonomical variability could be explained only if functionally similar organisms were occupying equivalent niches.
Chickens act as a reservoir for enteric human pathogens, especially *Salmonella*. Recently, various *Salmonella* serotypes such as Enteritidis, I,\[5\],12:i:-; Typhimurium; Heidelberg; Hadar; Mbandaka; Montevideo; Agona; and Infantis have been found to be associated with *Salmonella* outbreaks ([@B41], [@B42]). Those rampant multistate *Salmonella* outbreaks due to transmission from live poultry reveal the necessity of pursuing studies aimed at control of *Salmonella* in poultry. The presence of enteric pathogens in poultry was controlled by using antibiotic feed additives ([@B43]). With the recent FDA regulations designed to limit the use of antimicrobials in the food supply due to public health concerns, use of such antibiotic feed additives is currently highly controlled. It is pertinent to develop alternatives such as prebiotics and probiotics that could manipulate the microbial community in chicken and thus competitively exclude enteric pathogens. The pioneering work by Nurmi and Rantala in 1973 demonstrated prevention of growth of *Salmonella* by competitive exclusion in the adult chicken microbiome whereas the microbiome from young birds was incapable of preventing the growth of *Salmonella* ([@B17]). The recent outbreaks suggest that this subject should get renewed attention as there is evidence indicating that more *Salmonella* serotypes have been adapting to chickens and causing a potential threat to public health ([@B41]). Since our results show that feral-chicken gut microbiota could colonize germfree chickens, our results raise the possibility that such a complex community might exclude the colonization of pathogens such as *Salmonella* in these birds. Our model could also be used for determining the mechanistic effect of microbiota subcomponents by conducting polyassociation studies by inoculating simple to complex defined gut bacterial species. The gnotobiotic chicken model developed here could also be used for determining the safety and efficacy of new probiotic species by conducting monoassociation or polyassociation studies.
MATERIALS AND METHODS {#s4}
=====================
Experiment, sampling, and *Salmonella* detection. {#s4.1}
-------------------------------------------------
Feral chickens were obtained locally near Brookings, SD, USA. The feral flock was once a captive flock of mixed breed and has been feral for no less than 8 years. The birds forage on a small grain farm and in surrounding grasslands. Feral chickens were sampled during a routine slaughter for personal meat consumption by the land owners. Gut samples from six birds were collected from the viscera following slaughter. The intestine was ligated at distal ileum and distal colon, maintained in ice, and transported immediately to the laboratory. Protocols used in this study for sample collection were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at South Dakota State University, Brookings, SD. For processing, one portion of the sample for sequencing was frozen at −80°C, the remainder of each sample was transferred to a Coy anaerobic chamber, and the contents were expelled into 50-ml sterile conical tubes. Samples were diluted 1:10 (wt/vol) using anaerobic brain heart infusion broth supplemented with volatile fatty acids and vitamins (BHI-M), mixed by repeated pipetting, and aliquoted into cryovials. Anaerobic dimethyl sulfoxide (DMSO) was added at 18% (final concentration) and stored at −80°C until inoculation into young chickens. We used the detection protocol described in the FDA Bacteriological Analytical Manual (<https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm070149.htm>) for determining the absence of *Salmonella* in the feral-chicken cecal samples. Briefly, 5 g of fecal sample from each chicken was added to peptone water and incubated aerobically at 37°C overnight. We then transferred 1 ml peptone mixture to 24 ml selective enrichment broth with tetrathionate brilliant green broth (TTB) and incubated the reaction mixture at 37°C for 24 h. We then streaked these samples on xylose lysine tergitol-4 (XLT-4) agar plates and incubated the reaction mixture aerobically at 37°C for 24 h. Absence of black colonies in the plates after 24 h of incubation was considered to represent absence of *Salmonella*. For preparing the inoculant for inoculation into germfree chickens, *Salmonella*-free samples were thawed and samples of stock from 6 feral chickens were pooled at equal volumes and further diluted 1:10 using anaerobic phosphate-buffered saline (PBS).
Gnotobiotic chickens were reared using a modified protocol that was described previously ([@B17]). Eggs of White Leghorn chickens were acquired from a commercial hatchery, treated with Sporicidin disinfectant solution (Contec, Inc.) and sterile water, and incubated in an incubator (pretreated with Sporicidin) at 37°C and 55% humidity. Humidity was maintained using a 1% (wt/vol) aqueous solution of potassium permanganate. After 19 days of incubation, eggs were removed from the incubator and candled for viability confirmation. Viable eggs were transferred to a biosafety cabinet and dipped in Sporicidin solution for 15 s and then wiped with a sterile cloth saturated with sterile water. Eggs were then transferred to autoclaved egg trays, placed in sterile autoclave bags, and transferred immediately to the port of the isolator unit. Eggs were sprayed with 5% peracetic acid and, after 20 min of exposure, were transferred inside the isolators. Eggs were maintained at 37°C and 65% humidity until hatching occurred on day 21.
Following hatching, birds were provided sterilized water *ad libitum* and a gamma-irradiated starter diet (LabDiet 5065, Irradiated) designed to meet the nutrient requirements of young chickens ([Table 1](#tab1){ref-type="table"}) and monitored daily. On day 3 posthatch, birds (*n* = 16) were inoculated orally with 300 µl of pooled cecal contents. Seven birds were euthanized using cervical dislocation on day 9, and nine birds were euthanized on day 18 posthatch. The cecal contents were collected for DNA isolation and stored at −20°C until use.
######
Nutritional composition and energy content of the LabDiet 5065 irradiated diet
Parameter \%
--------------------------- ------
Composition
Protein 22.1
Fat (ether extract) 4.2
Fat (acid hydrolysis) 5.2
Fiber (maximum) 2.8
Nitrogen-free extract 55.6
Minerals 5.3
Energy source
Protein 25.4
Fat (ether extract) 10.7
Carbohydrates 63.9
Total Energy (kcal/g) 3.48
To assess the sterility of the isolator, swabs were collected on day 2 posthatch from the egg shells, droppings, and isolator floor and transferred to anaerobic transport media ([@B44]) and removed from the isolator. The swabs were then streaked on BHI-M agar plates and incubated aerobically at 37°C. The plates were examined for the presence of bacterial colonies after 24 h and 48 h of incubation.
Genomic DNA isolation from the cecal contents. {#s4.2}
----------------------------------------------
Genomic DNA was isolated using a Powersoil DNA isolation kit (Mo Bio Laboratories Inc., CA). Briefly, approximately 100 mg of cecal contents was transferred to bead tubes and samples were homogenized for 2 min using a TissueLyser (Qiagen, Germantown, MD). DNA isolation was performed according to the manufacturer's protocol, and DNA was eluted in 50 µl nuclease-free water. The quality of genomic DNA samples was assessed using NanoDrop One (Thermo Fisher Scientific, Wilmington, DE), and the samples were quantified using a Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA). Samples were stored at −20°C until use.
Microbial DNA enrichment and shotgun metagenomics sequencing. {#s4.3}
-------------------------------------------------------------
Selective enrichment of bacterial genomic DNA was performed using a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., MA) following methods previously published by our group ([@B31]). Briefly, 0.5 µg of genomic DNA was treated with 80 µl of MBD2-Fc-bound magnetic beads in the presence of binding buffer and incubated at room temperature for 15 min with rotation. After incubation, beads were separated by keeping the tubes on a magnetic rack for 5 min. The supernatant containing microbial DNA was transferred to a fresh tube. The DNA was further purified using Agencourt AMPure XP beads (Beckman Coulter) and stored at −20^0^C.
For shotgun metagenome sequencing, the enriched genomic DNA from pooled feral samples, seven samples from day 9 and nine samples from day 18 posthatch gnotobiotic chickens, was used. The concentrations of genomic DNA samples were adjusted to 0.3 ng/µl. Samples were then processed using a Nextera XT DNA sample prep kit (Illumina Inc., San Diego, CA) according to the manufacturer's protocol. Purified products with unique barcodes were normalized using the bead normalization protocol of the manufacturer, and equal volumes of normalized libraries were pooled and diluted in hybridization buffer. The diluted libraries were subjected to heat denaturation prior to loading into the sequencer. Illumina paired-end sequencing was performed on the MiSeq platform using 2-by-250 paired-end sequencing chemistry.
Sequence data processing. {#s4.4}
-------------------------
The raw data files were demultiplexed and converted to fastq files using Casava v.1.8.2 (Illumina, Inc., San Diego, CA, USA). Reads aligning to chicken genome were identified using Bowtie 2 v2.3.0 ([@B45]). The reads were mapped against the chicken genome using Bowtie 2, and trimming was performed using SolexaQA++ (~V~3.1.7.1) with a phred score of 20 ([@B46]). The raw reads were then analyzed against the MG-RAST version 4.0.3 pipeline ([@B47]). The quality control steps in MG-RAST included dereplication, ambiguous base filtering, and length filtering. The taxonomical abundance was analyzed using MG-RAST with the best-hit classification approach using the RefSeq database, and parameters were limited to a minimum E value of 10^−5^, minimum identity of 60%, a minimum abundance of 50, and a minimum alignment length of 30 amino acids. The functional abundance was analyzed using hierarchical classification in MG-RAST and the SEED Subsystems database, and parameters were limited to a minimum E value of 10^−5^, minimum identity of 60%, a minimum abundance of 50, and a minimum alignment length of 30 amino acids. The OTU abundance tables were downloaded from MG-RAST and were used for downstream statistical analysis.
For functional analysis, after host read removal and trimming, the reads were assembled using Spades V3.11.1 with the "---meta" flag ([@B48]). The contigs that were less than 500 bp in length were removed from this study. Thereafter, we annotated the remaining contigs in MG-RAST server version 4.0.3 and functional profiling was performed at different hierarchical levels (E value = 10^−5^, minimum percentage identity of 60%, and a minimum alignment length of 100 amino acids). The heat map was constructed in the Morpheus server (<https://software.broadinstitute.org/morpheus>) with a Euclidean distance matrix and average clustering method.
Statistical analysis. {#s4.5}
---------------------
The beta diversity between the feral-chicken inoculum and the day 9 and day 18 samples was estimated using the Morisita-Horn index in Explicet software ([@B49]). The PCoA analysis of taxonomical diversity was performed using Calypso version 8.72 ([@B50]). The differences between the day 9 and day 18 samples with respect to the phylum-level distributions were calculated in GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA) using the nonparametric Mann-Whitney U test. Significant differences were recorded at *P* values of \<0.05. Genus-level distribution tables were analyzed using Explicet software.
Data availability. {#s4.6}
------------------
The data sets used in the current study are available in the NCBI SRA database under accession number [PRJNA415593](https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA415593).
J.S. conceived and designed the experiments. M.T., S.W., S.G., L.A., and A.S. performed the experiments. R.K. analyzed the data. J.S., K.C.D., E.N., S.C., T.W., and T.J. contributed reagents and materials. M.T., R.K., J.S., and K.C.D. wrote the manuscript. All of us reviewed and approved the manuscript.
This work was supported in part by the USDA National Institute of Food and Agriculture, Hatch projects SD00H532-14 and SD00R540-15, and a grant from the South Dakota Governor's Office of Economic Development awarded to J.S.
We declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
[^1]: **Citation** Thomas M, Wongkuna S, Ghimire S, Kumar R, Antony L, Doerner KC, Singery A, Nelson E, Woyengo T, Chankhamhaengdecha S, Janvilisri T, Scaria J. 2019. Gut microbial dynamics during conventionalization of germfree chicken. mSphere 4:e00035-19. <https://doi.org/10.1128/mSphere.00035-19>.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Vagal paragangliomas (VPs) are rare neuroendocrine tumors arising from the neural crest-derived paraganglionic tissue surrounding the vagus nerve. Paragangliomas (PGs) have classically been said to account for 0.6% of head and neck tumors, and, first described in 1935, VPs are the most rare subtype of head and neck PGs, contributing less than 5% of their total \[[@B1]\]. VPs typically present as a neck mass associated with hoarseness or pulsatile tinnitus \[[@B2]\]. PGs of the head and neck may undergo malignant transformation in 6% to 19% of cases; VPs are the most likely to become malignant and to metastasize \[[@B3]\]. There is potential for PGs to be metabolically active in secreting catecholamines, but this feature is present in only 2% of VP cases \[[@B3]\]. Management options include an observational approach, stereotactic radiosurgery, and surgical excision \[[@B1], [@B3]\]. Herein a unique case of vagal nerve paraganglioma is presented, where a neck mass was associated with severe fits of coughing upon palpation.
2. Case Report {#sec2}
==============
A 70-year-old female presented with a 4-week history of a neck mass. She had suffered a concurrent sporadic dry cough unrelated to eating, talking, or any particular movement; fits of coughing would often be so severe that she would vomit. She reported that touching the neck mass would trigger the cough. The frequency and severity of the cough were diminishing her quality of life. She reported concomitant intermittent hoarseness. She denied dysphagia or velopharyngeal insufficiency. She had no family history of paragangliomas or other neuroendocrine tumors, or any relevant past medical history. On examination, a 2.5 cm right-sided level two neck mass deep to the sternocleidomastoid was present, which was discrete, mobile, and nonpulsatile. Palpation of the mass immediately triggered coughing. Flexible fiberoptic laryngoscopy demonstrated normal true vocal fold appearance and movement bilaterally, with prominent retropharyngeal carotid arteries the only finding of note. The remainder of the examination was unremarkable apart from a mildly hoarse voice.
A blood panel was normal and 24-hour urine was negative for catecholamines. CT angiography of the neck demonstrated a heterogeneously enhanced irregular 2.4 × 2.2 × 2.2 cm mass within the right carotid sheath along the lateral aspect of the proximal internal and external carotid arteries with minimal splaying of these arteries (Figures [1(a)](#fig1){ref-type="fig"}, [1(b)](#fig1){ref-type="fig"}, and [1(c)](#fig1){ref-type="fig"}). The central area of the mass was of lower attenuation suggestive of necrosis. MRI displayed a 2.5 × 1.9 × 3 cm mass extending from the inferior margin of the right carotid to the level of the piriform sinuses within the carotid sheath which was intensely enhanced following contrast administration (Figures [2](#fig2){ref-type="fig"} and [3](#fig3){ref-type="fig"}). Differential diagnoses included vagal paraganglioma, schwannoma, neurofibroma, or a necrotic lymph node. The CT and MR images were reviewed at our institution\'s head and neck radiology conference, with the consensus favoring a schwannoma over a paraganglioma due to the relatively homogenous appearance and lack of flow voids within the mass. To differentiate between these two pathologies, Indium-111-labelled octreotide scintigraphy was recommended. Octreotide is a somatostatin analogue which accumulates within paragangliomas due to a high density of somatostatin type 2 receptors on their cell surface; schwannomas do not uptake octreotide \[[@B4]\]. The mass was intensely avid on octreotide scan at both 4 and 24 hours with no additional visible masses. Therefore VP was now unequivocally the most likely pathology. The patient was counseled on management options. Due to the impact of the cough on her quality of life, she elected for surgical removal of the mass.
The mass was excised through a 7 cm incision made two finger-breadths below the mandible, overlying the sternocleidomastoid. Right perifacial and level II lymph nodes were resected to improve access to the mass. The mass was dissected free from the internal and external carotid arteries and the internal jugular vein. The vagal nerve was intimately involved with the mass, thus requiring suture ligation and removal with the mass. The spinal accessory and hypoglossal nerves were identified and preserved. The pathology report described a completely excised encapsulated 3.5 × 2 × 1.5 cm tumor associated with a nerve segment and ganglion. The central 10% of the tumor showed tumor necrosis. Immunohistochemical staining for SDHB was positive, with intratumoral heterogeneity. Positive immunostaining for SDHB suggests that the patient does not have a germline mutation that caused her paraganglioma; however, the significance of intratumoral heterogeneity is unclear, and therefore genetic counseling and the option of genetic testing were recommended. All dissected lymph nodes were negative for malignancy.
On postoperative evaluation in the office, the patient reported complete resolution of her severe dry cough after surgery. She was extremely pleased with this result. Her glottic closure and vocal quality remained good in the early postoperative period; therefore she chose to defer intervention for her vocal fold paralysis, with the understanding that her hoarseness would worsen over time as vocal fold atrophy occurred. She had no dysphagia after surgery and continued to tolerate a regular diet without difficulty. Genetic counseling and the option of genetic testing were discussed with the patient, based on the suggestion of the interpreting pathologist, which the patient declined. She elected to pursue further follow-up visits and future voice rehabilitation with a local otolaryngologist in her community.
3. Discussion {#sec3}
=============
Paragangliomas arise from the paraganglia, which are small groups of neuroendocrine cells stemming from autonomic nervous system ganglia. Usually slow growing and benign, tumors of the paraganglia are most common in the adrenal medulla (pheochromocytoma), with 85% of extra-adrenal PGs in the abdomen, 12% in the thorax, and 3% in the head and neck \[[@B1]\]. Four genetic PG syndromes have been described, all with autosomal dominant transmission. Germline mutations are known in three, all in the gene complex encoding succinate dehydrogenase (SDH): with subunits SDHD, SDHC, or SDHB mutated, any of which predisposes PGs \[[@B3]\]. SDH is a mitochondrial enzyme complex with an important role in oxidative phosphorylation and intracellular oxygen sensing and signaling. It is thought that SDH mutations cause dysregulation of hypoxia-induced factors, thereby yielding a cellular response mimicking that of hypoxia, which is known to cause carotid paraganglial hypertrophy \[[@B5]\]. However, more than half of head and neck PGs are sporadic without an identifiable genetic cause.
Surgical excision is the classical treatment of choice for most VPs; however contemporary management is evolving toward more conservative measures due to the high associated morbidity \[[@B1]\]. VP resection almost always requires vagus nerve sacrifice with resultant speech, swallow, and sensory deficits \[[@B1], [@B3], [@B6]\]. Observation with serial imaging has been successfully employed, with the majority of lesions remaining radiologically stable, although neuropathy progression has occurred in a third of the cases \[[@B1], [@B3], [@B6]\]. Observation is especially valuable for asymptomatic older patients \[[@B1]\]. Stereotactic radiosurgery is a proven treatment option for VPs \< 3 cm in maximum dimension and should be offered as a potential first-line treatment \[[@B1]\].
In the presented case, the severity of symptoms resulted in the management undertaken. A report exists describing a patient with a cough associated with VP; however this was a mild chronic cough, complicated by gastroesophageal reflux, and cough was not triggered by palpation of the mass \[[@B7]\]. Bouts of syncopal coughing associated with an intracranial jugular foramen paraganglioma have been reported, with transient cough-related increases in intracranial pressure as the proposed mechanism \[[@B8]\].
4. Conclusion {#sec4}
=============
A neck mass associated with cough on its palpation has previously been described as pathognomonic for vagal nerve schwannoma \[[@B9]\], yet an alternate diagnosis was reached in this case. To the best of our knowledge this is the first description in the English literature of a VP presenting as a neck mass associated with a severe cough that could be triggered by palpation of the mass. This case is presented to aid physicians should they encounter a neck mass associated with cough. Vagal paraganglioma, although rare, should be part of the differential in such a presentation.
Disclosure
==========
This study was deemed IRB exempt. This case was presented at the Triological Society Annual Meeting at COSM, May 20-21, 2016, in Chicago, Illinois.
Conflicts of Interest
=====================
The authors declare no conflicts of interest.
{#fig1}
{#fig2}
{#fig3}
[^1]: Academic Editor: Abrão Rapoport
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A novel Coronavirus (2019-nCoV) was identified in hospitalized patients in Wuhan, China, in December 2019 and January 2020 \[[@CR1]\]. Coronaviruses are enveloped, non-segmented positive-sense RNA viruses belonging to the family Coronaviridae and the order Nidovirales. They are broadly distributed in humans and other mammals \[[@CR2]\]. The number of patients affected by COVID-19 is rapidly increasing almost all over the world.
The following procedure has been suggested for diagnosis of patients with suspected infection: performing real-time fluorescence (RT-PCR) to detect the positive nucleic acid of SARS-CoV-2 in sputum, throat swabs, and secretions of the lower respiratory tract samples \[[@CR3]\].
Preliminary data have shown that once infected, patients display an extremely variable clinical course, from mild symptoms (fever and cough) to bilateral interstitial pneumonia. In the most serious cases, infection progresses into acute respiratory distress syndrome (ARDS) with diffuse alveolar consolidations (diffuse patchy-like lesions) \[[@CR4]\]. Furthermore, recent reports have classified patients into different clinical phenotypes from 1 to 5, with increasing clinical severity up to pre-ARDS (phenotype 4) and ARDS (phenotype 5).
Symptoms are very not specific, especially at an early stage, since they are common to most upper and lower airway diseases. The specific etiological diagnosis is performed through laboratory methods, but the main concern is to direct the patient to the best care setting (home observation, medical department/infectious disease clinic, sub-intensive ward or ICU).
Imaging methods are employed to assess the degree of lung involvement. The typical computed tomography (CT) feature of a patient with acute COVID-19 infection is that of ground-glass opacities (GGO) or mixed GGO, consolidation and vascular enlargement \[[@CR5], [@CR6]\]; lesions are more likely to display a peripheral distribution and bilateral involvement, and to be lower lung predominant \[[@CR5], [@CR6]\]. Although chest CT seems to be the method of choice, it is burdened by the risk of radiation exposure and concerns related to infection risk and the transport of the critical patient.
Thoracic ultrasound is an easy-to-learn and reliable method \[[@CR7]--[@CR13]\]. No advanced machinery or software is required. In an emergency setting or at bedside, the use of pocket-size ultrasound seems to be optimal. It is essential for the clinician to know and implement personal safety procedures before starting the examination. In particular, the physician performing the ultrasound must wear appropriate PPE. In case of a COVID-19 + patient with respiratory symptoms, these items are a surgical mask, gloves, a waterproof gown, and goggles or a visor; in the case of high risk of exposure to aerosols (patients subjected to continuous positive airway pressure or non-invasive ventilation), the use of a FFP2/3 mask, a double pair of gloves, goggles or a visor and boots is recommended. Ultrasound machine and probes must be disinfected with alcohol-based disinfectants before and after ultrasound, and thus equipped with disposable protective materials (probe cover).
The probe can be used as a stethoscope to auscultate/scan any portion of the lung not covered by bones. In practice, there are ultrasound scan protocols in emergency settings that provide for a minimum of 6--8 scans per hemithorax on a patient on supine position (2/4 anterior, 2 lateral and 2 posterior) \[[@CR8], [@CR12], [@CR13]\]. One of the main limitations of thoracic ultrasound is that it cannot be used to examine the deep fields of the lung due to air interface and artifacts, but the physiopathology of COVID-19 disease provides for a predominant involvement of the pulmonary periphery \[[@CR5], [@CR6]\], thus favoring the application of the method.
The main ultrasound finding in COVID-19 pneumonia is that of an acute interstitial disease, which is represented by vertical hyperechoic artifacts that depart from the pleural line directed in depth. These are called B lines \[[@CR14], [@CR15]\]. If ≤ 3 B lines on a scan are physiological, especially in the middle-basal fields, the presence of a greater number is pathological, and the number of B lines correlates with disease severity (Figs. [1](#Fig1){ref-type="fig"}, [2](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}).Fig. 1Lateral chest scan. Single vertical hyperechoic artifact originating from the pleural line, called "B line"Fig. 2Posterior chest area scan. Vertical artifacts ("B lines"), isolated and non-confluentFig. 3--4Lateral chest scans. Vertical artifacts ("B lines"), numerous and coalescent at a stretch of pleural line of increased thickness
Indeed, the presence of some B lines spread over several scans seems to be an early finding of COVID-19, even in mildly symptomatic subjects; in the most serious cases such as those that are pre-ARDS or ARDS, the B lines end up filling the ultrasound image almost completely, until they merge, creating a single hyperechoic image identified as "white lung" and distortion and irregularity of the pleural line (Figs. [4](#Fig3){ref-type="fig"}, [5](#Fig4){ref-type="fig"}).Fig. 5Postero-basal chest scan. Lung field is occupied by numerous B lines that come together to form a single hyperechoic image. This aspect is named "white lung," referring to the ground-glass opacities found in the computed chest tomography
Initially, those ultrasound findings were related to cardiogenic pulmonary edema, in which the B lines are present bilaterally, with a base-apex gradient and with progressive involvement depending on the severity of the disease \[[@CR7]\]. Moreover, B lines are also found in other diseases such as bruises, chronic fibrotic lung diseases and ARDS. ARDS can be distinguished from cardiogenic pulmonary edema by the presence of multifocal and diffuse lung alterations, without a base--apex gradient, while chronic interstitial diseases differ by an extreme irregularity of the pleural line, which can be thickened and distorted, with the presence of focal subpleural interruptions (consolidations), and by the presence of uneven and irregular vertical artifacts (B lines) \[[@CR7], [@CR16], [@CR17]\].
The difference between the features of B lines found in cardiogenic interstitial edema and chronic fibrosing pathologies has recently led to new proposals for reclassification of the types of artifacts \[[@CR18], [@CR19]\]. The ultrasound pattern of COVID-19 pneumonia seems to be very similar to that found in patients suffering from chronic pulmonary fibrosis; both are based on a prevalent interstitial involvement. In the most severe and advanced forms, lung consolidations are present, representing pulmonary pathological areas that are no longer normally ventilated (Figs. [6](#Fig5){ref-type="fig"}, [7](#Fig6){ref-type="fig"}, [8](#Fig7){ref-type="fig"}, [9](#Fig8){ref-type="fig"}).Fig. 6Longitudinal posterior chest scan. Coarse subpleural thickening with very irregular marginsFig. 7Lateral chest scan. Coarse supleural thickening with thin aerial bronchogramsFig. 8Anterior chest longitudinal scan. Minute subpleural parenchymal thickeningFig. 9Posterior chest longitudinal scan. Small basal pleural effusion
In the early stage of COVID-19, lung changes are more localized, and mainly detected in the subpleural regions of one or both lungs (corresponding to patchy or segmental GGOs) (Figs. [2](#Fig2){ref-type="fig"}, [4](#Fig3){ref-type="fig"}). Later, viral infection and inflammatory response involve multiple lobes, leading to air loss and consolidations of some lesions surrounded by several B lines (Figs. [6](#Fig5){ref-type="fig"}, [7](#Fig6){ref-type="fig"}, [8](#Fig7){ref-type="fig"}). In more advanced and critical cases, ultrasound findings resemble other pneumonia features, such as diffuse consolidations in both lungs, multiple and diffuse B lines (white lung), and occasionally by small amounts of pleural effusion (Fig. [5](#Fig4){ref-type="fig"}; Table [1](#Tab1){ref-type="table"}) \[[@CR20]\].Table 1Suggested clinical and sonographic classification of COVID-19 pneumoniaClinical stageClinical findingsUltrasound findingsI stage: simple diseasePatients with viral upper respiratory tract infection not complicatedNormal ultrasound pattern: A linesII stage: mild pneumoniaPatient with pneumonia and no signs of severe pneumoniaFocal B linesIII stage: severe pneumoniaFever or suspected respiratory infection, and at least one of the following: respiratory rate \> 30 acts/min, severe breathing difficulties or SpO~2~ \< 90% in ambient airDiffuse and multifocal B linesInitial subpleuric consolidationsIV stage: acute respiratory distress syndrome (ARDS)Respiratory failure that is not related to heart failure or overloaded with liquids. 200 mmHg \< PaO~2~/FiO~2~ ≤ 300 mmHg (with PEEP or CPAP ≥ 5 cmH~2~O or not ventilated)Diffuse and multifocal B linesLung consolidationsWhite lung patternAdapted from <https://www.siaarti.it/SiteAssets/News/COVID19%20-%20documenti%20SIAARTI/Percorso%20COVID-19%20-%20Sezione%201%20%20-%20Procedura%20Area%20Critica.pdf>
Therefore, thoracic ultrasound seems suitable for screening for lung involvement of patients with suspected or ascertained SARS-COVID-19 infection due to its easy applicability. It can be also a relevant method for monitoring patients. Indeed, follow-up of the previously described ultrasound findings according to the pre-established scan scheme can document improvements in or the worsening of the disease. It can thus, together with clinical-laboratory data, prompt therapeutic modifications. In an intensive care unit (ICU) setting, ultrasound can be a support for lung recruitment maneuvers, by assisting clinicians in ascertaining the possible re-expansion of the atelectasis areas (from consolidation to aerial artifacts).
Furthermore, the ultrasound evaluation of diaphragm excursion can provide relevant information about the patient\'s ventilatory capacity and, indirectly, about lung compliance \[[@CR17], [@CR21]--[@CR23]\]. In this context, an ultrasound follow-up of the degree of diaphragmatic excursion could identify a worsening of the pneumonia requiring non-invasive or invasive ventilatory support, or on the contrary, an improvement, prompting a subsequent attempt to wean the patient off or downgrade the respiratory support (Fig. [10](#Fig9){ref-type="fig"}).Fig. 10Ultrasound evaluation of diaphragm excursion. An oblique subcostal scan should be performed by convex probe (scan for the gallbladder), by adjusting the depth of the image so as to completely highlight the liver margins, with the diaphragm represented as a hyperechoic line surrounding the liver. The M-mode option is activated by centering the line on the bed of the gallbladder and evaluating the degree of excursion of the diaphragm
In conclusion, thoracic ultrasound can be used as a screening method for lung involvement in any care setting, even at the patient's home. The pathological characteristics (B lines and consolidations) revealed by ultrasound and their extent correlate with the severity of the disease, and can be employed as a monitoring method. Ultrasound is a useful tool to support the ventilated patient in terms of assessing diaphragm mobility and supporting recruitment maneuvers.
**Publisher\'s Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The authors declare no conflicts of interest and no funding sources.
Informed consent to publish figures was obtained from patients.
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Introduction {#Sec1}
============
The Elliptic Curve Digital Signature Algorithm (ECDSA) \[[@CR13]\], first proposed in 1992 by Scott Vanstone \[[@CR26]\], is a standard public key signature protocol widely deployed. ECDSA is used in the latest library TLS 1.3, email standard OpenPGP and smart cards. It is also implemented in the library OpenSSL, and can be found in cryptocurrencies such as Bitcoin, Ethereum and Ripple. It benefits from a high security based on the hardness of the elliptic curve discrete logarithm problem and a fast signing algorithm due to its small key size. Hence, it is recognized as a standard signature algorithm by institutes such as ISO since 1998, ANSI since 1999, and IEEE and NIST since 2000.
The ECDSA signing algorithm requires scalar multiplications of a point *P* on an elliptic curve by an ephemeral key *k*. Since this operation is time-consuming and often the most time-consuming part of the protocol, it is necessary to use an efficient algorithm. The Non Adjacent Form (NAF) and its windowed variant (wNAF) were introduced as an alternative to the binary representation of the nonce *k* to reduce the execution time of the scalar multiplication. Indeed, the NAF representation does not allow two non-zero digits to be consecutive, thus reducing the Hamming weight of the representation of the scalar. This improves on the time of execution as the latter is dependent on the number of non-zero digits. The wNAF representation is present in implementations such as in Bitcoin, as well as in the libraries Cryptlib, BouncyCastle and Apple's Common-Crypto. Moreover, until very recently (May 2019), wNAF was present in all three branches of OpenSSL.
However, implementing the scalar multiplication using wNAF representation and no added layer of security makes the protocol vulnerable to side-channel attacks. Side-channel attacks were first introduced about two decades ago by Kocher et al. \[[@CR14]\], and have since been used to break many implementations, and in particular some cryptographic primitives such as AES, RSA, and ECDSA. They allow to recover secret information throughout observable leakage. In our case, this leakage corresponds to differences in the execution time of a part of the signing algorithm, observable by monitoring the cache.
For ECDSA, cache side-channel attacks such as [Flush&Reload]{.smallcaps} \[[@CR28], [@CR29]\] have been used to recover information about either the sequence of operations used to execute the scalar multiplication, or for example in \[[@CR8]\] the modular inversion. For the scalar multiplication, these operations are either a multiplication or an addition depending on the bits of *k*. This information is usually referred to as a double-and-add chain or the trace of *k*. A trace is created when a signature is produced by ECDSA and thus we talk about signatures and traces in an equivalent sense. At this point, we ask how many traces need to be collected to successfully recover the secret key. Indeed, from an attacker's perspective, the least traces necessary, the more efficient the attack is. This quantity depends on how much information can be extracted from a single trace and how combining information of multiple traces is used to recover the key. We work on the latter to minimize the number of traces needed.
The nature of the information obtained from the side channel attack allows to determine what kind of method should be carried out to recover the secret key. Attacks on ECDSA are inspired by attacks on a similar cryptosystem, DSA. In 2001, Howgrave-Graham and Smart \[[@CR12]\] showed how knowing partial information of the nonce *k* in DSA can lead to a full secret key recovery. Later, Nguyen and Shparlinski \[[@CR19]\] gave a polynomial time algorithm that recovers the secret key in ECDSA as soon as some consecutive bits of the ephemeral key are known. They showed that using the information leaked by the side channel attack, one can recover the secret key by constructing an instance of the Hidden Number Problem (HNP) \[[@CR4]\]. The basic structure of the attack algorithm is to construct a lattice which contains the knowledge of consecutive bits of the epheremal keys, and by solving CVP or SVP, to recover the secret key. This type of attack has been done in \[[@CR3], [@CR8], [@CR26], [@CR28]\]. However, these results considered perfect traces, but obtaining traces without any misreadings is very rare. In 2018, Dall et al. \[[@CR6]\] included an error-resilience analysis to their attack: they showed that key recovery with HNP is still possible even in the presence of erroneous traces.
In 2016, Fan, Wang and Cheng \[[@CR7]\] used another lattice-based method to attack ECDSA: the Extended Hidden Number Problem (EHNP) \[[@CR11]\]. EHNP mostly differs from HNP by the nature of the information given as input. Indeed, the information required to construct an instance of EHNP is not sequences of consecutive bits, but the positions of the non-zero coefficients in any representation of some integers. This model, which we consider in this article as well, is relevant when describing information coming from [Flush&Reload]{.smallcaps} or [Prime&Probe]{.smallcaps} attacks for example, the latter giving a more generic scenario with no shared data between the attacker and the victim. In \[[@CR7]\], the authors are able to extract 105.8 bits of information per signature on average, and thus require in theory only 3 signatures to recover a 256-bit secret key. In practice, they were able to recover the secret key using 4 error-free traces.
In order to optimize an attack on ECDSA various aspects should be considered. By minimizing the number of signatures required in the lattice construction, one minimizes the number of traces needed to be collected during the side-channel attack. Moreover, reducing the time of the lattice part of the attack, and improving the probability of success of key recovery allows to reduce the overall time of the attack. In this paper, we improve on all three of these aspects. Furthermore, we propose the first error-resilience analysis for EHNP and show that key recovery is still possible with erroneous traces too.
***Contributions:*** In this work, we reinvestigate the attack against ECDSA with wNAF representation for the scalar multiplication using EHNP. We focus on the lattice part of the attack, *i.e.*, the exploitation of the information gathered by the side-channel attack. We first assume we obtain a set of error-free traces from a side-channel analysis. We preselect some of these traces to optimize the attack. The main idea of the lattice part is then to use the ECDSA equation and the knowledge gained from the selected traces to construct a set of modular equations which include the secret key as an unknown. These modular equations are then incorporated into a lattice basis similar to the one given in \[[@CR7]\], and a short vector in it will contain the necessary information to reconstruct the secret key. We call "experiment" one run of this algorithm. An experiment succeeds if the algorithm recovers the secret key.
*A New Preprocessing Method.* The idea of selecting good traces beforehand has already been explored in \[[@CR27]\]. The authors suggest three rules to select traces that improve the attack on the lattice part. Given a certain (large) amount of traces available, the lattice is usually built with a much smaller subset of these traces. Trying to identify beforehand the traces that would result in a better attack is a clever option. The aim of our new preprocessing---that completely differs from \[[@CR27]\]---is to regulate the size of the coefficients in the lattice, and this results in a better lattice reduction time. For instance, with 3 signatures, we were able to reduce the total time of the attack by a factor of 7.
*Analyzing the Attack.* Several parameters intervene while building and reducing the lattice. We analyze the performance of the attack with respect to these parameters and present the best parameters that optimize either the total time or the probability of success.
First, we focus on the attack time. Note that when talking about the overall time of the attack, we consider the average time of a single experiment multiplied by the number of trials necessary to recover the secret key. We compare[1](#Fn1){ref-type="fn"} our times with the numbers reported in \[[@CR7], Table 3\] with method C. Indeed, methods *A* and *B* in \[[@CR7]\] use extra information that comes from choices in the implementation which we choose to ignore as we want our analysis to remain as general as possible. The comparison is justified as we consider the same leakage model, and compare timings when running experiments on similar machines. For 4 signatures, our attack is slightly slower[2](#Fn2){ref-type="fn"} than timings in \[[@CR7]\]. However, when considering more than 4 signatures, our attack is faster. We experiment up to 8 signatures to further improve our overall time. In this case, our attack runs at best in 2 min and 25 s. Timings for 8 signatures are not reported in \[[@CR7]\], and the case of 3 signatures was never reached before our work. In Table [1](#Tab1){ref-type="table"}, we compare our times with the fastest times reported by \[[@CR7]\]. We choose their fastest times but concerning our results we choose to report experiments which are faster (not the fastest) with, if possible, better probability than theirs.Table 1.Comparing attack times with \[[@CR7]\], for 5000 experiments.Number of signaturesOur attack\[[@CR7]\]TimeSuccess ($\documentclass[12pt]{minimal}
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\begin{document}$$\%$$\end{document}$)339 h0.2----41 h 17 min0.541 min1.558 min 20 s6.518 min16$\documentclass[12pt]{minimal}
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\begin{document}$$\approx 5$$\end{document}$ min2518 min227$\documentclass[12pt]{minimal}
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\begin{document}$$\approx 2$$\end{document}$ min29----
The overall time of the attack is also dependent on the success probability of key recovery. From Table [2](#Tab2){ref-type="table"}, one can see that our success probability is higher than \[[@CR7]\], except for 7 signatures. They have $\documentclass[12pt]{minimal}
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\begin{document}$$68\%$$\end{document}$ of success with their best parameters whereas we only reach $\documentclass[12pt]{minimal}
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\begin{document}$$45\%$$\end{document}$ in this case.
For the sake of completeness, we mention that in \[[@CR21]\], the authors use HNP to recover the secret key using 13 signatures. Their success probability in this case is around 54$\documentclass[12pt]{minimal}
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\begin{document}$$\%$$\end{document}$ and their overall time is close to 20 s, hence much faster. However, as their leakage model is different, we do not further mention their work.
*Finding the Key with Only Three Signatures.* Overall, combining a new preprocessing method, a modified lattice construction and a careful choice of parameters allows us to mount an attack which works in practice with only 3 signatures. However, the probability of success in this case is very low. We were able to recover the secret key only once with BKZ-35 over 5000 experiments. This result is difficult to quantify as a probability but we note that finding the key a single time over 5000 experiments is still much better than randomly finding a 256-bit integer. If we assume the probability is around $\documentclass[12pt]{minimal}
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\begin{document}$$0.02 \% $$\end{document}$, as each trial costs 200 s in average, we can expect to find the secret key after 12 days using a single core. Note that this time can be greatly reduced when parallelizing the process, *i.e.*, each trial can be run on a separate core. On the other hand, if we use our preprocessing method, with 3 signatures we obtain a probability of success of $\documentclass[12pt]{minimal}
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\begin{document}$$0.2\%$$\end{document}$ and a total time of key recovery of 39 h, thus the factor 7 of improvement mentioned above. Despite the low probability of success, this result remains interesting nonetheless. Indeed, the authors in \[[@CR7]\] reported that in practice, the key couldn't be recovered using less than 4 signatures and we improve on their result.Table 2.Comparing success probability with \[[@CR7]\], for 5000 experiments.Number of signaturesOur attack\[[@CR7]\]Success ($\documentclass[12pt]{minimal}
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\begin{document}$$\%$$\end{document}$)TimeSuccess ($\documentclass[12pt]{minimal}
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\begin{document}$$\%$$\end{document}$)Time30.239 h----4425 h 28 min1.541 min5202 h 42 min436 min6401 h 4 min351 h 43 min7452 h 36 min683 h 58 min8455 h 2 min----
*Resilience to Errors.* We also investigate the resilience to errors of our attack. Such an analysis has not yet been done in the setup of EHNP. It is important to underline that collecting traces without any errors using any side-channel attack is very hard. Previous works used perfect traces to mount the lattice attack. Thus, it required collecting more traces. As pointed out in \[[@CR7]\], more or less twice as many signatures are needed if errors are considered. In practice, this led \[[@CR7]\] to gather in average 8 signatures to be able to find the key with 4 perfect traces. We experimentally show that we are still able to recover the secret key even in the presence of faulty traces. In particular, we find the key using only 4 faulty traces, but with a very low probability of success. As the percentage of incorrect digits in the trace grows, the probability of success decreases and thus more signatures are required to successfully recover the secret key. For instance, if $\documentclass[12pt]{minimal}
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\begin{document}$$2\%$$\end{document}$ of the digits are wrong among all the digits of a given set of traces, it is still possible to recover the key with 6 signatures. This result is valid if errors are uniformly distributed over the digits. However, we have a better probability to recover the key if errors consist in 0-digit faulty readings, *i.e.*, 0 digits read as non-zero. In other words, the attack could work with a higher percentage of errors, around $\documentclass[12pt]{minimal}
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\begin{document}$$4\%$$\end{document}$, if we could ensure from the side channel attack and some preprocessing methods that none of the non-zero digits have been flipped to 0.
***Organization:*** Sect. [2](#Sec2){ref-type="sec"} gives background on ECDSA and the wNAF representation. In Sect. [3](#Sec6){ref-type="sec"}, we explain how to transform EHNP into a lattice problem. We explicit the lattice basis and analyze the length of the short vectors found in the reduced basis. In Sect. [4](#Sec10){ref-type="sec"}, we introduce our preprocessing method which allows us to reduce the overall time of our attack. In Sect. [5](#Sec13){ref-type="sec"}, we give experimental results. Finally, in Sect. [6](#Sec14){ref-type="sec"}, we give an error resilience analysis.
Preliminaries {#Sec2}
=============
Elliptic Curves Digital Signature Algorithm {#Sec3}
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The ECDSA algorithm is a variant of the Digital Signature Algorithm, DSA, \[[@CR17]\] which uses elliptic curves instead of finite fields. The parameters used in ECDSA are an elliptic curve *E* over a finite field, a generator *G* of prime order *q* and a hash function *H*. The private key is an integer $\documentclass[12pt]{minimal}
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\begin{document}$$k\leftarrow _R\mathbb {Z}_q$$\end{document}$ and compute \[*k*\]*G*. Let *r* be the *x*-coordinate of \[*k*\]*G*. If $\documentclass[12pt]{minimal}
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\begin{document}$$s=0$$\end{document}$, select a new nonce *k*. Finally, the signature is given by the pair (*r*, *s*).
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\begin{document}$$v_2=r\cdot s^{-1}\bmod q$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$(x,y)=[v_1]G+[v_2]p_k$$\end{document}$. Finally, the signature is valid if $\documentclass[12pt]{minimal}
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\begin{document}$$x\equiv r \pmod q$$\end{document}$.
We consider a 128-bit level of security. Hence $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha , q$$\end{document}$ and *k* are 256-bit integers.
WNAF Representation {#Sec4}
-------------------
The ECDSA algorithm requires the computation of \[*k*\]*G*, a scalar multiplication. In \[[@CR10]\], various methods to compute fast exponentiation are presented. One family of such methods is called window methods and comes from NAF representation. Indeed, the NAF representation does not allow two non-zero digits to be consecutive, thus reducing the Hamming weight of the representation of the scalar. The basic idea of a window method is to consider chunks of *w* bits in the representation of the scalar *k*, compute powers in the window bit by bit, square *w* times and then multiply by the power in the next window. The window methods can be combined with the NAF representation of *k*. For any $\documentclass[12pt]{minimal}
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\begin{document}$$k \in \mathbb {Z}$$\end{document}$, a representation $\documentclass[12pt]{minimal}
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\begin{document}$$k = \sum _{j=0}^{\infty }k_j 2^j$$\end{document}$ is called a NAF if $\documentclass[12pt]{minimal}
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\begin{document}$$k_j \in \{0, \pm 1\}$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$k_jk_{j+1} =0$$\end{document}$ for all $\documentclass[12pt]{minimal}
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\begin{document}$$j \ge 0$$\end{document}$. Moreover, every *k* has a unique NAF representation. The NAF representation minimizes the number of non-zero digits $\documentclass[12pt]{minimal}
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\begin{document}$$k_j$$\end{document}$. It is presented in Algorithm 1.
The NAF representation can be combined with a sliding window method to further improve the execution time. For instance, in OpenSSL (up to the latest versions using wNAF 1.1.1b), the window size usually chosen was $\documentclass[12pt]{minimal}
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\begin{document}$$w = 3$$\end{document}$, which is the value we set for all our experiments. The scalar *k* is converted into wNAF form using Algorithm 2. The sequence of digits $\documentclass[12pt]{minimal}
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\begin{document}$$m_i$$\end{document}$ belongs to the set $\documentclass[12pt]{minimal}
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\begin{document}$$ \{0, \pm 1, \pm 3, \dots , \pm (2^w -1)\}$$\end{document}$. Let *k* be the sum of its non-zero digits, renamed $\documentclass[12pt]{minimal}
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\begin{document}$$k_i$$\end{document}$. More precisely, we get $\documentclass[12pt]{minimal}
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\begin{document}$$k = \sum _{j=1}^{\ell }k_j 2^{\lambda _j},$$\end{document}$ where $\documentclass[12pt]{minimal}
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\begin{document}$$\ell $$\end{document}$ is the number of non-zero digits, and $\documentclass[12pt]{minimal}
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\begin{document}$$\lambda _j$$\end{document}$ represents the position of the digit $\documentclass[12pt]{minimal}
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\begin{document}$$k_j$$\end{document}$ in the wNAF representation.
### Example 1 {#FPar1}
In binary, we can write $\documentclass[12pt]{minimal}
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\begin{document}$$ 23 = 2^4 + 2^2 + 2^1 + 2^0 = (1,0,1,1,1)$$\end{document}$ whereas in NAF-representation, we have $\documentclass[12pt]{minimal}
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\begin{document}$$ 23 = 2^5 - 2^3 - 2^0 = (1, 0, -1,0, 0, -1). $$\end{document}$ With $\documentclass[12pt]{minimal}
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\begin{document}$$w=3$$\end{document}$, the wNAF representation gives $\documentclass[12pt]{minimal}
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\begin{document}$$ 23 = 2^{4} + 7 \times 2^0 = (1, 0, 0, 0, 7). $$\end{document}$
Lattice Reduction Algorithms {#Sec5}
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A $\documentclass[12pt]{minimal}
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\begin{document}$$\mathbb {Z}$$\end{document}$-lattice is a discrete additive subgroup of $\documentclass[12pt]{minimal}
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\begin{document}$$\mathbb {Z}^n$$\end{document}$. It is usually specified by a *basis matrix* $\documentclass[12pt]{minimal}
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\begin{document}$$B \in \mathbb {Z}^{n \times n}$$\end{document}$. The lattice *L*(*B*) generated by *B* consists of all integer combinations of the row vectors in *B*. The determinant of a lattice is the absolute value of the determinant of a basis matrix: $\documentclass[12pt]{minimal}
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\begin{document}$$\det L(B) = |\det B|$$\end{document}$. The discreteness property ensures that there is a vector $\documentclass[12pt]{minimal}
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\begin{document}$$v_1$$\end{document}$ reaching the minimum non-zero value for the euclidean norm. Let us write $\documentclass[12pt]{minimal}
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\begin{document}$$||v_1||_2 = \lambda _1$$\end{document}$. Let $\documentclass[12pt]{minimal}
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\begin{document}$$\lambda _i$$\end{document}$ be the $\documentclass[12pt]{minimal}
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\begin{document}$$i^{th}$$\end{document}$ successive minimum of the lattice. The [LLL]{.smallcaps} algorithm \[[@CR15]\] takes as an input a lattice basis, and returns in polynomial time in the lattice dimension *n* a reduced lattice basis whose vectors $\documentclass[12pt]{minimal}
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\begin{document}$$b_i$$\end{document}$ satisfy the worst-case approximation bound $\documentclass[12pt]{minimal}
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\begin{document}$$||b_i||_2 \le 2^{(n-1)/2} \lambda _i$$\end{document}$. In practice, for random lattices, [LLL]{.smallcaps} obtains approximation factors such that $\documentclass[12pt]{minimal}
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\begin{document}$$||b_1||_2 \le 1.02^n \lambda _1$$\end{document}$ as noted by Nguyen and Stehlé \[[@CR18]\]. Moreover, for random lattices, the Gaussian heuristic implies that $\documentclass[12pt]{minimal}
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\begin{document}$$\lambda _1 \approx \sqrt{n/(2\pi e)} \det (L)^{1/n}.$$\end{document}$
The [BKZ]{.smallcaps} algorithm \[[@CR22], [@CR24]\] is exponential in some given block-size $\documentclass[12pt]{minimal}
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\begin{document}$$\beta $$\end{document}$ and polynomial in the lattice dimension *n*. It outputs a reduced lattice basis whose vectors $\documentclass[12pt]{minimal}
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\begin{document}$$b_i$$\end{document}$ satisfy the approximation $\documentclass[12pt]{minimal}
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\begin{document}$$||b_i||_2 \le i \gamma _\beta ^{(n-i)/(k-1)}\lambda _i$$\end{document}$ \[[@CR23]\], where $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma _\beta $$\end{document}$ is the Hermite constant. In practice, Chen and Nguyen \[[@CR5]\] observed that BKZ returns vectors such that $\documentclass[12pt]{minimal}
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\begin{document}$$b_1 \le (1+\epsilon _\beta )^n \lambda _1$$\end{document}$ where $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon _\beta $$\end{document}$ depends on the block-size $\documentclass[12pt]{minimal}
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\begin{document}$$\beta $$\end{document}$. For random lattices, they get $\documentclass[12pt]{minimal}
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\begin{document}$$1+\epsilon _\beta = 1.01$$\end{document}$ for a block-size $\documentclass[12pt]{minimal}
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\begin{document}$$\beta = 85$$\end{document}$.
Attacking ECDSA Using Lattices {#Sec6}
==============================
Using some side-channel attack, one can recover information about the wNAF representation of the nonce *k*. In particular, it allows us to know the positions of the non-zero coefficients in the representation of *k*. However, the value of these coefficients are unknown. This information can be used in the setup of the Extended Hidden Number Problem (EHNP) to recover the secret key. For many messages *m*, we use ECDSA to produce signatures (*r*, *s*) and each run of the signing algorithm produces a nonce *k*. We assume we have the corresponding trace of the nonce, that is, the equivalent of the double-and-add chain of *kG* using wNAF. The goal of the attack is to recover the secret $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ while optimizing either the number of signatures required or the total time of the attack.
The Extended Hidden Number Problem {#Sec7}
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The Hidden Number Problem (HNP) allows to recover a secret element $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha \in \mathbb {Z}_q$$\end{document}$ if some information about the most significant bits of random multiples of $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha \pmod q$$\end{document}$ are known for some prime *q*. Boneh and Venkatesan show how to recover $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ in polynomial time with probability greater than 1/2. In \[[@CR11]\], the authors extend the HNP and present a polynomial time algorithm for solving the instances of this extended problem. The Extended Hidden Number Problem is defined as follows. Given *u* congruences of the form$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} a_i \alpha + \sum _{j = 1}^{\ell _i}b_{i,j}k_{i,j} \equiv c_i \pmod q, \end{aligned}$$\end{document}$$where the secret $\documentclass[12pt]{minimal}
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\begin{document}$$0 \leqslant k_{i,j} \leqslant 2^{\eta _{ij}}$$\end{document}$ are unknown, and the values $\documentclass[12pt]{minimal}
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\begin{document}$$b_{i,j}$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$c_i$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$\ell _i$$\end{document}$ are known for $\documentclass[12pt]{minimal}
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\begin{document}$$1 \leqslant i \leqslant u$$\end{document}$ (see \[[@CR11]\], Definition 3), one has to recover $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ in polynomial time. The EHNP can then be transformed into a lattice problem and one recovers the secret $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ by solving a short vector problem in a given lattice.
Using EHNP to Attack ECDSA {#Sec8}
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From the ECDSA algorithm, we know that given a message *m*, the algorithm outputs a signature (*r*, *s*) such that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \alpha r = sk - H(m) \pmod q. \end{aligned}$$\end{document}$$The value *H*(*m*) is just some hash of the message *m*. We consider a set of *u* signature pairs $\documentclass[12pt]{minimal}
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\begin{document}$$(r_i, s_i)$$\end{document}$ with corresponding message $\documentclass[12pt]{minimal}
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\begin{document}$$m_i$$\end{document}$ that satisfy Eq. ([2](#Equ2){ref-type=""}). For each signature pair, we have a nonce *k*. Using the wNAF representation of *k*, we write $\documentclass[12pt]{minimal}
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\begin{document}$$k = \sum _{j =1}^{\ell } k_j 2^{\lambda _j}$$\end{document}$, with $\documentclass[12pt]{minimal}
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\begin{document}$$k_j \in \{\pm 1, \pm 3, \dots , \pm (2^w -1)\}$$\end{document}$ and the choice of *w* depends on the implementation. Note that the coefficients $\documentclass[12pt]{minimal}
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\begin{document}$$k_j$$\end{document}$ are unknown, however, the positions $\documentclass[12pt]{minimal}
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\begin{document}$$\lambda _j$$\end{document}$ are supposed to be known via some side-channel leakage. It is then possible to represent the ephemeral key *k* as the sum of a known part, and an unknown part. As the value of $\documentclass[12pt]{minimal}
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\begin{document}$$k_j$$\end{document}$ is odd, one can write $\documentclass[12pt]{minimal}
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\begin{document}$$k_j = 2k_j' + 1$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$$ -2^{w-1} \leqslant k_j' \leqslant 2^{w-1} -1$$\end{document}$. Using the same notations as in \[[@CR7]\], set $\documentclass[12pt]{minimal}
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\begin{document}$$d_j = k_j' + 2^{w-1}$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$$0 \le d_j \le 2^w -1$$\end{document}$. In the rest of the paper, we will denote by $\documentclass[12pt]{minimal}
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\begin{document}$$\mu _j$$\end{document}$ the window-size of $\documentclass[12pt]{minimal}
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\begin{document}$$d_j$$\end{document}$. Note that here, $\documentclass[12pt]{minimal}
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\begin{document}$$\mu _j = w$$\end{document}$ but this window-size will be modified later. This allows to rewrite the value of *k* as$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} k = \sum _{j=1}^{\ell }k_j 2^{\lambda _j} = \bar{k} + \sum _{j=1}^{\ell }d_j 2^{\lambda _j +1}, \end{aligned}$$\end{document}$$with $\documentclass[12pt]{minimal}
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\begin{document}$$\bar{k} = \sum _{j=1}^{\ell }2^{\lambda _j} - \sum _{j=1}^{\ell }2^{\lambda _j + w}$$\end{document}$. The expression of $\documentclass[12pt]{minimal}
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\begin{document}$$\bar{k}$$\end{document}$ represents the known part of *k*. By substituting *k* in Eq. ([3](#Equ3){ref-type=""}), we get a system of modular equations:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \alpha r_i - \sum _{j = 1}^{\ell _i} 2^{\lambda _{i, j}+1} s_i d_{i, j} - (s_i\bar{k_i} - H(m_i)) \equiv 0 \pmod q \end{aligned}$$\end{document}$$where the unknowns are $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ and the $\documentclass[12pt]{minimal}
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\begin{document}$$d_{i,j}$$\end{document}$. The known values are $\documentclass[12pt]{minimal}
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\begin{document}$$\ell _i$$\end{document}$, which is the number of non-zero digits in *k* for the $\documentclass[12pt]{minimal}
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\begin{document}$$i^{th}$$\end{document}$ sample, $\documentclass[12pt]{minimal}
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\begin{document}$$\lambda _{i,j}$$\end{document}$, which is the position of the $\documentclass[12pt]{minimal}
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\begin{document}$$j^{th}$$\end{document}$ non-zero digit in *k* for the $\documentclass[12pt]{minimal}
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\begin{document}$$i^{th}$$\end{document}$ sample and $\documentclass[12pt]{minimal}
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\begin{document}$$\bar{k}$$\end{document}$ defined above. Equation ([4](#Equ4){ref-type=""}) is then used as input to EHNP, following the method explained in \[[@CR11]\]. The problem of finding the secret key is then reduced to solving the short vector problem in a given lattice presented in the following section.
Constructing the Lattice {#Sec9}
------------------------
Before giving the lattice basis construction, we redefine Eq. ([4](#Equ4){ref-type=""}) to reduce the number of unknown variables in the system. This will allow us to construct a lattice of smaller dimension. Again, we use the same notations as in \[[@CR7]\].
*Eliminating One Variable.* One straightforward way to reduce the lattice dimension is to eliminate a variable from the system. In this case, one can eliminate $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ from Eq. ([4](#Equ4){ref-type=""}). Let $\documentclass[12pt]{minimal}
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\begin{document}$$E_i$$\end{document}$ denote the $\documentclass[12pt]{minimal}
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\begin{document}$$i^{th}$$\end{document}$ equation of the system. Then by computing $\documentclass[12pt]{minimal}
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\begin{document}$$r_1E_i - r_iE_1$$\end{document}$, we get the following new modular equationsUsing the same notations as in \[[@CR7]\], we define $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _{j,i} = 2^{\lambda _{1, j}+1}s_1r_i $$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma _{i,j} = -2^{\lambda _{i, j}+1}s_i r_1$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$ \gamma _i = r_1(s_i \bar{k}_i - H(m_i)) + r_i(s_1\bar{k}_1 - H(m_1)) $$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$2 \leqslant i \leqslant u$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$1 \leqslant j \leqslant \ell _i$$\end{document}$. Even if $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ is eliminated from the equations, if we recover some $\documentclass[12pt]{minimal}
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\begin{document}$$d_{i,j}$$\end{document}$ values from a short vector in the lattice, we can recover $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ using any equation in the modular system ([4](#Equ4){ref-type=""}). We now use Eq. ([5](#Equ5){ref-type=""}) to construct the lattice basis.
*From a Modular System to a Lattice Basis.* Let $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {L}$$\end{document}$ be the lattice constructed for the attack, and we have $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {L} = \mathcal {L}(\mathcal {B})$$\end{document}$ where the lattice basis $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B}$$\end{document}$ is given below. Let $\documentclass[12pt]{minimal}
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\begin{document}$$m= \max _{i, j} \mu _{ij}$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$1\leqslant j \leqslant \ell _i$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$2\leqslant i \leqslant u$$\end{document}$. We set a scaling factor $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \in \mathbb {N}$$\end{document}$ to be defined later. The lattice basis is given byLet $\documentclass[12pt]{minimal}
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\begin{document}$$n= (u-1) + T +1 = T+u$$\end{document}$, with $\documentclass[12pt]{minimal}
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\begin{document}$$T = \sum _{i=1}^u \ell _i,$$\end{document}$ be the dimension of the lattice. The $\documentclass[12pt]{minimal}
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\begin{document}$$u-1$$\end{document}$ first columns correspond to Eq. ([5](#Equ5){ref-type=""}) for $\documentclass[12pt]{minimal}
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\begin{document}$$2\le i \le u$$\end{document}$. Each of the remaining columns, except the last one, corresponds to a $\documentclass[12pt]{minimal}
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\begin{document}$$d_{ij}$$\end{document}$, and contains coefficients that allow to regulate the size of the $\documentclass[12pt]{minimal}
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\begin{document}$$d_{ij}$$\end{document}$. The determinant of $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {L}$$\end{document}$ is given by $\documentclass[12pt]{minimal}
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\begin{document}$$ \det \mathcal {L} = q^{u-1} \left( \varDelta 2^m\right) ^{u-1} 2^{\sum _{i, j}(m - \mu _{i,j})} 2^{m-1}. $$\end{document}$
The lattice is built such that there exists $\documentclass[12pt]{minimal}
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\begin{document}$$w \in \mathcal {L}$$\end{document}$ which contains the unknowns $\documentclass[12pt]{minimal}
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\begin{document}$$d_{i, j}$$\end{document}$. To find it, we know there exists some values $\documentclass[12pt]{minimal}
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\begin{document}$$t_2, t_2, \dots , t_u$$\end{document}$ such that if $\documentclass[12pt]{minimal}
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\begin{document}$$v = \left( t_2, \dots , t_u, d_{1, 1}, \dots , d_{u, \ell _u}, -1 \right) $$\end{document}$, we get $\documentclass[12pt]{minimal}
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\begin{document}$$w = v\mathcal {B},$$\end{document}$ and$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} w = (0, \dots , 0, d_{1, 1}2^{m - \mu _{1, 1}}-2^{m-1}, \dots , d_{u, \ell _u}2^{m - \mu _{u, \ell _u}}-2^{m-1}, -2^{m-1}). \end{aligned}$$\end{document}$$If we are able to find *w* in the lattice, then we can reconstruct the secret key $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$. In order to find *w*, we estimate its norm and make sure *w* appears in the reduced basis. After reducing the basis, we look for vectors of the correct shape, *i.e.*, with sufficiently many zeros at the beginning and the correct last coefficient, and attempt to recover $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ for each of these.
*How the Size of*$\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ *Affects the Norms of the Short Vectors.* In order to find the vector *w* in the lattice, we reduce $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B}$$\end{document}$ using LLL or BKZ. For *w* to appear in the reduced basis, one should at least set $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ such that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} ||w||_2 \leqslant (1.02)^n (\det L)^{1/n}. \end{aligned}$$\end{document}$$The vector *w* we expect to find has norm $\documentclass[12pt]{minimal}
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\begin{document}$$||w||_2 \leqslant 2^{m-1}\sqrt{T+1}$$\end{document}$. From Eq. ([6](#Equ6){ref-type=""}), one can deduce the value of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ needed to find *w* in the reduced lattice:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \varDelta \geqslant \frac{(T+1)^{(T+u) /(2(u-1))} 2^{\frac{1 + \sum \mu _{i, j} - (u+T)}{u-1}}}{q (1.02)^{\frac{(T+u)^2}{u-1}}}:=\varDelta _{th}. \end{aligned}$$\end{document}$$In our experiments, the average value of $\documentclass[12pt]{minimal}
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\begin{document}$$\ell _i$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$1\leqslant i \leqslant u$$\end{document}$ is $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{\ell } = 26,$$\end{document}$ and thus $\documentclass[12pt]{minimal}
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\begin{document}$$T = u \times \tilde{\ell }$$\end{document}$ on average. Moreover, the average value of $\documentclass[12pt]{minimal}
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\begin{document}$$\mu _{ij}$$\end{document}$ is 7 and so on average $\documentclass[12pt]{minimal}
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\begin{document}$$\sum {\mu _{ij}} = 7 \times u \times \tilde{\ell }$$\end{document}$. Hence, if we compute $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta _{th}$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$u = 3, \dots , 8$$\end{document}$, with these values, we obtain $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta _{th} \ll 1$$\end{document}$, which does not help us to set this parameter. In practice, we verify that $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta =1$$\end{document}$ allows us to recover the secret key. In Sect. [5](#Sec13){ref-type="sec"}, we vary the size of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ to see whether a slightly larger value affects the probability of success.
*Too Many Small Vectors.* While running [BKZ]{.smallcaps} on $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B}$$\end{document}$, we note that for some specific sets of parameters the reduced basis contains some undesired short vectors, *i.e.*, vectors that are shorter than *w*. This can be explained by looking at two consecutive rows in the lattice basis given above, say the $\documentclass[12pt]{minimal}
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\begin{document}$$j^{th}$$\end{document}$ row and the $\documentclass[12pt]{minimal}
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\begin{document}$$(j+1)^{th}$$\end{document}$ row. For example, one can look at rows which correspond to the $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma _{i, j}$$\end{document}$ values but the same argument is valid for the rows concerning the $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _{j, i}$$\end{document}$. From the definitions of the $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma $$\end{document}$ values we have $\documentclass[12pt]{minimal}
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\begin{document}$$ \sigma _{i,j+1} = -2^{\lambda _{i,j+1} +1} \cdot s_i r_1= -2^{\lambda _{i,j+1} +1} \cdot ( \frac{ \sigma _{i,j} }{-2^{\lambda _{i,j} +1}} ).$$\end{document}$ So $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma _{i,j+1} = 2^{\lambda _{i,j+1} -\lambda _{i,j}} \cdot \sigma _{i,j} .$$\end{document}$ Thus the linear combination given by the $\documentclass[12pt]{minimal}
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\begin{document}$$(j+1)^{th}$$\end{document}$ row minus $\documentclass[12pt]{minimal}
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\begin{document}$$ 2^{\lambda _{i,j+1} -\lambda {i,j}} $$\end{document}$ times the $\documentclass[12pt]{minimal}
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\begin{document}$$j^{th}$$\end{document}$ row gives a vector$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} ( 0\, , \, \cdots , 0 \, , -2^{\lambda _{i,j+1} -\lambda _{i,j} +m - \mu _{i,j}} \, ,\, 2^{m-\mu _{i,j+1}}\, ,\, 0\, , \, \cdots , 0). \end{aligned}$$\end{document}$$Yet, this vector is expected to have smaller norm than *w*. Some experimental observations are detailed in Sect. [5](#Sec13){ref-type="sec"}.
*Differences with the Lattice Construction Given in* \[[@CR7]\]*.* Let $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B'}$$\end{document}$ be the lattice basis constructed in \[[@CR7]\]. Our basis $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B}$$\end{document}$ is a rescaled version of $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B'}$$\end{document}$ such that $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {B} = 2^m \varDelta \mathcal {B'}$$\end{document}$. This rescaling allows us to ensure that all the coefficients in our lattice basis are integer values. Note that \[[@CR7]\] have a value $\documentclass[12pt]{minimal}
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\begin{document}$$\delta $$\end{document}$ in their construction which corresponds to $\documentclass[12pt]{minimal}
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\begin{document}$$1/\varDelta $$\end{document}$. In this work, we give a precise analysis of the value of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$, both theoretically and experimentally in Sect. [5](#Sec13){ref-type="sec"}, which is missing in \[[@CR7]\].
Improving the Lattice Attack {#Sec10}
============================
Reducing the Lattice Dimension: The Merging Technique {#Sec11}
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In \[[@CR7]\], the authors present another way to further reduce the lattice dimension, which they call the merging technique. It aims at reducing the lattice dimension by reducing the number of non-zero digits of *k*. The lattice dimension depends on the value $\documentclass[12pt]{minimal}
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\begin{document}$$T = \sum _{i=1}^{u}\ell _i$$\end{document}$, and thus reducing *T* reduces the dimension. To understand the attack, it suffices to know that after merging, we obtain some new values $\documentclass[12pt]{minimal}
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\begin{document}$$\ell '$$\end{document}$ corresponding to the new number of non-zero digits and $\documentclass[12pt]{minimal}
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\begin{document}$$\lambda _j'$$\end{document}$ the position of these digits for $\documentclass[12pt]{minimal}
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\begin{document}$$1 \leqslant j \leqslant \ell '$$\end{document}$. After merging, one can rewrite $\documentclass[12pt]{minimal}
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\begin{document}$$k = \bar{k} + \sum _{j=1}^{\ell '}d_j'2^{\lambda _j' +1}$$\end{document}$, where the new $\documentclass[12pt]{minimal}
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\begin{document}$$d_j'$$\end{document}$ have a new window size which we denote $\documentclass[12pt]{minimal}
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\begin{document}$$\mu _{j}$$\end{document}$, *i.e.*, $\documentclass[12pt]{minimal}
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\begin{document}$$0 \leqslant d_j' \leqslant 2^{\mu _j} - 1$$\end{document}$.
We present our merging algorithm based on Algorithm 3 given in \[[@CR7]\]. Our algorithm modifies directly the sequence $\documentclass[12pt]{minimal}
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\begin{document}$$\{\lambda _j\}_{j=1}^{\ell }$$\end{document}$, whereas \[[@CR7]\] work on the double-and-add chains. This helped us avoid some implementation issues such as an index outrun present in Algorithm 3 \[[@CR7]\], line 7. To facilitate the ease of reading of (our) Algorithm 3, we work with dynamic tables. Let us first recall various known methods we use in the algorithm: $\documentclass[12pt]{minimal}
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\begin{document}$$push\_back(e)$$\end{document}$ inserts an element *e* at the end of the table, *at*(*i*) outputs the element at index *i*, and *last*() returns the last element of the table. We consider tables of integers indexed in $\documentclass[12pt]{minimal}
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\begin{document}$$[ 0; S - 1 ]$$\end{document}$, where *S* is the size of the table.
A useful example of the merging technique is given in \[[@CR7]\]. From 3 to 8 signatures the approximate dimension of the lattices using the elimination and merging techniques are the following: 80, 110, 135, 160, 190 and 215. Each new lattice dimension is roughly $\documentclass[12pt]{minimal}
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\begin{document}$$54 \%$$\end{document}$ of the dimension of the lattice before applying these techniques, for the same number of signatures. For instance, with 8 signatures we would have a lattice of dimension 400 on average, far too large to be easily reduced. For the traces we consider, after merging the mean of the $\documentclass[12pt]{minimal}
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\begin{document}$$\ell _i$$\end{document}$ is 26, the minimum being 17 and the maximum 37 with a standard deviation of 3. One could further reduce the lattice dimension by preprocessing traces with small $\documentclass[12pt]{minimal}
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\begin{document}$$\ell _i$$\end{document}$. However, the standard deviation being small, the difference in the reduction times should not be affected too much.
Preprocessing the Traces {#Sec12}
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The two main pieces of information we can extract and use in our attack are first the number of non-zero digits in the wNAF representation of the nonce *k*, denoted $\documentclass[12pt]{minimal}
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\begin{document}$$\ell $$\end{document}$ and the weight of each non-zero digit, denoted $\documentclass[12pt]{minimal}
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\begin{document}$$\mu _{j}$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$1\leqslant j \leqslant \ell $$\end{document}$. Let $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {T}$$\end{document}$ be the set of traces we obtained from the side-channel leakage representing the wNAF representation of the nonce *k* used while producing an ECDSA signature. We consider the subset $\documentclass[12pt]{minimal}
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\begin{document}$$S_{a} = \{t \in \mathcal {T} | \max _{j} \mu _{j} \leqslant a, 1\leqslant j \leqslant \ell \}$$\end{document}$. We choose to preselect traces in a subset $\documentclass[12pt]{minimal}
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\begin{document}$$S_a$$\end{document}$ for small values of *a*. The idea behind this preprocessing is to regulate the size of the coefficients in the lattice. Indeed, when selecting *u* traces for the attack, by upper-bounding $\documentclass[12pt]{minimal}
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\begin{document}$$m = max_{i,j} \mu _{i,j}$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$2\leqslant i \leqslant u$$\end{document}$, we force the coefficients to remain smaller than when taking traces at random.
We work with a set $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {T}$$\end{document}$ of 2000 traces such that for all traces $\documentclass[12pt]{minimal}
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\begin{document}$$11 \le \max _{j} \mu _{ j} \le 67$$\end{document}$. The proportion of signatures corresponding to the different preprocessing subsets we consider in our experiments are: $\documentclass[12pt]{minimal}
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\begin{document}$$2 \%$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$S_{11}$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$18 \%$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$S_{15}$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$44 \%$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$S_{19}$$\end{document}$. The effect of preprocessing on the total time is explained in Sect. [5](#Sec13){ref-type="sec"}.
Performance Analysis {#Sec13}
====================
*Traces from the Real World.* We work with the elliptic curve $\documentclass[12pt]{minimal}
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\begin{document}$$\mathtt {secp256k1}$$\end{document}$ but none of the techniques introduced here are limited to this specific elliptic curve. We consider traces from a [Flush&Reload]{.smallcaps} attack, executed through hyperthreading, as it can virtually recover the most amount of information.[3](#Fn3){ref-type="fn"}
To the best of our knowledge, the only information we can recover are the positions of the non-zero digits. We are not able to determine the sign or the value of the digits in the wNAF representation. In \[[@CR7]\], the authors exploit the fact that the length of the binary string of *k* is fixed in implementations such as OpenSSL, and thus more information can be recovered by comparing this length to the length of the double-and-add chain. In particular, they were able to recover the MSB of *k*, and in some cases the sign of the second MSB. We do not consider this extra information as we want our analysis to remain general.
We report calculations ran on error-free traces where we evaluate the total time necessary to recover the secret key and the probability of success of the attack. Our experiments have two possible outputs: either we reconstruct the secret key $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha $$\end{document}$ and thus consider the experiment a success, or we do not recover the secret key, and the experiment fails. In order to compute the success probability and the average time of one reduction, we run 5000 experiments for some specific sets of parameters using either Sage's default BKZ implementation \[[@CR25]\] or a more recent implementation of the latest sieving strategies, the General Sieve Kernel (G6K) \[[@CR1]\]. The experiments were ran using the cluster Grid'5000 on a single core of an Intel Xeon Gold 6130. The total time is the average time of a single reduction multiplied by the number of trials necessary to recover the key. The number of trials necessary to recover the secret key corresponds the number of experiments ran until we have a success for a given set of parameters. For a fixed number of signatures, we either optimize the total time or the success probability. We report numbers in Tables [3](#Tab3){ref-type="table"}, [4](#Tab4){ref-type="table"} when using BKZ.[4](#Fn4){ref-type="fn"} Table 3.Fastest key recovery with respect to the number of signatures.Number of signaturesTotal timeParametersProbability of success ($\documentclass[12pt]{minimal}
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\begin{document}$$\%$$\end{document}$) BKZ Preprocessing $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$339 h35$\documentclass[12pt]{minimal}
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\begin{document}$$ \, \approx 2^{3}$$\end{document}$0.24 1 h 1725$\documentclass[12pt]{minimal}
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\begin{document}$$S_{15}$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\approx 2^{3}$$\end{document}$0.558 min 2025$\documentclass[12pt]{minimal}
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\begin{document}$$S_{19}$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$6.563 min 5520$\documentclass[12pt]{minimal}
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\begin{document}$$S_{all}$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$772 min 4320$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$17.582 min 2520$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$29 Table 4.Highest probability of success with respect to the number of signatures.Number of signaturesProbability of success ($\documentclass[12pt]{minimal}
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\begin{document}$$\%$$\end{document}$)ParametersTotal time BKZ Preprocessing $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$30.235$\documentclass[12pt]{minimal}
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\begin{document}$$\approx 2^{3}$$\end{document}$ 39 h4435$\documentclass[12pt]{minimal}
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\begin{document}$$\approx 2^{3}$$\end{document}$25 h 2852035$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$2 h 4264035$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$1 h 0474535$\documentclass[12pt]{minimal}
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\begin{document}$$ \approx 2^{3}$$\end{document}$2 h 3684535$\documentclass[12pt]{minimal}
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\begin{document}$$\approx 2^{3}$$\end{document}$5 h 02
*Comments on G6K:* We do not report the full experiments ran with G6K since using this implementation does not lead to the fastest total time of our attack: around 2 min using 8 signatures for BKZ and at best 5 min for G6K.
However, G6K allows to reduce lattices with much higher block-sizes than BKZ. For comparable probabilities of success, G6K is faster. Considering the highest probability achieved, on one hand, BKZ-35 leads to a probability of success of $\documentclass[12pt]{minimal}
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\begin{document}$$45 \%$$\end{document}$, and a single reduction takes 133 min. On the other hand, to reach around the same probability of success with G6K, we increase the block-size to 80, and a single reduction is only around 45 min on average. This is an improvement by a factor of 3 in the reduction time.
*Only 3 Signatures.* Using $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \approx 2^3$$\end{document}$ and no preprocessing, we recovered the secret key using 3 signatures with BKZ-35 only once and three times with BKZ-40. When using pre-processing $\documentclass[12pt]{minimal}
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\begin{document}$$S_{11}$$\end{document}$, BKZ-35 and $\documentclass[12pt]{minimal}
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\begin{document}$$0.2\%$$\end{document}$. Since all the probabilities remain much less than $\documentclass[12pt]{minimal}
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\begin{document}$$1 \%$$\end{document}$ an extensive analysis would have taken too long. Thus, in the rest of the section, the number of signatures only varies between 4 and 8. However, we want to emphasize that it is precisely this detailed analysis on a slightly higher number of signatures that allowed us to understand the impact of the parameters on the performance of the attack and resulted in finding the right ones allowing to mount the attack with 3 signatures.
*Varying the Bitsize of*$\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$. In Fig. [1](#Fig1){ref-type="fig"}, we analyze the total time of key recovery as a function of the bitsize of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$. We fix the block-size of [BKZ]{.smallcaps} to 25 and take traces without any preprocessing. We are able to recover the secret key by setting $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta =1$$\end{document}$, which is the lowest theoretical value one can choose. However, we observed a slight increase in the probability of success by taking a larger $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$. Without any surprise, we note that the total time to recover the secret key increases with the bitsize of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ as the coefficients in the lattice basis become larger.Fig. 1.Analyzing the overall time to recover the secret key as a function of the bitsize of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$. We report the numbers BKZ-25 and no preprocessing. The optimal value for $\documentclass[12pt]{minimal}
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\begin{document}$$2^3$$\end{document}$ except for $\documentclass[12pt]{minimal}
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\begin{document}$$2^5$$\end{document}$.
*Analyzing the Effect of Preprocessing.* We analyze the influence of our preprocessing method on the attack time. We fix BKZ block-size to 25. The effect of preprocessing is influenced by the bitsize of $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ and we give here an analyze for $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \approx 2^{25}$$\end{document}$ since the effect is more noticeable.
The effect of preprocessing is difficult to predict since its behavior varies depending on the parameters, having both positive and negative effects. On the one hand, we reduce the size of all the coefficients in the lattice, thus reducing the reduction time. On the other hand, we generate more potential small vectors[5](#Fn5){ref-type="fn"} with norms smaller than the norm of *w*. For this reason, the probability of success of the attack decreases since the vector *w* is more likely to be a linear combination of vectors already in the reduced basis. For example, with 7 signatures we find in average *w* to be the third or fourth vector in the reduced basis without preprocessing, whereas with $\documentclass[12pt]{minimal}
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\begin{document}$$S_{11}$$\end{document}$ it is more likely to appear in position 40.
The positive effect of preprocessing is most noticeable for $\documentclass[12pt]{minimal}
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\begin{document}$$u=5$$\end{document}$, as shown in Fig. [2](#Fig2){ref-type="fig"}. For instance, using $\documentclass[12pt]{minimal}
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\begin{document}$$u = 4$$\end{document}$ lowers the overall time by a factor up to 5.7. For $\documentclass[12pt]{minimal}
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\begin{document}$$u = 5$$\end{document}$, we gain a factor close to 3 by using either $\documentclass[12pt]{minimal}
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\begin{document}$$S_{19}$$\end{document}$. For $\documentclass[12pt]{minimal}
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\begin{document}$$u > 5$$\end{document}$, using preprocessed traces is less impactful. For large $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta $$\end{document}$ such as $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \approx 2^{25}$$\end{document}$, we still note some lower overall times when using $\documentclass[12pt]{minimal}
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\begin{document}$$S_{15}$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$S_{19}$$\end{document}$, up to a factor 2. When the bitsize gets smaller, reducing the size of the coefficients in the lattice is less impactful.Fig. 2.Overall time to recover the secret key as a function of the preprocessing subset for 4 and 5 traces. The other parameters are fixed: $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \approx 2^{25}$$\end{document}$ and BKZ-25.
*Balancing the Block-size of BKZ.* Finally, we vary the block-size in the BKZ algorithm. We fix $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \approx 2^3$$\end{document}$ and use no preprocessing. We plot the results in Fig. [3](#Fig3){ref-type="fig"} for 6 and 7 signatures. For other values of *u*, the plot is very similar and we omit them in Fig. [3](#Fig3){ref-type="fig"}. Without any surprise, we see that as we increase the block-size, the probability of success increases, however the reduction time increases significantly as well. This explains the results shown in Table [3](#Tab3){ref-type="table"} and Table [4](#Tab4){ref-type="table"}: to reach the best probability of success one needs to increase the block-size in BKZ (we did not try any block-size greater than 40), but to get the fastest key recovery attack, the block-size is chosen between 20 and 25, except for 3 signatures where the probability of success is too low with these parameters.Fig. 3.Analyzing the number of trials to recover the secret key and the reduction time of the lattice as a function of the block-size of BKZ. We consider the cases where $\documentclass[12pt]{minimal}
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\begin{document}$$u=7$$\end{document}$. The dotted lines correspond to the number of trials, and the continued lines to the reduction time in seconds.
Error Resilience Analysis {#Sec14}
=========================
It is not unexpected to have errors in the traces collected during side-channel attacks. Obtaining error-free traces requires some amount of work on the signal processing side. Prior to \[[@CR6]\], the presence of errors in traces was either ignored or preprocessing was done on the traces until an error-free sample was found, see \[[@CR2], [@CR9]\]. In \[[@CR6]\], it is shown the lattice attack still successfully recovers the secret key even when traces contain errors. An error in the setup given in \[[@CR6]\] corresponds to an incorrect bound on the size of the values being collected. In our setup, a trace without errors corresponds to a trace where every single coefficient in the wNAF representation of *k* has been identified correctly as either non-zero or not. The probability of having an error in our setup is thus much higher. Side-channel attacks without any errors are very rare. Both \[[@CR21]\] and \[[@CR6]\] give some analysis of the attacks [Flush&Reload]{.smallcaps} and [Prime&Probe]{.smallcaps} in real life scenarios.
In \[[@CR7]\], the results presented in the paper assume the [Flush&Reload]{.smallcaps} is implemented perfectly, without any error. In particular, to obtain 4 perfect traces and be able to run their experiment and find the key, one would need to have in average 8 traces from [Flush&Reload]{.smallcaps} -- the probability to conduct to a perfect reading of the traces being 56% as pointed out in \[[@CR21]\]. In our work, we show that it is possible to recover the secret key using only 4, even erroneous, traces. However, the probability of success is very low.
Recall that an error in our case corresponds to a flipped digit in the trace of *k*. Table [5](#Tab5){ref-type="table"} shows the attack success probability in the presence of errors. We ran used BKZ-25 and $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta \approx 2^3$$\end{document}$ with traces taken from $\documentclass[12pt]{minimal}
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\begin{document}$$S_{all}$$\end{document}$. We average over 5000 experiments. We write $\documentclass[12pt]{minimal}
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\begin{document}$$\ll 1$$\end{document}$ when the attack succeeded less than five times over 5000 experiments, thus making it difficult to evaluate the probability of success.Table 5.Error analysis using BKZ-25, $\documentclass[12pt]{minimal}
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\begin{document}$$S_{all}$$\end{document}$.Number of signaturesProbability of success (%)0 error 5 errors 10 errors 20 errors 30 errors40.28$\documentclass[12pt]{minimal}
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\begin{document}$$\ll 1$$\end{document}$00054.580.860.18$\documentclass[12pt]{minimal}
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\begin{document}$$\ll 1$$\end{document}$0619.525.261.260.14$\documentclass[12pt]{minimal}
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\begin{document}$$\ll 1$$\end{document}$733.5410.823.420.32$\documentclass[12pt]{minimal}
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\begin{document}$$\ll 1$$\end{document}$835.1413.264.700.58$\documentclass[12pt]{minimal}
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The attack works up to a resilience to $\documentclass[12pt]{minimal}
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\begin{document}$$2\%$$\end{document}$ of errors. Indeed, for $\documentclass[12pt]{minimal}
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\begin{document}$$u = 6$$\end{document}$, we recovered the secret key with 30 errors, *i.e.,* 30 flipped digits over $\documentclass[12pt]{minimal}
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\begin{document}$$6 \times 257$$\end{document}$ digits.
*Different Types of Errors.* There exists two possible types of errors. In the first case, a coefficient which is zero is evaluated as a non-zero coefficient. In theory, this only adds a new variable to the system, *i.e.*, the number $\documentclass[12pt]{minimal}
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\begin{document}$$\ell $$\end{document}$ of non-zero coefficients is overestimated. This does not affect the probability of success much. Indeed, we just have an overly-constrained system. We can see in Fig. [4](#Fig4){ref-type="fig"} that the probability of success of the attack indeed decreases slowly as we add errors of this form. With errors only of this form, we were able to recover the secret key up to nearly $\documentclass[12pt]{minimal}
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\begin{document}$$4 \%$$\end{document}$ of errors, (for instance with $\documentclass[12pt]{minimal}
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\begin{document}$$u=6$$\end{document}$, using BKZ-35). The other type of errors consists of a non-zero coefficients which is misread as a zero coefficient. In this case, we lose information necessary for the key recovery and thus this type of error affects the probability of success far more importantly as can also be seen in Fig. [4](#Fig4){ref-type="fig"}. In this setup, we were not able to recover the secret key when more than 3 errors of this type appear in the set of traces considered.Fig. 4.Probability of success for key recovery with various types of errors when using $\documentclass[12pt]{minimal}
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*Strategy.* If the signal processing method is hesitant between a non-zero digit or 0, we would recommend to favor putting a non-zero instead of 0 to increase the chance of having an error of type $\documentclass[12pt]{minimal}
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\begin{document}$$0\rightarrow $$\end{document}$ non-zero, for which the attack is a lot more tolerant.
Conclusion and countermeasures {#Sec15}
==============================
In the last decades, most implementations of ECDSA have been the target of microarchitectural attacks, and thus existing implementations have either been replaced by more robust algorithms, or layers of security have been added.
For example, one way of minimizing leakage from the scalar multiplication is to use the Montgomery ladder scalar-by-point multiplication \[[@CR16]\], much more resilient to side-channel attacks due to the regularity of the operations. However, this does not entirely remove the risk of leakage \[[@CR28]\]. Additional countermeasures are necessary.
When looking at common countermeasures, many implementations use blinding or masking techniques \[[@CR20]\], for example in BouncyCastle implementation of ECDSA. The former consists in blinding the data before doing any operations, and masking techniques randomize all the data-dependent operations by applying random transformations, thus making any leakage inexploitable.
However, it is important to keep in mind these lattices attacks as they can be applied at any level of an implementation that leaks the correct information.
In order to have a fair comparison with our methodology, the times reported in \[[@CR7]\] with which we compare ourselves have to be multiplied by the number of trials necessary for their attack succeed, thus increasing their total time by a lot. Using 5 signatures, their best total time would be around 15 h instead of 18 min.
For 4 signatures, no times are reported without method *A*. Thus, we have no other choice than to compare our times with theirs, using *A*. Yet their time for 4 signatures without *A* should at least be the time they report with it.
In practice, measurements done during the cache attack depend on the noise in the execution environment, the threat model and the target leaky implementation. For instance, [Flush&Reload]{.smallcaps} ran from another core would be noisy. [Prime&Probe]{.smallcaps} would give the same information, with a more generic scenario. In an SGX scenario, it would recover the largest amount of information but in a user/user threat model it would be too noisy to lead to practical key recovery.
In \[[@CR7]\], the authors use an Intel Core i7-3770 CPU running at 3.40 GHz on a single core. In order for the time comparison to be meaningful, we ran experiments with a machine of comparable performance to estimate the timings of a single reduction. As we obtained similar timings with an older machine than used in \[[@CR7]\], the variations we find when comparing ourselves to them solely come from the lattice construction and the reduction algorithm being used rather than hardware differences.
In the sense of vectors exhibited in ([7](#Equ7){ref-type=""}).
We would like to thank Nadia Heninger for discussions about possible lattice constructions, Medhi Tibouchi for answering our side-channel questions, Alenka Zajic and Milos Prvulovic for providing us with traces from OpenSSL that allowed us to confirm our results on a deployed implementation, Daniel Genkin for pointing us towards the Extended Hidden Number Problem, and Pierrick Gaudry for his precious support and reading. Experiments presented in this paper were carried out using the Grid'5000 testbed, supported by a scientific interest group hosted by Inria and including CNRS, RENATER and several universities as well as other organizations.
| {
"pile_set_name": "PubMed Central"
} |
All relevant data are within the paper.
Introduction {#sec001}
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Think of a pen held upright on a table with a finger, and imagine to slowly lifting your finger until the pen suddenly falls in an arbitrary direction as depicted in [Fig. 1A-B](#pone.0118224.g001){ref-type="fig"}. What has this in common with financial market dynamics? Both are systems in which whenever a small fluctuation makes the system cross a critical point, the system moves into one or more definite states. The phenomenon where a system goes from a symmetric but disordered or random state (the pen can fall into any given direction) into an ordered state in which the symmetry is broken and the pattern is well defined (the pen is falling in one specific direction) is what characterizes a phase transition in physics. In this paper we develop a theoretical framework for financial markets based on this phenomenological reasoning: the pen in the symmetric state is equivalent to price fluctuations around their fundamental value, whereas the pen falling (the symmetry is broken) is equivalent to price trends towards herding seen in a bubble phase or anti-bubble phase of the market.
{ref-type="fig"} depicts symmetric state of a pen with no clear direction taken. The state is symmetric since there is no a priori preferred direction in which the pen would fall. [Fig. 1B](#pone.0118224.g001){ref-type="fig"} describes the pen falling in one specific direction corresponding to a symmetry break when the finger is lifted. The symmetry break happens because a small fluctuation in the positioning of the pen inevitable happens when lifting the finger, leading to a change of the system from one state (pen held upright on a table with a finger) to another (pen suddenly falls in an arbitrary direction).](pone.0118224.g001){#pone.0118224.g001}
The key question we face is to explain when and why financial symmetry breaking is taking place, corresponding to the financial system moving from one state (or mood), where randomness in price movements obey no-arbitrage conditions, into another state in which the price takes the route towards bubble or anti-bubble formation giving rise to arbitrage conditions. The first mood is the *fundamental state*, while the second is the *speculative state*.
A large body of literature has explored markets seen as complex systems with bounded rational interacting agents. A possible, while non-exhaustive list of references includes Refs. \[[@pone.0118224.ref001]--[@pone.0118224.ref009]\]. The common feature of these studies concerns the way they model dynamical interactions of agents' choice, which are stylized within a 'mean-field' approach where the intensity of choice is equivalent to a temperature. In more depth, complex systems are assumed as "... dissipative structures that import free energy and export entropy in a way that enables them to self-organize their structural content and configuration, subject to boundary limits" Ref. \[[@pone.0118224.ref008]\]. In such a reasoning, markets are assumed as thermodynamically systems where energy (order) and entropy (disorder) are struggling against each other moving the markets from one state to another based on their inner "temperature".
In our paper we take the same "anti-reductionism" (or "metaphysical") perspective, where financial systems are not merely the sum of single parts but, instead, they reflect very complex dynamics that can be understood only by inspecting the system as a whole, enweaving micro-to-macro connections within a consistent economic framework that takes into account linear and non-linear interactions. Instead, conventional economic theory is often unable to describe such dynamics being based on a "reductionist" approach, in which the micro-economic behavior of a representative agent is first stylized using rational, bounded rational or behavioral frameworks, then next used in a synthesis of macro behaviors. In other terms, the state (or mood) of the system is the macroscopic result of many microscopic decisions.
The "dissipative" financial market structure we have in mind is characterized by two macro-configurations shaped by the spatiotemporal interactions of single agents: (1) a *speculative state*, in which single traders take the same direction (all buy or sell), leading to market trends of bubbles and anti-bubbles; (2) a *fundamental state*, where traders put different buy and sell orders leading the price path to move around its fundamental value.
Agents are assumed to be bounded rational, using a limited set of information including past price history (technical analysts) and expectation on future dividends (fundamentalists). Investment strategies are dynamically changed based on realized returns within a game theoretical scheme with Nash equilibria. In such a setting, the market payoff reflects an intrinsic financial symmetry that guarantees invariance inside the system until the symmetry is broken, then leading the market towards speculative scenarios. State transitions appear whenever the "temperature" of the market crosses a critical transition point, which marks the speculative vs. fundamental behaviors.
Our main innovation is to give a formal, theoretical-based measure to such a market temperature.
The collective "choice" of the system which we refer to as "aggregate decision making" is conducive to answer how prices are formed over time and describes states of the market, which Rational Expectations Hypothesis (REH) or even the bounded REH cannot address. Indeed, once asset prices and dividends are both included in the decision making process, the REH suggests agents should make their trading decisions based on dividends only, but the matter becomes far from trivial if you try to explain generally what will be the collective outcome of *N* people's decision making. Are decisions based on the price of the asset in anticipation of future price behavior (speculative state), or are rather the anticipation of future dividends the only driver of agents' trading decisions (fundamentalist state)?
Our framework is able to give an answer to this question by coarse-graining the agents' interactions through the well-known Ginzburg-Landau (GL) theory in physics, used to describe superconducting transition in terms of a complex order parameter field. The adaptation of the GL theory to our problem allows us to bypass the excessively complicated microscopic description of moods in the markets, by means of an "order parameter" that modulates transitions between fundamental and speculative states without examining the micro-dynamics of single agents and their interaction impacts on the price path and connected market transitions.
We proceed as follows. We first describe the multi-agent-based model used to describe the payoff functions of single agents and related impacts on market returns. Then we describe the rationale of market phase transitions through the GL theory, next reporting and commenting results from simulation study. We conclude summarizing our main contributions also outlining our future research agenda.
Multi-Agent Based Modeling {#sec002}
==========================
The theoretical framework used to formalize the speculative and the fundamental states refers to the "\$-Game" multi-agent based modeling approach proposed in Ref. \[[@pone.0118224.ref010]\], in which single agents make their investment decisions based on dividends and asset prices with the objective to maximize their profit payoff function.
The \$-Game is an extension of the Minority Game (MG), introduced in Ref. \[[@pone.0118224.ref011]\] and implemented in many studies on market price dynamics Refs. \[[@pone.0118224.ref012]--[@pone.0118224.ref014]\]. The basic MG scheme consists in a repeated game where the players choose 1 out of 2 alternatives (buy or sell) at each time step based on past information, and the winning agents are those in the minority group. Such a scheme was introduced following the leading principle in Physics for which, in order to solve a complex problem one should first identify essential factors at the expense of trying to describe all aspects in detail.
Similarly, the \$-Game is our "minimal" model to describe and predict financial market dynamics. Although simple in principle, the \$-Game yields rich system dynamics, the complexity of which can be acted upon by the choice of system parameters (memory length, liquidity, etc.). As will be shown below, this thereby creates a dilemma in terms of the investment strategies of the participants, and the pure cases of speculative vs. fundamentalist states will appear as special cases of the general theory. Differently from MG, in the \$-Game the best strategies are not always targeting the minority but are shifting opportunistically between the minority and the majority.
The mathematical definition of the model includes *N* players (or agents) that simultaneously take part in a one-asset financial market over a time horizon of $\mathcal{T}$ periods. At each *t* period, with t \< $\mathcal{T}$, each player *i* chooses an action *a* ~*i*~(*t*) ∈ {−1, 1}, where the action −1 is a "sell" order and the action 1 is a "buy" order. In submitting buy and sell orders, agents can use: (1) technical analysis strategies, trying to profit from *past* price trends, and (2) fundamental analysis strategies, based on expectation of *future* dividends. The aggregate choices that look at the past lead to a *pure speculative state* (technical analysis strategies), and the aggregate choices that look at the future lead to a *pure fundamental state* (fundamental analysis strategies). Players are assumed to be bounded rational, in the sense of using only a limited informational set (past price trends and dividends) to make their decisions, with no short-sales constraints.
Speculators {#sec003}
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In the speculative state a majority of the agents are technical analysts (chartists) who only analyze past realization of prices, with no anchor on fundamental economic analysis. Each player observes the history of past *m* ∈ ℕ price movements in making decision of whether to buy or sell an asset. Therefore, *m* denotes the size of the agent's memory or, putting in other terms, the length of the signal used in the decision making process. To take decisions the players have at their disposal a fixed number of *s* strategies, which are by construction randomly assigned at the beginning of the game. Thus, a specific strategy *j* tells whether to buy or sell an equity depending on the past price history of up and down moves, represented as 1 (up) and 0 (down), respectively.
At each time *t* the *i*-th player uses his/her *best* (in terms of payoff see [Eq. 1](#pone.0118224.e018){ref-type="disp-formula"} below) strategy taking an action $a_{i}^{*}\left( t \right)$ of either buying $a_{i}^{*}\left( t \right) = + 1$ or selling $a_{i}^{*}\left( t \right) = - 1$ (the \* is used to denote it is the best strategy). It follows that in general a given strategy $a_{i}^{j}$ is a mapping from the set of histories of size *m* to {−1, +1}.
[Table 1](#pone.0118224.t001){ref-type="table"} shows an example of a given strategy for *m = 3*. For all possible histories of up and down market moves over the last *m* time steps, the strategy suggests a specific action to take at time *t*, namely $a_{i}^{j}\left( {\overset{\rightarrow}{h}\left( t \right)} \right) = \pm 1$ with$\overset{\rightarrow}{h}\left( t \right) \in \left\{ {0,1} \right\}^{m}$. For instance, if the market went down over the last m = 3 days, the strategy in [Table 1](#pone.0118224.t001){ref-type="table"} suggests to buy the stock (000→ +1). If instead the market went down over the last two days and then up today, the same strategy suggests to sell (001→-1). Note again in [Table 1](#pone.0118224.t001){ref-type="table"} that, since the price has 2 possible moves (up or down) we have a space of possible past paths equal to 2^*m*^ = *8* bit sequences of 0's and 1's each one corresponding to a specific action {−1, +1} suggested by the *j* strategy. This signifies that the space of all possible strategies is given by$2^{2^{m}} = 256$ alternatives corresponding to each of the 8 bit sequences.
10.1371/journal.pone.0118224.t001
###### Example of speculative trading strategy.
{#pone.0118224.t001g}
History $\overset{\rightarrow}{h}\left( t \right)$ Action $a_{i}^{j}(t)$
---------------------------------------------------- -----------------------
000 1
001 −1
010 −1
011 1
100 −1
101 −1
110 1
111 −1
The table shows an example of a technical trading strategy for an investor with a history of past price movements of *m* = 3 time steps. For all possible histories of up and down market moves over the last *m* steps, the strategy suggests a specific action to take at time *t*, namely buy ($a_{i}^{j}\left( t \right)\operatorname{} = \operatorname{}1$) or sell ($a_{i}^{j}\left( t \right)\operatorname{} = \operatorname{} - 1$).
While a single strategy recommends an action for all possible histories (of length *m*), we also allow for agents to adopt different strategies over time. Namely, agents keep a record of the overall payoff each strategy would have yielded over the entire market history (i.e. not limited to *m* past periods) using a rolling window of size m, and use this record to update which strategy is the most profitable see [Eq. (1)](#pone.0118224.e018){ref-type="disp-formula"} below. This renders the game highly non-linear: as the price behavior of the market changes, the best strategy of a given agent changes, which then can lead to new changes in the price dynamics.
The action corresponding to the best strategy taken by agent *i* at time *t* is denoted by$a_{i}^{*}\left( t \right) = \pm 1$, while $\left( {a_{i}^{*}\left( t \right)} \right)_{i} \in \left\{ {- 1, + 1} \right\}^{N \times T}$ is the *action profile* of the entire market population, where ${\overset{\rightarrow}{a}}^{*}\left( t \right) = \left( {a_{1}^{*}\left( t \right),\ldots,a_{N}^{*}\left( t \right)} \right)_{i} \in \left\{ {- 1, + 1} \right\}^{N}$ corresponds to the action played by the *N* agents in period *t*.
The payoff π of the *i*-th agent's *j*-th strategy, $a_{i}^{j}\left( t \right)$, in period *t* is determined as follows: $$\pi\left\lbrack {a_{i}^{j}\left( t \right)} \right\rbrack = a_{i}^{j}\left( {t - 1} \right)r\left( t \right)$$
In [Eq. (1)](#pone.0118224.e018){ref-type="disp-formula"} *r(t)* denotes the return of the market between time *t*−1 and *t*. The payoff in the \$-Game therefore describes the gain/loss obtained by an investment strategy executed at time *t−*1 depending on the market return in the following time step *t*. I.e. \$-Game agents are investors that try to predict and profit from future market movements. *r(t)* can in turn be expressed in terms of the global order imbalance, $\sum_{k = 1}^{N}a_{k}^{*}\left( t \right)$, divided by the liquidity of the market λ, as discussed below.
As proven in Refs. \[[@pone.0118224.ref015], [@pone.0118224.ref016]\] traders' actions impact significantly on price returns and liquidity through a positive autocorrelation in equilibrium imbalances reflected in a positive predictive relation between imbalances and future returns. Price return *r(t)* from *t−*1 to *t* can be then assumed as proportional to the order imbalance, leading to the following $$r\left( t \right) \equiv \text{ln}p\left( t \right) - \text{ln}p\left( {t - 1} \right) = \lambda^{- 1}\sum_{k = 1}^{N}a_{k}^{*}\left( t \right)$$ with *p*(∙) denoting the price of the stock and λ is a parameter describing the liquidity of the market with λ ∝ *N*. Note that the price goes in the direction of the sign of the order imbalance according to Refs. \[[@pone.0118224.ref017], [@pone.0118224.ref018]\].
Therefore the payoff function in [Eq. (1)](#pone.0118224.e018){ref-type="disp-formula"} can be re-expressed in terms of the return function in [Eq. (2)](#pone.0118224.e020){ref-type="disp-formula"} as: $$\pi\left\lbrack {a_{i}^{j}\left( t \right)} \right\rbrack = a_{i}^{j}\left( {t - 1} \right)\sum_{k = 1}^{N}a_{k}^{*}\left( t \right)/\lambda$$
To summarize: in the \$-Game technical analysis trading strategies are based on a rewarding scheme for strategies that at time *t---*1 predicted the proper direction of the return of the market *r*(*t*) in the next time step *t*. The larger the move of the market, the larger the gain/loss depending on whether the strategy properly/improperly predicted the market move. If the agent correctly anticipates the right direction of the market, the profit will be positive and equal *to N/λ* in both the extremes where either all agents sell or buy \[-*N*; +*N*\], having in fact (-1 ×---*N* ≡ *+1 × +N = N)/λ* from [Eq. (2)](#pone.0118224.e020){ref-type="disp-formula"}.
Fundamentalists {#sec004}
---------------
Differently from pure speculators, fundamentalist make their investment decisions based on fundamental economic analysis. Each player makes forecasts on future price based on expected dividends *D(t)* over the entire time horizon, thereby obtaining a fundamental price *p* ~*f*~(*t*) estimation equivalent to the expectation of all future dividends discounted at a constant risk-free rate *ρ*, at which investors can buy or sell the stock: $$p_{f}\left( t \right) = E_{t}\left\lbrack \int_{t}^{T}e^{- \rho(\tau - t)}D\left( \tau \right)d\tau \right\rbrack$$ with *E* ~t~\[*D*(τ)\] = *D*(t) which implies in the continuum a variation in *p* ~*f*~ from *t* to *t* + *dt* as a martingale with σ constant and *dZ* denoting the standard Brownian motion: $$dp_{f}\left( t \right) = \sigma dZ$$
As result, a change in the fundamental price reflects the arrival of news regarding future cash flows of the stock.
Buy and sell actions made by fundamentalists at time *t*, $a_{i}^{f}\left( t \right)$, are based on the value of *p*(t) vs. *p* ~*f*~ *(t)* reflecting the common rule of thumb to sell when price is high (higher than the fundamental), and buy when price is low (lower than the fundamental): $$a_{i}^{f}\left( t \right) = \left\{ \begin{array}{l}
{- 1\ if\ p\left( t \right) > p_{f}\left( t \right)} \\
{+ 1\ if\ p\left( t \right) < p_{f}\left( t \right)} \\
\end{array} \right.$$
Furthermore, in order to limit the sell orders whenever *p*(t) ≫ *p* ~*f*~ *(t)*, which would rather indicate a speculative scenario\[[@pone.0118224.ref022]\], we assume a vanishing use of the fundamental strategy according to a Poisson process of the form γ*e* ^−γ^ with $\gamma = \frac{p\left( t \right) - p_{f}\left( t \right)}{D\left( t \right)}$. This assumption provides a maximum probability in choosing the fundamental strategy in correspondence with a price variation from its fundamental almost equivalent to the dividend yield, which makes sense given that the fundamental price is the expectation of future dividends.
The payoff function for fundamentalists is the same as that of speculators (see Eqs. [(1)](#pone.0118224.e018){ref-type="disp-formula"}--[(3)](#pone.0118224.e021){ref-type="disp-formula"}), the only change is the action selection mechanism given by ([Eq. 6](#pone.0118224.e025){ref-type="disp-formula"}).
Fine-grained market dynamics {#sec005}
----------------------------
The formulation of the decision making process of speculators and fundamentalists via the \$-Game leads to a financial market modeling based on 5 parameters: *N*---The number of market participants which expresses the "physical" size of the market.*m*---The memory length of the signal used by the agents in their decision making process when they act as speculators. This is expressed in terms of the past number of days the agents look at when they decide whether to buy or sell an asset.*s*---The number of strategies held by the agents when they act as speculators. By construction the *s* strategies of each agent is chosen randomly in the total pool of $2^{2^{m}}$ strategies at the beginning of the game.λ---The liquidity parameter of the market. λ will be assumed proportional to *ND*(*t*)---The future expectations about the dividends paid over the entire time horizon which is assumed to be constant in time *t* for simplicity, hence D(t) ≡ D.
These 5 parameters are mixed together in a reflexive, non-linear \$-Game-based market dynamics where each agent uses his/her best strategy at every time step giving rise to a complex system dynamics where, as the market changes, the best strategies of the agents change too, and as the strategies of the agents change, they thereby change the market.
The dynamics of the \$-Game is driven by a nonlinear feedback mechanism because each agent used his/her *best* strategy (fundamental/technical analysis) at each time step. The sign of the order imbalance, $\sum_{i = 1}^{N}a_{i}^{*}\left( {\overset{\rightarrow}{h}\left( t \right)} \right)$, in turn determines the value of the last bit *b(t*) at time *t* for the price movement history$\overset{\rightarrow}{h}\left( {t + 1} \right) = \left( {b\left( {t - m + 1} \right),b\left( {t - m} \right),\ldots,b\left( t \right)} \right)$. The dynamics of the \$-Game can then be expressed in terms of an equation that describes the dynamics of *b(t)* as: $$b\left( {t + 1} \right) = {\Theta\left\lbrack {\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( t \right)} \right)} \right\rbrack}$$ where Θ is a Heaviside function taking the value 1 whenever its argument is larger than 0 and otherwise 0, and $h\left( t \right) = \sum_{j = 1}^{m}b\left( {t - j + 1} \right)2^{j - 1}$ is now expressed as a scalar instead of a vector. The nonlinearity of the game can be formally seen from: $$a_{i}^{*}\left( {h\left( t \right)} \right) = a_{i}^{\{{{j|}\text{max}_{j = 1,\ldots,s}{\lbrack{\prod{\{{a_{i}^{j}{({h{(t)}})}}\}}}\rbrack}}\}}\left( {h\left( t \right)} \right)$$ with $$\prod\left\{ {a_{i}^{j}\left( {h\left( t \right)} \right)} \right\} = \sum_{k = 1}^{t}a_{i}^{j}\left( {h\left( {k - 1} \right)} \right)\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( k \right)} \right)$$
Inserting the Eqs. [(9)](#pone.0118224.e033){ref-type="disp-formula"} and ([10](#pone.0118224.e038){ref-type="disp-formula"}) in Eq. ([8](#pone.0118224.e032){ref-type="disp-formula"}) one obtains an expression that describes the \$-Game in terms of just one single equation for *b*(*t*) depending on the values of the 5 base parameters variables (*m;* s; *N*; λ; *D*(*t*)) and the random variables $a_{i}^{j}$ (i.e. their initial random assignments).
A major complication in the study of this system of equations happens because of the non-linearity in the selection of the best strategy, and the higher the number of *s* the complexity of the system gets bigger and bigger. The case of s = 2 is however simple to deal with, since one only need to know the relative payoff $q_{i} \equiv \pi\left\lbrack a_{i}^{1} \right\rbrack - \pi\left\lbrack a_{i}^{2} \right\rbrack$ between the two strategies Refs. \[[@pone.0118224.ref019]--[@pone.0118224.ref020]\]. Indeed, as proven in following proof for this special case the \$-Game is in Nash equilibrium with only technical analysis strategies (with no cash nor asset constraints) akin to that of Keynes' "Beauty Contest", where it becomes profitable for the subjects to guess the actions of the other participants. The optimal state is then one for which all subjects cooperate and take the same decision (either buy/sell) leading the price into a bubble state where it deviates exponentially in time from its fundamental. All subjects profit from further price increases/decreases in the bubble state, but it requires coordination among the subjects to enter and stay in such a state, see the following proof:
We would first like to point out an important difference compared to traditional game theory since in our game the agents have *no* direct information of the action of the other players. The only (indirect) information a given agent have of other agent's action through the aggregate actions of the past, i.e. the past price behavior. Let the action of optimal strategy $a_{i}^{*}$ be expressed in terms of the relative payoff, *q* ~*i*~, so as to formulate $\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( t \right)} \right)$ as follows
{#pone.0118224.e038g}
∑
i
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1
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a
i
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Inserting [Eq. (10)](#pone.0118224.e039){ref-type="disp-formula"} into [Eq. (7)](#pone.0118224.e032){ref-type="disp-formula"} and take the derivative of *b* in t + 1
{#pone.0118224.e039g}
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Looking inside the bracket of the sum in (11), it follows that a change in $\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( t \right)} \right)$ can occur either because the optimal strategy changes and the two strategies for a given *h*(*t*), $a_{i}^{1}\left( {h\left( t \right)} \right)$ and $a_{i}^{2}\left( {h\left( t \right)} \right)$, differ one each other (first term in the bracket). Furthermore, a change in $\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( t \right)} \right)$ can arise also because the optimal strategy changes its prediction for the given *h*(*t*) (second and third terms in the bracket).
The change in time of the relative payoff *q* ~*i*~ is computed as follows
{#pone.0118224.e044g}
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Using $h\left( t \right) = \sum_{j = 1}^{m}b\left( {t - j + 1} \right)2^{j - 1}$ and inserting (12) in (11) one obtains:
{#pone.0118224.e046g}
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If $\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( {t - 1} \right)} \right),\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( {t - 2} \right)} \right),\ldots,\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( {t - m} \right)} \right)$ have all the same sign, the right-hand-side of [Eq. (13)](#pone.0118224.e052){ref-type="disp-formula"} becomes 0, thus proving that a constant bit *b(t)*, corresponding to either an exponential increase or decrease in price, is a Nash equilibrium.
Phase Transitions in a Coarse-Grained Financial System {#sec006}
======================================================
From the point of view of physics, the mathematical modelling of the agents' decision making process described via the *b(t)* dynamics can be understood as a "magnetism". Such magnetism is determined by the "spins" represented by the strategies, in which the interaction between different spins (the products of $\sum_{i = 1}^{N}a_{i}^{*}\left( {h\left( \cdot \right)} \right)$ and$a_{i}^{j}$) are mixed with "free field" terms, namely the only terms without a payoff function (see [Eq. (13)](#pone.0118224.e052){ref-type="disp-formula"}). Spin models are widely used to describe the dynamics of traders in financial markets by several researchers implementing statistical physics to inspect complex dynamics in finance. See for e.g., Refs \[[@pone.0118224.ref034], [@pone.0118224.ref035]\].
We would like to emphasize such a spins' analogy of interaction in the description of a complex financial system, in which speculators and fundamentalists interact in a non-linear way with the objective to maximize their payoff function. It suggests another and more general perspective in order to understand the market dynamics based on the competition between $a_{i}^{j}$ and$a_{i}^{f}$, namely between technical analysis trading strategies and fundamental analysis trading strategies. Note that both technical and fundamental strategies can be active for different traders at the same time depending on the optimal strategies the agents possess at a given instant of time. Just like there is an interaction between spins in a magnet, there is an indirect interaction between market participants through their decision making, since the impact of one agents decision making can influence the future decision making of other agents through the price impact.
Taking this view, the financial market can be conceived as a thermodynamic system where its different states are characterized via the so-called free energy *F*, a concept in physics used to quantify the energy transferred by one system to another. The free energy plays a central role in physics, since its *minimum* determines how the state of the system will appear, and can be written as $$F = E - \mathcal{T}S$$ with *E* the internal energy of the system, $\mathcal{T}$ the temperature and 𝒮 the entropy which one can think of as representing how much disorder there is in a given system. From the definition of *F* we can see that the state of a system is determined by a struggle between two different forces, one representing "order", this is the *E* term, and the other term representing "disorder" given by the $\mathcal{T}$𝒮 term.
A similar struggle of "forces" can be envisioned in the financial market between speculators and fundamentalists, where the general tendency to create either a positive/negative price trend corresponds to "order" state, whereas either the lack of consensus or the mean reversion to the fundamental price value will destroy such order thus moving the system into "disorder" state. Transitions and fluctuations in financial market are scrutinized in a number of recent contributions such as Ref. \[[@pone.0118224.ref036]\], in which transition between economic states is studied focusing on stock correlations, and in Ref. \[[@pone.0118224.ref037]\], where macroscopic "phase-flipping" phenomena are modeled in a dynamical network setting.
In terms of market macro-dynamics, such a reasoning stresses the importance to look at interactions among agents when trying to explain whether aggregate decisions are based on past price trend (speculative state), or on expected future dividends (fundamental state). The classical order (speculative) vs. disorder (fundamentalist) phase transition problem in physics, is turned over to fundamentalist (disorder) vs. speculative (order) mood (phase) transitions dilemma, that we propose to disentangle by picking up the general properties of the system through an adaptation of the well-known Ginzburg-Landau theory. It is with such a view that we observe the pen right at the borderline of falling and imagine a price fluctuation around its fundamental (fundamental state) until the pen breaks the symmetry by taking a clear direction as the price moves into bubble or anti-bubble mood (speculative state). The key point is to explain such dynamics through the "temperature" factor in [Eq. (14)](#pone.0118224.e052){ref-type="disp-formula"} which moves the system from one mood to another.
The Ginzburg-Landau theory {#sec007}
--------------------------
The central importance of the temperature factor can be seen in [Eq. (14)](#pone.0118224.e052){ref-type="disp-formula"}, in which we note that for temperature $\mathcal{T}$ = 0 the minimum of the energy *E* is therefore also the minimum of the free energy *F*. However as soon as $\mathcal{T}$ \> 0, the finite temperature will introduce fluctuations in the system introducing thereby a non-zero contribution to the entropy *S*. The larger the temperature $\mathcal{T}$ the larger this tendency, until at a certain temperature *Tc* above which order has completely disappeared, and the system is in a disordered state. The GL theory explains how such a phase transition can be expressed in terms of an order parameter thus describing the general properties of the system without examining their microscopic properties. We do the same thing by exploring the macro-mechanisms of market transitions, while maintaining consistency in the micro-foundation of individual decision making.
Mathematically, the free energy *F* in [Eq. (14)](#pone.0118224.e052){ref-type="disp-formula"} is assumed to depend on the temperature and the magnitude of the order parameter *m* upon which we can expand the series as follows:
{#pone.0118224.e058g}
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It should be noted that [Eq. (15)](#pone.0118224.e058){ref-type="disp-formula"} does not contain odd terms (*m*, *m* ^3^, ...) in the expansion due to a symmetry argument: there is no difference in the free energy for a spin up, respectively spin down system. As we will see below a similar financial symmetry exists expressed by the fact that there is no difference in the profit from a long, respectively short position.
Assume:
{#pone.0118224.e059g}
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Taking furthermore the derivative in order to find its extreme, we end up with the equation for a minimum of *F*($\mathcal{T}$, *m*), hence determining the state of the system: $$\frac{\partial F\left( {\mathcal{T},m} \right)}{\partial m} = 2a\left( {\mathcal{T} - Tc} \right)m + 2bm^{3},$$ that has the following solutions:
{#pone.0118224.e062g}
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Solution 1) describes a disordered state, while solution 2) is the solution describing an ordered state. The second solution determines the value of the critical exponent *β* when one expresses the order parameter as a function of the temperature near the critical point: $$\left. \left. 2\text{’} \right)\ m\left( \mathcal{T} \right)\rightarrow\Psi\left( \mathcal{T} \right) = \left( {Tc - \mathcal{T}} \right)^{\beta}\mspace{9mu}\mathcal{T} < Tc \right.$$ thus obtaining the so-called "mean field" or GL exponent of the transition β = 0.5. The GL theory offers through a power expansion a functional description of the free energy by integrating over the microscopic degrees of freedom, while constraining their average to *m*($\mathcal{T}$). By doing so, the phenomenological parameters assume an unknown functional dependence on the original microscopic parameters, as well as on the temperature, in this way accounting for the entropy of the short distance fluctuations lost in the coarse-graining (micro-to-macro) procedure.
Mood transitions and the order parameter {#sec008}
----------------------------------------
The beauty of the GL theory is that one can describe phase transitions, without handling the microscopic description of interactions, simply via a power expansion of the order parameter. The structure of our market price dynamics lends itself to be treated in a similar vein, being conceivable as a complex system exhibiting an overall payoff function, as if it were the free energy of a thermodynamic system. Single particle movements are equivalent to the non-linear agent interactions (since one agent's decision making can influence other agent's decision making through price impact) that translate into a macro-dynamical environment in which: the ordered state corresponds to a speculative state, the price dynamics is going (up/down);the disordered state is instead corresponding to the fundamental state, in which the price moves randomly in a mean reversion process towards its fundamental value ([Eq. (21)](#pone.0118224.e069){ref-type="disp-formula"}), thus destroying the trend in the ordered state.
To make the analogy with our discussion above ([Eq. (14)](#pone.0118224.e052){ref-type="disp-formula"}), we introduce what we call the "Market Payoff" (*MP)* given by two terms in *MP* = *P−$\mathcal{T}$S*.
- *P* is the total profit of the ordered state which for $\mathcal{T}$ = 0 corresponds to a continuous up/down trend of the market.
- *S* is an entropy term that destroys the ordered state, and $\mathcal{T}$ is the "temperature" which move the system from order to disorder and vice versa.
As discussed beforehand, the payoff of a strategy in the \$-Game is equivalent to its profit, and agents use the same strategy over time in a Nash equilibrium. Therefore, the total profit *P* at time *t* for the system of traders can be written as: $${P\left( t \right) = \sum\limits_{i}\pi\left\lbrack {a_{i}^{*}\left( t \right)} \right\rbrack = \sum_{i = 1}^{N}\sum_{l = 1}^{N}a_{i}^{*}\left( {t - 1} \right)a_{l}^{*}\left( t \right)\mspace{9mu}\text{with}\ i \neq l},$$ where we note that the interactions among traders is, in a sense, "long-ranged", since trader *l*'s action at time *t* has an impact on trader *i*'s profit from the action he/she took at time t---1.
In the GL theory the micro-to-macro thermodynamically description of the free energy needs consistency between particles micro- and macro-dynamics of the system, and this is obtained via the order parameter. Similar to the general case, for describing the macro-mechanisms of mood transitions we need an order parameter to expand the "Market Profit", which also should maintain consistency in the micro-foundation of individual decision making.
As already observed, our trading setup is, in essence, the analog of a "magnetism" determined by single strategies chosen by the traders, who act as "atomic spins" moving in two possible directions, up (+1) or down (-1). As a result, the system as a whole moves between (and within) the two extremes all up (+*N*) and all down (-*N*), as in the physicist two-dimensional square-lattice Ising model, where the order parameter to describe phase transition is measured by the magnetism, which is just the averaged value of the spins. The Ising model is a model of ferromagnetism in which the energy *E = −J* ∑~\<*i*,\ *j*\>~ *s* ~*i*~ *sj* with *s* ~*i*~ and *s* ~*j*~ representing the atomic "spins" of a material. The \<\>---notation in the summation indicates that the sum is to be taken over all nearest neighbors pair of spins. Each spin itself can be thought of as a mini magnet. In the two-dimensional Ising model the spin *s* ~*i*~ = +1 if the spin is "up" and *s* ~*i*~ = -1 if the spin is "down". Taking the coupling strength between spins *J* positive, the minimum energy *E* ~*min*~ of the system is simply given by either all spins up *s* ~*i*~ ≡ +1), or all spins down (*s* ~*i*~ ≡ -1). Although our trading setup is very similar to the Ising model, there is a major difference that refers to the "interaction" mechanism: in our framework, it is "long-ranged" (trader *l*'s action at time *t* has an impact on trader *i*'s profit from the action he/she took at time *t*---1), whereas the interaction is local (it only concerns nearest-neighbors) for the Ising model. Therefore, the order parameter we suggest to use in our market structure is the order imbalance: $${ο\left( \mathcal{T} \right) = \frac{1}{N}\sum_{i = 1}^{N}a_{k}^{*}\left( t \right)},$$ which gives us a simple way to understand phase transitions characterized by the following two extreme scenarios: the case of $ο\left( \mathcal{T} \right) = \frac{1}{N}\sum_{i = 1}^{N}a_{k}^{*}\left( t \right) = \pm 1$ that corresponds to pure order in the system with all used strategies taking the same direction (buy or sell) and giving rise to a bubble or anti-bubble price path: this is the pure speculative state;the case of $ο\left( \mathcal{T} \right) = \frac{1}{N}\sum_{i = 1}^{N}a_{k}^{*}\left( t \right) = 0$ that corresponds, instead, to pure disorder in the system, with half of the population of used strategies ("spins") taking one action and the remaining taking the opposite action, thus giving rise to a price path around its fundamental: this is the pure fundamentalist state.
Financial symmetry {#sec009}
------------------
Applying now the GL idea and expanding *MP* in terms of *o(* $\mathcal{T}$), one ends up with the very same conditions ([Eq. (18)](#pone.0118224.e062){ref-type="disp-formula"}) to determine *o(* $\mathcal{T}$), but since the objective of traders is to maximize their payoff function, the state of the system is determined by its maximum (maxima), instead of the minimum (minima), as it was the case for *MP(* $\mathcal{T}$, *o)*.
Formally, by taking the derivative of *MP(* $\mathcal{T}$, *o)* and solving for *o(* $\mathcal{T}$) we obtain:
{#pone.0118224.e077g}
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Solution 1) gives *MP(* $\mathcal{T}$, *o) ≡* 0 for o($\mathcal{T}$) = 0, corresponding to no price trend. This is the fundamental state in which randomness in price movements leads to no-arbitrage condition translating into a financial symmetry with a sort of mean-invariance within the system maintaining a general equilibrium in the market.
Solution 2) is with *MP(* $\mathcal{T}$, *o) \>* 0 for o($\mathcal{T}$) \> 0 or o($\mathcal{T}$) \< 0, corresponding to the speculative state with up or down price movements leading to a bubble or anti-bubble state breaking the financial symmetry and with agents getting positive profits by going long or short in the market.
Transitions from fundamental to speculative state are thus modulated through the "temperature" parameter, which play a central role in the macro-dynamic of the market. Indeed, financial symmetry is broken whenever the parameter crosses a critical transition point, *Tc*, which move the system from fundamental (with *MP(* $\mathcal{T}$, *o) ≡* 0) to speculative state (*MP(* $\mathcal{T}$, *o) \>* 0.
[Fig. 2](#pone.0118224.g002){ref-type="fig"} illustrates the expansion of *MP* (y-axis) as a function of *o*(x-axis) for the two cases: i) the $\mathcal{T}$ ≥ *Tc* solution (i.e. the disordered state corresponding to no trend in the price path with *o(* $\mathcal{T}$) = 0) can be seen as the maximum of the solid line, whereas the two $\mathcal{T}$ \< Tc solutions (i.e. the ordered state corresponding to a specific trend in the price path with *o(* $\mathcal{T}$) ≠ 0) can be found as the maxima of the dashed line. Note that when financial symmetry is "restored" in the market, the system is in equilibrium with no difference between profits from going long (buy) or short (sell) in the market (the maximum is *MP(* $\mathcal{T}$, *o) ≡* 0). When instead financial symmetry is broken, due to a market temperature being less than the critical transition point ($\mathcal{T}$ \< *Tc)*, the system finds its optima either from going long *o(* $\mathcal{T}$) \> 0) or from going short *o(* $\mathcal{T}$) \< 0) with the same maximum at (*MP(* $\mathcal{T}$, *o)* \> 0.
{#pone.0118224.g002}
The "market temperature" {#sec010}
------------------------
One of the main implication of our GL-based theory of mood transitions in the market is the existence of a nontrivial transition from a "high temperature" symmetric state, where traders don't create a trend over time, to a "low temperature" state, characterized by trend following with a definite trend in the price trajectories (up or down). We will define in the following a "temperature" linked to the randomness of the agent's actions and suggest how it should be measured accordingly.
In our model setup, randomness enters the \$-Game through the initial conditions in the assignments of the *s* strategies to the *N* traders in the game. In order to create a given strategy one has to assign randomly either a 0 or a 1 for each of the 2^*m*^ different price histories; therefore the total pool of strategies increases as $2^{2^{m}}$ versus *m*. However, many of these strategies are closely related: take for e.g. [Table 1](#pone.0118224.t001){ref-type="table"} changing just one of the 0's to a 1, and note that this thereby creates a strategy which is highly correlated to the one seen in the table.
Refs. \[[@pone.0118224.ref011], [@pone.0118224.ref023]\] showed how to construct a small subset of size 2^*m*^ of independent strategies out of the total pool of $2^{2^{m}}$ strategies. As suggested in Refs. \[[@pone.0118224.ref024], [@pone.0118224.ref020]\], a qualitative understanding of this problem can be obtained by considering the parameter$\alpha = \frac{2^{m}}{N}$. Along the same line of reasoning, Ref. \[[@pone.0118224.ref025]\] pointed out, however, that the ratio $\alpha = \frac{2^{m}}{N \times s}$ seems more intuitive, since this quantity describes the ratio of the total number of relevant strategies to the total number of strategies held by the traders. Based on this intuition, and considering the presence of all relevant technical trading strategies (2^m^) together with *the* fundamental strategy, we then introduce the following measure for the market temperature in a speculative vs. fundamental moods transition financial system: $$\mathcal{T} = \frac{2^{m} + 1}{N \times s},$$ where the "+1" in the numerator is because of the fundamental strategy together with the 2^m^ uncorrelated speculative strategies.
The relation of $\mathcal{T}$ to the fluctuations of the system becomes clear when one consider the fact that the variance of a small sample is larger than the variance of a large sample. This statement is called "the law of small numbers" in Psychology/Behavioral Finance and was introduced by Tversky and Kahneman (Refs. \[[@pone.0118224.ref026]--[@pone.0118224.ref028]\]). In our setup, we have a similar behavior: when the sample of strategies *s* held by the *N* traders is small with respect to the total pool of relevant strategies (reflecting in low denominator of $\mathcal{T}$), this corresponds indeed to the large fluctuations, large temperature case. Vice versa a large sample of *s* strategies held by the *N* traders (increasing denominator of $\mathcal{T}$), therefore corresponds to a small temperature case as seen from the definition of $\mathcal{T}$.
Simulation Study {#sec011}
================
Experimental design {#sec012}
-------------------
In our numerical experiment, we run Monte Carlo simulations based on the 5 parameters of our *in silico* financial system (*N*, s, λ, *D*(*t*)). A number of 200 simulations of the \$-Game were run where each realization of the game were obtained for up to 200 × 2^m^ time steps. Numerically, we performed the experimental design in two main blocks of parameters: 1) *s* = 2; 2) s = 18. In this way we take into account both the case of small number of relevant strategies as well as the case of large number of strategies, since they substantially impact on our $\mathcal{T}$ parameter, as discussed in the previous section. For both the blocks, simulations were run starting from a stock price of 100 and varying parameters as follows: *N* = 11, 101. The two cases represent a thin and a large market.*m = 3*, *5*, *8*. The three values represent the number of past days traders use when forming their expectation and subsequently taking an action at time *t*; the parameter ranges from a short to a relatively long memory of the past;λ = 10, 100. We consider two levels of market liquidity which has an impact on price returns according to (2). The smaller the value of λ the larger the price impact of a given fixed value of the order imbalance.D(*t*) = 10, 20, 30, 40, 50, 60, 70, 80, 90, 100. We used ten possible scenarios for the expected dividends ranging from 10 to 100 percent of the dividend yield at the start of the simulation experiment (the price is set at 100). To simplify the model, the parameter is taken constant in time *t*.
The speculative and fundamental states are determined via the price path. Specifically, the system starts in a fundamental state with a *p* ~*f*~(*t)* ≡ 100 and is defined to maintain financial symmetry whenever price fluctuations remain within a 50 percentage range (at maximum) of the fundamental price *p* ~*f*~(*t)* converging to it at the end of the experiments. As discussed in the theoretical description of the model, people are assumed to trade shares of the company based on their expectations of future dividends, and the numerical experiment ends with the full price reflecting the expected dividends payout. This constraint is in line with those used in a large number of studies running experiments on stock trades (Refs. \[[@pone.0118224.ref029]--[@pone.0118224.ref033]\]).
The speculative state is instead determined whenever *m* successive price changes had occurred. [Fig. 3](#pone.0118224.g003){ref-type="fig"} shows three different results representing typical market behavior corresponding to fundamental price behavior, as well as speculative behavior in an increasing/decreasing market.
{#pone.0118224.g003}
Results {#sec013}
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Figs. [4](#pone.0118224.g004){ref-type="fig"} and [5](#pone.0118224.g005){ref-type="fig"} show histograms representing respectively speculative behavior (blue) or fundamentalist behavior (red) as outcomes of our trading setup. The histograms in [Fig. 4](#pone.0118224.g004){ref-type="fig"} represent simulations performed with s = 2 whereas the histograms in [Fig. 5](#pone.0118224.g005){ref-type="fig"} were done for simulations with *s* = 18. We first notice the somewhat surprising fact that the dividends D(*t*) as well as the liquidity of the market λ, only seem to have a quite limited impact on the final state of the market. In particular, for the smallest *m* values (*m* = 3; 5), increasing dividends appear to have a somewhat stabilizing effect allowing for slightly more fundamental value states. The same stabilizing trend appears to be at play as one increase the liquidity of the market, but again, this tendency appears to be very weak. A much clearer tendency is seen with respect to increasing speculation when increasing the number of traders *N*, respectively, decreasing the amount of information *m* used in the decision making of the technical analysis trading strategies. A larger number of strategies *s* assigned to the traders is also seen to enhance speculation (compare Figs. [4](#pone.0118224.g004){ref-type="fig"} and [5](#pone.0118224.g005){ref-type="fig"}).
{#pone.0118224.g004}
{#pone.0118224.g005}
Let us now explore how a qualitative behavior of this trading experiment can be predicted depending on our market temperature parameter $\mathcal{T}$. The fact that $\mathcal{T}$ determines the outcome of the trading behavior can be easily seen by changing the nominator and denominator by the same factor, which then should lead to invariant behavior in terms of trading decisions. This means that for example the case of short-run memory and thin market (*m* = 3; *N* = 11) should give rise to a $\mathcal{T}$ = 0.8182 s^−1^ trading behavior. Such a trading behavior for a given λ and *s* should fall in between the medium-range memory and large market (*m* = 5; *N* = 101) (i.e. $\mathcal{T}$ = 0.3267 s^−1^) and long-range memory and large market (*m* = 8; *N* = 101) (i.e. $\mathcal{T}$ = 2.5446 s^−1^) cases. From Figs. [3](#pone.0118224.g003){ref-type="fig"} and [4](#pone.0118224.g004){ref-type="fig"} this is seen indeed to be the case. Similarly comparing Figs. [4](#pone.0118224.g004){ref-type="fig"} and [5](#pone.0118224.g005){ref-type="fig"} it is seen that increasing (/decreasing) *N* and decreasing (/increasing) *s* by the same amount leads to two systems behaving similarly in terms of investment profile (compare *N* = 101 rows in [Fig. 4](#pone.0118224.g004){ref-type="fig"} to *N* = 11 rows in [Fig. 5](#pone.0118224.g005){ref-type="fig"}). [Table 2](#pone.0118224.t002){ref-type="table"} reports all values for $\mathcal{T}$ corresponding to small (*s* = 2) and high (*s* = 18) number of relevant strategies, respectively, showing how memory and number of traders impact on market temperature, and in turns on speculative vs. fundamental behaviors. Note that memory length seems to play a major role in moving the system between the two states of speculative, respectively fundamentalist behavior. The larger the *m* the higher the temperature, thus moving the aggregate market behavior towards a more fundamental investment oriented state. These results clearly underscore the importance of the parameter $\mathcal{T}$ when it comes to the understanding of the aggregate decision making in the model.
10.1371/journal.pone.0118224.t002
###### $\mathcal{T}$ values.
{#pone.0118224.t002g}
*s* = 2 *N*
--------- ---------- -----------
***m*** ***11*** ***101***
***3*** 0.409 0.045
***5*** 1.500 0.163
***8*** 11.682 1.272
***s* = 18** ***N***
-------------- --------- -------
***3*** 0.045 0.005
***5*** 0.167 0.018
***8*** 1.298 0.141
The table reports the values of the ratio the $\mathcal{T}$ ratio $\frac{2^{m} + 1}{N \times s}$ run in our simulation experiment with *s* = 2 and *s* = 18. For these two scenarios the corresponding matrices derive by crossing *N* with *m* with *N* = 11, 101 and *m* = 3, 5, 8.
Concluding Remarks {#sec014}
==================
As discussed in Ref. \[[@pone.0118224.ref038]\], financial systems are complex adaptive system, in which the micro interactions translate into macro dynamics through bottom-up mechanisms, followed by top-down feedback between the macro and the micro. In this paper we introduced a novel theoretical framework to describe financial market macro-dynamics in which single agents interact in non-linear and complex ways. The classical order vs. disorder phase transition problem in physics is used here to explain the fundamentalist vs. speculative mood transitions in the markets, that we propose to disentangle through a Ginzburg-Landau-based power expansion. As observing the pen right at the borderline of falling, we imagine a price fluctuation around its fundamental until the pen breaks the symmetry by taking a clear direction as the price moves into bubble or anti-bubble mood.
Our Ginzburg-Landau-based theory of market mood dynamics explains a nontrivial transition from a "high temperature" symmetric state, with no price trends over time, to a "low temperature" state, with up or down price trends. The key parameter that moves the markets from one state to another is the "temperature parameter", which we derive based on the randomness of the agent's actions, the number and the memory length of traders.
In our simulation exercise we have shown how a qualitative understanding could be found depending on just our temperature parameter, thus bypassing the excessively complicated microscopic description of moods in the markets. Indeed, the temperature parameter modulates transitions between fundamental and speculative states without examining the micro-dynamics of single agents and their interaction impacts on the price path and connected market transitions.
The main message we offer in this paper is that through a phenomenological explanation of complex market dynamics, we are able to describe when markets as a whole are expected to change from fundamental to speculative states and vice versa, only by focusing on three variables: (1) the number of market traders (*N*), (2) their trading strategies (*s*) and (3) their past price movements memory (*m)*. Together these variables are assembled into the ratio $\frac{2^{m} + 1}{N \times s}$ which is at the core of the complex market dynamics.
It should be noted that our method provides a framework to test the stability of a given market state and understand the influence of an external perturbation. One way to try to drive the market from one state to another would for example be to perturb (by e.g. forcing a large up/down price move) artificially the market when the agents have driven it into a fundamental state (or vice versa into the fundamental state when presently in a speculative state). Whether the agents would respond by driving the market further along the direction of the perturbation, or on the contrary, drive the market back to its unperturbed state is far from trivial. In Ref. \[[@pone.0118224.ref021]\] it was shown how considering decoupled strategies (see Ref. \[[@pone.0118224.ref021]\] for definition) was one way to get information about how the system of agents would reply to perturbations.
The proposed general framework can be used to describe the human decision making in a certain class of experiments performed in a trading laboratory, allowing us to predict the outcome of such type of trading experiments in terms of when to expect a fundamental versus a speculative state. Here we focused only on the theoretical description of the model, but in our future research agenda we will use our findings to the implementation of trading experiments performed in a trading laboratory.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JVA. Performed the experiments: RS JVA MS. Analyzed the data: JVA MS. Contributed reagents/materials/analysis tools: JVA RS. Wrote the paper: JVA RS.
| {
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In late December 2019, several clusters of pneumonia cases of unknown aetiology were reported in the city of Wuhan, People's Republic of China. Investigation of respiratory samples of these cases identified a novel coronavirus (CoV) as the causative agent \[[@CR1], [@CR2]\]. Nucleotide sequence analysis of the viral RNA genome revealed it to be closely related to that of the betacoronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS) in Asia Pacific (and Canada) in 2002--2003. From China, the new virus, which was designated SARS-CoV‑2, has rapidly spread throughout the world with a total of 5,697,334 confirmed cases and 355,758 deaths as of 28 May 2020 \[[@CR3]\]. These figures are likely a gross underestimation of the actual impact of SARS-CoV‑2 due to limited virological testing and underreporting. The primary routes of SARS-CoV‑2 transmission are respiratory droplets and direct contact. Approximately 80% of SARS-CoV‑2 infections are relatively mild (with flu-like symptoms) or even asymptomatic. Some 15% of cases result in severe disease (so-called COVID-19) characterised by pneumonia and dyspnoea, while \~5% of SARS-CoV-2-infected individuals experience critical illness (i.e. acute respiratory distress syndrome (ARDS), septic shock, (multi‑)organ failure) and require intensive care.
Like the original SARS-CoV, host cell penetration by SARS-CoV‑2 relies on the interaction of the viral spike (S) protein with angiotensin-converting enzyme 2 (ACE2) \[[@CR4]\]. ACE2 is a monocarboxypeptidase present on the surface of a wide variety of different cell types, including epithelial cells lining the respiratory tract, cardiac fibroblasts, cardiomyocytes, endothelial cells and vascular smooth muscle cells (VSMCs) \[[@CR5], [@CR6]\]. In the lungs, ACE2 expression is mainly found in alveolar macrophages and in the surfactant-producing type II pneumocytes and to a lesser extent in bronchial and tracheal epithelial cells \[[@CR5]\]. ACE2 is a paralogue of angiotensin-converting enzyme (ACE). Both these enzymes are Zn^2+^-dependent transmembrane proteins involved in the production of vasoactive peptides \[[@CR7], [@CR8]\]. However, ACE and ACE2 generally have opposite effects, thus functioning as counter-regulatory factors within the renin-angiotensin system (RAS).
ACE converts angiotensin I (AngI/angiotensin-(1--10)) into angiotensin II (AngII/angiotensin-(1--8)) (Fig. [1](#Fig1){ref-type="fig"}). Binding of AngII to the AngII type‑1 receptor (AT1R) has pro-inflammatory, pro-oxidative, pro-apoptotic and pro-fibrotic effects, increases vascular tone and leakage (Fig. [1](#Fig1){ref-type="fig"}) and is involved in pathophysiology of many different tissues and organs \[[@CR9]\]. Stimulation of AT1R on the surface of VSMCs by AngII results in the activation of signalling pathways that promote VSMC contraction. Excessive AngII-AT1R signalling also induces the proliferation, migration and growth of VSMCs, promotes vascular remodelling and contributes to initiation and progression of atherosclerosis by inducing endothelial dysfunction \[[@CR9]\]. Activation of AT1Rs in cardiac myocytes induces cellular hypertrophy, while binding of AngII to AT1Rs on the surface of cardiac fibroblasts results in cardiac fibrosis by stimulating the synthesis of extracellular matrix proteins, including collagen type I and III, and by inducing proliferation and migration of cardiac fibroblasts \[[@CR9]\]. Unbalanced AT1R signalling in the lungs is associated with airway inflammation, bronchial hyper-responsiveness, fibrosis and pulmonary hypertension. AngII is also an important driving force in the inflammatory cascade and alveolar epithelial injury associated with ARDS \[[@CR10], [@CR11]\]. Apart from binding to AT1R, AngII can also bind to the Ang II type‑2 receptor (AT2R) (Fig. [1](#Fig1){ref-type="fig"}). Stimulation of this receptor has effects that are largely opposite to those induced by AngII-AT1R signalling. Under pathophysiological conditions, AngII-AT1R signalling is generally dominant over AngII-AT2R signalling.Fig. 1Overview of major ACE and ACE2 substrates and products, the G‑protein-coupled receptors (GPCRs) activated by these products, the biological consequences of the stimulation of these GPCRs and the mode of action of ACE inhibitors (*ACEI*) and angiotensin II type‑1 receptor blockers (*ARB*). Besides ACE and ACE2, other proteolytic enzymes are also involved in the processing of renin-angiotensin system components, e.g. AngII can also be generated from AngI by chymase (*CHY*; secreted by mast cells) and Ang1--7 can be produced from AngI by neprilysin (also known as neutral endopeptidase (*NEP*)) \[[@CR8], [@CR13]\]
ACE2 can cleave AngI to generate angiotensin-(1--9) (Ang1--9) and AngII to produce angiotensin-(1--7) (Ang1--7) (Fig. [1](#Fig1){ref-type="fig"}). The conversion of AngII into Ang1--7 by ACE2 not only reduces detrimental AngII/AT1R signalling but also generates the ligand for the Mas receptor (MasR), activation of which opposes the effects of AT1R stimulation by AngII (Fig. [1](#Fig1){ref-type="fig"}). Recently, Ang1--7 was shown also to exert beneficial effects by binding to the AT1R and inducing β‑arrestin-biased signalling through this receptor \[[@CR12]\]. Ang1--9 is another ACE2 reaction product involved in counterbalancing AngII/AT1R signalling by stimulating AT2R signalling (Fig. [1](#Fig1){ref-type="fig"}).
The recognition of the adverse effects of unopposed AngII-AT1R signalling led to the development of ACE inhibitors (ACEIs) and AT1R blockers (ARBs). As shown in Fig. [1](#Fig1){ref-type="fig"}, ACEIs inhibit the production of AngI and thereby indirectly prevent harmful AT1R signalling. Analysis of plasma vasoactive peptide levels has shown a moderate decrease in AngII and a large increase in Ang1--7 (e.g. generated from AngI by neprilysin) following ACEI treatment \[[@CR8]\], Thus, ACEIs not only suppress detrimental AngII-AT1R signalling but also promote beneficial Ang1--7 signalling. ARBs, on the other hand, do not block AngII production but selectively and competitively inhibit the binding of AngII to AT1R (Fig. [1](#Fig1){ref-type="fig"}). The blood plasma of patients receiving ARBs contains strongly elevated AngII and moderately increased Ang1--7 levels \[[@CR8]\].
Due to their well-established anti-hypertensive, anti-oxidative, anti-inflammatory, anti-fibrotic and anti-hypertrophic effects, ACEIs and ARBs have found widespread use in clinical practice. As a consequence, a large percentage of cardiovascular patients, including those suffering from systolic heart failure (HFrEF) receive ACEIs or ARBs to lower their blood pressure by reducing systemic vascular resistance (and to slow down adverse cardiac remodelling). In Europe, ACEIs are part of the first-line treatment of (a)symptomatic HFrEF patients unless contraindicated or not tolerated \[[@CR14]\]. In the latter case, ACEIs are commonly replaced by ARBs. In the United States either ACEIs or ARBs are recommended in the first-line pharmacological treatment of HFrEF \[[@CR15]\]. The European Society of Cardiology guidelines recommend the use of AT1R-neprilysin inhibitors (ARNIs) instead of ACEIs or ARBs when patients remain symptomatic in spite of optimal treatment with ACEIs/ARBs, beta-adrenergic receptor antagonists and mineralocorticoid receptor blockers. The guidelines of the American College of Cardiology and American Heart Association recommend the prescription of ARNIs to chronic symptomatic NYHA class II and III HFrEF patients who tolerate ACEIs/ARBs to further reduce morbidity and mortality. The replacement of ACEIs/ARBs by ARNIs is supported by a recent meta-analysis showing that ARNIs are superior to ACEIs and ARBs in mediating reverse cardiac remodelling in HFrEF patients \[[@CR16]\].
By analogy to what happens with SARS-CoV, it is widely assumed that infection of (airway epithelial) cells results in a strong reduction of ACE2 expression at the plasma membrane but does not affect ACE (surface) levels \[[@CR17]\]. In the same study, the ARB losartan attenuated SARS-CoV S‑protein-induced lung injury in a mouse model. Moreover, serum AngII levels are significantly elevated in COVID-19 patients and exhibit a linear positive correlation to viral load and lung injury as determined by oxygenation index \[[@CR18]\]. This led to the hypothesis (hypothesis 1) that SARS-CoV-2-mediated downregulation of ACE2 disturbs the balance between ACE/AngII/AT1R and ACE2/Ang1--7/MasR signalling in the lungs and thereby contributes to the development of ARDS in COVID-19 patients (Fig. [2](#Fig2){ref-type="fig"}). If so, ACEIs and ARBs may have a beneficial effect on the course of COVID-19 by reducing ACE/AngII/AT1R signalling and thereby restoring the local balance between the activities of the ACE/AngII/AT1R and ACE2/Ang1--7/MasR axes (Fig. [3](#Fig3){ref-type="fig"}). Support for this idea comes from a study by Henry et al. showing favourable effects of ACEI treatment during hospitalisation of patients with (non-corona)viral pneumonia \[[@CR19]\]. Also, several research groups have demonstrated that ACEIs and ARBs can (1) attenuate the lung injury caused by (experimental) ARDS and (2) reduce pulmonary arterial hypertension \[[@CR10], [@CR11]\].Fig. 2SARS-CoV‑2 uptake by receptor-mediated endocytosis directly and indirectly lowers ACE2 surface expression, reduces conversion of AngII into Ang1--7 and hence causes a misbalance in the activities of the ACE/AngII/AT1R and ACE2/Ang1--7/MasR axes. This, together with SARS-CoV-2-induced cell death, inflammation and thromboembolism, is responsible for the severe lung injury seen in critically ill COVID-19 patients. A disintegrin and metalloprotease 17 (*ADAM17*) and ACE secretase are membrane-bound proteases involved in the production, by enzymatic cleavage, of soluble (*s*) ACE2 and ACE, respectively. AngII-AT1R signalling increases ADAM17 expression, leading to a reduction in surface ACE2 levels due to sACE sheddingFig. 3Schematic representation of hypothesis 1. ACE inhibitors (*ACEIs*) and angiotensin II type‑1 receptor blockers (*ARBs*) alleviate COVID-19 by inhibiting the harmful ACE/AngII/AT1R axis, which gained dominance due to SARS-CoV-2-induced downregulation of ACE2
A number of scientists \[[@CR20]\] has raised the opposite idea (hypothesis 2) that ACEI and ARB pharmacotherapy may aggravate SARS-CoV-2-induced lung disease by increasing ACE2 surface expression on airway epithelial cells (Fig. [4](#Fig4){ref-type="fig"}). Several animal studies have indicated an increase of ACE2 expression following treatment with ACEIs or ARBs although, especially for ACEIs, decreases or no alterations in ACE2 expression have also been reported (reviewed in \[[@CR21]\]). Information on the effect of these anti-hypertensive drugs in humans is limited to the analysis of duodenal biopsies. Collectively, the data show a more consistent upregulation of ACE2 by ARBs than by ACEs \[[@CR21]\] in various tissues. However, whether ACE2 expression is upregulated by either of these drugs in the primary target cells of SARS-CoV‑2 in human lungs is not yet known.Fig. 4Schematic representation of hypothesis 2. ACE inhibitors (*ACEIs*) and angiotensin II type‑1 receptor blockers (*ARBs*) aggravate COVID-19 by upregulating ACE2, causing increased SARS-CoV‑2 entry and replication
The hypotheses presented above are not mutually exclusive. In patients that are on ACEIs/ARBs before infection with SARS-CoV‑2, ACEI/ARB-mediated upregulation of ACE2 in airway epithelium could increase the risk of becoming infected with SARS-CoV‑2 and may facilitate initial virus propagation/spread. In patients that receive ACEIs/ARBs once they are infected with SARS-CoV‑2, these drugs could mitigate the extent of lung injury by inhibiting detrimental AngII/AT1R signalling and hence restoring the balance with beneficial Ang1--7/MasR signalling.
Recently, a number of observational studies were performed to investigate the influence of ACEI and ARB pharmacotherapy on SARS-CoV‑2 infections \[[@CR22]--[@CR28]\]. None of the studies showed use of these anti-hypertensive/cardioprotective drugs to be associated with an increased risk of (1) SARS-CoV‑2 infection, (2) severe COVID-19 or (3) SARS-CoV-2-related in-hospital death. Conversely, in the small-scale study (205 patients) of Bean and colleagues, a lower rate of death or transfer to a critical care unit within 7 days was found in patients on an ACE-inhibitor \[[@CR22]\]. Likewise, in the study of Mehra and co-workers involving 8910 COVID-19 patients, the use of ACEIs was associated with better survival \[[@CR25]\]. Moreover, in the small-scale studies of Meng et al. \[[@CR26]\] and Zhang et al. \[[@CR28]\], favourable effects of ACEIs/ARBs on, respectively, severity of disease and all-cause mortality were observed. Although each of these studies arrives at the same conclusion, namely that ACE/ARB pharmacotherapy is unlikely to be harmful for COVID-19 patients (and may even have beneficial effects), small-scale studies have a high risk of chance associations and with observational studies there is always the possibility of confounding. Because of this and some other weaknesses (e.g. use of different virological tests within one study, lack of information about the continuation of ACE/ARB treatment during hospitalisation, bias resulting from selective virological testing), randomised controlled trials will be required to definitively answer the question whether RAS inhibitors or other blood-pressure-lowering drugs (e.g. ARNIs, beta blockers, calcium channel blockers, thiazide diuretics) are harmful to COVID-19 patients. Ideally, these trials should also investigate the effect of different combinations of anti-hypertensive drugs and the timing of anti-hypertensive drug administration on the course of COVID-19. Of special interest is the effect of ARNIs on COVID-19, as these drugs cause a large rise in plasma AngII levels but do not increase the concentration of Ang1--7 in blood plasma \[[@CR8]\].
Apart from further exploring the possible benefit of chemical renin-angiotensin-aldosterone system inhibitors in COVID-19 patients, another promising approach might consist of the treatment of COVID-19 with human recombinant soluble ACE2. This biological drug could inhibit virus entry into host cells by competing with the binding of the S protein to membrane-bound ACE2 and at the same time increase Ang1--7 levels at the expense of AngII.
A.A.F. de Vries declares that he has no competing interests.
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Introduction {#s1}
============
Implantation of implantable cardioverter defibrillators (ICDs) and cardiac resynchronization therapy-defibrillators (CRT-Ds) has significantly increased after the positive results of some landmark primary prevention trials showing the efficacy of ICD therapy in reducing mortality.^[@EUP408C1]--[@EUP408C3]^ Defibrillation threshold (DFT) testing at implant is routinely done to ensure that the ICDs/CRT-Ds deliver appropriate amounts of energy and are functioning appropriately. The left ventricular ejection fraction (LVEF) of ICD patients implanted for either primary or secondary prevention can range from normal or near-normal (\>45%) to severely impaired (\<25%). Typically, patients with a low LVEF also have underlying cardiac disease that has progressed to an advanced degree. Among various other clinical parameters, depressed LVEF has been shown to be a potential predictor of high DFTs in patients implanted with unipolar or bipolar defibrillation systems.^[@EUP408C4]--[@EUP408C8]^
While clinical predictors of high DFTs have been extensively studied in patients receiving ICDs/CRT-Ds, stratification of DFTs by LVEF, one of the most commonly used indices for cardiac impairment, has never been done before. Accordingly, this analysis was undertaken to assess the change in DFT estimates as the LVEF goes from being normal to impaired in patients who are implanted with left-sided, active pectoral defibrillation lead systems.
Methods {#s2}
=======
This retrospective analysis included data from three different multicentre, prospective, randomized studies that were reviewed and approved by the appropriate Human Research Ethical Committees of each of the participating medical centres.^[@EUP408C9]--[@EUP408C11]^ Study-specific objectives, inclusion, and exclusion criteria for all the three studies are listed in *Table [1](#EUP408TB1){ref-type="table"}*. Patients were enrolled by the study site after appropriate informed consent was obtained. The patient population consisted of 230 patients who were implanted with any FDA-approved Atlas^®^, Epic^®^, Current^®^, and Promote^®^ ICDs/CRT-Ds and a compatible dual-coil defibrillation lead system. All patients who met the inclusion criteria in these three studies and underwent DFT testing using SVC coil turned ON were included in this analysis.
######
Study inclusion/exclusion criteria
Study 1^[@EUP408C9]^ Study 2^[@EUP408C10]^ Study 3^[@EUP408C11]^
----------------------------------------- ------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------
Objective To compare the DFT efficacy between 50/50% tilt and tuned defibrillation waveforms To compare DFT efficacy between SVC coil ON and OFF un-tuned defibrillation waveforms To compare DFT efficacy between the 2.5, 3.5, and 4.5 ms membrane time constant-based defibrillation waveforms
Inclusion criteria Patient is a candidate for ICD implantation Patient is a candidate for ICD implantation Patient is a candidate for ICD/CRT-D implantation
Patient is able to tolerate DFT testing Patient has had an echocardiogram, multiple gated acquisition (MUGA), or cath procedure within 6 months of ICD implant Patient has a compatible transvenous defibrillation lead system
Patient is able to tolerate DFT testing Patient has had an echocardiogram, MUGA, or cath procedure within 6 months of ICD implant
Patient is able to tolerate upper limit of vulnerability-guided DFT testing
Exclusion criteria Patient has a mechanical valve in the tricuspid position Patient has a mechanical valve in the tricuspid position Patient has a mechanical valve in the tricuspid position
Patient is pregnant Patient has a chronic defibrillation lead, which will not be removed Patient has epicardial defibrillation electrodes
Patient is \<18 years old Patient has a right-sided ICD implant Patient is pregnant
Patient is pregnant Patient is \<18 years old
Patient is \<18 years old
Defibrillation threshold testing {#s2a}
--------------------------------
Two of three studies required the use of a binary search protocol and 72% of the data contributing to this analysis came from those studies.^[@EUP408C9],[@EUP408C10]^ One of the three studies required the use of a binary search protocol guided by upper limit of vulnerability and 28% of the data contributing to this analysis came from that study.^[@EUP408C11]^ The defibrillation waveform for all patients was programmed to the optimal pulse width settings based on a theoretical 3.5 ms membrane time constant using a commercially available chart of optimal defibrillation pulse width (Phase 1/Phase 2) durations.^[@EUP408C12]^ The RV coil was programmed as the anode for the first phase and the SVC coil was always turned on. Ventricular fibrillation was induced by T-wave shock, burst-pacing, or 'DC (direct current) Fibber' through the ICDs. For all the methods, DFT estimate was established only after observation of a failed shock.
Analysis {#s2b}
--------
DFT estimates were stratified into four different LVEF groups (≤25%, 26--35%, 36--45%, and ≥46%). A linear model in which the LVEF group is treated as a factor was used to analyse the data. A *P*-value \< 0.05 was considered statistically significant.
Results {#s3}
=======
There were 230 patients included in this analysis (*Table [2](#EUP408TB2){ref-type="table"}*). The average age, LVEF, NYHA class, and gender distribution grouped by LVEF range is shown in *Table [3](#EUP408TB3){ref-type="table"}*. The mean DFT voltage for LVEF ≤25% was 425.8 ± 117.6 V, 26--35% was 417.5 ± 121.1 V, 36--45% was 394.1 ± 133.3 V, and ≥46% was 395.2 ± 115 V (*Table [3](#EUP408TB3){ref-type="table"}*). Similarly, the mean DFT energies for LVEF ≤25% was 8.6 ± 4.9 J, 26--35% was 8.4 ± 5.1 J, 36--45% was 7.6 ± 4.4 J, and ≥46% was 7.5 ± 4.2 J (*Table [3](#EUP408TB3){ref-type="table"}*). DFTs (voltage and energy) trended higher for lower LVEF but this trend is not statistically significant (*P* = 0.58 for DFT voltage and *P* = 0.69 for DFT energy). Only 3% of the patients (*n* = 7) had a DFT of \>20 J and all of these high-DFT patients had an LVEF \<35% (*Figure [1](#EUP408F1){ref-type="fig"}*). Of these seven high-DFT patients, a \>10 J safety margin could not be achieved in three patients.
{#EUP408F1}
######
Patient population (*n* = 230)
-------------------- -------------------
Age 66.6 ± 12.4 years
Gender 81% males
NYHA class
I 12.6%
II 40%
III 25.2%
IV 1.3%
Unknown 20.9%
Ischaemia 74.3%
Implant indication
Primary 63%
Secondary 33%
Unknown 4%
Hypertension 54%
Amiodarone usage 9.1%
-------------------- -------------------
######
Patient population and DFT estimates grouped by LVEF
LVEF range LVEF (%) Gender Age (years) NYHA class Impedance (Ω) DFT voltage (V) DFT energy (J)
-------------------- ------------ ---------- ------------- ------------ --------------- ----------------- ----------------
≤25% (*n* = 102) 20.7 ± 4.0 85% male 65 ± 12.2 2.5 ± 0.6 40.9 ± 6.6 425.8 ± 117.6 8.6 ± 4.9
26--35% (*n* = 90) 32.7 ± 4.3 82% male 67 ± 12.2 2.0 ± 0.7 41.2 ± 6.3 417.5 ± 121.1 8.4 ± 5.1
36--45% (*n* = 17) 41.2 ± 3.4 71% male 67 ± 11.6 1.8 ± 0.7 40.1 ± 6.2 394.1 ± 133.3 7.6 ± 4.4
≥46% (*n* = 21) 54.9 ± 5.2 62% male 74 ± 12.4 1.3 ± 0.6 40.5 ± 5.2 395.2 ± 115.0 7.5 ± 4.2
*P* = not significant.
All of the patients (*n* = 7) with DFT \> 20 J were men with an LVEF ≤ 35%. In this group, five patients received an ICD/CRT-D for primary prevention, four had ischemic cardiomyopathy, four had hypertension, and four had undergone previous ablation for sustained ventricular tachycardia. Two of these patients were below the age of 50 years, three of them were between 50 and 65 years, and two were above 65 years of age. Similarly, each of the patients (*n* = 4) with DFT \> 25 J had received an ICD/CRT-D for primary prevention; two had ischaemic cardiomyopathy and two had hypertension. One of the patients was younger than 50 years, two were between 50 and 65 years, and one was above 65 years.
A multiple variable regression estimation model constructed to estimate the effect of age, gender, NYHA class, LVEF, implant indication, type of study, and method of VF induction on DFTs revealed that gender was the only significant predictor of higher DFTs in this patient population, with men having higher DFTs than women (*P* = 0.02). The mean DFT in men was greater than that in women by 58.7 V (15.2%) and 2.3 J (31.3%).
Discussion {#s4}
==========
This is the first analysis that attempts to stratify the DFT estimates by LVEF in patients tested with biphasic, tuned waveforms that are optimized based on the high-voltage lead impedance. The primary results indicate that both DFT voltage and energy increase as LVEF decreases, but the difference in DFT energy between each adjacent LVEF group is very small and, even between the highest and the lowest LVEF groups is minimal (approximately 1 J).
In previous studies, the association of LVEF and DFT has been inconsistent.^[@EUP408C4],[@EUP408C7],[@EUP408C8],[@EUP408C13]--[@EUP408C18]^ Burke *et al*.^[@EUP408C13]^ analysed DFTs in 50 ICD/CRT-D patients. Although the mean LVEF in CRT-D group was significantly lower than that of the ICD group (23 ± 5% for the CRT group vs. 31 ± 10% for the control group), the mean DFTs of the two groups were not significantly different (10.2 ± 6.1 J for the CRT group vs. 9.5 ± 5.0 J for the control group). Similarly, Cuoco Jr. *et al*.^[@EUP408C14]^ found no significant difference in DFT between ICD and CRT-D groups (*n* = 537). In the ASSURE study,^[@EUP408C15]^ Doshi *et al*. showed that patients receiving CRT-D devices do not have higher defibrillation energy requirements when compared with ICD patients. Val-Mejias *et al*.^[@EUP408C16]^ found no difference in the DFT estimates between ICD/CRT-D patients implanted with primary and secondary prevention indications, in spite of significant differences in the LVEF between the two indication groups. In an analysis of 128 patients who received Ventak ICDs, Horton *et al*.^[@EUP408C17]^ did not find LVEF to be a significant factor in predicting high DFT. However, several studies have shown that LVEF was one of the significant predictors of a high DFT. In their review of 1139 patient records with all manufacturer\'s devices, Russo *et al*.^[@EUP408C18]^ found that 71 patients (6.2%) had high DFTs (\<10 J safety margin). Lower LVEF had a borderline predictive value for the need for system revision owing to lack of a 10 J safety margin (*P* = 0.054). Similarly, Shukla *et al*.^[@EUP408C17]^ analysed 968 patients with Medtronic devices and found that patients with higher threshold (≥18 J) had lower LVEF, a worse functional class, less frequently done bypass surgery, amiodarone and history of more frequent VF. Both Lubinski *et al*.^[@EUP408C7]^ (*n* = 168) and Pinski *et al*.^[@EUP408C8]^ (*n* = 125) found that low LVEF was a significant predictor of high DFT. In an older study involving 128 patients who received epicardial defibrillators, high LVEF was found to be an important determinant of improved defibrillation efficacy.^[@EUP408C4]^
In this study, only 7 of the 230 patients had a DFT \> 20 J which is slightly lower than the incidence reported in other studies that employed fixed tilt waveforms.^[@EUP408C5]--[@EUP408C8],[@EUP408C19],[@EUP408C20]^ This could be because the DFT protocol in some of these studies was neither uniform nor was it followed consistently and the definition of high DFT was different from the current study. Interestingly, all the 'high DFT' patients in our study had an LVEF of \<35% suggesting that the occurrence of high DFT is not a common problem in patients with normal to near-normal LVEF. It should be noted that the results from the current study were obtained with fixed pulse-width waveforms that are optimally tuned per impedance and assumed cardiac membrane time constant. Fixed pulse-width waveforms have been shown to provide lower voltage and energy DFTs than fixed-tilt waveforms, particularly when DFT is higher than 400 V. This might explain our lower DFT per LVEF range as well as our lower incidence of 'high DFT'.^[@EUP408C21],[@EUP408C22]^ It should be noted that concerns regarding DFTs between 20 and 26 J may not be as great when a device with maximum delivered energy capability of 36 J is used because a 10 J safety margin would be available.
This analysis should be interpreted under the light of certain limitations. First, this is a retrospective analysis, hence there is an unequal number of patients in the four stratified LVEF groups. Second, DFT estimates in all the patients were obtained with a left-sided, active pectoral pulse generator that utilized biphasic, tuned waveforms with SVC coil turned ON. We cannot assure that similar results would be observed if the different waveforms, generator pocket location, shocking vector, or lead configurations are used. In addition, the impact of infiltrative cardiomyopathy (i.e. sarcoidosis, amyloidosis, etc.) could not be assessed because there were no patients in the cohort with those diseases. The impact of kidney disorders could not be evaluated because data reflecting renal function was not collected in any of the three studies.
Conclusion {#s5}
==========
This analysis shows that across a very broad range of LVEF, changes in DFT are minimal. No patient with a near-normal to preserved LVEF had an occurrence of high DFT, and among the patients with severely impaired LVEF only a few (3%) had high DFTs. These results should reassure implanters that patients with severely impaired LVEF implanted with left-sided ICD/CRT-D devices employed with tuned defibrillation waveforms and dual-shocking leads will not necessarily have elevated DFTs.
**Conflict of interest:** J.E.V.-M. has received research support and fellows program support from St. Jude Medical. A.O. is an employee of and holds stock in St. Jude Medical.
Funding {#s6}
=======
The studies mentioned in this article were funded by St. Jude Medical. Funding to pay the Open Access publication charges for this article was provided by St. Jude Medical.
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{.A256}
{.A257}
{.A258}
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1. Introduction {#sec1-bioengineering-04-00089}
===============
There is a global public health problem relating to physical inactivity, which has been attributed to approximately 3.2 million annual deaths \[[@B1-bioengineering-04-00089]\]. In 2012, only 41% of men and 31% of women met the minimum recommendations for UK physical activity levels \[[@B2-bioengineering-04-00089]\]. The problem of inactivity presents a considerable concern to the general public; however, these effects are even more serious for those who have suffered an amputation. Multiple studies indicate that the likelihood of participating in physical activities, following an amputation, decrease if they did not participate in activities prior to the amputation \[[@B3-bioengineering-04-00089]\]. Current physical activity recommendations for an adult in the UK suggest that at least 150 min of moderate intensity exercise should be carried out, on a weekly basis. Typically recommended activities include brisk walking and cycling \[[@B2-bioengineering-04-00089]\]. The limb-absent population is no exception to these guidelines. Those who do return to leisure or sports activities tend to opt for less strenuous activities, such as swimming and fishing, where either a prosthesis is not needed or is not functionally required for participation. A review of 12 independent studies indicates that 68% of the amputee population is generally inactive \[[@B3-bioengineering-04-00089]\]. Following an upper-limb amputation, leisure pursuits become more sedentary and indoor in nature \[[@B4-bioengineering-04-00089]\]. Moreover, arms and hands are required for most daily activities, as well as being essential for communication and showing affection \[[@B5-bioengineering-04-00089],[@B6-bioengineering-04-00089]\]. Loss of an upper limb therefore results in serious restrictions to function, sensation and appearance \[[@B7-bioengineering-04-00089]\].
Upper-limb amputations occur less frequently than lower-limb amputations and typically result from traumatic injury in healthy, young adult, male individuals \[[@B8-bioengineering-04-00089],[@B9-bioengineering-04-00089]\]. Studies suggest that the average age of upper-limb amputees, at amputation, varies between 20 and 36 years \[[@B10-bioengineering-04-00089],[@B11-bioengineering-04-00089],[@B12-bioengineering-04-00089],[@B13-bioengineering-04-00089]\]. Estimates from 2005 suggest that the prevalence of major upper-limb loss in the USA was 41,000 \[[@B14-bioengineering-04-00089]\] and 280 in the UK \[[@B15-bioengineering-04-00089]\]. Following a major upper-limb amputation, multidisciplinary rehabilitation is required \[[@B16-bioengineering-04-00089]\].
In rehabilitation through sports, cycling is considered one of the best forms of exercise for amputees, since it is a muscle-strengthening activity and involves relatively low joint loadings \[[@B17-bioengineering-04-00089]\]. There are various opportunities for lower-limb amputees to participate in cycling, including rigid leg sport-prostheses and hand-cycles; however, there are only limited options for upper-limb amputees. In order to allow for these people to engage in an active lifestyle, there is a need for a low-cost upper-limb sports-prosthetic device. The NHS service specification for people of all ages with limb loss includes a provision for recreational prosthetic appliances and components, in order to meet the clinical needs and rehabilitation goals of an individual \[[@B18-bioengineering-04-00089]\]. If the individual previously participated in cycling, a cycling terminal device could allow these activities to be integrated into the rehabilitation process. This article describes the design and development of an upper-limb cycling prosthesis for rehabilitation and recreational purposes.
2. Materials and Methods {#sec2-bioengineering-04-00089}
========================
2.1. Current Designs and Limitations {#sec2dot1-bioengineering-04-00089}
------------------------------------
Participation and interest in Para-Sports has increased drastically in recent years. The London Paralympic Games in 2012 were televised in more countries than ever before and attracted the biggest ever audience \[[@B19-bioengineering-04-00089]\]. These events have been valuable to increase public awareness of the capabilities of amputee athletes and the disabled population in general. Studies have noted that participation in sports and recreation is important for persons with limb deficiency for their reintegration into the community \[[@B20-bioengineering-04-00089]\]. Opportunities to participate in leisure and competitive sports have improved through a growing public interest in physical fitness and accompanying sports organizations for the disabled \[[@B20-bioengineering-04-00089]\]. Advances in prosthesis design, componentry and fabrication have also been an essential part of this development. The use of a functional prostheses is quite difficult for most proximal amputations, which are associated with higher rejection rates \[[@B12-bioengineering-04-00089]\]. Only 37% of upper-limb amputees use their prosthesis regularly in the long term, with 19% being occasional users \[[@B4-bioengineering-04-00089]\]. A third of people with a limb deficiency only require a cosmetic prosthesis, for basic functions in daily life \[[@B21-bioengineering-04-00089],[@B22-bioengineering-04-00089]\]. These are considered 'passive function prostheses', as they are used for steadying and supporting \[[@B23-bioengineering-04-00089]\]. However, some upper-limb amputees are likely to favour a so-called activity limb, which is dedicated for sports-related activities. Others may prefer an artificial limb with different terminal devices that can be interchanged, depending on their lifestyle, occupation and leisure activities \[[@B21-bioengineering-04-00089]\]. In addition to standard aspects of prosthesis evaluation (comfort, fit, weight, hygiene, etc.), the demands of the desired activity and the environment in which the sport is to be played need to be considered. Sport-specific design efforts have produced a range of upper-limb activity limbs, suitable for sports such as basketball and skiing \[[@B24-bioengineering-04-00089]\].
When controlling bicycle handlebars, there are three key considerations: control, flexibility and release. The human hand offers very good control, many degrees of flexibility and release by simply letting go. When wearing a prosthesis, this is not possible. Current designs approach this problem with varying degrees of success, often sacrificing one factor for another. Some designs approach this issue by offering a rudimentary design, with a rigid connection to the handlebar. Professional devices (appropriate for the Paralympic level) consist of a customised socket, to match the residual limb, so that the rider can easily detach from the handlebar. However, amateur and semi-professional variants of this design involve the rider being attached, or 'strapped on', to the handlebar at the beginning of the race. In the event of a crash or accident, these designs rely on mechanical failure to release the rider. Failure of a prosthesis during either recreational use or competition can result in physical injury and severe psychological repercussions \[[@B20-bioengineering-04-00089]\].
To combat this, some designs offer flexible connections, such as a socket and pin which replicates wrist movements, whilst maintaining a fixed connection to the handlebar. In these cases, transradial amputees tend to wear a loose-fitting flexible socket, from which the residuum can easily be removed in the event of an accident. Although improving prosthetic flexibility, socket and pin connectors are still limited by a mono-directional release mechanism \[[@B24-bioengineering-04-00089]\]. The rider is required to push their arm up and forwards to release. In the event of a crash, it is unlikely that this will always be possible. Facing a crash with an arm still attached to the bicycle, the amputee may lose the opportunity to break their fall. The arm could even release from the socket, leaving the rider to land on their residual limb.
Alternative designs use a ball-connector that sits in a socket, attached to the handlebar. This solution offers greater control and even more flexibility. The ball is typically secured using a simple friction fit, offering greater movement to adjust riding positions and an easy release mechanism. However, this design is often perceived to offer too much flexibility for some users, since freedom of movement results in limited steering control \[[@B25-bioengineering-04-00089]\]. Moreover, commercially available products such as Mert's Hands start at \$2000 \[[@B26-bioengineering-04-00089]\]. Alternatively, \$10,000 are required to purchase a transradial upper-extremity prosthesis with a functional 'split hook' device for below-the-elbow amputees \[[@B27-bioengineering-04-00089]\]. The lack of affordable and commercial cycling prostheses presented an opportunity to develop an upper-limb sports prosthesis, which could viably outperform what the market currently offers.
2.2. Novel Design Concept {#sec2dot2-bioengineering-04-00089}
-------------------------
To devise a novel concept for an effective release mechanism, fundamental knowledge in regard to the correct and recommended behaviour during a crash was required. Some crashes are simply too violent and sudden to react to, especially involving collisions with another vehicle. Nonetheless, experts suggest that crashing is an important skill for cycling and that it must be practiced in order to be performed safely \[[@B28-bioengineering-04-00089]\]. In 2014, 16% of reported cycling accidents resulted from a loss of control. Moreover, driver or rider errors contributed to 73% of cases \[[@B29-bioengineering-04-00089]\]. Based on these findings, the team decided that two safety mechanisms would be required; one for instances in which the rider has partial control and can initiate the release themselves and another which can release automatically in situations too sudden for the rider to react to. When a rider loses control, they often have only a few moments to react. The correct behaviour in these situations can significantly reduce the risk of serious injury. The rider's hands need to get "off the bars and out" from the handlebars, to separate from the bicycle \[[@B28-bioengineering-04-00089]\]. Hands and forearm should make first contact with the ground; however, it is imperative that the arm is not held outstretched, which can result in a broken collarbone \[[@B30-bioengineering-04-00089]\]. Instead, the arm must be free to protect head and body during impact \[[@B27-bioengineering-04-00089]\]. Based on these recommendations, the concept of the upper-limb prosthetic was designed to enable the rider to free their arm and initiate a rolling action, in the event of a crash.
The specification and requirements for an 'ideal' prosthesis were developed in consultation with prosthetists. Standardised and commercially available components were used to reduce the overall manufacturing costs, enable simple servicing and ensure that the new design could be integrated with existing terminal devices. The proposed emergency release mechanism allows the prosthetic hand to release by sliding parallel to the handlebars, in either direction, depending on which way the rider is going to fall. A sliding bar serves as a simulated hand, which is able to recreate the desired motion during a crash. Spring plungers were included in the design to prevent the slider from releasing accidentally. Unless sufficient force is applied to overcome the plungers, the bar is rigidly connected to the handlebar. The force required by the rider to initiate the sliding motion will most likely vary from rider to rider. Two adjustable spring plungers were implemented to provide a wider range of operational forces. The second plunger acts as a redundancy, in case one fails unexpectedly. The release mechanism, shown in [Figure 1](#bioengineering-04-00089-f001){ref-type="fig"}, demonstrates the transitions from an initially locked position to sliding out of the housing.
If enough sideways force is applied by the rider, the hand begins to slide out by overcoming the force required to push the internal springs of the plungers up. The second plunger is overcome twice, as shown in [Figure 1](#bioengineering-04-00089-f001){ref-type="fig"}b, preventing accidental release. If enough force has been applied to overcome the second spring plunger, the slider releases from the middle-block entirely, as shown in [Figure 1](#bioengineering-04-00089-f001){ref-type="fig"}c.
The sliding release mechanism allows the user to 'let go' of the handlebar and protect head and body during impact. The limitation of this design is that the mechanism will not be effective during a head-on collision. In such crash scenarios, the rider cannot be realistically expected to apply a side-ways motion to release.
A secondary release mechanism was designed, intended to free the rider from the handlebar in accidents too sudden and/or violent to which to react. A so-called pylon provides a structural connection between the residual limb liner and end-effector (i.e., slider hand). The pylon thereby simulates the amputee's forearm. A bicycle seat clamp was used to secure two halves of the pylon by radial friction forces, which require an axial force to release the tubes from the clamp. This clamp connection can be reassembled easily following an incident and does not rely on mechanical failure to release.
2.3. Experimental Measurement Setup {#sec2dot3-bioengineering-04-00089}
-----------------------------------
An important consideration for the mechanism was the selection of appropriately sized spring plungers, which in turn provides the range of forces appropriate for most users to initiate release. To determine this, potential rider force data needed to be collected. An experiment was designed and executed, which involved a digital force meter, an immobilised vice and an able-bodied volunteer. The volunteer was a 23-year-old, healthy, male individual. The age and sex of our volunteers match the demographic of individuals most frequently affected by upper-limb amputations.
A vice was secured to a table and attached to a digital force gauge (CFG+, Mecmesin, West Sussex, UK). The other end of the force gauge was attached to an inelastic cable, which the volunteer tied around the palm of their hand. The experimental setup is shown in [Figure 2](#bioengineering-04-00089-f002){ref-type="fig"}. The force gauge was set to record maximum force. The volunteer was asked to initiate a sudden lateral motion, away from the force gauge. The experiment was repeated 3 times. It was determined that a mean force of approximately 200 N could be applied by our volunteer.
To determine the forces required to initiate release of the secondary mechanism, the experiment in [Figure 2](#bioengineering-04-00089-f002){ref-type="fig"} was repeated. The volunteer's arm was replaced with the two pylon halves, secured with the bicycle seat clamp. One end of the pylon was connected to the inelastic cable, while the volunteer pulled on the other end to simulate an axial force. The force gauge was set to record maximum force to determine how much force is required to release the pylon halves. It is difficult to estimate what forces will be experienced during a sudden accident; however, the volunteer was unable to release the pylon halves beyond a maximum force of 500 N. The radial friction forces of the bicycle seat clamp were increased to approximately 700 N. This prohibits the user from initiating the secondary release accidentally. External forces, experienced during an accident, are required for the pylon halves to separate.
The specification of a prosthesis is often tailored to the individual. The volunteer had agreed to participate in further experimentation to evaluate the prosthesis. The spring plungers used for the design prototype were selected to match the requirements from the results of the experiment described above. In this case, the prosthesis was intended for a male subject in his early 20 s. This procedure simulates the customisation process that is likely to occur when a prosthetist modifies a prosthesis for their patient. It is worth noting that the able-bodied individual has intact musculature and a potentially longer moment arm than an amputee. However, by referring to the maximum force applied by the able-bodied volunteer, we believe that the maximum force required for an amputee subject (in the same demographic) is likely to lie below this value. The technique used for selecting spring plungers is therefore suitable to account for most inter-person variability.
To determine the force required to push the spring plungers to release the slider, Equation (1) was used (in reference to [Figure 3](#bioengineering-04-00089-f003){ref-type="fig"}). $$F_{S} = \frac{F_{E}}{\tan\left( \frac{\theta}{2} \right)}$$
The angle of the V-shaped groove determines the sliding force required to release the plunger. $F_{S}$ is the force applied by the rider, which from the previous test was determined to be around 200 N. $F_{E}$ is the compression force of the spring plungers. This information was provided by the supplier of the spring plungers (Norelem, Birmingham, UK).
The vector sliding forces sum to produce a total sliding force. Therefore, the force required from each spring plunger must be halved. The optimal angle of grooves achievable in manufacturing was determined to equal 60°, as a result of using a 30° cutting tool. This gave a target $F_{E}$ of 57 N, shown in Equation (2). $$100 = \frac{F_{E}}{\tan\left( \frac{60}{2} \right)}$$
M12 spring plungers were used, giving a range of: $F_{E}\left( {{Min}.} \right) = 19~N~{and}~F_{E}\left( {{Max}.} \right) = 74\ N$. The total force required to initiate release can therefore be adjusted between the calculated range of: 141.8--256.3 N. A hex key is required to adjust the tension on the spring plungers. This range is likely to be sufficient for the mechanism to work effectively with male young adults. A wide selection of spring plungers can be used to achieve various ranges of forces, for different user demographics. Factors influencing the maximum force an amputee can exert may be affected by activity levels, body types and age. Moreover, the extent of the transradial amputation, i.e., length of the residual limb past the elbow joint, is likely to play a key factor. To aid detachment of the arm when stationary, a quick-release mechanism was included, as shown in [Figure 4](#bioengineering-04-00089-f004){ref-type="fig"}. This allows the user to pull and twist a lever to retract the spring plungers.
Twisting the lever allows the user to adjust the protruding distance of the spring plungers, which results in a substantial reduction in force required to release the slider. To ensure that the slider was properly constrained when attached, the design utilises a dovetail concept between the slider and middle-block, demonstrated in [Figure 5](#bioengineering-04-00089-f005){ref-type="fig"}.
A key advantage of the dovetail mechanism is that it provides a secure connection between the slider and the middle-block, which can only release by applying a lateral force. A summary of the forces required to release the two emergency mechanisms is shown in [Table 1](#bioengineering-04-00089-t001){ref-type="table"}.
2.4. Final Design {#sec2dot4-bioengineering-04-00089}
-----------------
The final prosthetic assembly is shown in [Figure 6](#bioengineering-04-00089-f006){ref-type="fig"}. Starting at the handlebar attachment, there is a slider block with a quick-release mechanism and spring plungers. The rest of the design consists of a restricted universal joint, male and female pyramid adapters, secondary clamp release, and cast attachment. An exploded view is shown in [Figure 7](#bioengineering-04-00089-f007){ref-type="fig"}.
The design is intended to compliment commercially available products, such as the *Iceross* Sport Liner. These liners are fitted with a locking mechanism, which involves a pin connecting to a plastic cast adapter, to form a secure and comfortable connection. Components (a)--(c) in [Figure 7](#bioengineering-04-00089-f007){ref-type="fig"} will be attached to the bicycle handlebar, while components (d)--(n) will be attached to the rider.
[Table 2](#bioengineering-04-00089-t002){ref-type="table"} shows a breakdown of the manufacturing details relating to the components in [Figure 7](#bioengineering-04-00089-f007){ref-type="fig"}. Most parts were manufactured 'in-house' by workshop technicians at the School of Engineering.
Pyramid connectors are standardised components used in a variety of prosthetic designs. These parts are very expensive and were therefore manufactured 'in-house' to further reduce costs. The estimated cost for materials and technician time for the device prototype is £500. Economies of scale and improved manufacturing processes can further reduce the production costs associated with this design concept. The manufactured prototype (shown in [Figure 8](#bioengineering-04-00089-f008){ref-type="fig"}) weighs 800 g and measures 415 mm.
3. Results {#sec3-bioengineering-04-00089}
==========
The manufactured upper-limb cycling prosthesis was tested in a controlled lab environment to demonstrate the efficacy of the device during normal operation and to determine the reliability of the safety release mechanism, in the event of an emergency. The handlebar bracket fits all standard 7/8 inch (22 mm) diameter bars. A custom arm cast, shown in [Figure 9](#bioengineering-04-00089-f009){ref-type="fig"}, was used to allow the able-bodied volunteer to simulate a prosthetic hand.
The quick-release could be applied with one hand to attach and detach the slider 'hand' during mounting and dismounting. The test-rider was successfully able to stabilise on the bicycle. The spring plungers were perceived to provide ample resistance and, when locked, allowed the rider to have full control of the handlebars to steer and complete basic turning manoeuvres. The volunteer was then stabilised by helpers and asked to release the slider from the handlebars by applying a sideways force. The test-rider was able to free his arm quickly, freeing it to protect head and body during a sideways fall. This matches the calculations of the upper resistance limit of about 200 N. If the user desired a lower resistance limit, the spring plungers could be simply adjusted by twisting the quick-release (to further retract the spring plunger) or using a hex key to adjust the tension on the spring plungers.
A new prosthesis is often evaluated by a prosthetist using a subject-reported experience. The assessment is intended to evaluate factors including: prosthesis suspension, socket comfort, socket fit, reliability, functionality, usefulness, weight, appearance and shape \[[@B25-bioengineering-04-00089]\]. The factors are rated on a scale of: not at all, a little, somewhat, quite a lot, a lot; relating to how restricting they are in participating in activities. Since the experiment was conducted with an able-bodied subject, the factors of reliability, functionality, usefulness, appearance/shape were evaluated, as shown in [Table 3](#bioengineering-04-00089-t003){ref-type="table"}.
The results from the subject-reported experience are encouraging. Usefulness, reliability and functionality factors were perceived as 'not at all' and 'a little' restricting to participate in activities. The appearance/shape is acceptable with its functional design; however, the weight is perceived as too heavy and restricting.
4. Discussion {#sec4-bioengineering-04-00089}
=============
For prosthetic devices that are intended for recreation and sports activities, cosmesis is of lower priority than functionality. However, the additional mass of the design poses a potential concern. Research indicates that the metabolic cost during walking is unchanged up to a mass of 1.3 kg using a transfemoral prosthesis \[[@B31-bioengineering-04-00089]\]. There is little known about the metabolic effects of prosthesis mass on higher level activities, such as cycling. Based on the results from the subject-reported experience, the foremost issue seems to be weight. The current prototype weighs 800 grams and would require an amputee to exert quite a lot of force through their residual limb. A major development for the arm design could involve an experiment to precisely quantify the forces an amputee would be able to exert during the general use of the prosthesis. This data could then be used to precisely allocate a number of angular turns of the quick-release for specific user demographics. Additionally, some of the parts could be machined out of lighter materials, such as aluminium or titanium alloy, instead of steel. Additive manufacturing techniques could even enable various parts to be 3D-printed. Extensive testing will be required to simulate and experimentally determine the mechanical properties of these components.
The qualitative evaluation is very subjective, due to only having one volunteer. This information can therefore only provide a rough indication of how an amputee user may assess the prosthesis. However, these impressions may serve as a valuable reference for future studies, where we hope to test and evaluate the prosthesis with 2--5 upper-limb amputee volunteers.
The length of the current design can be altered by cutting longer or shorter aluminium tubes to better suit the rider's forearm length. A more advanced solution could involve a design which adapts its length through a gear and pinion mechanism to create a 'one-size-fits-all' design. Children may require a new prosthesis once every three or four years until age 21 \[[@B27-bioengineering-04-00089]\]. This mechanism can be used to adapt the prosthesis pylon length to match the development of the user.
The novel mechanism proposed in this article will likely require users to attend training sessions to learn and practice applying the mechanism safely, in a controlled environment. However, it is not uncommon that persons using a new prosthesis for sports require extensive training to practice the safe use, maintenance and adjustment of the device \[[@B20-bioengineering-04-00089]\].
Due to the rigid design, the rider may experience shocks when riding over uneven surfaces. The significant biomechanical forces and repetitive nature of turning motions is likely to strain the interface with the residual limb. Interface materials such as silicone liners, gel liners, or hypobaric socks could be used to provide additional padding to help absorb and disperse pressure and shear forces. Moreover, the middle pylon could be replaced with a full suspension system to overcome this issue. When cycling on rough terrain, shocks and impacts exerted through the rider's arm could be absorbed and dissipated through a spring-based damping system. However, further work is required to determine the likely range of forces experienced during an average journey, e.g., riding on a dirt road or bike path.
5. Conclusions {#sec5-bioengineering-04-00089}
==============
A comprehensive assessment of the current drawbacks of transradial prosthetics in the field of cycling was presented. Based on these findings, a novel upper-limb cycling prosthesis was designed and developed. The design includes two release mechanisms, including a sliding mechanism for falls and minor collisions and a clamping mechanism for head-on collisions. The former requires the rider to exert about 200 N of force, while the latter releases if over 700 N are applied. The required forces can be customised to match the preferences of the rider. The design prototype was manufactured and tested by a volunteer test-subject to demonstrate that the design offers functionality and stability during normal operation and allows the rider to reliably activate the safety release mechanism, in a simulated emergency scenario. The total cost of the prototype is estimated at £500. The development of the device proposed in this article could allow people with upper-limb amputations to participate in cycling for rehabilitation or recreational purposes.
We thank all authors and peer reviewers for their invaluable contributions to this article. In particular, we thank John Head and the University of Salford for providing experimental resources. Lastly, we wish to acknowledge the outstanding technical support from the School of Engineering Workshop, University of Warwick.
Akira Tiele conceived the design concept. Akira Tiele, Shivam Soni-Sadar, Jack Rowbottom, Shilen Patel, Edward Mathewson and Samuel Pearson further developed the design and performed the experiments. David Hutchins supervised the project, supported by John Head and Stephen Hutchins. All authors were involved in the writing and editing of the article.
The authors declare no conflict of interest.
{#bioengineering-04-00089-f001}
{#bioengineering-04-00089-f002}
{#bioengineering-04-00089-f003}
{#bioengineering-04-00089-f004}
{#bioengineering-04-00089-f005}
{#bioengineering-04-00089-f006}
{#bioengineering-04-00089-f007}
{#bioengineering-04-00089-f008}
{#bioengineering-04-00089-f009}
bioengineering-04-00089-t001_Table 1
######
Release mechanism summary.
Crash Scenario Release Mechanism Required Force
-------------------- ------------------- ----------------
Falling Left/Right Slider 200 N
Head-on Collision Clamp 700 N
bioengineering-04-00089-t002_Table 2
######
Manufacturing details for upper-limb cycling prosthesis.
Part Name Description Material Manufacturing Process Qty
-------------------- ---------------------------------------------------------- -------------- ------------------------------------------ -----
Slider End-effector, which slides laterally if force is applied Steel Milled from solid steel block 1
Middle-block Housing secures slider/connects to handlebar Steel Milled from solid steel block 1
Spring plungers Provide variable locking forces to secure slider Steel Manufactured by Norelem 2
Quick-release Allows for easier release and adjustment of slider Steel Manufactured by Thorn Cycles 1
Handlebar brackets Connect slider housing to handlebar ABS Fused deposition modelling 1
Universal joint Simulates wrist and allows limited movement CrV Manufactured by Faithfull 1
Restrictor Limits excessive universal joint movement Neoprene Manufactured by RS Pro 1
Pyramid Connector Male/female joint adapters Steel Machined using manual lathe and CNC mill 4
Pylon Rod that provides structural support Al Metal turned 2
Seat Clamp Secures two pylon halves Carbon fibre Manufactured by IMUST 1
Plastic Cast Interface to *Iceross* Liner ABS Fused deposition modelling 1
*Iceross* Liner Protects residual limb and connects to plastic cast Silicone Manufactured by Ossur 1
bioengineering-04-00089-t003_Table 3
######
Qualitative evaluation---subject-reported experience.
Evaluation Criteria Scale Reasoning
--------------------- ------------- --------------------------------------------------------------
Reliability A little Releases well, but will require training to apply quickly
Functionality A little Functions well, but needs to be tested in real-life scenario
Usefulness Not at all Very useful to increase participating in activities
Weight Quite a lot Heavier than expected
Appearance/Shape Somewhat Design is very functional, but appropriate for activities
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#section1-2192568217699377}
============
Adolescent idiopathic scoliosis (AIS) affects 1-3% of children between 10 and 16 years of age.^[@bibr1-2192568217699377]^ In progressive scoliosis, a good correction of the deformity can be achieved with current surgical techniques, but the postoperative spinal motion is significantly diminished due to the fusion of the spine.^[@bibr2-2192568217699377]^ Moreover, patients who underwent fusion surgery report a lower physical function as compared to nonscoliotic controls at long-term follow-up, which could be attributed to the invasive surgery and stiffening of the spine.^[@bibr3-2192568217699377]^ To overcome these negative consequences of extensive fusion procedures, recent research has been aimed at developing techniques that allow scoliosis correction without fusion.^[@bibr4-2192568217699377],[@bibr5-2192568217699377]^
A new less-invasive and potentially fusionless scoliosis correction system is a posterior concave periapical distraction device (the ApiFix system, ApiFix Ltd, Misgav, Israel), which spans 3 to 4 motion segments ([Figure 1](#fig1-2192568217699377){ref-type="fig"}). The device is a unilateral construct that connects 2 periapical pedicle screws through polyaxial mobile ball-and-socket joints with a rod. The device is inserted on the concave side of the curve. The rod includes a ratchet that can elongate when the patient performs exercises after surgery, especially with side bending toward the convexity. By side bending, the ratchet allows elongation of the rod and thereby corrects the scoliotic curve ([Figure 1](#fig1-2192568217699377){ref-type="fig"}). The ratchet does not allow for shortening when the patient stands upright again and thus it preserves the correction. The poly-axial joints allow the pedicle screws to have a range of motion (ROM) of 30° (15° per pedicle screw) in flexion, extension, and axial rotation ([Figure 2](#fig2-2192568217699377){ref-type="fig"}). Floman et al^[@bibr6-2192568217699377]^ recently demonstrated the effectiveness of the periapical distraction device by reducing the Cobb angle from 43°-53° to 22°-33° in a small case series. The authors stated that the new device is a less rigid construct than traditional rigid pedicle screw-rod instrumentation and as a result, preserves a more physiologic spinal ROM after treatment with this device.^[@bibr6-2192568217699377]^
{#fig1-2192568217699377}
{#fig2-2192568217699377}
Nevertheless, it is unknown whether spinal motion is indeed preserved after treatment with this new device. This is important, because rigid spinal instrumentation has been shown to fail in long term if fusion is not achieved and spinal loads are transferred to the instrumentation.^[@bibr7-2192568217699377]^ Also, when spinal flexibility remains intact after treatment with this new device, this could have beneficial effects on the postoperative physical function in comparison to traditional long fusion surgery. Before large clinical studies are started with this fusionless scoliosis correction device, it should be analyzed whether spinal motion is indeed minimally constrained to prevent the transfer of loads to the implant. Therefore, the goal of this study was to analyze the effect of the posterior concave periapical nonfusion distraction device on the in vitro biomechanics of the spine.
Materials and Methods {#section2-2192568217699377}
=====================
Prior to the study, research agreements were signed by all parties to ensure the rights of academic freedom of the VU University Medical Center and consequently an independent publication of the research results.
Specimens and Specimen Preparation {#section3-2192568217699377}
----------------------------------
Seventeen thoracic spines were harvested from fresh frozen (−20°C) human cadavers. After radiographic evaluation and exclusion of specimens with bridging osteophytes or collapsed intervertebral disc spaces, 6 human thoracic spines (mean age 79.2 years, range 65-86 years) were included. Additionally, we included 6 thoracic spines of immature domestic pigs (mean weight 72 kg, range 64-80 kg) obtained from a local abattoir. This allowed for a better comparison with the flexible spine of a young adolescent. In the human spine, the morphology of the facet joint alters below T12-L1. This change in facet orientation, and as a result a change in biomechanics, in porcine spines already occurs more proximally at T10-T11.^[@bibr8-2192568217699377]^ To allow for a comparison between the human and porcine specimens, we used the levels T5-T12 of the human specimens and T3-T10 of the porcine specimens.
Testing Conditions {#section4-2192568217699377}
------------------
The spines were tested in 3 phases: untreated (for baseline analysis), instrumented with the (unilateral) posterior concave periapical nonfusion distraction device and finally, after bilateral segmental rigid pedicle screw-rod rod instrumentation ([Figure 3](#fig3-2192568217699377){ref-type="fig"}). The pedicle screws of the periapical distraction were implanted unilaterally at levels T7 and T10 in the human specimens and at levels T5 and T8 in the porcine specimens.
{#fig3-2192568217699377}
The internal ratchet of the periapical distraction device can be set in 3 positions: 1-way, locked and unlocked, with the latter 2 positions aiming to prevent and correct, respectively, any adverse overcorrection if needed. For this study, we set the ratchet in the locked position, thus fixing the rod length. We consider that the locked ratchet position represents the end situation after rod elongation and scoliosis correction, where the system effectively functions as a unilateral posterior rigid rod with a mobile connection to the polyaxial screws.
In half of the specimens the implant was placed on the right side and in the other half of the specimens on the left side, to exclude a possible effect of implant location. In the final third test the periapical distraction device was replaced by a bilateral segmental pedicle screw-rod system (Medtronic, CD Horizon Legacy Spinal System, Minneapolis, MN, USA) across the same motion segments as the periapical distraction device (T7, T8, T9, and T10 in the human specimens and at levels T5, T6, T7, and T8 in the porcine specimens) ([Figure 3](#fig3-2192568217699377){ref-type="fig"}). Proximal and distal to the instrumented segments there were 2 untreated adjacent spinal motion segments (ie, 2 intervertebral discs). For the third phase of testing with the rigid instrumentation, the pedicle screw holes in the proximal and distal instrumented vertebra of the investigated periapical distraction device were reused by implanting pedicle screws with a 1 mm larger diameter and 2 to 5 mm greater length (depending on the size of the vertebral body) to ensure adequate bone purchase of the screws using the same pedicle screw holes. No screw loosening was observed during or after testing based on manual inspection of the instrumentation and analysis of the load displacement curves.
Biomechanical Testing {#section5-2192568217699377}
---------------------
The test setup and analysis has been described and validated previously.^[@bibr9-2192568217699377][@bibr10-2192568217699377][@bibr11-2192568217699377][@bibr12-2192568217699377][@bibr13-2192568217699377]-[@bibr14-2192568217699377]^ Briefly, before testing, a compressive axial preload of 250 N was applied for 1 hour to obtain physiological conditions in the intervertebral disc.^[@bibr9-2192568217699377]^ Throughout testing, no compressive load was applied to prevent buckling of the spine.^[@bibr15-2192568217699377]^ The spinal segments were placed horizontally in a custom made spinal motion simulator ([Figure 4](#fig4-2192568217699377){ref-type="fig"}) in which pure moments in flexion-extension (FE), lateral bending (LB), and axial rotation (AR) can be applied to the spine using a hydraulic materials testing machine (Instron, model 8872; Instron and IST, Norwood, MA, USA). This spinal motion simulator allows for the biomechanical analysis of long, multisegment, spinal specimens.
{#fig4-2192568217699377}
The human spinal segments were tested at up to −4 and +4 N m and the porcine spinal segments at up to a moment of −2 and +2 N m to allow comparison with previous work.^[@bibr8-2192568217699377]^ Each movement direction was tested for 3 subsequent cycles. The third cycle was used for analysis. During loading, the displacement of 3 infrared-emitting LEDs, rigidly attached to each of the vertebral bodies, was recorded by an optoelectronic 3-dimensional movement registration system (Optotrak 3020, Northern Digital Inc, Waterloo, Ontario, Canada). A custom Matlab (Mathworks Inc, Natick, MA, USA) program was used to calculate the load-displacement curves and ROM of the loaded and coupled directions (rotation about an axis with additional motions about 1 or 2 other axes^[@bibr16-2192568217699377]^) of the bridged and adjacent levels.
Statistical Analysis {#section6-2192568217699377}
--------------------
Effects of type of instrumentation were tested separately for the untreated cranial motion segment, bridged motion segments (average of 3 bridged spinal motion segments), and for the untreated caudal motion segment, using a 1-way repeated-measure analysis of variance with the surgical condition as the within-subject factor. Multiple comparisons based on Bonferroni-corrected paired *t* tests were applied for analysis among testing conditions. *P* values less than .05 were considered statistically significant. SPSS for Mac version 23.0 (IBM Corp, Armonk, NY, USA) was used for statistical analyses.
Results {#section7-2192568217699377}
=======
No device failures were observed during testing.
Bridged Segments {#section8-2192568217699377}
----------------
In the loaded direction, the (locked) periapical distraction device resulted in a significant decrease in ROM of FE (human, −40.0%, −1.1°, *P* = .009; porcine, −55.9%, −4.1°, *P* \< .001) and LB (human, −18.2%, −0.7°, *P* = .020; porcine, −17.9%, −1.7°, *P* = .007) of the bridged motion segments as compared with the untreated segments ([Tables 1](#table1-2192568217699377){ref-type="table"} and [2](#table2-2192568217699377){ref-type="table"}; [Figure 5](#fig5-2192568217699377){ref-type="fig"}). No significant alteration in the ROM of AR of the bridged motion segments was observed.
######
Human Spines: Change in Range of Motion (ROM) in Flexion-Extension (FE), Lateral Bending (LB), and Axial Rotation (AR), With Mean and SD Expressed as Percent (%).^a^
ROM (% Change) Posterior Concave Distraction Device vs Untreated Rigid PS vs Untreated Rigid PS vs Posterior Concave Distraction Device
-------------------- --------------------------------------------------- ----------------------- -------------------------------------------------- ----------- ---------- ------------ ----------- ---------- ----------
FE
Proximal adjacent −16.1 23.3 .454 −0.8 33.9 1.000 18.3 50.6 1.000
Bridged −**40.0** **18.0** **.009** −**80.9** **27.3** **.002** −**68.2** **45.1** **.042**
Distal adjacent −0.6 12.1 1.000 −6.3 21.8 1.000 −5.7 11.4 .816
LB
Proximal adjacent −8.2 10.1 .313 −17.1 22.4 .362 −9.7 30.0 1.000
Bridged −**18.2** **10.0** **.020** −**75.0** **19.1** **.001** −**69.4** **20.5** **.001**
Distal adjacent −6.0 6.3 .199 −3.2 11.9 1.000 3.0 14.1 1.000
AR
Proximal adjacent −12.2 12.9 .208 −18.1 8.7 .011 −6.8 22.0 1.000
Bridged −**0.1** **2.6** **1.000** −**71.3** **17.2** **\<.001** −**71.3** **18.0** **.001**
Distal adjacent 14.3 10.2 .056 2.5 23.4 1.000 −10.3 22.9 .966
^a^Data of the 3 bridged motion segments is averaged. Boldfaced values indicate statistical significance (*P* \< .05). Rigid PS: bilateral rigid pedicle screw-rod instrumentation.
######
Porcine Spines: Change in Range of Motion (ROM) in Flexion-Extension (FE), Lateral Bending (LB), and Axial Rotation (AR), With Mean and SD Expressed as Percent (%).^a^
ROM (% Change) Posterior Concave Distraction Device vs Untreated Rigid PS vs Untreated Rigid PS vs Posterior Concave Distraction Device
-------------------- --------------------------------------------------- ----------------------- -------------------------------------------------- ----------- ---------- ---------- ----------- ---------- ----------
FE
Proximal adjacent 2.1 7.4 1.000 1.0 6.3 1.000 **−**1.0 2.8 1.000
Bridged **−55.9** **7.1** **\<.001** **−94.0** **23.0** **.001** **−86.4** **46.6** **.018**
Distal adjacent **−**7.7 5.5 .056 **−**8.3 7.1 .105 **−**0.6 6.4 1.000
LB
Proximal adjacent **−**4.7 6.9 .455 **−**5.8 3.9 .045 **−**1.1 6.8 1.000
Bridged **−17.9** **7.7** **.007** **−92.2** **26.2** **.001** **−90.4** **28.0** **.002**
Distal adjacent **−**4.1 4.8 .268 **−**6.8 7.7 .244 **−**2.8 6.4 .970
AR
Proximal adjacent 0.7 10.4 1.000 **−**9.4 22.1 1.000 **−**10.0 14.6 .459
Bridged **−**3.9 3.3 .104 **−86.9** **23.2** **.001** **−86.3** **21.8** **.001**
Distal adjacent 17.9 18.2 .183 **23.9** **15.4** **.038** 5.1 17.3 1.000
^a^Data of the 3 bridged motion segments is averaged. Boldfaced values indicate statistical significance (*P* \< .05). Rigid PS: bilateral rigid pedicle screw-rod instrumentation.
{#fig5-2192568217699377}
Rigid pedicle screw-rod instrumentation caused a larger and significant decrease in ROM of FE (human, −80.9%, −2.2°, *P* = .002; porcine, −94.0%, −6.9°, *P* = .001), LB (human, −75.0%, −2.77°, *P* = .001; porcine, −92.2%, −8.8°, *P* = .001), and AR (human, −71.3%, −4.4°, *P* \< .001; porcine, −86.9%, −9.3°, *P* = .001) of the bridged motion segments as compared with the untreated spine ([Tables 1](#table1-2192568217699377){ref-type="table"} and [2](#table2-2192568217699377){ref-type="table"}; [Figure 5](#fig5-2192568217699377){ref-type="fig"}).
In the human specimens, coupled motions of the bridged motion segments were not significantly affected after instrumentation with the periapical distraction device ([Table 3](#table3-2192568217699377){ref-type="table"}; [Figure 6](#fig6-2192568217699377){ref-type="fig"}). In the porcine specimens, ROM of coupled LB during FE loading increased (+122.7%, +1.9°, *P* = .007), while ROM of coupled AR during LB decreased (−20.5%, −1.0°, *P* = .009) after instrumentation with the periapical distraction device ([Table 4](#table4-2192568217699377){ref-type="table"}; [Figure 6](#fig6-2192568217699377){ref-type="fig"}).
######
Human Spines: Change in Coupled Range of Motion (ROM) in Flexion-Extension (FE), Lateral Bending (LB), and Axial Rotation (AR), With Mean and SD Expressed as Percent (%).^a^
Coupled ROM (% Change) Posterior Concave Distraction Device vs Untreated Rigid PS vs Untreated Rigid PS vs Posterior Concave Distraction Device
------------------------ --------------------------------------------------- ----------------------- -------------------------------------------------- ----------- ---------- ---------- ----------- ---------- ----------
FE
Coupled AR −11.5 24.6 .908 −**39.7** **16.4** **.006** −**31.9** **19.9** **.033**
Coupled LB 8.0 35.7 1.000 −28.1 31.1 .234 −33.4 27.7 .095
LB
Coupled AR 1.1 17.5 1.000 −18.1 26.6 .467 −19.0 32.4 .628
Coupled FE 5.7 30.1 1.000 −27.6 27.3 .169 −31.5 31.2 .170
AR
Coupled LB 4.6 11.4 1.000 −46.6 61.3 .366 −48.9 62.0 .334
Coupled FE −13.3 12.1 .130 −**31.9** **19.6** **.031** −21.5 31.8 .476
^a^Data of the 3 bridged motion segments is averaged. Boldface values indicate statistical significance (*P* \< .05). Rigid PS: bilateral rigid pedicle screw-rod instrumentation.
{#fig6-2192568217699377}
######
Porcine Spines: Change in Coupled Range of Motion (ROM) in Flexion-Extension (FE), Lateral Bending (LB), and Axial Rotation (AR), With Mean and SD Expressed as Percent (%).^a^
Coupled ROM (% Change) Posterior Concave Distraction Device vs Untreated Rigid PS vs Untreated Rigid PS vs Posterior Concave Distraction Device
------------------------ --------------------------------------------------- ----------------------- -------------------------------------------------- ----------- ---------- ------------ ----------- ---------- ------------
FE
Coupled AR −0.5 21.0 1.000 −**42.5** **23.1** **.019** −42.2 33.6 .083
Coupled LB **122.7** **53.4** **.007** −**37.0** **25.0** **.045** −**71.7** **28.5** **.005**
LB
Coupled AR −**20.5** **9.4** **.009** −**76.2** **15.9** **\<.001** −**70.0** **12.6** **\<.001**
Coupled FE 46.5 44.4 .151 −**39.9** **23.5** **.026** −**59.0** **32.3** **.020**
AR
Coupled LB −9.0 12.4 .407 −**85.6** **23.1** **.001** −**84.2** **17.2** **\<.001**
Coupled FE −2.8 22.7 1.000 −29.7 31.8 .212 −27.7 26.4 .151
^a^Data of the 3 bridged motion segments is averaged. Boldfaced values indicate statistical significance (*P* \< .05). Rigid PS: bilateral rigid pedicle screw-rod instrumentation.
Rigid pedicle screw-rod instrumentation caused a significant decrease in ROM of coupled AR (−39.7%, −0.39°, *P* = .006) during FE loading, and conversely, in ROM of coupled FE (−31.9%, −0.42°, *P* = .031) during AR loading in the human spines of the bridged motion segments ([Table 3](#table3-2192568217699377){ref-type="table"}; [Figure 6](#fig6-2192568217699377){ref-type="fig"}). In the porcine spines, rigid pedicle screw-rod instrumentation caused a significant decrease in all coupled ROM except ROM of coupled FE during AR loading ([Table 4](#table4-2192568217699377){ref-type="table"}; [Figure 6](#fig6-2192568217699377){ref-type="fig"}).
Adjacent Segments {#section9-2192568217699377}
-----------------
Other than a decrease in ROM of AR (−18.1%, −1.19°, *P* = .011) of the proximal adjacent motion segment in the human specimens and an increase in ROM of AR (+23.9%, +0.88°, *P* = .038) of the distal adjacent segment in the porcine specimens after rigid pedicle screw-rod instrumentation, no significant adjacent segment effects were observed ([Tables 1](#table1-2192568217699377){ref-type="table"} and [2](#table2-2192568217699377){ref-type="table"}; [Figure 5](#fig5-2192568217699377){ref-type="fig"}).
Discussion {#section10-2192568217699377}
==========
Recently, a posterior concave periapical distraction device was presented for fusionless surgical correction of adolescent idiopathic scoliosis.^[@bibr6-2192568217699377]^ This device is designed to maintain spinal motion by using mobile poly axial pedicle screw-rod joints. In fusion surgery, rigid spinal instrumentation has been shown to fail if fusion is not achieved and spinal loads are transferred to the instrumentation.^[@bibr7-2192568217699377]^ A spinal implant designed for fusionless scoliosis correction should therefore minimally constrain spinal motion to prevent the transfer of loads to the implant, thereby reducing the risk of implant failure. The present in vitro biomechanical study was designed to determine the effect of the new periapical distraction device on spinal range of motion in comparison to traditional rigid pedicle screw-rod instrumentation.
This study shows that, across the bridged motion segments, the periapical distraction device roughly halved spinal ROM of FE (human −40.0% and porcine −55.9%), while LB was only slightly affected (human −18.2% and porcine −17.9%) and AR was unaffected. In comparison, the rigid pedicle screw-rod instrumentation resulted in a far larger decrease in ROM of FE (human −80.9% and porcine −94.0%), LB (human −75.0% and porcine −92.2%), and AR (human −71.3% and porcine −86.9%).
The periapical distraction device was tested with the internal ratchet set in "locked" position. This ratchet position prevented rod elongation with each subsequent load cycle (each spine was loaded for 3 cycles) and thus prevented the creation of a scoliotic deformity while testing. In this mode, the periapical distraction device has a fixed length and functions as a unilateral posterior tether (ie, lengthening or shortening was not possible) attached to the pedicle screws with mobile ball and socket joints with large degrees of freedom. This is the situation that would occur after correction of the deformity. The fixed length of the device explains the decrease in ROM of FE after instrumentation of the spine. The decrease in ROM of LB (human, −18.2%, −0.7°; porcine, −17.9%, −1.7°) due to the periapical distraction device was less than expected. With the ratchet of the device set in "locked" position, a larger decrease in LB was anticipated. The relatively mild reduction in ROM may be explained by the phenomenon of coupled motion. The thoracic spine has been shown to exhibit significant coupled motion under LB loading.^[@bibr17-2192568217699377]^ It appears that the mobile polyaxial rod-screw joint design of the periapical distraction device allows coupled AR and FE during LB loading of the bridged segments in a magnitude comparable to the untreated spine with only minor changes ([Figure 6](#fig6-2192568217699377){ref-type="fig"}). We speculate that, because of this, the instrumented spinal segments can find the path of motion with the least resistance (ie, a combination of LB, AR, and FE) to allow most of the LB without requiring a change in length of the periapical distraction device. Combined with the unilateral rod design, which is less rigid than a bilateral rod system, this may explain why there was only a relatively small and less than expected decrease of ROM of LB.
Although a causal relationship has yet to be established, adjacent segment degeneration after spinal fusion has been attributed to altered motion and disruption of the anatomy of the adjacent level.^[@bibr18-2192568217699377]^ In this study, the periapical distraction device did not significantly affect adjacent segment motion. In contrast, after rigid pedicle screw-rod instrumentation the ROM of AR decreased by 18.1% in the cranial adjacent segment of the human spines and increased by 23.9% in the caudal adjacent segment of the porcine spines. This could possibly be the result of an altered spinal alignment due to the instrumentation, although care was taken to customize the rod curvature to each individual specimen.^[@bibr19-2192568217699377]^ Also, proximal adjacent segment biomechanics can be affected by damage of spinal ligaments and joint capsules of the proximal facet joint due to pedicle screw placement.^[@bibr20-2192568217699377],[@bibr21-2192568217699377]^
Other options for fusionless surgery for adolescent idiopathic scoliosis such as vertebral body stapling or anterior spinal tethering have been investigated.^[@bibr4-2192568217699377],[@bibr5-2192568217699377]^ Current research on the use of vertebral body stapling shows a wide range of results of the effects on the ROM of FE (11% decrease to no decrease), LB (4% to 40% decrease) and AR (6% to 60% decrease) and therefore it is difficult to conclude if and how stapling alters postoperative spinal ROM.^[@bibr22-2192568217699377][@bibr23-2192568217699377]-[@bibr24-2192568217699377]^
We took great care to study the implant in vitro in a simulated end situation after rod elongation and scoliosis correction to evaluate possible safety concerns regarding its motion preserving properties. However, this and other preclinical studies examining new treatment options for AIS are limited due to the unavailability of cadaveric AIS specimens or an animal model that combines all characteristics of AIS (ie, a lateral deviation, rotation and alterations of the sagittal profile). Thus, the effectiveness of the periapical distraction device in correcting scoliosis could not be tested. The disadvantage of human cadaveric specimens is their high age and associated state of degeneration. Therefore, we excluded specimens with bridging osteophytes or collapsed intervertebral disc spaces. Additionally, we also included thoracic spines from immature pigs, which have previously been shown to be good biomechanical models for the human spine and are more flexible to represent the adolescent spine.^[@bibr8-2192568217699377],[@bibr25-2192568217699377]^
In conclusion, although its effectiveness in scoliosis correction has still only been demonstrated in a small case series, this study demonstrates that the spinal ROM of the bridged segments after spinal instrumentation with the novel posterior concave periapical non-fusion distraction device is partially diminished and adjacent segment biomechanics were not significantly altered.^[@bibr6-2192568217699377]^ Consequently, a more physiological spinal ROM is expected after scoliosis correction with the periapical distraction device as compared with rigid spinal instrumentation. Thus, it is expected that the spinal flexibility of patients treated with this device is not significantly altered, which could have beneficial effects on the post-operative physical function in comparison with traditional long fusion surgery. Also, the risk of implant failure is deemed low as implant loads are expected to be minimal. Although no destructive forces were applied to the specimens in this study, no device failures were seen. We do not know how the implant will function over time in vivo with continued loading and motion. However, while clinically important, failure testing was beyond the scope of this study. Thus, patients treated with this new device should have regular and prolonged follow-up.
A research grant and implants were provided to the research institution by ApiFix Ltd (Misgav, Israel). Medtronic Netherlands (Heerlen, The Netherlands) provided implants to the research institution. The authors did not receive any personal financial or in-kind support; all support was transferred directly to the research institute.
**Declaration of Conflicting Interests:** The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
**Funding:** The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: A research grant and implants were provided to the research institution by ApiFix Ltd (Misgav, Israel).
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
============
Pluripotent stem cells provide a valuable system to explore intrinsic and extrinsic requirements for self-renewal *in vitro*. Murine embryonic stem cells (ESCs) are derived from epiblasts of late blastocysts ([@DEV124602C2]; [@DEV124602C4]; [@DEV124602C29]). Their potential to produce all tissues, including gametes, when injected into host embryos defines them as naive pluripotent ([@DEV124602C16]). ESCs can be propagated in medium containing foetal calf serum and leukaemia inhibitory factor (LIF) ([@DEV124602C21]; [@DEV124602C26]). In these conditions, developmentally advanced cells can be distinguished and cultures exhibit heterogeneous expression of markers for naive pluripotency, such as *Nanog*, *Rex1* (*Zfp42*), *Stella* (*Dppa3*), *Pecam1* and *Klf4* ([@DEV124602C5]; [@DEV124602C8]; [@DEV124602C10]; [@DEV124602C11]; [@DEV124602C13]; [@DEV124602C24]). A culture regime was subsequently developed based upon inhibition of the MEK/ERK pathway and GSK3, known as '2i' ([@DEV124602C29]). ESCs propagated in 2i exhibit more homogeneous expression of naive pluripotency markers ([@DEV124602C16]; [@DEV124602C27]). Comparative profiling of ESCs propagated in serum/LIF versus 2i/LIF confirmed these differences ([@DEV124602C13]).
Generation of chimaeras from ESCs is used extensively to create transgenic mouse lines ([@DEV124602C23]) or to test the potency of putative pluripotent stem cells ([@DEV124602C3]). This is generally achieved by providing 8-20 ESCs to a host morula or blastocyst. An inoculum of fewer donor cells tends to produce chimaeras less efficiently ([@DEV124602C1]). A probable explanation of this phenomenon is that only a proportion of the injected cells can integrate into the embryo. In support of this, a maximum of three ESCs per chimaera were observed to produce progeny contributing significantly to the adult animal ([@DEV124602C25]). Based upon experimental enrichment of ESCs expressing markers of naive pluripotency, it might be assumed that the ESCs permitted to contribute to the embryo are those residing in the naïve state ([@DEV124602C8]; [@DEV124602C24]).
The capacity of the morula environment to alter the developmental trajectory of lineage-specified cells isolated from blastocysts was a surprising revelation ([@DEV124602C9]). Whether the embryonic niche can exercise a similar effect on lineage-priming ESCs is currently unknown. Understanding how the environment can influence exit from pluripotency and its potential reversion is important for the design of *in vitro* differentiation protocols and interpretation of transplantation studies. The recent advances in transgenic reporters and live imaging open the possibility to explore how incoming ESCs incorporate into chimaeras and determine the fate of those that are rejected.
In this study, we exploit two culture regimes: serum/LIF (SL) and 2i/LIF (2iL) to provide ESCs that are more (SL) or less (2iL) heterogeneous for markers of naive pluripotency. ESCs are injected into host embryos at the 8-cell stage. By tracking the process of chimaera formation, spatial and temporal trends for integration or exclusion can be uncovered. We also use a validated destabilised GFP reporter of the zinc finger protein Rex1 (Rex1-GFPd2), which correlates closely with naive pluripotency *in vivo* and *in vitro* ([@DEV124602C17]; [@DEV124602C28]). This enables separation of SL-cultured ESCs into naive pluripotent (Rex1^+^) and developmentally advanced (Rex1^−^) populations prior to injection. In addition, GFP fluorescence enables assessment of the pluripotency status of integrating or excluded cells during chimaera formation.
Our results uncover some interesting phenomena. Firstly, a large proportion of SL-cultured ESCs is dramatically eliminated by apoptosis within the first few hours after injection. Coincidentally, surviving ESCs appear to undergo compensatory proliferation. Secondly, 2iL-cultured ESCs continue to proliferate throughout the experiment, but undergo increased apoptosis during the second day of culture, in concert with the second lineage segregation event of the host embryo. Finally, although the majority of eliminated cells appear to have begun exit from pluripotency, Rex1^−^ cells can occasionally upregulate GFP expression during development, but this is not a conditional prerequisite for integration into the epiblast.
RESULTS {#s2}
=======
ESCs cultured in 2iL out-perform those from SL conditions during chimaera formation {#s2a}
-----------------------------------------------------------------------------------
To test the hypothesis that ESCs in the state of naive pluripotency preferentially integrate into chimaeras, we used two alternative culture conditions. ESCs propagated in SL for at least four passages exhibited a substantial level of heterogeneity, both morphologically and by immunohistochemistry ([Fig. 1](#DEV124602F1){ref-type="fig"}A). Those expanded using 2iL formed more compact, rounded colonies and a higher proportion expressed pluripotency markers Sox2 and Nanog ([Fig. 1](#DEV124602F1){ref-type="fig"}B). Fig. 1.**Comparison of ESCs cultured in conventional versus ground-state conditions.** Morphology and immunohistochemistry of ESCs cultured for 2 days in (A) conventional, serum/LIF (SL) or (B) ground-state, 2i/LIF (2iL) conditions. Left panels: bright field; second and third panels: immunoreactivity to Sox2 (green) and Nanog (white), respectively; right panels: overlay of Sox2 and Nanog. (C) Scheme for the experimental strategy: 8-cell embryos were injected with fluorescently labelled ESCs and chimaeras transferred to an immobilising grid for live imaging for 2 days. (D) Bar plot of the average numbers of ESC deaths, divisions and resulting viable ESCs by the end of captured development. (E) Plot of the average numbers of viable ESCs per embryo over time (hpc). Grey bars reflect s.e.m. between the curves of the five embryo groups (profiles per embryo injected with 3-7 ESCs). See [Table S1](Table S1) for full data. Scale bars: 100 µm in A,B.
ESCs labelled with ubiquitous tdTomato-H2B ([@DEV124602C14]) were used to facilitate tracking in chimaeras. This reporter localises to chromatin and therefore serves as an ideal identifier of nuclear fragmentation associated with cell death and separation of chromosomes during mitosis ([Fig. S1A,B](Fig. S1A,B)). Each pre-compacted 8-cell embryo was injected with 3-7 ESCs ([Table S1](Table S1)); pooled data from two experiments were separated into five groups based on the number of injected ESCs per embryo (3, 4, 5, 6 or 7 cells). Embryos isolated at mid-day on the third day after mating were assumed to approximate 60 h post coitum (hpc). They were tracked by live imaging from about 10 h post-flushing until the late blastocyst stage, 36-38 h later ([Fig. 1](#DEV124602F1){ref-type="fig"}C). The emergence of morphology consistent with cell death or division was recorded every 20 to 30 min. The first 10 h after flushing was not recorded to minimise fluorescence exposure and promote healthy development. However, analysis of movies revealed condensed and fragmented ESC nuclei assumed to have undergone apoptosis at the start of imaging ([Fig. S1C](Fig. S1C)), indicating that cell death occurred soon after injection. Apoptotic cell counts during 60-70 hpc were extrapolated by adding ESC deaths detected before imaging. To score the incidence of death, division and location of injected ESCs, chimaeras were filmed in 4D (three physical dimensions and time). Using Fiji (ImageJ) TrackMate manual tracking, a total of 46 embryos across two experiments were analysed: 18 SL-injected embryos (producing 16 chimaeras) and 28 2iL-injected embryos (28 chimaeras).
Analysis of time-lapse movies produced a dataset containing all ESC deaths and divisions scored temporally for each embryo. The numbers of viable ESCs were determined per embryo ([Fig. 1](#DEV124602F1){ref-type="fig"}D and [Table S1](Table S1)). Injected ESCs and their progeny are referred to as 'ESCs' hereafter, for simplification. Comparison of the percentage increase of viable 2iL and SL ESCs across embryos at the end of culture revealed a statistically significant difference ([Table S1](Table S1); *P*=0.0265). Embryos injected with 2iL ESCs incorporated a higher number of viable ESCs (137.4±41.3%; mean±s.d.) compared with those injected with SL ESCs (34.9±20.2%; [Fig. 1](#DEV124602F1){ref-type="fig"}E). The survival rate of 2iL ESCs within the embryo remained significantly higher compared with SL ESCs for the duration of recorded development ([Fig. 1](#DEV124602F1){ref-type="fig"}E; *P*\<0.0001).
SL ESCs exhibit substantial cell death within hours of injection {#s2b}
----------------------------------------------------------------
The differential survival of SL versus 2iL ESCs must arise from quantitative differences in cell death, proliferation, or both. To ascertain the underlying cause, the dynamics and distribution of ESC death was investigated further. Incidence of ESC death and division differed between the five embryonic groups injected with 3, 4, 5, 6 or 7 ESCs; however, no consistent correlation between the number of starting ESCs and their subsequent rate of proliferation or elimination could be assigned ([Figs S2-S4](Figs S2-S4)). Therefore, for the remaining analysis the behaviour of ESCs across the five individual embryo groups was averaged. The morphological manifestation of nuclear fragmentation or condensation resulting in eventual loss of the cell ([Fig. 2](#DEV124602F2){ref-type="fig"}A) is characteristic of apoptosis ([Fig. S1A](Fig. S1A)). Immunohistochemistry of ESC nuclear fragments from apoptotic events revealed reactivity for cleaved caspase 3 ([Fig. 2](#DEV124602F2){ref-type="fig"}B). SL ESCs displayed an acute 'death wave' within 10 h of injection ([Fig. 2](#DEV124602F2){ref-type="fig"}C); 27.6% of the injected SL ESCs died within this time window, compared with only 4.3% of 2iL ESCs ([Table S2](Table S2)). The transcriptional profile of ESCs in 2iL closely resembled that of the preimplantation epiblast, whereas SL ESCs are generally more divergent ([@DEV124602C2]). By the end of culture, the death rate for 2iL ESCs was higher than for SL ESCs ([Fig. 1](#DEV124602F1){ref-type="fig"}D), but this was a consequence of increased survival during the first 10 h. ESCs from 2iL did, however, exhibit a peak of elimination from around 100-110 hpc ([Fig. 2](#DEV124602F2){ref-type="fig"}D). Thus, the incidence of ESC death accumulation is dynamically different between the two groups (*P*\<0.0001), reflecting divergent properties of donor cells imposed by their culture history. Fig. 2.**Differential ESC death dynamics during embryonic development.** (A) Selected progressive snap shots of ESC death events, visualised by disintegration of nuclei during live imaging. (B) ESC nuclear fragments (red) colocalise with cleaved caspase 3 (Casp3, green) in two representative embryos injected with SL ESCs. (C) Bar plot of average numbers of ESC deaths accumulated in the first ∼10 h of development. See [Table S2](Table S2) for full data. (D) Accumulation in developmental time of average number of ESC deaths per embryo. (E) Cumulative distribution plot (CDF) of the ESC deaths per embryo, showing temporal distribution of accumulated ESC deaths from total ESC deaths per embryo. Grey bars reflect s.e.m. between the five curves, where each curve is the temporal profile per embryo injected with 3-7 ESCs. (F) Results of the *in vitro* control experiment for cell death: 300,000 ESCs plated per well in SL or 2iL (18 wells per condition) and counted 24 h later. Each black dot displays percentage viable ESCs of total cells counted per well. Each box plot is overlaid with the raw data, distributed along *x*-axis for clarity. Red line represents average value and grey box 1 s.d. *P*=0.1372 \[non-significant (ns)\]. Scale bars: 50 µm in A; 30 µm in B.
To evaluate the incidence of ESC death, we examined the cumulative distribution function (CDF). This represents the temporal distribution of the accumulated ESC deaths per embryo. This analysis showed that SL ESC deaths accumulated at a strikingly higher rate early in chimaera formation, whereas such behaviour was not apparent in 2iL ESCs ([Fig. 2](#DEV124602F2){ref-type="fig"}E). Approximately half (48%) of the deaths for SL ESCs occurred by 82 hpc compared with only 12% for 2iL ESCs. After the initial death wave, SL ESCs exhibited lower levels of death ([Fig. 2](#DEV124602F2){ref-type="fig"}D,E). Interestingly, at the blastocyst stage (100-110 hpc) an increase in death was observed for both conditions ([Fig. 2](#DEV124602F2){ref-type="fig"}D,E). This second death wave coincided with the apoptosis previously reported in ICM cells during sorting of the host epiblast and primitive endoderm (PrE) lineages, whereby 'mis-positioned' cells might be eliminated ([@DEV124602C15]; [@DEV124602C18]). Asserting the importance of the embryonic environment in the choreography of cell mortality, no significant difference in cell death frequency was observed between SL and 2iL ESCs after 24 h of culture *in vitro* ([Fig. 2](#DEV124602F2){ref-type="fig"}F). We propose that the embryo may actively eradicate 'unsuitable' ESCs based on their differentiation status.
SL ESCs undergo compensatory proliferation during the early phase of cell death {#s2c}
-------------------------------------------------------------------------------
ESC mitoses within chimaeras were scored ([Fig. 3](#DEV124602F3){ref-type="fig"}A, [Fig. S1B](Fig. S1B)). That development of host embryos was not irreparably damaged by the imaging procedure was confirmed by birth of live animals following transfer of filmed embryos to recipient mice ([Fig. 3](#DEV124602F3){ref-type="fig"}B-D). From 17 embryos injected with SL-cultured cells and imaged for 39.5 h, six mice were born, two of which exhibited red fluorescence. This was lower than expected, based on the number of chimaeras at the blastocyst stage (16/18). Therefore, we cannot eliminate the possibility that repeated exposure to fluorescent excitation during the culture period adversely affected the injected ESCs in postimplantation stages. Fig. 3.**Differential ESC division dynamics during embryonic development.** (A) Selected progressive images of ESC division events, visualised by condensation of fluorescently tagged chromatin and appearance of two smaller cells at the next time point during live imaging. (B) Pups born from injected embryos transferred to recipients after imaging for 40 h. (C) Red fluorescent image of chimaeric pups. (D) Adult chimaera from pup shown in C. (E) Accumulation in developmental time of average ESC divisions per embryo. (F) Cumulative distribution plot (CDF) of ESC divisions per embryo, exhibiting temporal distribution of accumulated ESC divisions from total per embryo. Grey bars reflect s.e.m. between curves, where each curve is the temporal profile per embryo injected with 3-7 ESCs. (G) Result of *in vitro* control experiment (shown in [Fig. 2](#DEV124602F2){ref-type="fig"}F) with respect to cell division; each black dot displays total number of ESCs in a single well in SL or 2iL 24 h after plating. Total cell number includes viable ESCs plus non-viable (apoptotic) cells. The total number of ESCs is significantly different in SL versus 2iL groups, \**P*\<0.0001. Scale bar: 50 µm in A.
The fraction of newly generated cells from the total injected was calculated. The number of cell divisions per embryo was higher for 2iL than SL ESCs (*P*=0.0056) ([Fig. 3](#DEV124602F3){ref-type="fig"}E, [Table S3](Table S3)). Normalisation of the mitotic accumulation by the total number of ESC division events in each group (CDF) revealed fluctuations of the proliferation rate for SL ESCs ([Fig. 3](#DEV124602F3){ref-type="fig"}F). A 24 h *in vitro* chase experiment indicated that 13.4% more ESCs had divided in 2iL compared with in SL ([Fig. 3](#DEV124602F3){ref-type="fig"}G; *P*=0.00055). Hence, the comparative reduction in SL ESC divisions throughout chimaera formation is likely to be caused by their overall reduced proliferation rate, in combination with the persistence of fewer cells after the initial wave of elimination ([Fig. 2](#DEV124602F2){ref-type="fig"}C, [Table S2](Table S2)).
SL ESCs displayed a transient increase in division rate during the early phase of integration, peaking between 70 and 82 hpc ([Fig. 3](#DEV124602F3){ref-type="fig"}F), coinciding with the first ESC elimination phase ([Fig. 2](#DEV124602F2){ref-type="fig"}E). This suggests that two population states emerge from the pool of injected SL ESCs: one directed to undergo apoptosis and another that is permitted to propagate. This behaviour is characteristic of the 'compensatory proliferation' observed in competition assays, where 'more fit' cells undergo a surge in division, which is interpreted as a means to compensate for elevated cell death of 'less-fit' cells ([@DEV124602C20]). By contrast, 2iL ESC divisions occurred uniformly throughout recorded development ([Fig. 3](#DEV124602F3){ref-type="fig"}F).
Spatial distribution of cell death and proliferation during chimaera integration {#s2d}
--------------------------------------------------------------------------------
Each embryo was analysed at every time point for the location of ESC death and division events ([Fig. 2](#DEV124602F2){ref-type="fig"}A and [Fig. 3](#DEV124602F3){ref-type="fig"}A). Analysis of embryos injected with either SL or 2iL ESCs revealed that donor cell death occurred in both the inner and outer area of the embryo during the first day of imaging, with the majority localising to the inside for SL and vice versa for 2iL ([Fig. 4](#DEV124602F4){ref-type="fig"}A). During subsequent development, it was restricted predominantly to the epiblast and TE lineages for SL ESCs, but was observed in all lineages for 2iL ESCs, with the majority occurring in the epiblast ([Fig. 4](#DEV124602F4){ref-type="fig"}A). Fig. 4.**Localisation of** **ESCs during chimaera formation.** (A) Pie charts displaying percentage of SL or 2iL ESC deaths recorded at different locations in embryos. Top panels depict ESC localisation during day 1 of development prior to cavitation; bottom panels display day 2. Percentages are exclusive of deaths or divisions not clearly assigned to an embryonic location because of restricted visibility (20-30% of events). (B) Pie charts displaying percentages of SL or 2iL ESC divisions recorded at different locations in embryos, as for cell death events in A. (C-E) Representative images of ESCs localising to epiblast (C, 30/30), primitive endoderm (D, 2/30) or trophectoderm (E, 3/30) in late blastocyst chimaeras. Two left panels are maximum projections; remaining images are single planes. tdTomato-H2B, red; Sox17, blue; Cdx2, green. Scale bars: 30 µm in C-E.
The distribution of divisions for both SL and 2iL ESCs was similar during the first day ([Fig. 4](#DEV124602F4){ref-type="fig"}B). No division was observed for any cell that did not integrate. After cavitation, the majority of divisions tended to occur predominantly in the epiblast for both 2iL and SL ESCs ([Fig. 4](#DEV124602F4){ref-type="fig"}B).
Positional fate of integrated ESCs {#s2e}
----------------------------------
Following filming, chimaeras were processed for immunohistochemistry. The majority contained progeny of injected ESCs solely in the epiblast lineage ([Fig. 4](#DEV124602F4){ref-type="fig"}C). Although 3/30 (two SL and one 2iL) contained a total of 7 ESCs located in the PrE domain ([Fig. 4](#DEV124602F4){ref-type="fig"}D), none of them expressed the early PrE marker Sox17. Similarly, 7 ESCs were found in the TE region of 2/30 chimaeric embryos (2iL), but no Cdx2 expression was apparent ([Fig. 4](#DEV124602F4){ref-type="fig"}E). These results suggest that viable progeny of ESCs do not acquire extraembryonic lineage identity in response to environmental stimuli during development of preimplantation chimaeras, at least within the context of this study.
Eradication of injected ESCs coincides with progression towards differentiation {#s2f}
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Markers of naive pluripotency characteristic of the E4.5 epiblast are more specifically enriched in 2iL-cultured ESCs, compared with SL ESCs, which tend to cluster towards the postimplantation epiblast ([@DEV124602C2]). We hypothesise that the SL cells succumbing to elimination from chimaeras are developmentally more advanced. To test this, we imaged embryos injected with purified ESCs expressing high or low levels of the pluripotency marker Rex1. Previous work demonstrated that in SL, ESCs with a GFP knock-in at the *Rex1* locus constitute a mixture of Rex1-GFP^high^ and Rex1-GFP^low^ populations that can be isolated by flow cytometry ([@DEV124602C13]). The Rex1^−^/Oct4^+^ population, comprising up to 50% cultured ESCs, was suggested to resemble early postimplantation epiblast ([@DEV124602C13]; [@DEV124602C24]).
Rex1-GFP^low^ (Rex1^−^) and Rex1-GFP^high^ (Rex1^+^) ESCs were separated by fluorescent cell sorting (top and bottom 5%, respectively) before injection into embryos (3-8 cells per embryo), which were either incubated or imaged for 2 days. Subsequent analysis revealed that embryos injected with Rex1^−^ ESCs form chimaeras with significantly fewer ESC progeny compared with those generated from Rex1^+^ ESCs ([Fig. 5](#DEV124602F5){ref-type="fig"}A, [Table S4](Table S4)). In each of two separate experiments, a proportion of embryos injected with Rex1^−^ ESCs was found to be non-chimaeric (14/22 and 8/19; [Fig. 5](#DEV124602F5){ref-type="fig"}A, [Fig. S5](Fig. S5)), whereas Rex1^+^ ESCs contributed robustly (10/12 and 17/17; [Fig. 5](#DEV124602F5){ref-type="fig"}A, [Fig. S5](Fig. S5)). Fig. 5.**ESCs commencing differentiation are preferentially eliminated from host embryos.** (A) Number of ESCs, sorted from SL cultures for presence (pos) or absence (neg) of Rex1-GFP, incorporated into injected embryos at the blastocyst stage (113 hpc). 8/22 Rex1^−^ chimaeras and 10/12 Rex1^+^ chimaeras are displayed as black dots above baseline. A second experiment, conducted with mKO ESCs, is presented in [Fig. S5](Fig. S5). Each boxplot is overlaid with raw data, where each black dot represents data from a single embryo; red line shows mean value. (B) Bar plot for average numbers of ESC deaths, divisions and resulting viable ESCs by the end of culture (see [Table S1)](Table S1)). (C) Bar plot of average numbers of ESC deaths accumulated in first 7 h of development; for full details, see [Table S2](Table S2). (D) Plot of average numbers of viable ESCs per embryo; grey bars reflect s.e.m. between embryos. (E) Immunohistochemistry of embryos injected with mKO ESCs sorted for high (Rex1^+^) GFP expression after 2 days of culture (see [Fig. S6](Fig. S6) for details of sorting). 16/17 chimaeras exhibited a mixture of positive and negative Rex1-GFP cells, ranging from a single Rex1^−^ cell in the epiblast (top embryo, single planes) to a significant proportion of the epiblast (bottom embryo, max projection). (F) Immunohistochemistry images (max projections) of three representative embryos for the three outcomes generated from Rex1^−^ ESC injections. Top panels show Rex1^+^ epiblasts in embryos injected with Rex1^−^ ESCs (4/19). Middle panels illustrate chimaeras from Rex1^−^ ESCs not expressing GFP (7/19). Bottom panels show embryos that lost Rex1^−^ ESCs during culture (8/19). Scale bars: 30 µm in E,F.
[Fig. 5](#DEV124602F5){ref-type="fig"}B and [Table S1](Table S1) show total numbers of ESC deaths, divisions and viable donor cells per injected embryo. Rapid elimination of around half the donor cells was observed within the first 7 h of culture in Rex1^−^ ESC-injected embryos ([Fig. 5](#DEV124602F5){ref-type="fig"}C, [Table S2](Table S2)), whereas only 14% of Rex1^+^ ESCs were lost during this period. By the late blastocyst stage, Rex1^+^-injected embryos contained significantly more viable ESCs than the Rex1^−^ group (*P*=0.0021; [Fig. 5](#DEV124602F5){ref-type="fig"}B,D), because Rex1^−^ ESCs were more extensively eliminated and divided less frequently throughout the experiment ([Table S3](Table S3); [Fig. 5](#DEV124602F5){ref-type="fig"}D). This corroborates the hypothesis that developmentally advanced (Rex1^−^) ESCs are selectively eliminated during chimaera formation.
Rex1^−^ ESCs can upregulate Rex1 *in vivo* {#s2g}
------------------------------------------
Inspection of chimaeras from Rex1^+^ ESC injections revealed that 16/17 epiblasts contain a mixture of Rex1-GFP^−^ and Rex1-GFP^+^ ESC progeny ([Fig. 5](#DEV124602F5){ref-type="fig"}E). The number of Rex1-GFP^−^ cells ranged from a single cell ([Fig. 5](#DEV124602F5){ref-type="fig"}E, top panel) to a significant proportion of the ESC-derived epiblast ([Fig. 5](#DEV124602F5){ref-type="fig"}E, bottom panel). This is consistent with downregulation of Rex1 at the onset of implantation and exit from naive pluripotency. Chimaeras generated from Rex1^−^ ESC injections occasionally (4/19 cases) exhibited Rex1-GFP fluorescence at the blastocyst stage ([Fig. 5](#DEV124602F5){ref-type="fig"}F, top panel). In the remaining Rex1^−^ chimaeras (7/19), donor cell progeny not expressing Rex1 persisted in the epiblast ([Fig. 5](#DEV124602F5){ref-type="fig"}F, middle panel). These cells may represent 'epiblast' that has advanced beyond the Rex1^+^ stage. In 8/19 Rex1^−^ ESC-injected embryos, no surviving ESC progeny were detected ([Fig. 5](#DEV124602F5){ref-type="fig"}F, bottom panel).
The 4 Rex1-GFP^+^ chimaeras from 19 Rex1^−^ ESC-injected embryos are unlikely to be solely attributable to contamination of the fluorescent cell sorting prior to injection, because only 0.64% total contaminants were detected in simultaneous purity checks of 10,000 cells from the injected population ([Fig. S6](Fig. S6)). To investigate potential embryo-induced Rex1 upregulation further, we injected bi-allelic Rex1^−^ ESCs into 8-cell-stage embryos (*n*=19) and imaged for 2 days ([Fig. S7A-D](Fig. S7A-D)). Live chimaera imaging showed that Rex1^−^ to Rex1^+^ conversion occurred at low rates (2/19, [Fig. S7A,B](Fig. S7A,B), [Table S5](Table S5)). As one control, 5 ESCs were explanted per well of a 96-well plate from each sorted population and cultured for 12 days in SL ([Fig. S7E,F](Fig. S7E,F)). Although the results indicated that 6/60 Rex1^−^ ESCs formed small colonies, GFP was not observed, in contrast to the majority (21/24) originating from Rex1^+^ ESCs ([Fig. S7E](Fig. S7E)). Consistent with this, it was reported that Rex1^−^ ESCs cannot produce undifferentiated colonies in SL culture, even when plated in high density from less-stringent Rex1-GFP^−^ cell sorting; however, a few undifferentiated colonies emerged in 2i ([@DEV124602C13]). We therefore plated Rex1^−^ sorted cells into 2iL at high and low density ([Fig. S7F,G](Fig. S7F,G)). To increase the potential for reversion, they were also plated on mitotically inactivated murine embryonic fibroblasts ([@DEV124602C10]). Rex1^−^ ESCs could form Rex1^+^ colonies within 7 days of culture in 2iL at low frequencies (0.1-1.4%; [Fig. S7F,G](Fig. S7F,G)). Comparing this rate with chimaera experiments ([Fig. S7F](Fig. S7F), [Fig. 5](#DEV124602F5){ref-type="fig"}A and [Table S5](Table S5)), we observed more Rex1 reversion within the embryo (3.3%), although the difference was not statistically significant.
Elimination of ESCs is independent of differential c-Myc levels in preimplantation chimaeras {#s2h}
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Previous studies demonstrate that postimplantation epiblast cells with low c-Myc expression preferentially undergo apoptosis ([@DEV124602C7]; [@DEV124602C20]). Cell selection is apparently triggered by heterogeneity of c-Myc expression between neighbouring cells in the postimplantation epiblast and in ESC cultures following several days in conditions driving exit from naive pluripotency and assumption of primed pluripotency. To determine whether differential c-Myc expression is responsible for elimination of differentiating ESCs during chimaera formation, embryos were injected, cultured for 7-10 h (during the wave of ESC elimination) and inspected for expression of c-Myc and pluripotency markers. Intensity of marker expression was quantified using ImageJ and Volocity. c-Myc protein was observed in both 2iL and SL ESCs ([Fig. 6](#DEV124602F6){ref-type="fig"}A,B), but at levels consistently below those in host embryos ([Fig. 6](#DEV124602F6){ref-type="fig"}C). Furthermore, c-Myc expression was lower for 2iL than SL ESCs *in vitro* and in chimaeras ([Fig. 6](#DEV124602F6){ref-type="fig"}A-D). We saw no consistent correlation between c-Myc and Rex1 or Nanog levels in ESCs ([Fig. 6](#DEV124602F6){ref-type="fig"}C), or any obvious difference between chimaeras of sorted Rex1^−^ or Rex1^+^ SL ESCs ([Fig. 6](#DEV124602F6){ref-type="fig"}D). To determine whether Nanog correlates with c-Myc expression in 2iL chimaeras, the intensity of Nanog and c-Myc of each ESC was measured as a percentage of the brightest ESC ([Fig. S8](Fig. S8)). Only a weak correlation could be detected (Pearson correlation *r*=0.481, *P*=0.001). Further evidence that differential c-Myc expression between donor ESCs and the embryo is unlikely to play a role in ESC selection in this context was provided by generation of chimaeric blastocysts from embryos injected with c-*Myc* null ESCs ([Fig. 6](#DEV124602F6){ref-type="fig"}E). Fig. 6.**Expression of** **c-Myc in ESCs and chimaeras.** (A) Immunohistochemistry of SL ESCs and (B) 2iL ESCs for c-Myc (green), Rex1-GFPd2 (white) or Nanog (white). SL ESCs are mKO2 Rex1-GFP, whereas 2iL ESCs are membrane-bound CFP Confetti. (C) Representative chimaeras from SL Rex1^+^ (top panels, *n*=10), SL Rex1^−^ (middle panels, *n*=10) and 2iL (bottom panels, *n*=10) ESC injections. Left-hand images show maximum projection of whole embryos; right-most images show higher magnification of single planes. (D) Ratio of c-Myc expression in ESCs relative to host cells in chimaeras. Each black dot represents relative expressions levels in a single chimaera. Each box plot is overlaid with raw data distributed along *x*-axis for clarity; red lines indicate average values; grey box is s.e. Intensity of fluorescence for all ESCs and host cells were measured manually on ImageJ (see the Materials and Methods). (E) Representative chimaeras whose ES-derived epiblasts express naive pluripotency markers from 13/14 embryos injected with EYFP c-*Myc* null ESCs. Scale bars: 50 µm in A,B,E; 20 µm in C.
Downregulation of pluripotency factors may mark cells for elimination {#s2i}
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To challenge further the hypothesis that differentiating cells are selected for elimination by the host embryo, we questioned whether reduced expression of pluripotency markers could presage elimination of donor ESCs. We quantified expression of Nanog, Oct4 and Sox2 at 10 h, 24 h and 48 h post-injection of ESCs ([Fig. 7](#DEV124602F7){ref-type="fig"}). Expression appeared higher in most ESCs compared with host cells, at all time points. However, we noted a comparative decrease in average expression of the naive pluripotency markers, Nanog and Sox2 at 24 h, which coincides with the onset of segregation of epiblast from PrE in the host embryo and the second wave of cell death of injected ESCs. Fig. 7.**Ratio of marker expression in donor ESCs relative to embryonic cells.** Intensity levels for expression of (A) Nanog, (B) Oct4 and (C) Sox2 in ESCs relative to average expression level for host embryo cells. Values at the top (A) indicate number of chimaeras inspected at different time points. Each black dot represents expressions levels for a single ESC relative to the host. Each box plot is overlaid with data distributed along *x*-axis for clarity; red lines indicate mean values, grey box is s.e. Intensity of fluorescence was detected using Volocity (see the Materials and Methods).
DISCUSSION {#s3}
==========
Challenging the regulative capacity of the developing mouse embryo by provision of supernumerary cells affords a means to explore the mechanisms by which cells are incorporated or rejected. That a selection procedure operates during this process has been retrospectively inferred from previous studies by inspecting fixed chimaeric embryos and adult tissues ([@DEV124602C19]; [@DEV124602C25]). To attempt to uncover a mechanism for this phenomenon, we used confocal live imaging to compare the behaviour of populations of largely undifferentiated (2iL) and mixtures of undifferentiated and developmentally advanced (SL) ESCs during integration. Strikingly, we recorded a dramatic and reproducible wave of cell death within the first few hours following injection of SL-cultured ESCs, which was not seen in 2iL chimaeras. Subsequently, more 2iL donor cells persisted ([Fig. 1](#DEV124602F1){ref-type="fig"}D,E), which is consistent with the idea that the developmentally advanced cells from SL cultures are preferentially eliminated. However, cell death during the second day of culture, particularly in 2iL chimaeras, increased dramatically, coincident with the onset of epiblast and PrE sorting in the host embryo ([Fig. 2](#DEV124602F2){ref-type="fig"}D). We speculate that this may result from overcrowding caused by a combination of reduced cell death during day 1 and increased division throughout the experiment in the 2iL inoculum ([Fig. 2](#DEV124602F2){ref-type="fig"}D and [Fig. 3](#DEV124602F3){ref-type="fig"}E,G). Furthermore, as development progresses in the absence of inhibitors, some 2iL ESCs may be exiting naive pluripotency, leading to higher elimination during day 2. This phenomenon is reflected in the relative downregulation of the naive pluripotency markers Nanog and Sox2, compared with the core pluripotency marker Oct4, that is observed in some cells following 24 h of culture ([Fig. 7](#DEV124602F7){ref-type="fig"}), which coincides with the onset of the second death wave ([Fig. 2](#DEV124602F2){ref-type="fig"}D,E).
A system of selective cell elimination in the early post-implantation epiblast is described to depend upon differential c-Myc expression ([@DEV124602C7]; [@DEV124602C20]). However, the existence of such a process has not been shown in the preimplantation embryo. Our results suggest that a selection mechanism exists in preimplantation development to eliminate preferentially more advanced cells by pro-apoptotic instruction ([Fig. 8](#DEV124602F8){ref-type="fig"}). However, this could not be attributed to differential c-Myc expression between host embryo and donor ESCs. Our results are consistent with previous work in which cell competition was not observed between naive pluripotent ESCs, but realised only once they had transited to the primed state ([@DEV124602C20]). The exact mechanism for donor ESC elimination by the host embryo remains to be elucidated. Fig. 8.**Model for incorporation of ESCs into early mammalian embryos.** (A) ESCs cultured in stringent ground state conditions (2i/LIF) represent a nearly homogeneous culture of undifferentiated cells. Most injected cells incorporate and contribute to the epiblast. (B) ESCs cultured in conventional conditions (serum/LIF) represent a heterogeneous culture of undifferentiated and more developmentally advanced cells. In the embryonic environment, a wave of elimination is observed within a few hours. (C) Rex1^−^ ESCs are acutely eliminated when placed into the embryonic environment. The outcome by E4.75 is either complete loss of ESC progeny or incorporation of Rex1^−^ ESCs into the blastocyst (at variable frequencies), or in rare cases (depicted here), upregulation of Rex1 in one or more cells during the late blastocyst stage.
We further tested our hypothesis that acutely eliminated cells in the SL inoculum are those in the process of exiting naive pluripotency by using ESCs expressing destabilised GFP under the control of Rex1. Although most incorporating ESCs were Rex1^+^, a proportion of Rex1^−^ ESCs was also detected in chimaeras generated from sorted Rex1^−^ ESCs (7/19 embryos; [Fig. 5](#DEV124602F5){ref-type="fig"}F, middle panel; 8/22, [Fig. 5](#DEV124602F5){ref-type="fig"}A and 11/20, [Fig. S5](Fig. S5)). These cells were also Nanog^−^ (not shown), so may be representative of peri-implantation epiblast, destined to survive and differentiate along with the host epiblast. The apparently healthy nuclear morphology and substantial number of donor cells present after the culture period is consistent with successful integration into the host epiblast. Alternatively, these cells may have been fated for elimination or colonisation of extra-embryonic lineages, if development had been prolonged. GFP was detected in 16% of cultured Rex1^−^ ESC-injected embryos ([Fig. 5](#DEV124602F5){ref-type="fig"}; [Fig. S7](Fig. S7)). This represents at least 3.3% of the total number of donor Rex1^−^ ESCs. Since the maximum yield of GFP^+^ cells from plating Rex1^−^ cells *in vitro* was only 1.4% ([Fig. S7F](Fig. S7F)), we infer that upregulation of GFP within chimaeras may occur by virtue of the permissive capacity of the embryonic environment to enhance the developmental potential of donor cells, as previously shown ([@DEV124602C9]).
In conclusion, our novel live imaging approach to study chimaera formation reveals that the culture history experienced by ESCs before transfer into the embryonic environment strongly influences their subsequent fate. ESCs previously grown in 2iL, reported to be the closest *in vitro* relative to the preimplantation epiblast ([@DEV124602C2]), integrate efficiently into host embryos, whereas the embryo rapidly eliminates most unwanted, developmentally more advanced cells. Interestingly, a minority of Rex1^−^ cells can become incorporated into host embryos, with or without concomitant upregulation of the GFP reporter. This revelation emphasises the importance of rigorously determining the potential of living tissue to regulate the behaviour of transplanted cells.
MATERIALS AND METHODS {#s4}
=====================
Experiments were performed in accordance with EU guidelines for the care and use of laboratory animals, and under the authority of appropriate UK governmental legislation. Use of animals in this project was approved by the Animal Welfare and Ethical Review Body for the University of Cambridge. Relevant Home Office licences are in place. All mice were maintained under a 14 h light:10 h dark cycle with food and water supplied *ad libitum*.
ESC lines {#s4a}
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ESCs expressing tdTomato-H2B ([@DEV124602C14]) were used for most experiments. For visualisation of naive pluripotency, these were electroporated with *Rex1*-*Gfpd2* construct ([@DEV124602C13]; [@DEV124602C28]). An alternative Rex1 reporter line (mKO2 Rex1-GFP) derived from *Rex1-Gfpd2* homozygous embryos transfected with Kusabira Orange was kindly provided by Carla Mulas (Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute). c-*Myc* null ESCs (clone 11: *c-myc^del/del^; N-myc^flox/flox^*) were derived from ES-D3 cells (R.S., unpublished results). Null cells were generated by transient transfection with Cre recombinase and single clone selection; cells constitutively express EYFP under the *Rosa26* promoter. Confetti ESCs were derived from R26R-Confetti embryos ([@DEV124602C22]), activated by Tat-Cre recombinase treatment and single clones selected.
ESC culture {#s4b}
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ESCs were routinely maintained on 0.1% gelatin-coated plates. Passaging by trypsinisation of ESCs grown in SL or 2iL was synchronised. Generally, cells were plated 300,000 per well in 6-well plates (Corning Life Sciences) with medium changes every 2 days. Culture medium for 2iL comprised N2B27 (Stem Cells NDif N2B27) supplemented with MEK inhibitor PD0325901 (1 μM, Stemgent), GSK3 inhibitor CH99021 (3 μM, Stemgent) and mouse LIF (25 ng/ml, produced in-house). SL ESCs were cultured in Glasgow minimal essential medium (GMEM, Sigma) supplemented with 10% foetal calf serum, 1 mM sodium pyruvate, 100 μM 2-mercaptoethanol, 1× nonessential amino acids and mouse LIF (10 ng/ml). Chromosome counts were performed as described previously ([@DEV124602C12]).
Generation of chimaeras {#s4c}
-----------------------
Embryos were harvested from F1 (C57BL/6×CBA) or C57BL/6×F1 crosses. Females were selected by morphological identification of oestrus ([@DEV124602C6]). Detection of a copulation plug on the following day confirmed mating. Embryo staging was based on the assumption that mating occurred at midnight, so that at 12 noon the next day embryos are assigned E (embryonic day) 0.5 or 12 hpc. E2.5 (60 hpc) embryos were flushed from oviducts in M2 (Sigma) and cultured in BlastAssist (Origio) under embryo-tested mineral oil (Sigma) at 37°C and 7% CO~2~ in air. ESCs (3-8) were injected via a laser-generated perforation in the zona pellucida using XYClone (Hamilton Thorne Biosciences). For experiments comparing naive pluripotent versus differentiating donor cells, ESCs were sorted for Rex1-GFPd2^high^ (top 5% of population) or Rex1-GFPd2^low^ (bottom 5% of population) expression using a Beckman Coulter MoFlo high-speed sorter immediately before injection.
Time-lapse image acquisition of chimaera development {#s4d}
----------------------------------------------------
Injected embryos were transferred to the environmental chamber of the spinning disk microscope (Andor Revolution XD System with a Nikon Eclipse Ti Spinning Disk) and imaged for 2 days. Twenty-one *z*-stacks per time step (20 or 30 min) were taken, with two channels (567 nm excitation for ESC visualisation, and bright field). Temperature (37°C), CO~2~ concentration (7%) and fluorescence exposure (148 ms of 567, 300 ms bright field) were standardised. Prior to each imaging experiment, the incubation chamber (Oko Lab) was allowed to stabilise to 37°C. The CO~2~ concentration was generated by an active mixer (Life Imaging Sciences) and humidified before supply to the sample. Embryos were immobilised using a 118×118 µm polyester mesh (Plastok Group) in a glass-bottomed dish (MatTek Corporation). An Andor 85 camera recorded images with magnification through a Plan Fluor 40×/1.3 NA oil lens. Each experiment was set up using Andor IQ Software. A multi-position map was created: every embryo was manually assigned an *x-y-z* location at its centre and visited (starting from the upper-most plane) by the 40× lens at each time point of data acquisition. Channels were sequentially acquired per *z*-section. Each image collected data in 502×501 (width×height) pixels, 2 μm per pixel.
Image analysis of time-lapse development {#s4e}
----------------------------------------
Embryos were tracked from two synchronised experiments and data pooled for analysis. For Rex1 ESC-injected embryos, one large-sample experiment was analysed. All time-lapse data were analysed manually, using an open-source plug-in for ImageJ (TrackMate). Each time point per embryo was scored for occurrence of death or division. Time and location of these events were recorded throughout. Tracked data was converted from time-steps to minutes; the beginning of each time-lapse movie was offset according to time post-injection, or time after 60 hpc. Hence *t*=0 is defined as 60 hpc. The data from two consecutive experiments performed under identical conditions were pooled.
Statistical analysis {#s4f}
--------------------
For each experiment, ESC death and division events were separated across time. The number of viable cells (*V~n~*) was calculated for each embryo at every time point, where a viable cell is defined as one that has not undergone cell death. Hence, at every time point, dead cells were subtracted from the pool of injected cells in each embryo, and newly generated cells were added: In the case of death of an ESC, the first time point of each experiment was omitted in the cumulative distribution functions (CDFs) as it displayed previous cell death events at unknown time points (thus skewing time distribution inaccurately). The pooled data from the two experiments was separated into five groups based on the number of injected ESCs per embryo (3-7 cells); for Rex1 tracked data, the embryos were placed in one group. For each embryo group, the total numbers of ESC deaths or divisions for each time point were aggregated and normalsed (i.e. an embryo group, containing *n* embryos injected with the same number of ESCs each, contains on average *x*/*n* cell death events in time point *y* per embryo, where *x* is the total number of deaths in *n* embryos at time point *y*). Using a single-embryo profile for the number of accumulated ESC events at each time point, the cumulative distribution function (CDF) was plotted as an average of all embryo groups. To determine whether data points in a set are normally distributed, a one-sample Kolmogorov--Smirnov test was used. When the data points were normally distributed, a two-way *t*-test was used at the 5% significance level. When data points were not normally distributed, a two-way Kolmogorov--Smirnov test was used at the 5% significance level.
Embryo transfer {#s4g}
---------------
Embryos injected with 3-7 tdTomato-H2B ESCs, imaged for 39.5 h were transferred to recipient F1 females rendered pseudopregnant by mating with vasectomised males 2.5 days previously. They were allowed to develop to term and offspring examined for fluorescence.
Immunohistochemistry {#s4h}
--------------------
Trypsinised ESCs, collected on glass slides by cytospin, and embryos were prepared for immunohistochemistry as previously described ([@DEV124602C11]). Primary antibodies were against Nanog (eBiosciences, 14-5761-80), Cdx2 (Cell Signaling Technology, 3977S or Abcam, 157524), Sox17 (R&D, AF1924), Sox2 (BioLegend, 656109 or eBioscience, 14-9811-80), cleaved caspase 3 (Cell Signaling, 9661), Oct4 (Santa Cruz, SC-8628), all used at 1:100 dilution; GFP (Life Technologies, A11122), used at 1:400; c-Myc \[Y69\] Abcam, ab32072 (lot no. GR184243-1) used at 1:200 dilution according to [@DEV124602C20]). Alexa-Fluor-conjugated secondary antibodies (Molecular Probes) were used at 1:500 dilution. Confocal images were acquired using a Leica TCS SP5 confocal microscope. Images were processed using Leica software, Imaris (Bitplane) and ImageJ (Fiji).
Quantification of immunofluorescence {#s4i}
------------------------------------
For c-Myc experiments, confocal *z*-stacks of images were analysed for intensity of marker expression, either manually (using ImageJ) in 2D for experiments involving small cell numbers (morulae) or semi-automatically in 3D using Volocity (PerkinElmer) software (for experiments requiring high-throughput data analysis). For manual quantification of immunofluorescence, one image plane from each ESC or host nucleus was selected using the 'Magic Wand'. The option 'measure' was selected, which outputs the measurement of the selected area (nuclei) for all channels. For semi-automatic quantification on Volocity, volumes of ESC and host nuclei were identified using the Oct4 channel and appropriate thresholding for partitioning of nuclei. ESCs were separated from host cells by sorting nuclear intensity of the red channel. Volocity measures and outputs all channel intensity for each selected volume. Manual inspection was applied to ensure all parts of nuclei were recorded without overlap between neighbours. Dividing ESCs were excluded from analysis because they displayed no marker expression.
We thank Samuel Jameson and the Stem Cell and Gurdon Institute animal facilities for mouse husbandry, Carla Mulas for providing mKO-Rex1-GFP ESC line and the homozygous Rex1-GFPd2 ESC line, Kenneth Jones for expert tissue culture assistance, Bernhard Strauss for instruction in embryo immobilisation, Charles Dumeau and Bill Mansfield for embryo transfers and animal photography. We are also indebted to Tristan Rodriguez and Austin Smith for valuable discussions, and Thorsten Boroviak and Sarra Achourri for helpful comments on the manuscript.
**Competing interests**
The authors declare no competing or financial interests.
**Author contributions**
P.H. provided expert advice and assistance for live-cell imaging experiments, A.R. performed ESC flow cytometry, T.K. and S.A. generated the tdTomato-H2B-Rex1-GFP ESC line and designed experiments utilising Rex1, R.S. and A.T. generated the c-Myc null ESC line, S.A. conducted most of the experiments, S.A. and J.N. prepared the manuscript. J.N. conceived the project.
**Funding**
This work was supported by a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust--MRC Cambridge Stem Cell Institute and by the Deutsche Forschungsgemeinschaft (DFG) \[SFB873\] and the Dietmar Hopp Foundation (to A.T.). Deposited in PMC for immediate release.
**Supplementary information**
Supplementary information available online at <http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.124602/-/DC1>
| {
"pile_set_name": "PubMed Central"
} |
The electrical control of the crystal or electronic phase of a material is a cornerstone of many of today's electronic devices.^[@ref1]^ Transistor devices that use ionic liquids (ILs) allow for the development of intense electric fields within an ultrathin electric double layer (EDL) that is formed at the interface between the IL and a thin film channel.^[@ref2]−[@ref5]^ It has been shown that these fields can lead to ion migration into and out of the film that results in structural changes or phase transformations.^[@ref6]−[@ref13]^ These changes have potential for applications in spintronic,^[@ref14],[@ref15]^ sensing,^[@ref16]^ bioinspired,^[@ref17]^ and neuromorphic^[@ref18]^ devices. Notably, the IL gating induced migration of ions can take place over very long distances up to microns, thus resulting in changes in physical properties of the entire bulk of a thin film.^[@ref19]−[@ref21]^ However, the modification of the thin film surface itself has been little studied,^[@ref22],[@ref23]^ even though small changes in the state of the surface can strongly influence the electrochemical reactions that can take place in IL gating.^[@ref24],[@ref25]^ The existence of the viscous IL hinders the application of many surface sensitive characterization techniques.^[@ref26],[@ref27]^ There has been preliminary work investigating the ordering, dynamics, and interactions of ions in ILs at neutral and electrified solid/liquid interfaces using conventional atomic force microscopy (AFM).^[@ref28]−[@ref30]^ Here, using photothermal excitation^[@ref31],[@ref32]^ we employ *in situ* AFM to investigate the evolution of the surface of SrCoO~*x*~ (SCO) thin films under IL gating. We find significant changes in the morphology of the edges of atomically smooth terraces at the surfaces of these films but the terraces themselves appear unchanged. With the help of X-ray photoemission spectroscopy (XPS), we show that subnanoscale ridge structures (RSs) that emerge at the terrace edges result from reaction with hydroxyl (OH^--^) groups dissolved in the ionic liquid. First-principles density functional calculations show that the terrace edges are more reactive than the terraces themselves, consistent with our experiments.
Results and Discussion {#sec2}
======================
IL Gate-Induced Terrace Edge Structure Modifications {#sec2.1}
----------------------------------------------------
In this study, 30 nm thick SrCoO~2.5~ films were epitaxially grown by pulsed laser deposition on both (001) TiO~2~-terminated, undoped, and 0.5 wt % Nb-doped SrTiO~3~ substrates. The films grow in a layer-by-layer growth mode, as shown by clear oscillations in reflection high-energy electron diffraction (RHEED) intensity (see [Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). The topographical evolution under IL gating was *in situ* mapped within an IL droplet which covered both the SrCoO~2.5~ thin film and a lateral Au gate electrode. The AFM cantilever was immersed in the ionic liquid EMIM-TFSI (1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide) while the surface was scanned with a gate voltage (*V*~G~) applied, as shown in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}a. We refer to EMIM-TFSI as IL in the rest of the paper. In order to clearly identify the evolution of the surface structure without damaging it, an AC (tapping) mode was used. The *in situ* AFM imaging was captured using photothermal excitation,^[@ref32]^ which allows for high-resolution imaging comparable to that obtained by scanning without the IL (see [Figure S2](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf) for typical images that reveal the terraces in TiO~2~-terminated SrTiO~3~ (100) in both air and IL).
) at a given *V*~G~.](nn0c02880_0001){#fig1}
[Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}b--g illustrates typical high-resolution AFM topographical images scanned while *V*~G~ was swept as follows: 0 → −1.8 → 0 → +1.8 → 0 V. Note that *V*~G~ is applied for ∼5 min before each AFM scan at a fixed *V*~G~. Initially, the pristine SrCoO~2.5~ surface shows clear (001) terraces ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}b, *V*~G~ = 0 V), consistent with a high quality epitaxial thin film (also see the RHEED results in [Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). The steps at the edges of the terraces are ∼4.0 Å high ([Figure S3](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)), which is consistent with the pseudotetragonal structure of SrCoO~2.5~ (*a*~t~ = 3.905 Å and *c*~t~/4 = 3.936 Å).^[@ref33]^ By applying *V*~G~ = −1.0 V for 5 min, the edges of each of the atomically flat terraces swell up, as shown in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}c. The mean height of these ridged structures is *h*~RS~ ∼ 1.3 Å (see [Supporting Information](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf) for a definition of the mean height and method used to extract it). To quantify the change in *h*~RS~ with *V*~G~, we summarize the evolution of *h*~RS~ versus *V*~G~ in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}h. As *V*~G~ is further decreased to −1.8 V, the RSs become more pronounced, as shown [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}d, and *h*~RS~ is increased to ∼3.5 Å. Subsequently, when *V*~G~ is switched to +0.5 V ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}e), the RSs are maintained with an almost unchanged mean height of ∼3.5 Å, indicating that the RSs induced by IL gating are nonvolatile. Nevertheless, with further increases of *V*~G~ to +1.0 and +1.8 V, *h*~RS~ is reduced to ∼3.2 and ∼2.5 Å, respectively, as shown in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}f,g. This confirms that the ridge structural changes are, at least in part, reversible. As distinct from the dramatic changes at the terrace edges, the flats portions of the terrace do not show any obvious response to the IL gating (the topographical profiles including RSs and terraces during a *V*~G~ cycle are shown in [Figure S3](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). The corresponding AFM phase images indicate that the chemical compositions of the terraces and edges undergo reversible and irreversible changes, respectively, during this gating cycle (see details in [Supporting Information](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf) and [Figures S4 and S5](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)).
In the following, the conductivity and crystal structure of the SrCoO~2.5~ thin films are investigated under IL gating. A thin film was fabricated into a transistor device in the form of Hall-bars with lateral gate electrodes located in the vicinity of the channel. The channel is 400 μm long and 100 μm wide (ref ([@ref21])). As *V*~G~ is varied from 0 to −1.8 V, the channel resistance is reduced from 4.78 × 10^6^ to 2.50 × 10^2^ Ω ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a), and the initially insulating SrCoO~2.5~ film becomes metallic. The resistance reverts back to 4.52 × 10^6^ Ω when *V*~G~ = +1.8 V is applied to the sample. By cycling *V*~G~ according to 0 → −1.8 → 0 → +1.8 → 0 V, the magnitude of the resistance of SrCoO~2.5~ could be reversibly manipulated back and forth by nearly 4 orders of magnitude.
![IL gate-induced SCO phase transition. (a) Gate voltage-dependent resistance of a 30 nm thick SCO thin film at room temperature; the resistance was measured while cycling *V*~G~ in the sequence: 0 → −1.8 → 0 → +1.8 → 0 V, as denoted by the blue arrows. Note that *V*~G~ is incremented/decremented in steps of 0.015 V: the resistance is recorded at each step (this takes ∼2 s) after *V*~G~ has been applied for 10 s (note that only every 10th data point is plotted). (b) X-ray diffraction patterns of an as-grown SrCoO~2.5~ thin film (black) and the same film after IL gating at *V*~G~ = −1.8 V (red); the Miller indices of the diffraction peaks are given in the figure (these are referenced to a pseudotetragonal unit cell^[@ref33]^).](nn0c02880_0002){#fig2}
The X-ray diffraction (XRD) results that are shown in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b reveal that a significant structural change accompanies the IL gate-induced insulator--metal transition. The IL gating effect is nonvolatile so that the XRD could be carried out *ex situ*. The XRD of the pristine (as-grown) SrCoO~2.5~ thin film clearly exhibits a characteristic quadrupled *c*-axis lattice constant expected for the brownmillerite phase which is the result of an ordered oxygen vacancy structure. Diffraction peaks corresponding to (002), (006), and (0010) of the brownmillerite phase SrCoO~2.5~ can clearly be identified. A constant gate voltage *V*~G~ = −1.8 V was then applied for 30 min through the IL to the device. The IL was then removed by washing the device in acetone followed by isopropyl alcohol (IPA). The (002), (006), and (0010) SrCoO~2.5~ diffraction peaks disappear, accompanied by shifts of the (004) and (008) diffraction peaks to higher 2-theta values. These latter two peaks correspond to the (001) and (002) peaks of the perovskite structure SrCoO~3~, respectively. Thus, the XRD results show that IL gating at *V*~G~ = −1.8 V induces a structural phase transition from SrCoO~2.5~ to SrCoO~3~, which is consistent with the insulator to metal phase transition observed via the transport studies discussed above. Although the gating processes for XRD and transport measurements are different, a constant *V*~G~ = −1.8 V is applied for 30 min for XRD while *V*~G~ is gradually swept from 0 to −1.8 V for the transport measurements, both processes induce the full phase transition from SrCoO~2.5~ to SrCoO~3~ ([Figure S6](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). From the XRD peak positions, we find that the out-of-plane lattice parameter (within the pseudotetragonal structure) of the initial SrCoO~2.5~ layer is reduced from *c*~t~/4 = 3.94 Å to *c*~t~ = 3.80 Å at *V*~G~ = −1.8 V. Scanning transmission electron microscopy (STEM) images and enlarged XRD patterns are given in [Figure S7](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf). However, such a shrinkage in the bulk lattice perpendicular to the surface of the film is not reflected in the AFM images and cannot account for the very large changes in the RSs at the terrace edges (also see [Figure S8](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf) for the STEM of ridge structure).
Interplay Between Hydroxyl and SrCoO~*x*~ {#sec2.2}
-----------------------------------------
The phase transition between SrCoO~2.5~ and SrCoO~3~ reflected in the XRD and transport data corresponds to the bulk of the thin film. Hence, to understand the mechanism for the surface evolution during IL gating, we turn to a surface sensitive technique, namely X-ray photoelectron spectroscopy (XPS), to characterize the chemical bonding at the surface averaged over many terraces and steps (the X-ray beam is 100 μm wide). XPS was carried out on the gated samples (*V*~G~ = −1.5 V, the full phase transition from SrCoO~2.5~ to SrCoO~3~ is obtained as shown in [Figure S6](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)) immediately after the RSs emerge (monitored by *in situ* AFM) and after the IL was removed by ultrasonic cleaning of the sample in acetone followed by IPA. [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}a depicts the measured O-1s core-level spectrum and a corresponding multiple-peak fit to the data for the pristine SrCoO~2.5~ sample. The fits to the XPS data use a Co---O peak that is centered at 530.3 eV and a Co---OH peak that is centered within the range 531.2 to 531.9 eV (these values are taken from refs ([@ref34] and [@ref35])). The main peak for the pristine SrCoO~2.5~ is located at ∼530.4 eV, indicating that Co---O bonding is dominant. In the sample that was gated at *V*~G~ = −1.5 V, in addition to the main peak at ∼530.4 eV, an additional shoulder peak at the higher binding energy of ∼532.0 eV is found, which is evidence for the existence of Co---OH bonds ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}b). The ratio, *R*, of the area within the Co---OH peak (*S*~Co--OH~) to that of the sum of the areas in the Co---O and Co---OH peaks (*S*~Co--O+Co--OH~) equals ∼0.25 in the gated sample, as compared with ∼0.07 in the pristine SrCoO~2.5~ film. The origin of OH^--^ ions found after gating might arise from any residual H~2~O dissolved in the IL. Note that for all studies, extreme care is taken to eliminate any water dissolved in the IL by drying the IL for at least half of a day in vacuum (better than 10^--6^ mbar) at 380 K. For the *in situ* AFM measurements, the IL and the sample are contained within a closed cell that is isolated from the surrounding air by a Viton O-ring seal, but some small amount of H~2~O will likely be absorbed by the IL during the experiment and when the IL is exposed briefly to the air during the application of the IL onto the sample before sealing the cell. Thus, there is always some small amount of residual water in the IL even after the baking procedure mentioned above (for example, ref ([@ref12]) estimates ∼0.01 vol % H~2~O in EMIM-TFSI after baking).
![Effect of water on the ridge structure. XPS O-1s core-level spectra of (a) pristine SrCoO~2.5~ and the same film after gating at *V*~G~ = −1.5 V using an IL without and with added H~2~O; (b) dried IL; (c) IL with 0.1 vol % H~2~O; (d) IL with 1.0 vol % H~2~O. The XPS spectra are fitted using spectra for Co---OH (red) and Co---O^[@ref34]^,^[@ref35]^ (blue). Corresponding AFM images after gating using an IL with (e) 0.1 vol % and (f) 1.0 vol % H~2~O; the scale bar is 400 nm. (g) Mean fwhm (left axis) and height (right axis) of the ridge structures induced by gating with an IL without and with added H~2~O of 0.1 vol % and 1.0 vol %.](nn0c02880_0003){#fig3}
To further confirm the role of hydroxyl groups in the formation of the RSs, we carried out gating experiments using ILs in which H~2~O, in concentrations of 0.1 vol % and 1.0 vol %, was added. The Co--OH peak intensities found in the XPS studies for these two samples are dramatically enhanced with *R* = 0.49 (0.1 vol % H~2~O) and *R* = 0.86 (1.0 vol % H~2~O), as shown in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}c,d. The AFM images of samples gated with 0.1 vol % and 1.0 vol % H~2~O-IL (*V*~G~ = −1.5 V) in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}e,f both show wider RSs as compared to the case for unadulterated IL (also see the profiles across the ridge structures shown in [Figure S9](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}g summarizes the mean height *h*~RS~ and fwhm (full width at half-maximum) of the RSs induced by IL gating with distinct H~2~O doping levels (the mean fwhm definition and calculation methods are described in [Supporting Information](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). After gating with *V*~G~ = −1.5 V (20 min), the *h*~RS~ in all three cases has a similar value of ∼4.0 Å. However, the mean fwhm of the RSs is increased from ∼20 to ∼94 nm when 0.1 vol % H~2~O is added to the IL. An even greater enhancement to ∼123 nm is found for the case of 1.0 vol % H~2~O. The simultaneous increase in both the mean fwhm of the RSs as well as *R* when using an IL with added H~2~O doping supports the hypothesis that the RSs are related to an interaction between OH^--^ ions and the surface of SCO under negative *V*~G~. Moreover, *R* = 0.21 is found after a gating procedure in which *V*~G~ is varied from 0 → −1.5 → 0 → +1.8 → 0 V, which implies that the resulting changes are only partially reversible ([Figure S10](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)).
Electrical Property of the Ridge Structures {#sec2.3}
-------------------------------------------
We now turn to investigate the electrical properties of the RS and terraces. To profile the conductivity of the RSs, we utilize conductive AFM (cAFM) mapping of the leakage current through a second set of similar SrCoO~2.5~ films that were prepared, however, on conducting Nb-doped SrTiO~3~ substrates. After the sample is gated at *V*~G~ = −1.5 V for 20 min, the IL is removed by rinsing in acetone and IPA, and then cAFM is performed to map the leakage current through the terraces and the RSs. As shown in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}a, one can clearly see the RSs at the terrace edges, which are almost the same as those measured in the IL (see [Figure S11](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). Compared with homogeneous low leakage current of pristine, insulating SrCoO~2.5~ ([Figure S12](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)), the corresponding leakage current image ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}b) illustrates that the RSs exhibit a lower leakage current (green) than that of the terraces (blue). This can more readily be seen in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}c where line profiles of the topography and leakage current (white solid lines in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}a,b, respectively) are compared. In addition, *V*~G~-dependent transport measurements were carried out on Hall bar devices that were patterned from samples prepared with several different thicknesses of SCO. In [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}d, the insulator-to-metal-to-insulator transition of a 30 nm thick sample, as *V*~G~ is varied according to 0 → −1.8 → 0 → +1.8 → 0 V, is almost fully reversible. The data shown here are the same data as those in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a but are plotted on a linear scale to magnify the resistance divergence after one *V*~G~ cycle. The resistance of a thinner 15 nm thick sample after a similar gating cycle shows a much higher resistance as compared with the initial state. Data for 10 and 5 nm thick samples are also given in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}d. It is clear that as the film thickness is decreased, the contribution of the insulating RSs becomes larger, which results in larger irreversible resistance changes. It is noteworthy that cAFM measurements can clearly distinguish the current variation (∼100 pA) between RS and terraces in the direction perpendicular to the film, suggesting that the increased irreversible behavior in transport measurements is mainly due to the more insulating RSs. H~2~O vol % dependent transport measurements were also performed ([Figure S13](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)); these results show more pronounced irreversible behavior as the water level was increased.
{#fig4}
Next we carry out first-principle density functional calculations to explore the role of the hydroxyl groups in forming the RSs. We established a slab model for calculating the binding energy (Δ*E*~OH~) of hydroxyl adsorbed at different sites located at various distances from a terrace edge, respectively (Top 1--3 in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a--c). Here, SrCoO~3~ is used in the calculations since hydroxyl is absorbed at negative gating voltages at which the phase transition from SrCoO~2.5~ to SrCoO~3~ has already taken place, as demonstrated in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}. As SrCoO~2.5~ thin films were grown in a layer-by-layer mode on the TiO~2~-terminated SrTiO~3~ substrate (see RHEED oscillations in [Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)), it is expected that the surface of the thin film should be CoO~2~-terminated. In the slab model used in the calculations, the terrace was constructed as an 8 × 1 (*x* × *y*) supercell with three SrCoO~3~ layers in the *z*-direction, while the model for the terrace edge includes an additional layer in which in the *x*-direction is only 6 cells wide. The two lower SrCoO~3~ layers were first fixed according to that of the calculated bulk structure and then the slab models were relaxed to obtain the ground state. Thereafter, neutral OH was added to the three positions Top 1, Top 2, and Top 3 corresponding to the three unique sites on the surface of the 6-cell wide terrace in the slab models (see [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a--c). After relaxation of the final structures, the OH binding energies at these sites, Δ*E*~OH~, are obtained. The calculated Δ*E*~OH~ (see [Methods](#sec4){ref-type="other"}) shows that OH clearly prefers to be adsorbed at the terrace edge; the binding energy at the Top 1 site at the terrace edge is −3.32 eV while Δ*E*~OH~ for the interior sites on the same terrace is significantly lower (−2.13 and −2.10 eV, respectively (see [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a--c)). We also calculate *E*~OH~ for OH^--^ using a similar slab model by electron doping and find that the results show the same trend as for neutral OH (see [Figure S14](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)).
{#fig5}
The local electronic density of states (DOS) for Co and OH, located at the edge (Top 1 model), are shown in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}d (neutral OH) and [Figure S14d](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf) (charged OH^--^). The OH is clearly insulating, meanwhile the DOS near the Fermi energy (*E*~F~) integrated over the 4 Co atoms underneath the hydroxyl (shown by the red dashed line in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a) is lower than that of the center 4 Co atoms away from the OH (shown by the blue dashed line in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a). Therefore, the quasi-one-dimensional columnar structure formed from 4 Co atoms and the absorbed OH is less conducting, which is consistent with our experimental findings.
Conclusions {#sec3}
===========
In summary, utilizing *in situ* AFM we find that IL gating of SrCoO~*x*~ thin films results in significant modifications of the surface. While the morphology of the atomically flat terraces is not much changed, the edges of the terraces develop ridges that we find are due to the reaction of hydroxyl groups dissolved in the ionic liquid. These ridges are much less conducting than the terraces. Our findings imply the possibility of manipulating surfaces using ionic liquid gating when the surface has distinct morphological structures or creating nanoscale structures/interfaces with disparate conductivity, for example, with terrace edges whose spacing and orientations can be controlled by the miscut angle^[@ref36]^ or with hinges, formed, for example, from surface reconstructions that minimize the surface energy (*e.g*., MgO (111) surface^[@ref37]−[@ref39]^). The observed terrace edge modification also shows the potential, for example, of nanostructured electrochemical interfaces in applications ranging from heterogeneous catalysis to biomedical and environmental sensing.^[@ref40],[@ref41]^
Methods {#sec4}
=======
SrCoO~2.5~ Thin Film Sample Preparation {#sec4.1}
---------------------------------------
SrCoO~2.5~ thin films were grown on STO and Nb-STO (001) substrates at 750 °C in an oxygen pressure of 5 × 10^--4^ mbar, by pulsed laser deposition (PLD).^[@ref21]^ Before the deposition, all of the substrates (Crystec GmbH) were treated with buffered hydrofluoric acid \[BOE 7:1 (HF:/NH~4~F = 12.5%:87.5%)\] for 1 min at room temperature and then annealed at 950 °C in oxygen atmosphere for 3 h to achieve TiO~2~-terminated atomic terraces. The thickness of the SrCoO~2.5~ films for all of the *in situ* AFM, cAFM, STEM, and XPS measurements was ∼30 nm while SrCoO~2.5~ samples with thicknesses of 5, 10, and 15 nm were used for the transport measurements. After deposition, the SrCoO~2.5~ films were cooled down to room temperature in the same oxygen pressure as that used for the growth.
*InSitu* AFM Characterization of IL Gating-Induced Ridge Structures {#sec4.2}
-------------------------------------------------------------------
The *in situ* AFM measurements were carried out in a Cypher (Asylum Research) atomic force microscope. An integrated blueDrive (Asylum Research) laser was used to actuate the oscillation of the cantilever (AC-55 TS, Olympus) in the IL. During AFM scanning, the cantilever, gate electrode plate (Au), and SCO sample were all covered by the IL ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}a), and the gate voltage was applied by an external source meter (Keithley 2400). The ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMIM-TFSI), was used for all IL gating experiments. The IL and the devices were separately baked at 380 K in high vacuum (10^--7^ mbar) for at least 12 h before the gating experiments were carried out. Except for the H~2~O-vol % dependent XPS measurements, dried IL was otherwise used for all measurements.
X-ray Photoelectron Spectroscopy {#sec4.3}
--------------------------------
The X-ray photoelectron spectroscopy measurements were carried out in a Thermo Fischer Scientific K-Alpha fitted with MAGCIS and a dual mode argon ion source. Before the measurements, the gated samples were rinsed by IPA, and then immediately transferred to the XPS chamber. To remove the residual IL and other contaminants, the surface was gently treated by argon cluster ions (∼80 molecules of Argon) that do not penetrate the oxide surface and allow chemical state information to be preserved after such a cleaning process.^[@ref42]^ Al Kα radiation with the X-ray source operated at 14 kV with a spot size of 100 μm was used.
Conductive AFM {#sec4.4}
--------------
Before the cAFM experiments, the SCO sample grown on a Nb-doped SrTiO~3~ substrate was gated (*V*~G~ = −1.5 V for 20 min), and the development of the RSs was monitored by *in situ* AFM (see [Figure S11](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf)). The Nb-doped SrTiO~3~ substrate was sealed by paraffin to avoid current leakage.^[@ref21]^ After gating, the sample was rinsed by acetone and IPA to remove the residual IL and paraffin. In the cAFM measurements, a constant voltage of −5.0 V was applied to the sample holder (the tip is grounded) and the current flowing through the sample (perpendicular to the surface) is mapped. A 500 MΩ resistor was connected in series with the sample to prevent saturation of the amplifier during the measurements, and a low-noise trans-impedance amplifier (ORCA module, Asylum Research) was used that allowed for a current measurement as low as ∼1.5 pA. A conductive diamond probe (AD-2.8-AS, Adama Innovations) was used for cAFM, and the radius of the tip was ∼10 ± 5 nm.
Transport Measurements {#sec4.5}
----------------------
The devices for transport measurements were prepared by photolithography and wet etching in the form of Hall-bars with lateral gate electrodes located in the vicinity of the channel. The channel was 400 μm long and 100 μm wide. Electrical contacts to the edges of the channel were formed from Au (60 nm)/Cr (10 nm) that was deposited by thermal evaporation.
First-Principles Calculations {#sec4.6}
-----------------------------
All calculations were performed by a density function theory (DFT) method as implemented in the Vienna *ab initio* Simulation Package (VASP).^[@ref43]^ The Perdew--Burke--Ernzerhof (PBE) pseudopotentials with the generalized gradient approximation (GGA) were used in the calculations.^[@ref44]^ All structures contain a vacuum space larger than 17 Å between successive slabs and were relaxed until the atomic forces on the atoms are less than 0.02 eV Å^--1^. Spin polarization was included in all calculations. The Co atoms were set to be ferromagnetic with a Hubbard U set to be 2.5 Ev.^[@ref45]^ The binding energy was calculated fromwhere *E*(slab + OH), *E*(slab), and *E*(OH) are the energies of the composite system, the clean slab, and the uncoordinated adsorbate, respectively.
The Supporting Information is available free of charge at [https://pubs.acs.org/doi/10.1021/acsnano.0c02880](https://pubs.acs.org/doi/10.1021/acsnano.0c02880?goto=supporting-info).SrCoO~2.5~ thin film sample preparation and RHEED patterns, mean height, and fwhm calculation of ridge structures; profiles of terraces as a function of gating voltages; surface chemical composition (phase) evolution; STEM images and XRD patterns of SCO induced by IL gating. XPS of O 1s core-level spectra of reverse gating SCO; electronic structures of charged hydroxyl on the SrCoO~3~ surface ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acsnano.0c02880/suppl_file/nn0c02880_si_001.pdf))
Supplementary Material
======================
######
nn0c02880_si_001.pdf
^†^ (B.C.) School of Physics, Shandong University, Jinan 250100, P.R. China.
^‡^ Y.Z. and B.C. contributed equally.
S.S.P.P. conceived and supervised the work. Y.Z. conducted the *in situ* AFM measurements. B.C. grew the samples and performed the transport and XRD measurements. Y.Z., B.C., and F.G. performed the XPS measurement. cAFM was carried out by Y.Z. and B.C. First-principles calculations were performed by H.Y. and analyzed by Y.Z, B.C., and H.Y. All authors discussed the results and Y.Z, B.C., and S.S.P.P wrote the paper with contributions from H.Y. and F.G.
The authors declare no competing financial interest.
We thank T. Ma for useful discussions and N. Schammelt and A. K. Srivastava for TEM sample preparation. Part of this research was supported by the DFG through SFB 762 and EU H2020 program "Phase Change Switch". Y.Z. acknowledges The International Max Planck Research School (IMPRS) for Science and Technology of Nano-Systems for support.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Demodicosis is one of the most frequent skin conditions in dogs. It is a parasitic disease caused by mites of the genus *Demodex* \[[@CR1], [@CR2]\]. A small number of mites are usually considered as a normal component of the dog's skin microbiota, but their proliferation leads to a potentially serious condition \[[@CR3], [@CR4]\]. Puppies acquire mites from their mother in the first days of their life through direct skin contact \[[@CR2]\]. The evolution from asymptomatic carriage to skin disease may be related to a particular cellular immunodeficiency allowing the multiplication of the mites, although the pathogenesis is not yet elucidated \[[@CR3]--[@CR5]\]. Canine demodicosis is classically divided into two main clinical manifestations, i.e. localised and generalised demodicosis. The localised form appears as patches of alopecia and mild erythema in limited areas of the body, usually in young dogs, although it may also affect older ones. It can regress spontaneously without treatment \[[@CR2]\]. Generalised demodicosis is more severe and can even be fatal if a secondary bacterial infection develops \[[@CR2]\]. It may evolve from the localised condition or occur spontaneously especially in older animals with underlying diseases \[[@CR5], [@CR6]\]. Recently, it has been proposed that localised demodicosis is characterised as no more than four lesions with a diameter of up to 2.5 cm, while canine generalised demodicosis is characterised by five or more affected areas, or by lesions covering an entire region of the body, and/or demodectic podal dermatitis involving two or more paws \[[@CR3], [@CR4], [@CR6], [@CR7]\]. In the case of generalised demodicosis, the affected areas are frequently erythematous, with comedones, hair loss, follicular papules to pustules, and scales. Secondary severe bacterial infections are frequent. Suspicion of demodicosis based on clinical signs has to be confirmed by the detection of mites in deep skin scrapings. Alternatively, skin biopsy or hair plucks may also be performed \[[@CR3], [@CR4], [@CR6]\].
Generalised demodicosis is a very challenging disease to treat effectively. Only a few drugs and formulations, either topical or systemic, are registered \[[@CR4], [@CR8]\]. Many treatment protocols in the field include off label use of macrocyclic lactone, providing variable efficacy with potential for toxicity, especially in dogs carrying *MDR-1* gene mutations \[[@CR3], [@CR9], [@CR10]\]. Recently, a new class of insecticides/acaricides, the isoxazolines, demonstrated very good efficacy against many ectoparasites of dogs and cats: fleas and ticks, but also mites, including *Otodectes cynotis*, *Sarcoptes scabiei*, *Lynxacarus radovskyi* and *Demodex canis* \[[@CR11]--[@CR18]\]. Among the isoxazolines, the efficacy of afoxolaner against *D. canis* has been demonstrated in one laboratory study involving naturally infested animals conducted in South Africa. In this study, afoxolaner was administered at fortnighly interval for one month then at a monthly interval for two additional months and demonstrated \> 99% reduction in mite counts \[[@CR11]\]. The purpose of the present field study was to assess the efficacy of monthly oral administration of afoxolaner in two different formulations, alone (NexGard®) and in combination with milbemycin oxime (NexGard Spectra®) against generalised canine demodicosis in the European pet dog population.
Methods {#Sec2}
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Design {#Sec3}
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This multi-center field study was held in France, Italy and Poland between January 2016 and March 2017, and was conducted in accordance with Good Clinical Practices as described in the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products, VICH Guideline 9 \[[@CR19]\].
Animals {#Sec4}
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Client-owned dogs of various breeds and of both sexes, weighing at least 2 kg, with a minimum age of 8 weeks and presenting clinical signs of generalised demodicosis were considered eligible for the study.
Criteria for inclusion were the presence of clinical signs of generalised demodicosis (i.e. erythema, hair loss, follicular casts and crust, and/or pyoderma) on 5 or more areas, or pododemodicosis on 2 or more paws and at least 5 live *Demodex* spp. mites (i.e. at least 1 alive mite/alopecic area). All dogs were subjected to a physical examination before treatment to be considered suitable for inclusion into the study. Clinical history and ongoing medications were recorded at inclusion.
Treatment {#Sec5}
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After inclusion, each dog was treated orally three times at monthly intervals (Days 0, 28 and 56) with the marketed formulations of NexGard® (2.7 mg/kg afoxolaner) or NexGard Spectra® (2.5 mg/kg afoxolaner and 0.5 mg/kg milbemycin oxime) according the European label instructions. The veterinarian could choose NexGard® or NexGard Spectra® based on the needs of the client, i.e. anthelmintic activity of milbemycin oxime. At least 30% of the dogs were required to be treated with NexGard Spectra®. Dogs were weighed before each treatment for appropriate dose determination.
Treated animals were observed for at least 5 min after each administration to ensure that the chew was swallowed. Personnel involved in the assessment of product efficacy were not blinded to treatment as there was no negative control group, and the primary efficacy variable was the comparison of the mite count with the initial pre-treatment count for each individual dog.
Owners were questioned at each visit about any abnormal observation seen during the study period. Dogs were managed under normal conditions by their owners. Out of the 50 dogs enrolled, 48 completed the study on Day 84.
Mite counts {#Sec6}
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Mite counts were performed on Days 0, 28, 56 and 84. Deep skin scrapings were performed in duplicate from five sites with skin lesions on the days of clinical evaluation. Skin scrapings were made of a 2 × 2 cm surface with a blade until capillary oozing occurred. The collected samples were placed onto a microscope slide and mixed with mineral oil and observed under microscope for total mite counts. Live adults, nymphs and larvae as well as dead mites and skeleton were counted. The same sites were scraped at each subsequent examination.
Clinical scoring {#Sec7}
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Both severity and extent of clinical signs consistent with generalised demodicosis were evaluated at inclusion and on each day of the mite counts. Five clinical signs were evaluated: alopecia, erythema, papules, pustules and scales/crusts. The severity of the clinical signs was scored as absent (0), mild (1), moderate (2), or severe (3). When present, the extent of the lesion was scored as "limited" \[seen on up to 1/3 of the (head + body) surface\]; "marked" \[seen on up to 2/3 of the (head + body) surface\]; and "generalised" \[seen all over the (head + body) surface\]. In addition, the intensity of pruritus was evaluated by the veterinarian according to a canine pruritus scale \[[@CR20]\] and scored from 0 (absent) to 10 (intense).
Statistical analyses {#Sec8}
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The statistical analysis was performed using SAS System® v.9.4 (SAS Institute Inc., Cary, NC, USA). For all statistical tests, a nominal significance level of 5% (*P* \< 0.05) was applied. No adjustment for multiple tests was performed.
The primary antiparasitic efficacy variable was the reduction of the number of live mites (adults and immature stages) on Day 84 compared to the baseline (pre-treatment). The average percentage reduction in mite counts was calculated using Abott's formula:
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\begin{document}$$ \mathrm{Efficacy}\ \left(\%\mathrm{mite}\ \mathrm{reduction}\right)=100\times \left[\left(\mathrm{C}-\mathrm{T}\right)/\mathrm{C}\right] $$\end{document}$$
where C is the arithmetic mean of the baseline count and T is the arithmetic mean of the Day 84 count. The difference between live mite counts on Days 28, 56 and Day 84 *versus* baseline was tested using a signed rank test.
In addition, the difference between percentage reductions in mite counts in two different classes of age (dogs younger than 18 months and dogs older than 18 months) was tested using a Wilcoxon Rank Sum test with continuity correction.
The secondary variable was the resolution of clinical signs. Lesion severity and extent scores were recorded for each dog at each time-point for each lesion (alopecia, erythema, pustules, papules and scales/crusts). The total skin lesion and total extent of the lesions were calculated for each dog as the sum of the skin lesions scores and extent of the lesions scores, respectively. These scores, as well as the pruritus score, were summarized by time-points. Differences in scores between Days 28, 56, 84, and baseline were tested using a Cochran-Mantel-Haenszel (CMH) test \[(*F*) Mean Score Difference test\].
Results {#Sec9}
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Inclusion {#Sec10}
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A total of 50 dogs (29 females and 21 males) weighing from 2.4 to 46.0 kg were enrolled in the study (14 dogs from France, 21 dogs from Italy and 15 dogs from Poland). Forty-four dogs were pure breed and only six were mixed breeds (Table [1](#Tab1){ref-type="table"}). The most prevalent breeds enrolled were American Staffordshire Terrier (10.2%), English Bulldog (10.2%), French Bulldog (8.2%), Pug (8.2%) and Yorkshire Terrier (8.2%). The dogs were 3 months to 15 years-old. Twenty-seven of them were younger than 18 months while 23 dogs were older. Little information was available on concomitant diseases at inclusion. One 6-year-old French Bulldog was reported with Cushing syndrome, one 2-year-old crossbreed Maremma Sheepdog had leishmaniosis and one 15-year-old poodle was displaying polyuria-polydipsia associated with breast neoplasia.Table 1Summary of dog information and clinical history when available. The total mite counts at enrolment (Day 0) and study end (Day 84) is indicatedDog nameAgeBreedSexLesions at enrolment and clinical historyMite countDay 0Day 84Cherry3 monthsPugFAlopecia, erythema, papules, pustules and scaling1820Kenzo4 monthsChihuahuaMSevere alopecia and papules2130Liner6 monthsStaffordshire Bull TerrierMAlopecia, erythema, papules, pustules and scaling1400Mia6 monthsCrossbreed PinscherFMultifocal alopecia without pruritus180Figa6 monthsWhippetFSevere alopecia and erythema4411Loca7 monthsFrench BulldogFAlopecia, erythema and scaling21630Shaya9 monthsAmerican Staffordshire TerrierFModerate lesions of demodicosis430Loki9 monthsBull TerrierMSevere lesions of demodicosis1210Jagoda9 monthsEnglish BulldogFAlopecia, erythema and scaling775Kaya11 monthsAmerica Staffordshire TerrierFSevere alopecia and erythema1100Baby10 monthsCrossbreed German ShepherdFAlopecia, papules and pustules310Zoe10 monthsBoston TerrierFMultifocal alopecia410Kora10 monthsDrathaarFAlopecia, erythema and scaling460Ares11 monthsYorkshire TerrierMSevere demodectic pododermatitis573Luna11 monthsAmerican Staffordshire TerrierFSevere demodectic pododermatitis3860Hollywood5 monthsAmerican Staffordshire TerrierFMultifocal alopecia with pruritus since 1 month390Borys5 monthsBeagleMDemodectic pododermatitis490Mya7 monthsCrossbreed Dogo ArgentinoFMultifocal alopecia420Odi8 monthsMongrelMDemodectic pododermatitis400Elsa1 yearPugFSevere alopecia130Argo1 yearDobermannMAlopecia and scaling; demodicosis diagnosed 6 months earlier and treated with amitraz970Ares1 yearAmerican Staffordshire TerrierMAlopecia, erythema, papules pustules and scaling360Klops1.1 yearPugMSevere alopecia erythema, papules, pustules and scaling23490Achille1.2 yearPitbullMAlopecia and scaling530Buch1.5 yearGerman ShepherdMAlopecia, erythema, papules, pustules18NCALaure2 yearsPointerFSevere erythema and moderate alopecia for one month770Benek2 yearsEnglish BulldogMSevere demodectic pododermatitis979Lili2 yearsYorkshire TerrierFDemodectic pododermatitis560Ibex3 yearsJack Russell TerrierFModerate alopecia and erythema360Kenzo3 yearsBasset Bleu de GascogneFAlopecia, erythema, pustules2560Carlitos4 yearsPugMSevere lesions of chronic demodectic pododermatitis250Gruby4 yearsEnglish BulldogMDemodectic pododermatitis4760Buza5 yearsEnglish BulldogFSevere alopecia and erythema696Brego5 yearWhite Swiss ShepherdMAlopecia, erythema and scaling390Meggy8 yearsYorkshire TerrierFDemodectic pododermatitis642Szajba8 yearsToy SchnauzerFAlopecia and scaling510Hector1 yearFrench BulldogMSevere alopecia, erythema and scaling1000Nari1 yearPodencaFSevere alopecia and erythema for 2 months2740Jazzie1.5 yearFrench BulldogFAlopecia, erythema, pustules2130Sonia10 yearMongrelFSevere demodectic pododermatitis464Angy10 yearYorkshire TerrierMAlopecia, erythema and scaling180Lilly15 yearsPoodleFDemodectic pododermatitis, polyuria, polydipsia, breast neoplasia, heart failure239NCACosmo2 yearsCrossbreed Maremma SheepdogMSevere alopecia, papule, pustules and pruritus; leishmaniosis7729Jacky3 yearsJack Russell TerrierFDemodectic pododermatitis220Shelly4 yearsMalteseFSevere alopecia, erythema and scaling623Moira4 yearsPinscherFAtopic dog with alopecia and scaling180Asia5 yearsEnglish BulldogFAlopecia, erythema and scaling930Leon6 yearsFrench BulldogMCushing syndrome and severe demodectic pododermatitis9253Rocky7 yearsLabrador RetrieverMSevere demodectic pododermatitis308Hoffman8 yearsPitbullMDemodectic pododermatitis21137*Abbreviations: F* female, *M* male, *NCA* no count available
Thirty-one dogs were treated with NexGard® and 19 dogs with NexGard Spectra®.
Mite counts {#Sec11}
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All dogs were confirmed to have more than five live *Demodex* mites before treatment with an arithmetic mean count of 183 mites/dog (range of 13--2349). Treatment with afoxolaner resulted in a rapid and significant reduction of the number of mites in all post-treatment counts (Table [2](#Tab2){ref-type="table"}). Overall, afoxolaner miticidal efficacy was shown to be 87.6%, 96.5% and 98.1% on Days 28, 56 and 84, respectively. At the end of the study, 75% of the dogs had no live mites. At this last time-point, the 12 dogs with a positive skin scraping had an arithmetic mean of 3.54 mites.Table 2*Demodex canis* mite count reduction in dogs treated three times at a monthly interval with oral afoxolanerDay 0Day 28Day 56Day 84Total number of dogs (*n*)50504948Mean mite count (*n*)18322.86.43.5Count range (*n*)13--23490--1910--650--53Reduction (%)na87.696.598.1Mite-free dogs (%)^a^ (no. of mite-free dogs/total no. of dogs)na12 (6/50)38.8 (19/49)62.5 (30/48)Signed rank (S)na-599-612-588Degrees of freedomna484847*P*-valuena\<0.0001\<0.0001\<0.0001NexGard-treated animals Number of dogs (*n*)31313029 Mean mite count (*n*)229.826.57.44.1 Count range (*n*)18--23490--1910--650--53 Reduction (%)na88.596.898.2 Signed rank (S)na-224-232.5-217.5 Degrees of freedom292928 *P-*valuena\<0.0001\<0.0001\<0.0001NexGard Spectra-treated animals Number of dogs (*n*)19191919 Mean mite count (*n*)106.616.84.82.7 Count range (*n*)13--3860--910--260--37 Reduction (%)na84.395.597.5 Signed rank (S)na-95-95-95 Degrees of freedomna181818 *P-*valuena\<0.0001\<0.0001\<0.0001*Abbreviation*: *na* not applicable^a^ Mite-free dogs: absence of mite (live or dead) at count
Specific analyses of the efficacy for juvenile (\< 18 months) or adult (\> 18 months) onset of demodicosis were conducted (Table [3](#Tab3){ref-type="table"}), including or excluding dogs with demodectic podal dermatitis. A significant difference was observed between the efficacy in the dogs younger than 18 months compared to the dogs older than 18 months in the overall treated population (*Z* = 375.5, *P =* 0.018), while no significant difference was observed between the same classes of age if dogs with demodectic podal dermatitis are excluded (*Z* = 375.5, *P =* 0.23).Table 3Percent efficacy of afoxolaner against *Demodex* spp. according to the age of the dogs and the presence of specific lesions of demodectic pododermatitisDog ageWilcoxon Rank Sum test\< 18 months\> 18 months*Z*-value*P-*valueEfficacy (%) against *Demodex* spp. in the overall treated population (*n*)98.6 (26)92.1 (22)375.50.018Efficacy against *Demodex* spp*.* excluding demodectic pododermatitis (*n*)98.6 (22)95.7 (12)1530.230*Abbreviation*: *n* number of dogs
Clinical scores {#Sec12}
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In order to evaluate the effects of afoxolaner on the clinical expression of demodicosis, all dogs that had received concomitant medications for the control of skin conditions (e.g. antibiotics, corticosteroids, antihistamines) were excluded from the clinical score analyses. Among the 17 excluded dogs, 8 were from the Nexgard® group and 9 from the NexGard Spectra® group. Treatments included chlorhexidine shampoos (9/17), systemic antibiotherapy (6/17) with cephalosporins or fluoroquinolone, oclacitinib (1/17) and food supplementation for immune system activation (beta-glucan) (2/17).
Alopecia and erythema were the two most frequent clinical signs affecting the enrolled animals with 100 and 88%, respectively, of the dogs harboring them (almost half of these dogs presented severe lesions). At the end of the study, 78.1 and 87.5% of the dogs had no alopecia or no erythema, respectively. Total skin lesion score and total extent score and pruritus score were significantly lower on Days 28, 56 and 84 compared to pre-treatment (Day 0) values (Table [4](#Tab4){ref-type="table"}). The evaluation of the prevalence of the individual lesion scores (severity) and the extent score for each of the five selected clinical signs (alopecia, erythema, papules, pustules and scaling) between Day 0 and Day 84 is presented in Table [5](#Tab5){ref-type="table"}.Table 4Cochran-Mantel-Haenszel (CMH) Mean Score Difference (*F*) test for the total skin lesions score, the total extent of the lesion score and the pruritus score at Day 28, Day 56 and Day 84 compared to Day 0 for 31^a^ dogs treated with afoxolaner who didn't received concomitant medications for the control of skin conditionsDay 28Day 56Day 84Total skin lesion scoreCMH row mean scores differ*F*~(1,\ 62)~ = 19.0*F*~(1,\ 63)~ = 38.2*F*~(1,\ 62)~ = 45.3*P-*value\<0.0001\<0.0001\<0.0001Total lesion extent scoreCMH row mean scores differ*F*~(1,\ 62)~ = 15.0*F*~(1,\ 63)~ = 33.0*F*~(1,\ 62)~ = 45.4*P-*value\<0.0001\<0.0001\<0.0001Pruritus scoreCMH row mean scores differ*F*~(1,\ 63)~ = 13.7*F*~(1,\ 64)~ = 27.6*F*~(1,\ 63)~ = 32.2*P-*value0.0002\<0.0001\<0.0001^a^17 animals were excluded from the analyses because of concomitant medications for the control of skin conditionsTable 5Individual lesion and extent scores (Day 0 and Day 84) for 31 dogs treated with afoxolaner who didn't received concomitant medications for the control of skin conditionsLesionSeverityExtentDay 0Day 84Day 0Day 84AlopeciaAbsent (%)078.1Absent (%)078.1Mild (%)18.218.8Limited (%)^a^54.521.9Moderate (%)33.33.1Marked (%)^b^39.40Severe (%)48.50Generalised (%)^c^6.10CMH row mean scores differ*F*~(1,\ 63)~ = 46.7; *P* \<0.0001CMH row mean scores differ*F*~(1,\ 63)~ = 42.3; *P* \<0.0001ErythemaAbsent (%)12.187.5Absent (%)12.187.5Mild (%)18.29.4Limited (%)^a^51.512.5Moderate (%)24.23.1Marked (%)^b^27.30Severe (%)45.50Generalised (%)^c^9.10CMH row mean scores differ*F*~(1,\ 63)~ = 38.1; *P* \<0.0001CMH row mean scores differ*F*~(1,\ 63)~ = 36.3; *P* \<0.0001PapulesAbsent (%)53.193.8Absent (%)53.193.8Mild (%)12.53.1Limited (%)^a^25.06.3Moderate (%)12.53.1Marked (%)^b^21.90Severe (%)21.90Generalised (%)^c^00CMH row mean scores differ*F*~(1,\ 62)~ = 13.8; *P* = 0.0002CMH row mean scores differ*F*~(1,\ 62)~ = 13.9; *P* = 0.0001PustulesAbsent (%)56.396.9Absent (%)56.396.9Mild (%)12.50Limited (%)^a^21.93.1Moderate (%)18.83.1Marked (%)^b^21.90Severe (%)12.50Generalised (%)^c^00CMH row mean scores differ*F*~(1,\ 62)~ = 14.2; *P* = 0.0002CMH row mean scores differ*F*~(1,\ 62)~ = 14.7; *P* = 0.0001Scaling crustsAbsent (%)39.496.9Absent (%)21.293.8Mild (%)15.23.1Limited (%)^a^51.56.3Moderate (%)24.20Marked (%)^b^21.20Severe (%)00Generalised (%)^c^6.10CMH row mean scores differ*F*~(1,\ 63)~ = 32.1; *P* \<0.0001CMH row mean scores differ*F*~(1,\ 63)~ = 33.5; *P* \<0.0001*Note*: 17 animals were excluded from the analyses because of concomitant medications for the control of skin conditions^a^Seen on 1/3 of the (head + body) surface^b^Seen on 2/3 of the (head + body) surface^c^Seen all over the head + body
Afoxolaner administration was thus associated with significantly lower clinical sign scores, lesion extent and pruritus score compared to Day 0 over the course of the treatment (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1Clinical evolution after monthly administrations of afoxolaner in two dogs with generalised demodicosis. **a** Pre-treatment lesions of erythema and alopecia in a 2-year-old Pointer. **b** Lesions after two treatments with afoxolaner (NexGard®) at a monthly interval (D56). **c** Pre-treatment lesions of multifocal alopecia and erythema in an 11-month-old American Staffordshire Terrier. **d** Lesions after two treatments with afoxolaner (NexGard Spectra®) at a monthly interval (D56)
Safety {#Sec13}
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Except for one dog vomiting a few hours after the first administration, no treatment related adverse event was observed in any dog. One dog from the Nexgard® group was removed at the owner's request because of an aggressive behavior that jeopardized the appropriate follow-up of the dog, and another dog from the same group previously diagnosed with cancer and chronic heart problems died from heart failure.
Discussion {#Sec14}
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This multi-center clinical field study demonstrated that monthly oral administrations of afoxolaner, both alone or in combination with milbemycin oxime, provided a rapid and significant reduction of the number of *Demodex* mites and of clinical signs of demodicosis in privately-owned dogs in Europe. The results obtained are consistent with the findings reported previously with afoxolaner \[[@CR11]\] or other isoxazolines \[[@CR14], [@CR15], [@CR21]--[@CR23]\].
No comparison between NexGard® and NexGard Spectra® was performed because the objective was the evaluation of afoxolaner activity independently of the formulation. It was assumed that the addition of milbemycin oxime would have no impact on the overall efficacy of afoxolaner against *Demodex* spp. mites. Indeed, the half-life of milbemycin oxime is very short (2--3 days) with no accumulation. A monthly dose of 0.5 mg/kg of milbemycin oxime would not provide a sufficient amount of active ingredient to improve the control of the disease.
The enrolled dog population reflected the profile of dogs usually presented for demodicosis in veterinary practices. A recent broad-spectrum survey conducted on a cohort of 431 dogs in California identified the Pitbull Terrier group (including American Staffordshire Terrier) as probably predisposed to demodicosis \[[@CR24]\] and this was also the most frequent breed group enrolled in the present study. Differentiation between juvenile- and adult-onset demodicosis is sometimes difficult. It is mainly driven by the presence of underlying conditions to manage in addition to the parasitic infestation in adult animals \[[@CR2], [@CR24]\]. For this reason, the treatment is often considered easier in younger dogs than in adults. In the present study, 27 dogs were younger than 18 months while 23 dogs were older. The efficacy on Day 84 was 98.6% for dogs under 18 months and 92.1% for older dogs, suggesting that afoxolaner can be used to treat all clinical types of demodicosis. The difference between the efficacies in the two classes of age is significant. This is in accordance with the difference in the course of the disease of adult-onset compared to juvenile-onset of demodicosis well described in the literature.
Out of the 48 dogs which completed the study, 14 had demodectic podal dermatitis. Seven of these dogs were among the dogs still harboring live mites at the end of the study. Demodectic podal dermatitis is more difficult to cure and the prognosis presupposes a longer course of treatment \[[@CR25]\]. In addition, demodectic pododermatitis is often related to dogs affected with underlying factors (diabetes mellitus, cancer, strong immunosuppression), which may need continuous protection against *Demodex* spp. proliferation \[[@CR2]\]. Interestingly, if dogs with demodectic podal dermatitis are excluded from the analysis comparing classes of age, no significant difference is observed between dogs older or younger than 18 months. This finding corroborates the difficulty of controlling demodectic pododermatitis.
The challenging question that remains is related to the duration of treatment. It is known to be highly variable and depending on individual features. In the American cohort study, juvenile demodicosis was treated within 4.5 months (range 0.25--15) for 86.4% of the dogs. Adult demodicosis was treated within 5.9 months (range 1--24) for 87.7% of the dogs \[[@CR24]\]. In veterinary practices, the treatment is stopped after complete remission of clinical signs and two negative skin scrapings performed at a monthly interval \[[@CR26]\]. However, according to some authors, dogs should not be considered cured unless no relapse occurs in the year following the end of the specific treatment \[[@CR2]\]. In the present study, 19 dogs (40%) had two consecutive negative skin scrapings at Day 56 and Day 84.
Long term compliance is a key factor for the control of chronic diseases \[[@CR27]\]. A treatment administrated at a monthly interval is in-line with the monthly follow up of the mite infestation and is expected to improve adherence to treatment.
The need for flea and tick prevention justifies long-term isoxazoline treatment and may prevent relapse/recurrence of demodicosis or even decrease the overall frequency of the disease \[[@CR24]\]. It would be of interest to assess the preventive efficacy of these molecules in young dogs predisposed to demodicosis. A long term epidemiological survey of breeds predisposed to demodicosis might help answering this question.
One hypothesis would be that the acaricidal efficacy of isoxazolines used for a sufficient period would eventually kill the whole population of *Demodex* spp. mites present on the body surface of a dog. In that case, no relapse would occur even in the context of demodicosis related to underlying conditions. A recent publication indicated that treatment with isoxazoline (afoxolaner or fluralaner) had a limited effect on cutaneous *Demodex* spp. populations of normal dogs over a 90 day period and thus does not eliminate the mite population on a dog. However, this study was based on a DNA detection using simple PCR with no quantification methods or evaluation of the viability of the mites \[[@CR28]\]. Those results should therefore be considered as not conclusive and additional studies are necessary to better understand the effect of isoxazoline on *Demodex* mite populations.
Conclusions {#Sec15}
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The high level of activity against *Demodex* spp. achieved with afoxolaner-based products offers new opportunities to veterinarians for the control of demodicosis. It provides new solutions combining safety, efficacy and ease-of-use for improved owner compliance. The potential of choosing a combination product including a nematodicide molecule allows veterinarians to adapt the treatment of demodicosis to specific epidemiological situations such as those encountered in heartworm or lungworm disease enzootic areas.
The authors would like to thank Patxi Sarasola for the final report and Anabel Blasco for the help with statistical analysis (ONDAX Scientific S.L., Spain).
Funding {#FPar1}
=======
Funding for the design, conduct of the study and data collection was provided by Boehringer Ingelheim Animal Health, Lyon France. Data analysis and interpretation was performed by ONDAX Scientific S.L., Spain, an independent contract research organisation. Writing of the manuscript was performed by LH and WL who are Boehringer Ingelheim Animal Health employees.
Availability of data and materials {#FPar2}
==================================
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.
Disclaimer {#FPar3}
==========
NexGard® or NexGard Spectra® are registered trademarks of Merial. All other brands are the property of their respective owners. This document is provided for scientific purposes only. Any reference to a brand or a trademark herein is for informational purposes only and is not intended for a commercial purpose or to dilute the rights of the respective owner(s) of the brand(s) or trademark(s).
WL, DL, ET, FB and LH designed the studies. JG, CD, OC, PB, VB, TB, DP, MM, JP, AC, DPH and MB enrolled the animals, conducted the study and assisted with the interpretation of the data. The original manuscript was prepared by LH and WL. All authors revised each version and read and approved the final manuscript.
Ethics approval {#FPar4}
===============
All animals enrolled in the study were privately owned dogs and an informed consent and agreement for participation in the study was obtained from each owner before enrollment of the dog.
Consent for publication {#FPar5}
=======================
Not applicable.
Competing interests {#FPar6}
===================
This clinical study was funded by Boehringer Ingelheim Animal Health, 29 Avenue Tony Garnier, 69007 Lyon, France. WL, DL, ET, FB and LH are employees of Boehringer Ingelheim Animal Health. JG, CD, OC, PB, VB, TB, DP, MM, JP, AC, DPH and MB are independent investigators contracted for this study and declare that they have no competing interests.
Publisher's Note {#FPar7}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Introduction {#s1}
============
Chemical communication through pheromones is widespread in nature. The classical definition of pheromonal function recognizes the signaling effects that influence behaviors and the primer effects that mediate physiological changes in conspecifics (Wilson and Bossert, [@B56]). The early examples of specific olfactory messengers in mammals are estrus suppression (Lee-Boot effect) (Lee and van der Boot, [@B23], [@B24]) and estrus synchrony (Whitten effect) (Whitten, [@B53], [@B54]), first observed in the house mouse (*Mus domesticus*) (reviewed in Koyama, [@B18]). The identification of these messengers has been based on detection in animal fluids, and the demonstration that synthetic versions of these molecules were capable of eliciting the appropriate biological effects (Jemiolo et al., [@B15]; Novotny et al., [@B35]; Ma et al., [@B26], [@B27]; Koyama, [@B18]). More recently, a novel olfactory effect was described, in which soiled bedding from male mice induced an increase in neurogenesis within the subventricular zone (SVZ) (Mak et al., [@B28]; Larsen et al., [@B22]; Oboti et al., [@B38]) and dentate gyrus (Mak et al., [@B28]) of recipient adult females. However, the identity of the relevant olfactants has remained unknown and these studies focused on females. There have been no reports concerning olfactory effects on neurogenesis or cell proliferation in the brains of males.
In the studies on the impact of male-soiled bedding on neurogenesis in females, the soiled bedding from dominant males induced higher levels of neurogenesis than the bedding from subordinate males (Mak et al., [@B28]). In our previous studies on olfactory communication, the compounds dehydro-exo-brevicomin (DHB), 2-*sec*-butyl-4,5-dihydrothiazole (SBT), E,E-α-farnesene and E-β-farnesene (Jemiolo et al., [@B15]; Ma et al., [@B27]) were shown to mediate the Whitten effect, i.e., the effect of inducing estrus in females (Koyama, [@B18]). These 3 compounds have also been detected at higher concentrations in the urine of dominant vs. subordinate males (Novotny et al., [@B33]). We thus hypothesized that these compounds may be excellent candidate chemosignals that stimulate neurogenesis in female mice.
Previous studies have focused on the influences of male-soiled bedding on neurogenesis in females. In earlier work, we showed that female-soiled bedding enhanced sperm density in males (Koyama and Kamimura, [@B20]). The influence was stronger (i) when males were exposed to the soiled bedding of females housed in groups than when they were exposed to the soiled bedding of the same number of females housed individually and also (ii) when the females were housed at higher density (unpublished data of SK). We have previously shown that 2,5-dimethylpyrazine (2,5-DMPZ) is secreted specifically by females that are kept in groups and it causes the suppression of their estrous cycles (Lee-Boot effect) (Lee and van der Boot, [@B23], [@B24]; Novotny et al., [@B35]; Ma et al., [@B26]). This suppression is stronger when females are kept at higher density (Coppola and Vandenbergh, [@B9]). These studies showed that males are affected by female-soiled bedding. The studies also suggested that, if female-soiled bedding affects the males\' reproductive condition, neurogenesis may also be influenced as gonadal hormones are known to be related to neurogenesis (Galea et al., [@B13]). As 2,5-DMPZ is known to be secreted at higher concentration by females housed in groups, and as group-housed females have a stronger impact on male sperm density, we hypothesized that this compound may affect neurogenesis in males.
Materials and methods {#s2}
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Animals
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C57BL/6 mice (120 females and 53 males, which includes the odor donor mice) were utilized. Mice were bred in-house to ensure that they had no prior contact with adult male scent after they are weaned. All the mice were housed in individually ventilated isolation cages, connected to a tube providing filtered air from outside of the building, so there was no odor contamination between cages. Mice were used at 11--12 weeks of age to determine the influences of exposure to pheromones and soiled beddings on cell proliferation in the adult brain. The initial ages of the mice used to measure pheromone influence as a function of developmental stage were as follows: 24, 30, 34, 37, 51, and 84 days. Ages of the mice used as the odor donors varied between 3 and 6 months (adult but not old mice). The study was approved by the Indiana University Institutional Animal Care and Use Committee (IACUC). Food and water were provided *ad libitum*. Corn Cob bedding (\#7097 Teklad Corn Cob Bedding, ¼." Harlan Laboratories, Inc., Indianapolis) was used as bedding. The odor-receiver females were housed at 2 mice/cage to avoid the influence of isolation (Koyama, [@B16],[@B17]; Bartolomucci et al., [@B1]) and large group housing (Lee and van der Boot, [@B23], [@B24]; Novotny et al., [@B35]; Ma et al., [@B26]; Koyama, [@B18]). The odor-donor mice were housed at 4 mice/cage. 2,5-Dimethylpyrazine, a chemical that induces Lee-Boot effect (Novotny et al., [@B35]; Ma et al., [@B26]; Koyama, [@B18]), was not detectable in the female mouse-bedding odor generated with this housing condition (see Odor Collection and Analysis for details on analyses of soiled bedding).
Exposure to soiled bedding and synthetic pheromones
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To expose mice to soiled bedding, mice were moved every 2 days for 7 days (4 times) to cages where odor-donor mice had been housed. Control mice were moved to clean cages. The bedding was used by odor-donor mice for 7 days. The period of exposure of 7 days was adapted from the studies of Mak et al. ([@B28]) and Larsen et al. ([@B22]). Those studies showed that both shorter (2 days) and longer (2 weeks) exposure periods did not induce enhanced neurogenesis. We also conducted a preliminary experiment exposing females to the bedding of odor donor males that were used for 2, 4, and 7 days, or to clean bedding. We determined that exposure to the bedding soiled by odor donor males for longer periods of time induced higher levels of cell proliferation in the SVZ of female mice (data not shown). We determined the concentrations of pheromones included in these soiled beddings using GC-MS (see Odor Collection and Analysis below) and found that the concentration of the farnesene isomers and SBT were increased in the bedding exposed to odor donors for longer periods (data not shown). These pheromones are normally volatile and odoriferous substances, however, their equilibrium concentrations in the air in the mouse cages is primarily controlled by forming complexes with the major urinary proteins (MUPs) (Zidek et al., [@B58]; Sharrow et al., [@B42]; Beynon and Hurst, [@B3], [@B4]) that permit a slow release. The increased (measured) concentrations of pheromones in the headspace as a function of time are presumably due to (i) enhanced surface area in the bedding "coated" with urine; and (ii) perhaps to even larger degree, pheromone release from the MUPs due to the actions of microbial proteases \[see Novotny ([@B36]) for review\]. Based on these preliminary experiments, the concentration of pheromone solutions for exposure of mice to synthetic pheromones was determined by the concentration of farnesene we detected in the soiled bedding used by odor donor mice for 7 days. For exposure to synthetic pheromones and benzothiazole, aqueous 250 ppm solutions (50 μ L) (or water for control) were directly deposited on the nostrils of mice (Jemiolo et al., [@B15]; Ma et al., [@B27]) twice daily for 7 days. The concentration of other pheromones was adjusted to the same concentration (250 ppm). This application frequency was based on the methods previously utilized, which successfully stimulated or suppressed estrus in females (Ma et al., [@B27]). The estrous status of the females was not controlled, but was evaluated at the time of perfusion. There were females of each estrous stage included in the samples but there was no obvious correlation between estrous status and proliferation of cells in the SVZ (data not shown).
Marking and counting the proliferating cells
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On the 8th day, mice were injected with 5-bromo-2-deoxyuridine (BrdU, B5002, Sigma-Aldrich) (300 mg/kgbw) 4 times every 2 h and intracardially perfused with paraformaldehyde 2 h after the last BrdU injection. Brains were post-fixed overnight at 4°C and stored in 15% sucrose in 0.2 M phosphate buffer until sectioning (16 μm, from Bregma distance 1.54 to −0.22 mm) using a cryostat (Leica CM1850, Leica Microsystems GmbH, Germany). Each section on slides was 80 μm apart. The sections were stored at −20°C until use, stained with anti-BrdU antibodies (Accurate Chemical Co., \#OBT0030, 1:300, rat) and incubated with fluorescent secondary antibodies (Alexa Fluor 488, Invitrogen). Stained sections were imaged using Nikon Eclipse 80i (Nikon, Japan) at Indiana University METACyt core neuroscience labs for counting cells. BrdU^+^ cells were counted using Image-J software. The average of the number of BrdU^+^ cells on the left and right SVZ in 16 μm thick sections that were spaced 80 μm apart from Bregma distance 1.34 to −0.1, were analyzed. The length of the region along the ventricle was measured and the number of cells per millimeter was calculated, which was converted into the number of cells per unit length of SVZ at each Bregma distance. The total of these numbers were used as the data for each individual.
Immunohistochemistry and measurement of fluorescence intensity
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For observation of the expression of Sox2 in the proliferating cells at the SVZ, we conducted immunohistochemistry with anti-Sox2 (ab97959, rabbit, Abcam, Cambridge, MA) and anti-BrdU (Accurate Chemical Co., \#OBT0030, 1:300, rat). Sodium citrate (0.01 M) was used for the denaturation process (Tang et al., [@B45]). Sections were washed with 0.1 M PBS and then treated with sodium citrate buffer for 20 min in a steamer (\>99°C inside the steamer). After 20 min, the slides were incubated for 2 min in the sodium citrate buffer at room temperature to cool down. After the antigen recovery process, sections were washed with 0.1 M PBS before blocking with 10% normal goat serum in 0.1 M PBS for 1 h at room temperature. Then sections were kept at 4°C with the anti-BrdU and anti-Sox2 in 0.1 M PBS with 3% normal goat serum. On the second day, sections were washed with 0.1 M PBS, stained with secondary antibodies (goat anti-rat Alexa Fluor 488 and goat anti-rabbit Alexa Fluor594, 1:500, respectively, Invitrogen, Inc.) in 0.1 M PBS with 0.3% Triton X-100, and stained with Draq5 (\#4084, 1:1000, Cell Signaling Technology, Beverly, MA). Stained sections were imaged using a Leica SP5 scanning confocal (Leica Microsystems GmbH, Germany) at The IUB Light Microscopy Imaging Center to generate the confocal images in Figures [3](#F3){ref-type="fig"} and [6](#F6){ref-type="fig"}. Sox2^+^ cells were counted using Image-J software.
Odor collection and analysis
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Small magnetic stir bars provided with a polymer coating were used to absorb odor components. Stir bars (Twister™, 10 × 0.5 mm film thickness, 24 μL polydimethylsiloxane (PDMS) volume, Gerstel GmbH, Mülheim an der Ruhr, Germany) were also spiked with an internal standard (8 ng of 7-tridecanone) (Soini et al., [@B43]). Volatile and semi-volatile odor compounds were collected by passive diffusion (headspace mode) into the PDMS polymer by the sorption mechanism. To collect odors, the stir bars were placed in 0.5 mL polypropylene vials with drilled holes (0.5 mm, i.d.), placed in wire-mesh holders hung on the sides of the cages for up to 7 days.
The gas chromatograph-mass spectrometer (GC-MS) instrument was an Agilent 6890N gas chromatograph connected to a 5973i MSD mass spectrometer (Agilent Technologies, Inc., Wilmington, DE) with the Thermal Desorption Autosampler/Cooled Injection System (TDSA-CIS 4, Gerstel GmbH). A DB-5MS separation column (30 × 0.25 mm, i.d., 0.25 μm film thickness) capillary (Agilent, J&W Scientific, Folsom, CA) was employed.
Test compounds
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The farnesene mixture of isomers was obtained from Tokyo Kasei Kogyo Co., Ltd (Tokyo, Japan). Benzothiazole and 2,5-dimethylpyrazine were purchased from Sigma-Aldrich Chemical Company (Milwaukee, WI). Dehydro-*exo*-brevicomin (DHB) and SBT were synthesized in the Novotny laboratory and syntheses of them have been described in the literature (Wiesler et al., [@B55]; North and Pattenden, [@B32]). Test compounds were dissolved in OmniSolv® water (EM Science, Gibbstown, NJ).
Statistics
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Levene\'s test was used, confirming the homogeneity of variance in the data (*F* = 0.517, *df* = 7.4, *P* = 0.816), and the null hypothesis that all the variance are equal was accepted. ANOVA was used to compare the number of proliferating cells. For one-way ANOVA comparison with control conditions, Dunnett test was used for the *post-hoc* analyses. For two-way ANOVA comparisons with age differences and odor condition differences, Tukey\'s *post-hoc* analyses was used to determine the impact of pheromone at each age. For comparison of Sox2^+^ cells among the proliferation cells in the mice exposed to pheromone and control group, Mann-Whitney *U*-test was employed (Nachar, [@B31]). SYSTAT software was used for these statistical analyses. Curve fitting for the change of neurogenesis rate, depending on the concentration of pheromone solution, was conducted using MATLAB and R software.
Results {#s3}
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Aqueous solutions of male murine pheromones, the farnesene isomers (α and β, 1:2 ratio), SBT, DHB, and female murine pheromone 2,5-DMPZ were evaluated for their ability to promote cell proliferation in the SVZ of female mice. The concentration (250 ppm) used was approximately equivalent to the amount of the farnesenes present in male-soiled bedding used by odor donor males for 1 week and this concentration was previously shown to induce estrus in female mice (Jemiolo et al., [@B15]; Ma et al., [@B27]). Benzothiazole was chosen as a control compound because it is ubiquitously present both in female and male mouse urine. No active roles in murine chemical signaling have been identified for urinary benzothiazole (Novotny, [@B36]; Novotny et al., [@B37]). The rate of SVZ cell proliferation after exposure to either male or female-soiled bedding (used by odor donors for 7 days) was assessed.
In the SVZ of female mice, the number of BrdU^+^ cells in the SVZ of females exposed to male-soiled bedding (*n* = 7) was significantly higher than females exposed to clean bedding (*n* = 5), whereas females exposed to female-soiled bedding (*n* = 6) did not show a significant difference \[*F*~(2,\ 15)~ = 33.759, *P* \< 0.001; Dunnett test of *post-hoc* comparison with Clean bedding group, To male bedding group, *P* \< 0.001, To female bedding group, *P* = 0.166\] (Figure [1B](#F1){ref-type="fig"} left side of dashed line). The number of BrdU^+^ cells in the SVZ of females exposed to synthetic analogs of pheromones, the farnesenes (*n* = 8) and SBT (*n* = 6), was significantly higher than the females exposed to water (*n* = 4), whereas females exposed to DHB (*n* = 6), 2,5-DMPZ (*n* = 4), and benzothiazole (*n* = 5) did not show a significant difference \[*F*~(5,\ 27)~ = 14.695, *P* \< 0.001; Dunnett test of *post-hoc* comparison with Water group, Farnesene group and SBT group, *P* \< 0.001 respectively, DHB group, *P* = 0.114, benzothiazole group, *P* = 0.498, 2,5-DMPZ, *P* = 0.332\] (Figure [1A](#F1){ref-type="fig"} and right side of dashed line in Figure [1B](#F1){ref-type="fig"}). As the female murine pheromone 2,5-DMPZ suppresses estrus (Novotny et al., [@B35]; Ma et al., [@B26]; Koyama, [@B18]), we had hypothesized that it might suppress the rate of cell proliferation, however, this compound did not affect cell proliferation in females. It seems plausible that some of the pheromones from one sex may enhance the rate of cell proliferation in the SVZ of the opposite sex, with little or no effect on animals of the same sex.
{#F1}
The detection threshold of the farnesenes, SBT and DHB at the vomeronasal neuron is known to be as low as 10^−11^ to 10^−10^ M, 10^−10^ to 10^−9^ M, and 10^−10^ to 10^−9^ M respectively (Leinders-Zufall et al., [@B25]). In order to determine whether these levels also induced a detectable change in the rate of cell proliferation in the SVZ, we exposed females to a range of concentrations of the farnesenes and SBT, which had been shown to stimulate cell proliferation at 250 ppm (Figure [1](#F1){ref-type="fig"}). The rate of cell proliferation showed an exponential decay as the concentration decreased. We determined that the threshold concentration to induce enhanced cell proliferation was approximately 10^−6^ M for farnesene and 10^−5^ M for SBT (Figure [2](#F2){ref-type="fig"}). Although previous studies that showed the detection threshold at the vomeronasal neuron (Leinders-Zufall et al., [@B25]) utilized a slice preparation and our study used live animals, it is conceivable that a concentration higher than the detection threshold for the vomeronasal neurons is required to induce cell proliferation in the SVZ.
![**Influence of various concentrations of male mouse pheromones, farnesenes and SBT, on cell proliferation in the SVZ of adult female mice.** Arrows with "X" and "Y" show the threshold concentrations of SBT and farnesenes at the vomeronasal neurons *in vitro* (Leinders-Zufall et al., [@B25]), respectively. Each dot indicates individual result and white dots show the results of controls.](fnbeh-07-00101-g0002){#F2}
Sox2, a HMG (high-mobility-group) box transcription factor gene, is expressed highly in adult neural progenitors and serves as one of the markers of neural stem/progenitor cells (Von Bohlen und Halbach, [@B51]). We used immunofluorescence to determine if exposure to the male synthetic pheromone SBT might enhance the generation of neural progenitor cells. SBT was selected for its strong influence on cell proliferation. We found that the number of Sox2^+^ cells in the proliferating cells in the SVZ was higher in the females exposed to SBT than in the control females (Mann-Whitney *U*-test, *U* = 0, *P* = 0.05) (Figure [3](#F3){ref-type="fig"}).
{#F3}
We then measured the influence of male murine pheromones on cell proliferation in the SVZ of females at distinct developmental stages using SBT. We found that SBT enhanced cell proliferation in the SVZ of adult females (3-month-old) but not in the pre-pubertal (31-day-old, starting exposure to pheromone from 24-day-old), pubertal (37-day-old, starting exposure to pheromone from 30-day-old, which is the average age when the vagina opens) or young post-pubertal females (41-, 44- and 58-day-old; ages when exposure started were 7 days prior to these ages) (Figure [4](#F4){ref-type="fig"}). When the ages were grouped as being either near puberty (31- and 37-day old), as young post-pubertal females (41- and 44-day old), as young adults (58-day-old) or as adults (84-day-old), only the latter group showed a significant difference in cell proliferation in the SVZ between treated females and controls with the other groups not showing any significant differences \[two-way ANOVA, age *F*~(3,\ 27)~ = 5.604, *P* = 0.004, odor condition *F*~(1,\ 27)~ = 1.093, *P* = 0.305, age\*odor condition *F*~(3,\ 27)~ = 5.523, *P* = 0.004; Tukey\'s *post-hoc* test, age-matched pair-wise comparison of pheromone group vs. control group, near puberty, *n*~pheromone~ = 4, *n*~control~ = 4, *P* = 1; young post-pubertal, *n*~pheromone~ = 4, *n*~control~ = 4, *P* = 0.891, young adult, *n*~pheromone~ = 4, *n*~control~ = 4, *P* = 1, adult, *n*~pheromone~ = 64, *n*~control~ = 5, *P* = 0.006\].
{#F4}
Next, we tested whether a female murine pheromone (Novotny et al., [@B35]; Ma et al., [@B26]; Koyama, [@B18]) 2,5-DMPZ or male murine pheromones (SBT, the farnesene isomers, and DHB) might influence cell proliferation in the SVZ of males. We also exposed males to soiled bedding from either other males, from females or to clean bedding (control). We found that males exposed to either female-soiled bedding (*n* = 8) or male-soiled bedding (*n* = 7) showed significantly higher levels of cell proliferation in the SVZ compared to the males exposed to clean bedding (*n* = 6) \[ANOVA, *F*~(2,\ 18)~ = 8.634; Dunnett *post-hoc* comparison with clean bedding group, To male bedding group *P* = 0.037, To female bedding group, *P* = 0.001\] (Figure [5](#F5){ref-type="fig"}). The finding that the soiled bedding from males (the same sex) could stimulate cell proliferation in other males was distinctly different from the situation observed for females. Of the groups that were exposed to synthetic analogs of murine pheromones, only the group that was exposed to 2,5-DMPZ (*n* = 6) showed significantly higher cell proliferation in the SVZ whereas the male murine pheromones, the farnesenes, SBT, and DHB, did not stimulate or suppress cell proliferation in the SVZ of males \[ANOVA, *F*~(4,\ 24)~ = 32.963, *P* \< 0.001; Dunnett *post-hoc* comparison with Water group (*n* = 7), 2,5-DMPZ, *P* \< 0.001, the farnesenes (*n* = 6), *P* = 0.353, SBT (*n* = 6), *P* = 0.063, DHB (*n* = 4), *P* = 0.5\]. The number of BrdU^+^ cells in the SVZ of males exposed to the female pheromone 2,5-DMPZ (*n* = 4) that also stained with Sox2^+^ was not significantly different from that of control group males (*n* = 3) (Figures [6A,B](#F6){ref-type="fig"}) (Comparison of males exposed to 2,5-DMPZ and control group males, Mann-Whitney *U*-Test, *U* = 2.5, *P* = 0.156).
{#F5}
![**Sox2 expression in the proliferating cells in the SVZ of male mice. (A)** Expression of Sox2 in the BrdU^+^ cells (arrow heads) in the SVZ of male mice exposed to 2,5-DMPZ (above) and control group male mice (below). **(B)** Rate of BrdU^+^ cells doubled labeled with Sox2^+^ in the males exposed to 2,5-DMPZ (*n* = 4) and control group males (*n* = 3). Sox2^+^ cells that did not overlap with BrdU^+^ cells and nucleus staining \[arrow in **(A)**\] were not counted. Bars indicate median and interquartile range.](fnbeh-07-00101-g0006){#F6}
Discussion {#s4}
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To our knowledge, these findings represent the first observation that single, synthetic pheromones promote cell proliferation in the SVZ in mammals. Previous studies using soiled bedding as a source of pheromones have reported their stimulatory impact on neurogenesis in females (Mak et al., [@B28]; Larsen et al., [@B22]; Oboti et al., [@B38]), however, in this report we are presenting the first evidence that female pheromones strongly promote cell proliferation in the SVZ of males.
We have demonstrated that previously recognized male murine pheromones, the farnesenes and SBT, are capable of enhancing cell proliferation in the SVZ of female mice. DHB did not significantly stimulate cell proliferation in the SVZ of female mice, which indicates that not all of the male murine pheromones tested were capable of stimulating cell proliferation in the female SVZ. In addition, we found that the female murine pheromone, 2,5-DMPZ (Novotny et al., [@B35]; Ma et al., [@B26]) was capable of enhancing cell proliferation in the male SVZ. Our findings suggest that both male and female pheromones, when presented as their synthetic analogs at concentrations detected in soiled bedding, are capable of stimulating cell proliferation in the brains of the opposite sex. We also found that these pheromones do not have detectable effects on brains of the same sex, i.e., the female murine pheromone 2,5-DMPZ did not affect cell proliferation in the SVZ of female mice and the male murine pheromones, the farnesenes, SBT, and DHB, did not affect cell proliferation in the SVZ of male mice. Previous studies have shown that pheromones have suppressive impact on the reproductive system within the same sex, for example, female pheromones suppressed estrus in females (Lee and van der Boot, [@B23], [@B24]; Novotny et al., [@B35]; Ma et al., [@B26]) and dominant male odors suppressed sperm activity in subordinate males (Koyama and Kamimura, [@B19], [@B21]). Our findings suggest that, on neurogenesis, pheromones do not have suppressive impact on the same sex. However, males exposed to male-soiled bedding showed an increased proliferation in the SVZ, although this was not observed in females exposed to female-soiled bedding. This suggests that for males, the ability of pheromones to promote neurogenesis may not only occur between the sexes. Further studies on the sex differences in the impact of pheromones on neurogenesis may be of importance.
The cells proliferating in the SVZ are known to be astrocyte-like cells expressing glial fibrillary acidic protein (GFAP) that are capable of giving rise to neuroblasts that migrate to the olfactory bulb and become interneurons (Whitman and Greer, [@B52]). Sox2 is a transcription factor that is involved in the proliferation of neural stem cells in the SVZ and subgranular zone (SGZ) and thus serves as one of the neural stem/progenitor cell markers (Bylund et al., [@B8]; Ferri et al., [@B12]; Ghashghaei et al., [@B14]; Suh et al., [@B44]; Favaro et al., [@B11]; Mu et al., [@B30]; Von Bohlen und Halbach, [@B51]; Yao et al., [@B57]). Sox2 was detected in a higher percentage of the proliferating cells in the SVZ of females exposed to male murine pheromones than in the males exposed to female pheromones. Although there is a possibility that it is due to the small sample size, these findings suggest that the effect of pheromone-induced cell proliferation, i.e., the cell fate, in males and females may also differ. Future studies will be required to determine the number and identity of the mature neuronal types that are generated.
As summarized above, cell proliferation/neurogenesis in the SVZ of females was strongly stimulated by male pheromones and cell proliferation in the SVZ of males was strongly stimulated by female pheromones. What are the potential molecular mechanisms that underly these effects? In our previous studies, we determined that sperm density was enhanced by exposure to female-soiled bedding (Koyama and Kamimura, [@B20]) and male pheromones stimulated females to come into estrus (Jemiolo et al., [@B15]; Ma et al., [@B27]; Koyama, [@B18]). As gonadal hormones have been previously shown to influence neurogenesis (Galea et al., [@B13]), it is possible that pheromones act via this class of effectors to stimulate neural cell proliferation. Prolactin has been shown to regulate neurogenesis in the SVZ of females in response to male-soiled bedding, and its infusion into males was also capable of stimulating neurogenesis (Mak and Weiss, [@B29]). In addition, estrogen was able to promote neurogenesis in the dentate gyrus (Mak et al., [@B28]). These studies suggest that some pheromones act via the sex hormones and that these in turn stimulate cell proliferation in the reproductive organs and in the brain. Previous studies have suggested that the increased neurogenesis in females induced by male-soiled bedding was capable of enhancing female preference for dominant males (Mak et al., [@B28]) and for fostering mate recognition (Oboti et al., [@B39]), which may be critical for avoiding pregnancy block (Bruce effect) (Bruce, [@B7]). The infusion of prolactin in males has been associated with an improved recognition of pups (Mak and Weiss, [@B29]) and the improved recognition of a male to its own pups may reduce infanticide, which happens with unrelated offspring (Vom Saal and Howard, [@B50]). The ability of pheromones to act via sex horomones to promote cell proliferation/neurogenesis is an area that merits further investigation.
The time required for newly born progenitor cells in the SVZ to be incorporated in the olfactory bulb as interneurons is about 3 weeks. Therefore, it is unlikely that biological effects that occur more rapidly would be mediated by the pheromonally-induced neurogenesis. 2,5-DMPZ is a female murine pheromone that is secreted by females when they are housed at higher density and it suppresses their estrus (Lee and van der Boot, [@B23], [@B24]; Coppola and Vandenbergh, [@B9]; Novotny et al., [@B35]; Ma et al., [@B26]; Koyama, [@B18]). The male murine pheromones, SBT, the farnesenes, and DHB, will stimulate females to come into estrus and this will be mostly on the third day after exposure to male pheromones. The time interval from the start of regaining a short estrous cycle until delivery is \~22--26 days (3 days to come into estrus and 19 days of pregnancy lead to a total of 22 days and, if pregnancy starts at the next estrous day from some reason, it would be 26 days). This timeframe suggests that the newly incorporated interneurons could contribute to pup recognition, however, this interval is not consistent with a role in mate recognition, which takes place within 1 week of exposure. Thus, the enhanced neurogenesis may not be related to the Bruce effect, which is based on the memory of the mate, but may be involved in maternal/paternal recognition.
A previous study demonstrated that the bedding of dominant males increased the rate of neurogenesis in females, while that of subordinate males lacked this ability (Mak et al., [@B28]). Those investigators also demonstrated that females exposed to the bedding of dominant males showed a preference for dominant males in sociality tests, whereas females exposed to the bedding of subordinate males did not show any preference to either dominant or subordinate males. Dominant males secrete the murine pheromones, the farnesenes, SBT and DHB, at higher levels than subordinate males (Novotny et al., [@B33]). Together, these results suggest that the farnesenes and SBT could serve as the pheromones involved in establishing females\' preference for dominant males by stimulating their neurogenesis. Studies on the biology of house mice have shown that these mice establish social groups with a despotic type of social dominance (Uhrich, [@B47]) and a dominant male sires most of the offspring (Berry and Bronson, [@B2]). It is beneficial for female mice to be able to identify males of higher social status and to have a preference for these males to ensure their reproductive success.
In addition to the impact on males\' and females\' reproductive conditions and neural cell proliferation, these male murine pheromones are known to stimulate aggressive behaviors in other males as well (Novotny et al., [@B34]; Koyama, [@B18]; Rodriguez and Boehm, [@B41]; Tirindelli et al., [@B46]). It might be that these pheromones stimulate the neural circuit that stimulates GnRH-expressing neurons (Boehm et al., [@B5]) in both females and males. The stimulation of GnRH neurons may, in females, increase the secretion of estrogen and induce them to come into estrus, and, in males, increase the secretion of testosterone, which promotes aggressive behaviors (Bronson and Desjardin, [@B6]; Davidson and Levine, [@B10]). Therefore, the same pheromone molecules can produce multiple responses in the same neural circuits.
Previous studies have shown that exposure of females to male-soiled bedding has enhanced the integration of newborn neurons (Oboti et al., [@B38]). Integration of newborn neurons is known to be increased by various factors including exercise (running), an enriched environment (van Praag et al., [@B48],[@B49]) and mating (Portillo et al., [@B40]). One of our future goals will be to determine if the exposure to synthetic analogs of pheromones enhances the integration of neurons in males and females. The elucidation of the mechanisms by which specific compounds influence cell proliferation and neurogenesis, and eventually reproductive conditions in males and females should provide further insight as to how the odor environment controls and modulates the reproductive activities of animals. The use of individual murine pheromones in such studies should permit an improved understanding of the neural circuitry underlying this process.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Dr. T. R. Birkhead, FRS, from Sheffield University, Dept. Anim. Plant Sci., U.K., and Dr. E. Anton from UNC Chapel Hill, for critically reading the draft, Ms. Amanda Posto for her technical assistance in the Novotny laboratory, Ms. Bailey Midgett in the Lai laboratory and Mr. Jeremy R. Page in METACyt Neuroscience Laboratory for their assistance to Sachiko Koyama. We also thank Mr. George A. Wilson of Indiana University, Dept. Psychol. Brain Sci., for his help to Sachiko Koyama. This work was partially supported by the Lilly Chemistry Alumni Chair funds (Milos V. Novotny), by National Institute of Neurological Disorders and Stroke at the National Institutes of Health \[grant number, R01-NS039411\] (Cary Lai), and by the Gill Center for Biomolecular Science (Cary Lai).
[^1]: Edited by: Shigeyoshi Itohara, RIKEN Brain Science Institute, Japan
[^2]: Reviewed by: Masahiro Yamaguchi, The University of Tokyo, Japan; Jane Hurst, University of Liverpool, UK
| {
"pile_set_name": "PubMed Central"
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Background
==========
The medical evidence-base is increasing exponentially, making it virtually impossible for individual healthcare professionals to maintain an up-to-date and comprehensive knowledge of their field of practice \[[@B1]-[@B4]\]. In an attempt to address this problem, professional bodies and healthcare organizations have invested in the development of evidence-based resources that are available electronically. Like many other specialties, oncology practice has taken advantage of the growing use of web-based technology by developing online guideline and protocol systems. These have been developed across multiple jurisdictions but originate mostly from North America and Europe \[[@B5]\].
Electronic decision support has the capacity to improve the processes of care and patient outcomes in oncology practice. But the provision of computer support does not guarantee uptake, and utilization of systems at the point-of-care is crucial if systems are to improve the quality of clinical practice \[[@B6]\]. Despite the growing number of web-based oncology systems around the globe, there has been limited evaluation of the use and impact of electronic support systems in the cancer treatment setting. However, there is some evidence demonstrating electronic oncology systems are adopted readily by clinicians and are seen as integral in cancer treatment delivery \[[@B7]\]. Moreover, they have been shown to increase protocol compliance and reduce chemotherapy prescribing errors and adverse events \[[@B8]-[@B12]\].
In this article we focus specifically on an Australian web-based protocol system, eviQ (previously known as CI-SCaT). eviQ is a web-based repository of nearly 1,300 peer-reviewed cancer treatment protocols managed under the auspices of the Cancer Institute New South Wales (NSW) \[<https://www.eviq.org.au/>\]. The system underwent a major rebuild and rebranding in 2009 to better meet the specific needs of oncologists, nurses, primary health care physicians, pharmacists, and patients. Evaluations of the system to date have been primarily qualitative, with a focus on barriers to use at the point-of-care \[[@B11],[@B12]\]. However, the new platform increases opportunities to conduct longitudinal quantitative research based on web-logfile analysis. As such, the aim of this study is to describe the patterns of eviQ use by clinicians practising in the Australian healthcare setting.
Methods
=======
Study design
------------
This is a study of web-logfiles generated from eviQ over a 26-month period. The study period coincides with the launch of the new platform in October 2009 and covers the first two years of operation until December 2011. The previous platform (CI-SCaT) was taken offline on March 31 2010 to allow sufficient opportunity for users to transition and register on the eviQ website.
Study setting
-------------
In Australia, oncology protocols are delivered primarily in the ambulatory care (outpatient) setting at metropolitan hospitals (university-affiliated, tertiary referral centers covering geographic areas of around 75 square kilometers), regional centers (with catchments up to 1,200 square kilometers) and rural hospitals (with catchments up to 3,400 square kilometers).
eviQ is managed by the Cancer Institute NSW, a government funded agency established to improve cancer control in Australia's largest state, NSW. eviQ primarily targets health professionals involved in implementing cancer care by providing detailed and extensive instructions on how to deliver evidence-based treatments safely and appropriately. Treatment information encompasses adolescent and young adult care, cancer genetics, haematology, haemapoietic progenitor cell transplants, medical oncology, nursing, primary health, palliative care and radiation oncology. The site comprises over 1,300 protocols, developed by a consensus process involving specialist physicians, nurses, pharmacists and allied health practitioners from across Australia. Each protocol undergoes a comprehensive review every 1 to 2 years. While the primary eviQ target audience is health professionals, the site also publishes information tailored specifically to cancer patients and their carers.
Data sources and analysis
-------------------------
For the purposes of this evaluation we used two data sources, both of which have different capabilities in terms of understanding eviQ use. Outputs from both data sources were converted to Microsoft excel 2010 format for analysis. We report demographic variables for all registrants at the end of our study period (December 31 2011) and patterns of use during a typical month based on eviQ logfiles. For the latter, we examined rates of use across the last three months of the study period (October through December, 2011). Rates of overall use were lowest in December (1,145 hits/100 registrants) most probably due to the holiday period, but were comparable in October (1,499 hits/100 registrants) and November (1,418 hits/100 registrants). We selected October 2011 to represent a 'typical' month and all subsequent analyses focused exclusively on this period.
eviQ platform
-------------
The eviQ secretariat provided the research team with access to de-identified data from the eviQ platform. Demographic registrant and logfile data were obtained on-site at the Cancer Institute NSW in unit record format (stripped of personal identifiers such as usernames). The eviQ platform has the capacity to generate data on the characteristics of all registered users including registrant type (individual clinician or unit registration), health setting (primary care or hospital), health sector (public, private, or both), geographical location of practice, clinician group, years of oncology experience, and source of referral to the eviQ website. This information is reported by users upon registration and website registrants are prompted to update this information on an annual basis. Further, logfiles also monitor webhits, defined as one click anywhere on the eviQ website, that can be stratified by any of the aforementioned variables (e.g., clinician type, years of oncology experience) and the time at which the webhits occur (e.g., time of day, month, year). However, the current eviQ logfile reports are aggregated and do not have the capacity to determine the content accessed according to health professional groups.
As such, we report on the following:
Registrant characteristics
We report the characteristics of individuals and units identifying themselves as Australian health professionals upon site registration. We report the number of new health professional registrations by month for the period October 2009 to December 2011 and the demographic characteristics of all registrants at the end of the study period.
Patterns of eviQ use
We examined webhits during standard clinic hours (Monday to Friday between 08:00--18:00) compared with use outside clinic hours. This approach has been used previously in logfile analyses as a proxy for point-of-care use in Australian clinical practice \[[@B13]\]. Moreover, the majority of chemotherapy and radiotherapy cancer treatments are delivered during these times.
We stratified our analysis by registrant type (individual or unit registration), individual clinician group (medical, nursing, pharmacy and radiation therapists) and years of oncology experience. As is standard in logfile analysis, we report both the volume of webhits and hits/100 website registrants \[[@B14]\].
Google analytics
----------------
The eviQ secretariat provided the research team with access to their Google Analytics profile from which we extracted data of interest. Google Analytics' reports provide data on website traffic and allows for the examination of the intensity of eviQ use for all registrants including number of visits and unique visits to eviQ. A visit is defined as a registrant logging on to eviQ for up to four hours, unless the registrant terminates their visit by logging off or leaving the website. Google Analytics produces aggregated data relating to typical user sessions including number of pages accessed and time spent on eviQ during a typical visit for a defined period (e.g., visits in a typical week or month). Google Analytics also has the capacity to provide data on content accessed and time spent on specific eviQ pages (e.g., time spent on a particular chemotherapy protocol page).
Data from Google Analytics does not distinguish between different user groups (e.g., health professionals' vs. consumers), limiting the capacity to undertake analyses on how different groups access eviQ. However, given that health professionals comprise 92% (n = 14,800) of eviQ registrants, Google Analytics output will be heavily influenced by patterns of use by health professionals.
Using this data source, we report on the following during a typical month (October 2011):
Typical user sessions
We report the total number of eviQ visits, average number of pages/visit, average visit duration, and mode of access (mobile device or computer). Google Analytics output is averaged across all users such that we cannot calculate descriptive statistics such as median and range for these data.
Content accessed
We report the top 100 pages accessed and time spent on these pages. The top 100 pages were grouped according to their content into the following categories: login/registration; transition pages (such as tabs directing users to specific content); and cancer treatment content pages (chemotherapy protocols or supportive treatment information). We used the time spent on each of the 100 pages to calculate the range and median time spent on pages according to the abovementioned categories. To better understand the range of clinical content accessed by eviQ users, we also conducted an analysis of the top 100 cancer treatment content pages (excluding all login, registration, and transition pages). We present total, median and range of page views according to the following categories: medical oncology, haematology, radiation oncology, and supportive treatment information.
Ethics
------
Ethics approval to monitor eviQ utilization using eviQ web-logfiles and Google Analytics was obtained from the NSW Population and Health Services Research Ethics committee (approval number HREC/10/CIPHS/70).
Results
=======
Registrant characteristics
--------------------------
At December 31, 2011 there were 16,037 eviQ registrants, the majority of whom identified themselves as Australian health professionals (85.5%, n = 13,711), followed by consumers (7.7%, n = 1,237), and health professionals practising outside Australia (6.7%, n = 1,089).
Australian health professionals were registered as individuals (92.4%, n = 12,688) or units (7.6%, n = 1,043) \[Table [1](#T1){ref-type="table"}\]. Registrations for individual clinicians and units rose steadily over the study period with a median of 480 (range 237--889) new registrations each month (Figure [1](#F1){ref-type="fig"}).
{#F1}
######
Characteristics of eviQ Australian health professional registrants at the end of the observation period (December, 2011)
**Unit registration 1,043(7.6)** **Individual clinician 12,668(92.4)**
---------------------------------- ------------------- ---------------------------------- ---------------------------------------
**Health setting** *Primary health* 212(20.3) 3,009(23.8)
*Hospital* 831(79.7) 9,659(76.3)
**Health sector** *Public* 724(69.4) 9,005(71.1)
*Private* 259(24.8) 2,259(17.8)
*Both* 33(3.2) 632(5.0)
*Not specified* 27(2.6) 772(6.1)
**Location** *New South Wales* 378(36.2) 5,065(40.0)
*Victoria* 236(22.6) 2,577(20.3)
*Tasmania* 41(3.9) 405(3.2)
*Australian Capital Territory* 12(1.2) 280(2.2)
*Queensland* 212(20.3) 2,427(19.2)
*South Australia* 79(7.6) 856(6.8)
*Western Australia* 73(7.0) 873(6.9)
*Northern Territory* 12(1.2) 181(1.4)
**Clinican group** *Medical* \- 2,491(19.7)
*Nursing* \- 5,909(46.7)
*Pharmacy* \- 1,755(13.9)
*Radiation* \- 640(5.1)
*Other*^*a*^ \- 1,873(14.8)
**Professional experience** *\< 5 years* \- 6,348(50.1)
*5 -- 10 years* \- 1,608(12.7)
*\> 10 years* \- 2,756(21.8)
*Not specified* \- 1,956(15.4)
**Source of referral to eviQ** *Peer/colleague* \- 7,899(62.4)
*eviQ Education session* \- 877(6.9)
*Conference/Booth* \- 409(3.2)
*Internet search* \- 583(4.6)
*Cancer Institute communication* \- 1,401(11.1)
*Professional organisation* \- 985(7.8)
*Other* \- 481(3.8)
*Not specified* \- 33(0.3)
^a^Other: Academic, Allied Health, Clinical Information Manager, Medical Physicist, 'Other- not specified'.
The majority of individual clinician registrants nominated they were practising in the public hospital setting (71.1%) and this is where most unit registrations were also located (69.4%). Most of the individual and unit registrations originated from the state of NSW (40.0% and 36.2% respectively) \[Table [1](#T1){ref-type="table"}\].
The largest individual clinician group was nursing (46.7%) followed by medical doctors (19.7%), pharmacists (13.9%), and radiation therapists (5.1%). Approximately half of individual clinicians had less than five years' oncology experience (31.2% had less than 2 years and 18.9% had 2--5 years' experience). The most common eviQ referral sources were colleagues (62.4%), followed by Cancer Institute NSW communications (11.1%). Unit registrations are likely to represent a group of health professionals with varying years of clinical experience. As such, we did not report further on these registrations.
Patterns of eviQ use
--------------------
In a typical month (October 2011) there were a total of 169,647 webhits, 86.5% of which occurred during standard clinic hours (08:00--18:00; Monday to Friday). The volume of webhits was generally proportional to the number of registrations, with the largest registrant groups having the largest number of hits \[Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}\].
{#F2}
{#F3}
Not surprisingly, units had more than three times the webhit rate of individual clinicians (4,997 hits/100 versus 1,159 hits/100 site registrations) \[Figure [2](#F2){ref-type="fig"}\]. Further, pharmacists had at least 1.4 and up to three times the webhit rate (1,962 hits/100 site registrations) of other clinical groups such as medical doctors, nurses, and radiation therapists (1,373, 885 and 653 hits/100 site registrations, respectively) \[Figure [3](#F3){ref-type="fig"}\].
We also found that irrespective of professional group, clinicians with 5--10 years' experience had 1.5 times the webhit rate of clinicians with less than 5 years' experience (1,817 versus 1,184 hits/100 site registrations) and 1.4 times that of clinicians with more than 10 years of experience (1,817 versus 1,284 hits/100 site registrations).
### Typical user sessions
In October 2011, there were 20,611 eviQ site visits, 7,458 of which were by unique visitors. A typical visit was 7 minutes 33 seconds in duration and 9 pages were viewed. Most visits were accessed from a computer (99%, n = 20,326), with the remainder via a mobile device (1%, n = 285).
### Content accessed
In October 2011, there were 184,812 eviQ page views. This figure, based on Google Analytics page views, reflects data from all registrants which is why it is marginally higher than the total eviQ webhits quoted previously. The latter includes data from medical, nursing, pharmacy, radiation therapy and unit webhits only. The top 100 pages visited accounted for 77% (n = 142, 537) of all page views. With respect to page content, 7 were registration/login pages, 45 were transition pages (such as tabs directing users to specific content) and the remaining 48 were cancer treatment pages \[Table [2](#T2){ref-type="table"}\]. Registration/login and transition pages accounted for 85% (n = 122, 488) of the top 100 page views.
######
Profile of the 100 most commonly accessed eviQ pages in October 2011
**Page type** **n** **Page views** **Median time (range)**
-------------------------------- ------- ---------------- -------------------------
General pages
*Login/registration* 7 59,742 00:50 (00:26--01:53)
*Transition* 45 62,746 00:31 (00:11--01:46)
*Total* 52 122,488 00:31 (00:11--01:53)
Cancer treatment content pages
*Protocols* 39 16,483 02:27(00:51--04:46)
*Supportive/Other* 9 3,566 02:23 (00:28--02:51)
*Total* 48 20,049 02:27(00:28--04:46)
**Total (top 100 pages)** 100 142,537 00:57 (00:11--04:46)
Users spent nearly five times longer on cancer treatment content pages (median 02:27, range 00:28--04:46) compared to registration/login and transition pages (median 00:31, range 00:11--01:53) \[Table [2](#T2){ref-type="table"}\].
The most commonly accessed content pages (excluding login and transition pages) were medical oncology protocols (64/top 100 content pages); these were representative of the most common cancer types \[Table [3](#T3){ref-type="table"}\]. Haematology (16/100) and supportive treatment (18/100) pages were also frequently accessed; supportive treatment pages included drug calculators, patient information, and side effect management pages (e.g., antiemetic regimens, extravasation management, neutropenia management).
######
The 100 most commonly accessed cancer treatment content pages in October 2011
**Medical oncology (n = 64)** **n** **Median (range) page views/protocol** **Total page views**
------------------------------------------- ------- ---------------------------------------- ----------------------
*Breast* 18 343 (138--1143) 7,770
*Colorectal* 19 238 (144--720) 5,879
*Respiratory* 11 163 (140--714) 2,783
*Gynaecological* 5 195(140--318) 1,014
*Upper Gastrointestinal* 5 235(158--515) 1,388
*Other solid tumor* 6 198 (156--279) 1,230
**Haematology (n = 16)**
*Leukaemias* 4 177 (154--263) 770
*Lymphomas* 9 221(146--415) 2,155
*Myeloma* 3 241 (150--299) 690
**Radiation oncology (n = 2)**
*Radiation oncology protocols* 2 145.5(140--151) 291
**Supportive treatment & other (n = 18)**
*Drug calculators* 2 380(258--502) 760
*Management of side effects* 7 288(148--694) 2,108
*Patient information* 3 155(147--211) 513
*Other* 6 289 (145--432) 1,705
Discussion
==========
eviQ is an oncology protocol system that has been rated among the highest quality web-based oncology applications internationally \[[@B5],[@B15]\]. This paper details a comprehensive examination of the uptake and patterns of eviQ use by healthcare professionals. Despite the increasing popularity of systems of this kind, this is the first study to explore the way in which clinicians are using web-based oncology resources.
Our study demonstrates eviQ is used by the key professional groups involved in oncology care. Currently, we are unable to ascertain the proportion of the oncology workforce registered with eviQ due to an absence of comprehensive data on the number of health care professionals employed in oncology practice in Australia. However, the distribution of health professional registrations by state is directly comparable to the proportion of cancer cases by state which suggests that eviQ has been adopted by clinicians nationwide \[[@B16]\]. This is promising in terms of the potential impact of eviQ on cancer treatment delivery across Australia.
The uptake of eviQ is not surprising given our previous research showing the system is perceived by Australian clinicians to provide high quality support for the full spectrum of cancer care \[[@B11]\]. The system also rates highly in terms of usability and applicability of protocols to clinical practice \[[@B13],[@B14]\]. Our finding that the majority of registrants were referred to eviQ by their peers and colleagues is consistent with our previous work and demonstrates that the website is highly regarded by its users \[[@B11],[@B12],[@B17]\]. Our logfile analyses suggests that eviQ is used by clinicians at the point-of-care for cancer treatment delivery as most web activity (across all professional groups) occurred during standard clinic hours. Moreover, clinicians spent more time viewing specific chemotherapy protocols compared to other content and the cancer protocols represented the cancer burden for which chemotherapy is the standard of care (breast, colorectal, and respiratory cancers) \[[@B16],[@B18]\]. Clearly, not all web traffic during standard clinic hours will relate directly to point-of-care treatment and our previous research has shown eviQ is used around the clock as a reference tool to prepare for clinics and research various treatment options \[[@B11],[@B12]\].
Importantly, we found differences in the rates of eviQ use by health professional role and years of oncology experience. We found higher rates of use for oncology units compared with individual clinicians. This reflects real world clinical practice where numerous clinicians access the same computer and use a sole unit login during oncology clinics and the delivery of cancer treatment. Additionally, while pharmacists comprised one of the smallest individual clinician groups, they had the highest rates of use of any professional group. The more frequent use by pharmacists is likely due to their roles in the oncology treatment process: checking prescriptions/dose calculations and answering prescribing related questions on behalf of the multi-disciplinary team \[[@B11]\]. Additionally, clinicians with 5--10 years' oncology experience (across all professional groups) had the highest rates of eviQ use (versus \<5 and \>10 years' experience). It is highly likely clinicians with 5--10 years of experience are those working most actively at the point-of-care and making treatment decisions with less experienced staff working under their guidance. Further, our previous qualitative work demonstrated that the most senior oncology clinicians were the least reliant on eviQ to guide their practice \[[@B11]\].
In addition to better understanding the way in which different professional groups use eviQ our evaluation also highlights the utility of exploring use in multiple ways and the strengths and limitations of using specific indicators of use. To date crude webhits have been the standard eviQ metric. While this is a good indicator of the high level of web traffic, it tells little about the specifics of use. In contrast, rates are a valuable metric that allow for comparisons between different user groups. Our rate analyses could give the impression that individual level use is relatively low (an average of only 12 hits per month), however, one of the key limitations of our current analyses is the inability to examine the inter-registrant variability or to stratify analyses by frequent and non-frequent eviQ users.
The Google Analytics metrics (which account for extent of use) provide further detail on the content and time spent on eviQ during a typical session. However, logfile analyses are necessarily limited and should not be interpreted in isolation. Our program of work has made best use of other methodologies to address important questions relating to the use of computer support systems in oncology including system quality and clinicians perceptions about the utility of these systems \[[@B5],[@B11],[@B12],[@B15]\].
Conclusions
===========
Our study has shown eviQ is used widely, registrations continue to grow and the system is becoming an integral part of Australian oncology practice. Globally, it is recognised that the number of cancer cases will increase, so too will the number of available treatments, placing higher demands on the oncology workforce delivering care \[[@B19]\]. As such, systems like eviQ are likely to play a more significant role in the safe and effective administration of cancer treatments. Ongoing monitoring and evaluations are pivotal to understanding the contribution of web-based decision support systems in promoting efficient service delivery, standardizing care and improving patient outcomes.
Competing interests
===================
The authors report no financial disclosures. RLW is the Program Director of eviQ Cancer Treatments Online and SR is a Cancer Institute NSW employee, which poses a potential conflict of interest- Cancer Institute staff may have a conflict of interest in evaluating their own website. The objective nature of the data and having external authors (SAP, JL, NP) responsible for the study design, data analysis and interpretation have reduced the potential impact of this conflict of interest.
Authors' contributions
======================
JML contributed to design and conception of the study, data collection and analysis, interpretation and drafting of the manuscript. NP contributed to design and conception of the study, data collection and analysis, interpretation and drafting of the manuscript. SR contributed to data collection, interpretation and drafting of the manuscript. RLW contributed to interpretation and drafting of the manuscript. SP contributed to design and conception of the study, data analysis, interpretation and drafting of the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2407/13/112/prepub>
Acknowledgments
===============
This research was funded by a Cancer Institute New South Wales Translational Research Grant 09/THS/2-10). SP is a Cancer Institute New South Wales Career Development Fellow.
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1. Introduction {#sec1-ijerph-16-03084}
===============
National health expenditures are projected to grow at an average annual rate of 5.5 percent for 2018--2027 and represent 19.4 percent of gross domestic product in 2027. It is estimated that more than 95% of the trillions of dollars spent on health care in the United States each year funds direct medical services, even though 60% of preventable deaths are rooted in modifiable behaviors and exposures that occur in the community \[[@B1-ijerph-16-03084]\]. Effective coordination of health care, social services, public health, and community-based organizations could improve population health outcomes and advance health equity \[[@B2-ijerph-16-03084],[@B3-ijerph-16-03084],[@B4-ijerph-16-03084]\]. Some encouraging innovations are emerging, catalyzed in part by payers, delivery system reform, and the growth of value-based or shared-risk payment models, to support high-value community focused interventions. However, developing sustainable payment models to support such partnerships remain a challenge \[[@B5-ijerph-16-03084],[@B6-ijerph-16-03084],[@B7-ijerph-16-03084]\].
The Center for Medicare and Medicaid Services (CMS) is testing Accountable Care Organizations (ACOs) and shared savings models as part of its health care innovation program \[[@B8-ijerph-16-03084],[@B9-ijerph-16-03084],[@B10-ijerph-16-03084]\]. The Morehouse Choice Accountable Care Organization and Education System (MCACO-ES), or (M-ACO) is a physician-led, CMS Medicare Shared Savings Program (MSSP) ACO, that has developed and deployed an evolving model for clinical and operational integration among otherwise independently owned health care organizations, rendering primary care, specialty care other community-focused services to urban and rural populations in Georgia. Although committed to the development of an accountable care organization, the collaborators maintain a unique position to extend the definition in a manner that:Builds on the history, mission and value proposition for unparalleled community health improvement of M-ACO and its partners.Incorporates its distinguished training capacity to expand knowledge and understanding about value-based care to clinical care and health administration teams.Optimizes existing and new collaborative community relationships.Makes use of comprehensive, aggregated data and vast experience with underserved populations.Addresses health disparities and social determinants of health.
M-ACO's focus on measurable population and community health outcomes has a health equity lens that targets intervention of upstream factors impacting healthcare delivery (socioeconomic and social determinants), as well as individual factors (behavior; physiologic markers of disease, e.g. blood pressure; blood glucose). This model adopts the "Triple Aim" which strives to simultaneously improve population health, improve the patient experience of care, and reduce per capita cost. The Institute for Healthcare Improvement (IHI) developed the Triple Aim framework, and it has since become the organizing framework for the National Quality Strategy of the US Department of Health and Human Services (HHS) and for strategies of other public and private health organizations such as the CMS \[[@B11-ijerph-16-03084]\]
The primary objective of this paper is to present our research approach, early outcomes, lessons learned, and future directions for this physician-led and community-focused ACO. Supported by a comparatively larger body of literature, many ACOs represent large, hospital-led health systems, including those that "integrate" small group practices, often through purchase acquisition and other consolidation, whose health care practitioners then become employees of the large health system. Little or no research has been done on care delivery models that bring together independent safety-net, community-focused health care provider groups, enabled by robust centralized, communication and health technology platforms. Our early outcomes demonstrate key performance indicators related to care accessibility, clinical and quality outcomes improvement and earned shared savings delivered through programs that prioritize value-based interventions to high risk patients with multiple chronic conditions. The M-ACO participant organizations align in their innovation goals that continue to drive relevant and timely research in population health and advanced alternative payment reform models.
2. Materials and Methods {#sec2-ijerph-16-03084}
========================
2.1. M-ACO Participant Organizations {#sec2dot1-ijerph-16-03084}
------------------------------------
M-ACO participant organizations align on a collective mission-critical approach to data aggregation analysis and actionable intent to deliver high quality, equitable and cost efficient care, through community and school-based practice locations within preexisting evidence-based models as well as new care delivery models designed by the M-ACO. [Table 1](#ijerph-16-03084-t001){ref-type="table"} shows the list of M-ACO participant organizations that serve 169 rural and urban ambulatory practice locations primarily comprised of health care safety net organizations, and three mobile medical units with over 700 providers (of which 500 are primary care clinicians, including physicians, nurse practitioners and physician assistants)
2.2. Program Goals and Objectives {#sec2dot2-ijerph-16-03084}
---------------------------------
The program goals and objectives of M-ACO resonate with state and local health policy and healthcare reform intentions:Re-design Patient Care to attain "Triple Aim" goals \[[@B11-ijerph-16-03084]\]Research and engagement of providers and patients in predominantly underserved communitiesEducation and Training utilizing scalable, digital training modelsCommunity Health bridging biopsychosocial determinants to health outcomesPayment Reform with Aligned Incentives across independent health care organizations
2.3. Demographics of M-ACO Service Area {#sec2dot3-ijerph-16-03084}
---------------------------------------
In January 2018, the M-ACO engaged new partnerships and achieved over a 5-fold increase in its service area footprint from 33 ambulatory care health care centers to 169 rural and urban ambulatory locations, expanding its service role in the health care safety-net, moving M-ACO's total MSSP Medicare beneficiary attribution as appointed by CMS from approximately 5000 to 10,000. Current attribution as of this writing is approximately 15000, which includes a higher number of dual eligible older Medicare beneficiaries with disabilities who typically drive the highest medical cost expenditures as compared to other MSSP ACO cohorts and, in comparison, to national traditional Medicare beneficiary baselines. MSSP ACOs differ by constitution and characteristics, segmenting their Medicare beneficiaries, by severity: 1) ESRD 2) Disabled, 3) Aged Dual and 4) Aged Non Dual, averaging an annual average per capita spend of about \$10,000 per beneficiary \[[@B7-ijerph-16-03084]\] Since M-ACO's started in the MSSP on January 2013, the total attribution has tripled. Currently M-ACO has approximately 15,000 traditional Medicare and dual eligible attributables with a disproportionate share of the more costly dual and disabled beneficiaries that make up over 40% of all M-ACO attributed beneficiaries. Projected population growth rates in the M-ACO service area for those 50+ years of age is greater than double that of other age groups, and from the period 2000 until 2010, the 45--64 age cohort, or the "Baby Boomers", experienced an increase of almost 50 percent; the largest growth of any age group analyzed by the U.S. Census for the M-ACO service region \[[@B7-ijerph-16-03084]\]. By 2040, those aged 65+ will reach over 1.5 million in the Atlanta region, more than the entire Georgia 65 + population as of this writing. The Atlanta region is cited by the Center for Disease Control as an area experiencing significant health disparities that are significantly influenced by social determinants of health \[[@B12-ijerph-16-03084]\].
M-ACO partners care for a disproportionate share of high-need, complex populations with evolutionary psychosocial burden and endure extraordinary challenges in managing utilization, with comparatively limited resources, calling for a different approach to improving cost and health outcomes. Organizational re-design that is implemented among M-ACO partners, including those that "treat and teach" in the community, is aimed to provide for a distinct implementation methodology to maximize broad and deep competence and accountability in managing such populations under new standards and metrics, with performance results that can be translated to other non-Medicare populations \[[@B9-ijerph-16-03084]\] According to the Health Resources and Services Administration (HRSA), 85% of the M-ACO service area is considered a medically underserved area (MUA) with several health professional shortage areas (HPSAs) including primary care, dental care, and behavioral health services \[[@B13-ijerph-16-03084]\].
2.4. Hypothesis {#sec2dot4-ijerph-16-03084}
---------------
Our central hypothesis is that despite a disproportionate share of high-need patient population and the complexities associated with service fragmentation, the M-ACO health program redesign embedded in collaborative value-based performance principles, will achieve CMS targeted care, health, and cost objectives.
2.5. Approach {#sec2dot5-ijerph-16-03084}
-------------
M-ACO is guided by a strategic roadmap that reaffirms its shared goals and collaborative framework; based on the following 5 integrated success measures: 1) Promote financial viability that drives economic value to each ACO Participant organization to achieve positive net assets and operating margin to fund future advanced program enhancement and advanced technologies; 2) Enhance process improvement using evidence-based clinical and business practices; 3) Measurably improve service quality as defined by patients and their treatment plans (target improvement not less than 2% each year); 4) Maintain or improve physician and care team productivity that increase patients' overall access to care as seamlessly as possible under a Patient Centered Medical Home (PCMH) delivery model; and 5) Support or improve clinical quality as defined by the M-ACO, CMS and other payers ([Figure 1](#ijerph-16-03084-f001){ref-type="fig"}).
2.6. Implementing M-ACO Strategic Plan {#sec2dot6-ijerph-16-03084}
--------------------------------------
Three distinct, and linked strategies are used to implement the shared goals: 1) Integrated and centralized care coordination; 2) web accessible communication platform; 3) centralized health information technology data warehouse with interoperability. The data warehouse aggregates data from multiple sources (electronic health records, health information exchange and state databases) that is funneled into the centralized risk stratification model which appoints patients to the right resources for care management. The work of the M-ACO includes ongoing deployment and comprehensive assessment of independent and collective clinical integration, health information technology capabilities, care coordination sophistication, service defragmentation, patient satisfaction and safety, governance and compliance strength, and the existing affiliations with community partners. Particular emphasis is placed on the unique population that the M-ACO and its community-based participant organization locations serve, the financing of their care, and the various structures and governance upon which they depend. The integration model developed by the M-ACO incorporate the education programs of Morehouse School of Medicine (MSM), its academic health center partner, which includes MSM's primary care training capability, and its unique relationships among the M-ACO collaborators. In all respects, the application of benchmarks and standards are applied across all aspects of the organizational partners' clinical and administrative operations.
2.7. Centralized (Integrated) Care Coordination {#sec2dot7-ijerph-16-03084}
-----------------------------------------------
M-ACO serves a unique population in which more than half of the attributed Medicare beneficiaries have a disability or are eligible for Medicaid. Centralized care coordination (CCC) works to maximize clinical interventions by assisting with adherence to medications, treatment plans, and removing barriers to healthcare access \[[@B14-ijerph-16-03084],[@B15-ijerph-16-03084],[@B16-ijerph-16-03084],[@B17-ijerph-16-03084]\].
Care Coordination was initially fragmented, and lacked a cohesive process, with each organization working independently. To serve a population that is potentially at high risk for complications, care coordination needed to be standardized with defined roles that required commitment from all parties involved. The M-ACO Centralized Care Coordination is based on the Patient Centered Medical Home (PCMH) ([Figure 2](#ijerph-16-03084-f002){ref-type="fig"}). Standardization of care coordination begins at the point of care with the provider and care teams. Engaging the provider and health centers is just as important as engaging the patient. MCACO-ES discovered the biggest barrier to execution was related to the dissemination of information in a timely manner. Through the communication platform, specific campaigns and programs are provided through 1 to 3-minute short video clips with resources and toolkits available to all clinicians and care team.
The patient Centered Medical Home (PCMH) \[[@B14-ijerph-16-03084],[@B15-ijerph-16-03084],[@B16-ijerph-16-03084],[@B17-ijerph-16-03084]\] is at the core of the patient experience, and underpins the Team based Centralized Care Coordination (CCC). Similarly, the team based care transitions and the specialist referral pathway, integrate with the PCMH framework. This integrated delivery model is enabled by the communications platform and the centralized health information technology data warehouse, which are described below (see [Section 2.8](#sec2dot8-ijerph-16-03084){ref-type="sec"} and [Section 2.9](#sec2dot9-ijerph-16-03084){ref-type="sec"}).
2.8. Web Accessible Communications Platform {#sec2dot8-ijerph-16-03084}
-------------------------------------------
M-ACO's evolving model of a web accessible Communication Platform includes focused education campaigns on Annual Wellness Visits, Chronic Care Management Analytics, Medication Therapy Management, and Centralized Care Coordination. M-ACO also utilizes a centralized care coordination management platform in which care coordination activities are documented and tracked. Tools rendered from an enterprise data warehouse and advanced analytics platform, high utilizers, modifiable population cohorts, cost, utilization and extensive referral patterns are identified for care coordination intervention.
The standardized process is directly linked within the care coordination management system so that all care teams have direct access to care coordination activities in a central platform. The centralized care coordination process is focused on connecting risk stratified patients with primary care providers to prevent complications for dual eligible and ambulatory sensitive conditions resulting in hospitalizations (See [Table 2](#ijerph-16-03084-t002){ref-type="table"} and [Table 3](#ijerph-16-03084-t003){ref-type="table"}). The ultimate goal is to ensure patient-centered outcomes of centralized care coordination, including the use of mobile health technology, such as Health 360x in order to maximize clinical interventions of M-CACO providers by assisting with adherence to medications, treatment plans, and removing barriers to healthcare access \[[@B18-ijerph-16-03084],[@B19-ijerph-16-03084],[@B20-ijerph-16-03084]\].
This Data management pillar of the communication platform provides an evolving solution in working with multiple EMR systems within the ACO. The data received from various sources will go through a matching algorithm utilizing the EMPI methodology. The MCACO-ES data will flow in one direction from all known EMRs (i.e., NextGen, EPIC, E-Clinical Works, CMS and/or IDX), and be stored within a centralized Operational Data-Store (ODS). Once data is validated and aggregated, the summarized data will be moved into an additional database, which will allow for decision support and analytic reporting, when fully operationalized.
2.9. Enterprise Data Warehouse with Interoperability {#sec2dot9-ijerph-16-03084}
----------------------------------------------------
M-ACO informatics capability includes an integrated data warehouse, which supports Point of Care Data Integration, with HIPAA compliant data transfer from M-ACO participants and suppliers, through secure access to electronic medical records (EMR) and personal health information (PHI) data sources. The HIPAA compliant enterprise data warehouse and advanced analytics platform is configured to support the centralized care coordination and communications platform. As shown in [Figure 3](#ijerph-16-03084-f003){ref-type="fig"}, the M-ACO data will flow in one direction from all known electronic medical records (EMRs) of participating organizations, including NextGen, McKesson Practice Partners, E-Clinical Works, EPIC, CMS, and/or IDX, and stored within a centralized Operational Data-Store (ODS). After data is validated and aggregated, the summarized data is moved into an additional database instance, which will allow for decision support reporting to the M-ACO senior management. After the data goes through a series of quantitative and qualitative quality control checks, and the reports are approved, the data is sub-populated into aggregates for PCMH, Quality Coalition, and Quality and Cost Metrics (including Meaningful Use and Medicare Shared Savings).
2.10. Data Analysis {#sec2dot10-ijerph-16-03084}
-------------------
All data are reported as rates (percent or per 10,000 beneficiaries). Unit of analysis/summary was practice organization for [Table 1](#ijerph-16-03084-t001){ref-type="table"}, patients for [Table 2](#ijerph-16-03084-t002){ref-type="table"}, [Table 3](#ijerph-16-03084-t003){ref-type="table"} and [Table 4](#ijerph-16-03084-t004){ref-type="table"} and service for [Table 5](#ijerph-16-03084-t005){ref-type="table"}. Summary comparisons are shown between M-ACO and All Medicare Shared Savings Program (MSSP).
3. Results {#sec3-ijerph-16-03084}
==========
3.1. Medicare Beneficiary's Health Conditions in M-ACO Compared with All Medicare Shared Savings Program (MSSP) ACOs {#sec3dot1-ijerph-16-03084}
--------------------------------------------------------------------------------------------------------------------
Medicare beneficiaries in MCACO-ES have higher rates of multiple complex conditions. As shown in [Table 2](#ijerph-16-03084-t002){ref-type="table"}, Medicare beneficiaries in M-ACO MSSP have 3--5 times higher rates of End Stage Renal Disease (ESRD) and Disabled patients (including Dual Eligible and Non-Dual Eligible). These patient populations as well as the Aged Dual Eligible patients have health challenges that are complicated by social determinants, such as low socioeconomic status and a zip code or place of residence that has limited access to health resources. By policy design, Aged Non-Dual Eligible Medicare patients do not qualify for Medicaid, and therefore have less complexities based on social determinants. This group comprises less than half of the Aged M-ACO population compared with 80% for the MSSP ACOs. Per the data set, All MSSP ACOs include averaged data from 400 ACOs participating under the MSSP.
Similarly, M-ACO has higher rates per 10,000 beneficiaries of hierarchical chronic condition (CMS-HCC) that are associated with increased hospitalization and cost of care, such as diabetes with complications, chronic obstructive pulmonary disease, congestive heart failure, and major depression. ([Table 3](#ijerph-16-03084-t003){ref-type="table"}). M-ACO selected the most common chronic conditions (or HCCs) based on the volume of beneficiaries in comparison to the average MSSP ACO cohort.
3.2. MSSP Shared Savings Outcomes for M-ACO {#sec3dot2-ijerph-16-03084}
-------------------------------------------
Despite high rates of multiple chronic conditions and health disparities, MCACO-ES generated shared savings for several performance years. For Performance Year 2015, MCACO-ES received \$1,675,995 (based on a 50:50 sharing model with CMS). Shared savings is based on ACOs either meeting or remaining below the CMS-designated expenditures (or benchmark) per beneficiary. Another important factor includes triaging patients from the emergency room into primary care. Organizations that usually generate shared savings remain 5% below the benchmark cost reduction target and have positive risk adjustment factors. Risk adjustment factors are derived from patient demographics, such as age, gender and Medicare cohort and Medicare risk scores based on ICD-10 coding. M-ACO distribution of Shared savings:Proportion invested in infrastructure: 24%,Proportion invested in redesigned care processes/resources: 15%,Proportion of distribution to ACO participants: 61%.
3.3. M-ACO Compared with All MSSP ACO Quality Measures as Publicly Reported by CMS {#sec3dot3-ijerph-16-03084}
----------------------------------------------------------------------------------
M-ACO attained lower rates for several CMS Quality Measures, including four patient satisfaction measures, such as timely appointments and access to specialists and three quality measures including: influenza vaccination, and hypertension control (See [Table 4](#ijerph-16-03084-t004){ref-type="table"}). These quality measures along with twenty-three other measures are weighted and calculated by CMS to provide the M-ACO's overall quality score, which was 92.06% in 2017.
3.4. M-ACO Access Primary Care Services and Specialist Physicians Compared with All MSSP ACO as Publicly Reported by CMS {#sec3dot4-ijerph-16-03084}
------------------------------------------------------------------------------------------------------------------------
The number of primary care visits in M-ACO have increased by 35.4% since 2016 compared to the CMS national benchmark which increased by 14% in the same time period (See [Table 5](#ijerph-16-03084-t005){ref-type="table"}). Increases in the number of primary care services are congruent with increases in overall cost of care, due to patients being diagnosed with chronic conditions in the primary care setting. The data also reflects increased emergency room and inpatient utilization as a result. Patients not getting into primary care does not impact whether shared savings are earned and can have a negative impact on cost containment, especially among high risk patients in M-ACO.
4. Limitations {#sec4-ijerph-16-03084}
==============
The operational efficiencies of the enterprise data warehouse remain a work in progress, primarily due cost of implementation and to non uniform adoption across organizations.
Despite these challenges, the Communications Platform's focused education campaigns on Annual Wellness Visits, Chronic Care Management Analytics, Medication Therapy Management, and Centralized Care Coordination are informing the M-ACO improvement process across practice organizations`.`
4.1. HCC Coding Opportunity {#sec4dot1-ijerph-16-03084}
---------------------------
M-ACO HCC and Demographic risk scores are consistently lower, compared with CMS national means (see [Table 6](#ijerph-16-03084-t006){ref-type="table"}).
These data are informing 2019 HCC Coding Campaign (95% of Providers On-Boarded) and 37% HCC course completion, as well as 40% Annual Wellness Visit Completion, which increases billable care coordination management.
The data also informed care coordination targets for 2019: of 15,014 total attribution, 4725 are eligible for billable care coordination management (CCM); with a target set at 38% of the total eligible, and average expenditure of \$8,715.23 per non ESRD beneficiary, the total target expenditure for shared savings is \$83,903,729.00.
4.2. Performance and Impact based on 1) Populations of Interest and 2) Processes and Outcomes of Care {#sec4dot2-ijerph-16-03084}
-----------------------------------------------------------------------------------------------------
While M-ACO has made significant progress in improving the quality of care, challenges remain with regard to access to specialists, as well as several CMS quality measures for preventive care, hospitalization, and diabetes control. M-ACO's Centralized Care Coordination with use of patient engagement technology and telemedicine \[[@B18-ijerph-16-03084],[@B19-ijerph-16-03084],[@B20-ijerph-16-03084],[@B21-ijerph-16-03084]\], are opportunities to address these challenges. Evaluation for ongoing assessment of M-ACO's Care Coordination Agency Model tests the effectiveness of the Care Coordination Agency Model (control group) compared to those cohorts that do not receive care coordination (experiment group). Successful improvement strategies shown to improve patient health outcomes will measure the operational cost to provide care coordination services compared to the savings generated from improving health outcomes for patients with multiple chronic conditions. The model with optimal achievement will be deemed as the standardized care coordination protocol, which will be used to train care coordinators across M-ACO practices. Morehouse School of Medicine will assist with the development of dashboards to measure, track and analyze data collected from both models. Potential clinical impact of this approach among high risk ACO patients with diabetes from a single health center was recently demonstrated, using a sample of 41 patients in a NIH funded pilot study: 19 were diagnosed with Type 2 diabetes. 58% (11 patients) reported decreasing A1C since enrollment into centralized care coordination with a notable reduction in A1C from 13.5% to 6.8% within a span of only 3 months \[[@B21-ijerph-16-03084]\]. This original research was sponsored by the National Institutes on Minority Health and Health Disparities (NIMHD) at NIH, through funding for the Transdisciplinary Collaborative Center (TCC) for Health Disparities Research at Morehouse School of Medicine under the leadership of Dr. David Satcher, Founder and Senior Advisor of the Satcher Health Leadership Institute and 16th Surgeon General of the United States. The TCC highlights collaborative and health policy innovations that address upstream and modifiable risks to attaining health equity \[[@B22-ijerph-16-03084],[@B23-ijerph-16-03084],[@B24-ijerph-16-03084],[@B25-ijerph-16-03084]\]. The M-ACO will continue to explore and prioritize such interventions for testing and scaling across the ACO and similar practices serving high risk patient populations, including dual eligible Medicare beneficiaries.
In describing the framework for evaluating ACOs, Fisher et al \[[@B26-ijerph-16-03084]\] note the importance of tracking the impact of the accountable care models on subgroups of the population at greater risks, such as socioeconomically disadvantaged populations or people who are cared for by safety-net providers, anticipating greater challenges of meeting target savings or quality outcomes among such disadvantaged and high risk patients. Processes and outcomes of care such as patients' experience of care, including degree of activation, engagement in shared decision making, and decision quality are especially important in the evaluation framework \[[@B26-ijerph-16-03084]\]. Inclusion of such populations and patient experience outcomes are not generally described across ACOs \[[@B26-ijerph-16-03084],[@B27-ijerph-16-03084]\].
4.3. Future Directions to address Limitations in Data Integration, Performance and Impact {#sec4dot3-ijerph-16-03084}
-----------------------------------------------------------------------------------------
Data integration across disparate EMRs remain a challenge across healthcare. Recent technology advancement with FHIR (Fast Healthcare Interoperability Resource) enables apps to connect to electronic health record systems \[[@B28-ijerph-16-03084],[@B29-ijerph-16-03084]\]. Even as it continues to implement its enterprise data warehouse integration model, the M-ACO is exploring app enabled FHIR EMR accessibility using a patient engagement platform that is currently used by ACO member patients to monitor and track their health \[[@B18-ijerph-16-03084],[@B20-ijerph-16-03084],[@B21-ijerph-16-03084]\]. Such patient engagement application will enable data collection on patient experience and shared decision making, a critical dimension for evaluation and care outcomes.
5. Discussion {#sec5-ijerph-16-03084}
=============
Based on the MSSP model, the project objectives focused on 1) Promoting financial viability 2) Enhancing process improvement 3) Measurably improving service quality 4) maintaining or improving physician and team productivity 5) Improving quality as defined by CMS. Our findings show that despite high rates of multiple chronic conditions and health disparities, M-ACO, is delivering quality care and has achieved incentive payments in the Medicare Shared Savings Program. Given constant changes in the financing of health care services, M-ACO remains ready and committed to the mission of improving population health through evidence-based clinical and administrative models that prioritize the patient experience, while addressing social determinants and behavior risk modification. Already, M-ACO's strategic approach has improved the health status of its attributed population and it has been financially rewarded for such outcomes through shared savings incentive payments.
The value and contribution of this study is the unique opportunity to describe MSSP outcomes in a large safety net and predominant primary care practice of a diverse Medicare beneficiary patient population.
A comparison of MSSP ACO cohorts from 2012 and 2013 showed savings in the earlier cohort of primary care based MSSP, and less so for hospital based MSSP \[[@B27-ijerph-16-03084]\] However, these cohorts were less diverse with 82% white beneficiaries, and only 8.2% and 5.1% Black and Hispanic beneficiaries respectively; and only 8.7% recipients below the federal poverty level \[[@B27-ijerph-16-03084]\].
Future and pursuant work will focus on empirical validation of the summary comparisons presented in this manuscript by expanding data to allow inferential analyses that will employ among other approaches statistical methods like random effects and propensity scores to minimize bias and control for confounders.
6. Conclusions {#sec6-ijerph-16-03084}
==============
M-ACO will continue to seek ways to innovate care delivery, as we expand our practice geographic area, to ultimately deliver care to 100,000 beneficiaries with expanding coverage to rural and urban communities across Georgia. The Centralized Care Coordination model with attention to preventive care, care transitions and referral pathways are key targets for innovation. Integration of mobile health technology for patient engagement and retention, as well as telemedicine technology, will be tested to determine how these technologies improve quality care in the high-risk patients with complexities and social determinants, that we serve at the Morehouse Choice Accountable Care Organization and Education System.
We thank the CEOs, Medical Directors, Physicians and Staff of Morehouse Choice ACO Member Practices: Albany Area Primary Health Care: Shelly Spires; Tracey Rosenbaum (CMO); Atlanta Family Physicians: Ahmad Jingo; CareConnect Health: Ajay Gehlot; Community Health Care Systems: Carla Belcher; The Clinic For All: Patricia Ferguson; East Georgia Healthcare Center: Jennie Wren Denmark, Daryl McCartney, MD (CMO); Family Health Centers of Georgia: Michael Brooks (CEO); Keila Brown (CMO); Four Corners Primary Care Centers: Brian Williams (CEO); Donna Sinclair (CMO); Morehouse Healthcare: Valerie Montgomery Rice (CEO/President); Cheryl Franklin (Medical Director---HM); Michelle Nichols (Medical Director---BW) Roland Matthews (Chair OB/GYN); Medical Associates Plus: J.R. Richards; MedLink Georgia: Dave Ward; North Georgia Health Care Center: Delanie Hunter; Southside Medical Center: David Williams (CEO); Donna Sinclair (CMO). We thank Jeff Street of Via Consulting Group for work done on the Communication Platform and Data Flow technical design and relative support.
M.B. and E.O.O. are equal first author contributors. Conceptualization, E.O.O. and M.B.; Data curation, D.O., Y.S., A.Q., C.S. and E.A.-M.; Formal analysis, Y.S., A.Q., M.M., C.S. and E.A.-M.; Funding acquisition, E.O.O. and P.P.; Investigation, E.O.O., M.B. and P.P.; Methodology, E.O.O., M.B., D.O., Y.S., A.Q., and M.M.; Project administration, D.O.; Resources, E.O.O., M.B., P.P., V.M.R., D.W. and M.B.; Software, A.Q. and C.S.; Supervision, P.P., C.F. and A.Q.; Writing---original draft, E.O.O., M.B. and D.O.; Writing---review & editing, E.O.O., M.B., D.O., P.P., C.F., A.Q., C.S., V.M.R., D.W., M.B., E.A.-M., D.M. and D.D.
This research was partially supported by the National Institute on Minority Health and Health Disparities (NIMHD): U54MD008149, 8U54MD007588, and U54MD008173; National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002378. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare no conflict of interest.
{#ijerph-16-03084-f001}
{#ijerph-16-03084-f002}
{#ijerph-16-03084-f003}
ijerph-16-03084-t001_Table 1
######
M-ACO Participant Organizations and Health Center Locations Across Urban and Rural Georgia.
Organization Practice Type and Locations
------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Morehouse School of Medicine and Morehouse Healthcare Primary care and select multispecialty: Two Urban locations
Southside Medical Center FQHC: Eleven Urban and Rural locations including school-based health centers, Adult and Pediatric Primary care, specialty care, dental optometry, behavioral health and mobile medical and dental unit.
Family Health Centers of Georgia FQHC: Seven Urban locations including school-based health centers, Adult and Pediatric Primary care, specialty care, dental optometry, behavioral health and mobile medical and dental unit.
Four Corners Primary Care FQHC: Three Urban locations
CareConnect Health FQHC: Forty-one Rural locations, including school-based clinics, Dental, OB/GYB, Urgent Care Centers.
Medical Associates Plus FQHC: Eight Suburban and Rural locations including Adult and Pediatric Primary care, specialty care, dental optometry, behavioral health and pulmonary health
Community Health Care Systems FQHC: Thirteen Rural locations including Adult and Pediatric Primary care, podiatry, behavioral health and mobile medical unit
East Georgia Healthcare Center FQHC: Ten Rural locations including Adult and Pediatric Primary care, specialty care, dental and behavioral health
MedLink Georgia FQHC: Eighteen Rural locations including Adult and Pediatric Primary care, specialty care, dental optometry, behavioral health
Albany Area Primary Health Care FQHC: Twenty-six Rural locations including school-based health centers, Adult and Pediatric Primary care, specialty care, dental optometry, behavioral health and mobile medical and dental unit.
North Georgia Healthcare Center Independent Rural practice including Adult and Pediatric Primary care, specialty care, dental optometry, behavioral health and physical therapy
Atlanta Family Physicians Independent Urban practice
The Clinic For All Independent Urban Practice
ijerph-16-03084-t002_Table 2
######
Medicare Beneficiary Health Conditions (%) in MCACO-ES Compared to All MSSP ACOs.
Medicare Beneficiary MCACO-ES \* All MSSP ACOs
---------------------------- ------------- ---------------
ESRD 2.96% 0.69%
ESRD Dual Eligible 1.34% 0.24%
ESRD Non-Dual Eligible 1.62% 0.45%
Disabled 33.36% 11.72%
Disabled Dual Eligible 17.61% 5.56%
Disabled Non-Dual Eligible 15.75% 6.14%
Aged 63.68% 87.41%
Aged Dual Eligible 15.99% 6.19%
Aged Non-Dual Eligible 47.69% 80.86%
\* *p* \< 0.001.
ijerph-16-03084-t003_Table 3
######
Frequencies and Rates per 10,000 Beneficiaries by Disease Group (CMS-HCC) for Assigned Beneficiaries.
CMS-HCC Condition MCACO-ES All MSSP ACOs
----------------------------------------------------------------- ---------- ---------------
Diabetes w/Chronic complications 2134 1670
Chronic Obstructive Pulmonary Disease 1481 1318
Congestive Heart Failure 1202 1196
Morbid Obesity 1180 544
Diabetes without Complication 1162 1172
Vascular Disease 1120 1402
Major Depressive, Bipolar & Paranoid Disorder 930 737
Specified Heart Arrhythmias 898 1525
Rheumatoid Arthritis and Inflammatory Connective Tissue Disease 644 716
Seizure Disorders and Convulsions 500 295
ijerph-16-03084-t004_Table 4
######
Select ACO Quality Measures as publicly reported by CMS.
Select ACO Quality Measures M-ACO (%) All MSSP ACOs (%)
---------------------------------------------------- ----------- -------------------
Getting Timely Care, Appointments, and Information 72.20 80.60
How Well Your Providers Communicate 90.71 93.13
Access to Specialists 78.43 83.32
Shared Decision Making 70.36 75.85
Influenza immunization 62.37 72.52
Diabetes A1c poor control 23.94 16.74
Hypertension control (High BP control) 60.18 71.47
ijerph-16-03084-t005_Table 5
######
Primary Care Services as publicly reported by CMS. FQHC/RHC=Federally Qualified/Rural Health Clinic; FFS=Fee for Service.
Primary Care Services M-ACO All MSSP ACOs National Assignable FFS
-------------------------------------------------------------- ------- --------------- -------------------------
With a Primary Care Physician 8760 9711 10,120
With a Specialist Physician 1004 3811 3713
With a Nurse Practitioner/Physician Assistant/Clinical Nurse 3383 4348 4491
With a FQHC/RHC 762 1110 1427
ijerph-16-03084-t006_Table 6
######
HCC and Demographic Risk Scores by Medicare Category in M-ACO HCC compared with CMS (National Means).
Medicare Category M-ACO HCC Risk Score CMS-HCC Risk Score (National Mean) M-ACO Demographic Risk Score CMS Demographic Risk Score (National Mean)
------------------- ---------------------- ------------------------------------ ------------------------------ --------------------------------------------
ESRD 0.893 1.115 1.005 1.021
Disabled 0.865 1.282 0.987 1.057
Aged/Dual 0.750 1.805 0.946 1.562
Aged/Non-Dual 0.904 1.055 1.002 0.911
[^1]: Michelle Brown and Elizabeth Ofili are equal first author contributors.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Tuberculosis (TB) is a rampant disease with 8.8 million new cases reported in 2010 \[[@B1]\]. According to the World Health Organization (WHO) it is the leading infectious cause of mortality worldwide killing 1.45 million people \[[@B1]\]. Although the majority of the cases of TB occur in developing countries, recently an alarming increase in the incidence of TB has been reported in many of the developed countries. This resurgence of TB is being attributed to the increased incidence of alcohol and drug abuse, human immunodeficiency virus, immunosuppression \[[@B2]\], and the increased influx of immigrants from third-world countries \[[@B3]\].
Extrapulmonary manifestations of TB are also fairly common with skeletal system involvement being the most frequent \[[@B4]\]. Spinal tuberculosis, first described by Sir Percival Pott in the Eighteenth century, accounts for 50% of these cases \[[@B5]\] and results in immense morbidity and mortality. Although spinal TB presents clinically with a multitude of symptoms, back pain is the major symptom. Spinal TB can lead to bone destruction and vertebral collapse resulting in paravertebral abscesses and deformity. If prompt management is not employed, severe neurological symptoms ensue, which can lead to a debilitating consequence. In fact, spinal TB still remains the leading cause of non-traumatic paraplegia in developing nations \[[@B6],[@B7]\].
If cases of spinal TB are diagnosed early, anti-tubercular chemotherapy along with immobilization can be effective. It has been argued that if patients do not respond to conservative treatment or show progressive neurological symptoms, spinal deformity or instability, surgical intervention should be the mainstay of treatment \[[@B8],[@B9]\]. However, anterior resection or combined anterior and posterior resection can result in substantial morbidity \[[@B10]\]. As a result, percutaneous needle aspiration (PCNA) to drain tuberculous abscesses has gained increasing popularity in the management of spinal TB \[[@B4],[@B11]\]. The use of PCNA along with antituberculous drug therapy has shown encouraging results and seems to be an effective prospect in the management of spinal TB.
In this prospective study, we present our experience of conservative management of spinal TB from an endemic region, Pakistan. This is to reiterate the importance of managing spinal tuberculosis conservatively, keeping surgical intervention as the last resort.
Materials and Methods
=====================
1. Patients
-----------
During the four year study period from January 2006 to December 2009, 47 patients (M=14, F=33) attending Ziauddin University Hospital were included in the study. Prior to enrollment, an informed consent was taken from all patients. Tuberculosis of the spine was suspected on the basis of history followed by an X-ray ([Fig. 1](#F1){ref-type="fig"}) and a magnetic resonance imaging (MRI) of the spine. In our study, suspected MRI lesions were those which showed an isointense lesion on T1 or a high signal lesion on T2 involving the disc, body, paravertebral region and sometimes extending into the epidural space or psoas fascia ([Figs. 2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}). The patients were divided into either mild, or a moderate to severe group, on the basis of symptoms and signs. Mild patients were classified as those having any degree of pain and/or mild motor weakness. Moderate-severe patients had severe motor deficits or any degree of sphincter disturbance. All patients were initially subject to computed tomography (CT) guided PCNA for the confirmation of diagnosis. Simultaneously, antituberculous therapy (ATT) was also started and continued for at least 12 months (average, 12.3 months; maximum, 18 months) considering the endemic state of TB in this region.
Patients with mild symptoms were kept on ATT for the defined period irrespective of the outcome of PCNA. Patients with moderate-severe symptoms who responded to successful PCNA in terms of significant improvement of symptoms especially pain within 48 hours of the procedure were kept on ATT only.
2. Anti-tuberculous therapy
---------------------------
The standard ATT consisted of four drugs; isoniazid (5 mg/kg; maximum, 300 mg/day), rifampicin 15 mg/kg; maximum, 600 mg/day, ethambutol (15-25 mg/kg; maximum, 2 g/day), pyrazinamide (15-30 mg/kg; maximum, 2 g/day). Pyridoxine was also added to the regimen prophylactically. This regimen was instituted for three months followed by withdrawal of pyrazinamide for the rest of the period.
3. Percutaneous needle aspiration
---------------------------------
PCNA was performed using CT guidance, for which patients were placed in a prone or lateral position on the CT scanner table. Few pilot scans were taken beforehand to tailor the approach for safe insertion of the spinal needle and localization of the lesion. An 18 gauge spinal needle was inserted percutaneously, depth and route of which was dictated by CT scan. Necrotic material was aspirated as much as possible using a 10 mL syringe. If blocked, the needle was replaced with another needle until the aspirate stopped coming. The sample collected from PCNA was sent for an acid fast bacillus (AFB) smear as well as culture and sensitivity of *Mycobacterium tuberculosis*.
4. Surgery
----------
Patients with moderate-severe disease who did not show remarkable improvements within 48 hours after successful PCNA were candidates for surgery. These patients showed persistent symptoms despite PCNA and ATT. Patients in the same severity group with failed PCNA were also enrolled for operative management. This group did not respond to ATT alone and had persistent neurology. Spinal deformity or instability was not the indication for surgery in any of our patients.
The surgical procedure varied from anterio-lateral to posterior decompression. The prime objective of surgical procedure was to debride the necrotic material causing the pressure effect on the thecal sac while secondarily keeping removal of bone to a minimum. This resulted in no significant increase in instability or deformity with regards to the spine thus the need for instrumentation was eliminated. Moreover, all patients who underwent surgery for the cervical, the thoraco-lumbar junction or the lumbar region were prescribed an appropriate brace or collar for 6 months postoperatively.
5. Follow-up
------------
Patients were recalled at 6 week intervals for the first two visits followed by 3 monthly reviews until 12 months after the end of ATT. The mean follow-up period was 24.5 months (range, 24-36 months). They were managed and monitored for pain, erythrocyte sedimentation rate (ESR), and neurological status with special attention given to motor weakness. Follow up X-rays of the spine were done at 3 monthly intervals, or in those who could afford it, MRIs were completed at 6 monthly intervals. Imaging studies to monitor abscess recurrence were completed regularly until end of ATT treatment with a last imaging study 6 months after the completion of ATT. Smoothening of the vertebral end plates with or without fusion of vertebral bodies on X-ray ([Fig. 4](#F4){ref-type="fig"}) was considered as evidence of healing. Moreover, loss of high signal on T2 weighted MRI along with resorption of abscess ([Fig. 5](#F5){ref-type="fig"}) further confirmed the resolution of infection.
Results
=======
During the four year study period 47 patients (M:F=1:2.35) attending our center were included in the study. The mean age of individuals was 33.9±13.7 years. Presenting complaints included pain in 45 (95.7%), motor weakness in 40 (85.1%) and sphincter involvement in 6 (12.8%) patients. An MRI of the spine showed a paravertebral abscess in 37 (78.7%), disc and body destruction in 29 (61.7%) and an epidural abscess in 12 (25.5%) patients whereas, psoas abscess was seen in 4 (8.5%) patients. Two (4.2%) patients had concomitant hydrocephalous secondary to TB meningitis at the time of presentation and hence, were candidates for a ventriculoperitoneal (VP) shunt and extended ATT treatment up to 18 months.
Regarding the grade of motor weakness, 2 (5%) had power 5/5, 22 (55%) had 4/5, 14 (35%) had 3/5, 1 (2.5%) had 2/5, and 1 (2.5%) had power 1/5 graded on the basis of the patient\'s effort \[[@B12]\] at presentation.
The cervical spine was involved in 6 (12.8%), the cervico-dorsal spine in 1 (2.1%), the dorsal spine in 16 (34%), the dorso-lumbar spine in 4 (8.5%), the lumbar spine in 19 (40.4%) and the lumbo-sacral spine was involved in 1 (2.1%) patient. Ten (21.2%) patients had single level involvement, 35 (74.4%) patients have two levels, and 2 (4.2%) patients showed three or more levels of involvement. L1 was the most common level involved in our study with 10 (21.2%) patients affected at this level of the spine, irrespective of the number of levels and region of spine involved.
All patients were initially planned for PCNA; however four patients denied undergoing PCNA and hence, were continued on ATT only. PCNA was successfully performed on 39 (90.6%) patients while it failed in 4 (9.3%) patients. All patients underwent single aspiration except two. The first patient in this series had PCNA done in two sittings, which was done electively because of a large amount of bilateral abscesses. Another patient required repeat PCNA treatment one month later during the treatment phase due to a residual abscess. The total amount of necrotic material aspirated during successful PCNA varied from 0.5 mL to as much as 300 mL with a mean of 12.2 mL. Following successful PCNA, AFB smear was positive in 35 (89.7%) patients while the cultures turned out to be positive in all cases. All cultured bacilli were sensitive to first line anti-tuberculous drugs.
The mean ESR during presentation was 64.2±6.3 mm in the first hour, which fell to 37.3±3.85 mm in the first hour at 6 weeks on ATT. At the end of treatment, mean ESR was 14.2±2.6 mm, which corresponded with resolution of symptoms.
Decompressive surgery was performed in 9 (19.1%) patients, with 7 (14.9%) undergoing anterio-lateral decompression and 2 (4.2%) undergoing posterior decompression; however, instrumentation was not performed on any of our patients for the aforementioned reason. Out of these surgical candidates, 4 had failed PCNA attempts and 5 had indications despite successful PCNA including persistent motor weakness, pain and/or sphincter involvement. All surgical patients were prescribed appropriate spinal braces for 6 months postoperatively, in order to provide external support to the spine since instrumentation was avoided.
Forty-four (93.6%) patients in our study showed good compliance with ATT therapy and fully recovered without any residual weakness or pain. However, 3 (6.4%) patients were non-compliant with the treatment. Patients who demonstrated poor compliance deteriorated during the course. One compliant patient died of an unrelated medical condition before the completion of the regimen. Moreover, none of the patients showed any signs for the recurrence of TB, in the spine or any other region of the body, during the follow-up period.
Discussion
==========
According to the Global Tuberculosis Control WHO Report 2011 \[[@B1]\], Pakistan is amongst the highest burdened countries for tuberculosis with 150 per 100,000 new cases in 2010. This high incidence is due to a number of factors including low socio-economic conditions, over-crowding, multi-drug resistance and most importantly, non-compliance to ATT.
The mean age at presentation in our study was 33.9 years with a range of 12 to 68 years which is about a decade younger than that found in other studies \[[@B4]\]. An overwhelming number of patients in our study were female (M:F=1:2.35). This is in contrast to the extensively reported male predominance within the literature \[[@B3],[@B8],[@B13]\]. However, another study from the same region reported similar trends for younger individuals and gender predominance \[[@B14]\].
In our study the most common symptoms presented were pain and motor weakness. Axial pain of insidious onset is regarded as one of the most common symptoms of spinal tuberculosis. However, it can present itself with a wide range of symptoms and complications \[[@B15],[@B16]\]. Atypical presentations for TB of the spine include destruction of the posterior arch, isolated neural arch or disease without disc involvement ([Fig. 3](#F3){ref-type="fig"}). Other clinical features in the active stage of the disease are malaise, weight loss, anorexia, night sweats and low grade fever with stiff spine and sometimes kyphosis or cold spine \[[@B17]\]. This wide range of clinical presentations makes the diagnosis of spinal TB difficult.
The diagnosis can be made using an AFB smear, an AFB culture and the polymerase chain reaction (PCR) of *Mycobacterium tuberculosis*. The ESR and Mantoux test can be used to assist in the diagnosis. Radiological studies also play a key role in establishing the diagnosis of spinal TB.
The optimal technique for the diagnosis of a spinal infection and its sequel is MRI \[[@B18]\]. The difference between inflammatory and neoplastic pathologies may not be appreciated on plain X-rays or CT scans and hence, MRI is needed, which reveals TB as a contrast enhancing rim \[[@B19]\]. This appearance can be readily differentiated from a metastatic lesion first involving the pedicles, which are initially spared in spinal TB. In addition to MRI, a CT-guided biopsy has been regarded as an efficacious method for the establishment of diagnosis \[[@B11],[@B20]\]. Finally, adequate response to ATT can also be used to establish the diagnosis \[[@B21]\].
The AFB smear was prepared using Ziehl Neelsen stain and Lowenstein-Jensen medium was used for the culture of the organism. Although PCR is a quicker and more reliable way for detecting tuberculous bacillus, it was not used in our study considering its high cost and extensive positivity of the AFB smear and culture in our patients.
It has been reported that the dorsal spine is the most common site of involvement followed by the lumbar, the dorso-lumbar, and the cervical spine \[[@B14],[@B22]\]. However, in our study the dorsal spine was the second most frequent region involved following the lumbar spine, with L1 being the most frequently affected level irrespective of the number of levels involved. Tuberculosis frequently involves multiple spinal levels; as many as 17 vertebrae have been reported to be involved \[[@B14]\].
The success rate of PCNA in our study was 90.6%, which yielded a sufficient sample for the AFB and the culture. However, it has been reported that the sample is often inadequate \[[@B4]\]. We had a positive AFB smear in 89.7% and a positive culture in all successful PCNAs. However, a positive culture of 76% has been reported by Staatz et al. \[[@B23]\] using CT-guided PCNA. CT-guided PCNA has been shown to be successful in draining almost all abscesses from the neck to the sacroiliac joint \[[@B4],[@B24]\].
In our study only 9 (19.1%) patients underwent surgery; 4 of which had failed PCNA and 5 had persistent symptoms. Indications for surgery include failed conservative management, spinal deformity or instability, and/or persistent neurological symptoms; however, this surgical approach has a 2% to 11% mortality rate and a recurrence rate of 10% to 20% \[[@B25]\].
It has been claimed that patients with a neurological deficit show better recovery with surgery and chemotherapy as opposed to those on chemotherapy only \[[@B20]\]. According to the fourteenth report of the Medical Research Council Working Party regarding Tuberculosis of the Spine, surgical debridement had no significant therapeutic advantage over chemotherapy alone. Additionally, PCNA reduces damage to adjacent structures and also eliminates the risk of general anesthesia \[[@B24],[@B26]\]. Lastly, perioperative morbidity and mortality should not be overlooked.
Of the 47 patients, 93.6% (n=44) demonstrated good compliance with the treatment regimen and showed complete recovery at the end of treatment. All 3 patients who exhibited poor compliance deteriorated steadily and eventually stopped attending the clinic, furthermore they did not respond to hospital queries. This reiterates the importance of compliance with ATT. Additionally, Directly Observed Treatment Strategy can be used not only to improve compliance but also to prevent the development of multi-drug resistant forms of TB \[[@B1]\].
1. Limitations
--------------
The study was conducted on patients presenting to a tertiary care urban hospital. The sample does not include patients from rural areas of the country where TB spine is diagnosed at a much advanced stage. Most of our patients presented with symptoms of early stages of the illness without any gross deformity or instability. They were diagnosed in a timely fashion due to the high index of suspicion within the endemic region. Therefore, the results may not be generalized to regions where spinal TB is rare.
Conclusions
===========
A combined approach using clinical staging, PCNA, and ATT can minimize surgical intervention in most patients and thus avoid potential complications as well as further instability associated with it. The results of the conservative treatment consisting of PCNA and ATT for at least 12 months in compliant patients are excellent. Early diagnosis and treatment before the development of spinal deformity seems to be essential for better outcomes and survival. However, ATT remains to be the cornerstone of management with regards to spinal TB.
Dr. Anwar Ahmed and Dr. Irfan Lutfi, Department of Radiology, Ziauddin University, for their cooperation with PCNA and Radiological imaging during the study.
2010 NSA Annual Scientific Meeting, September 30 - October 2, 2010, Sunshine Coast, Queensland, Australia.
No potential conflict of interest relevant to this article was reported.
{#F1}
{#F2}
{#F3}
{#F4}
{#F5}
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
The biological function of proteins and cellular metabolism can be significantly modulated through post-translational modifications (PTMs) [@pone.0032805-Deribe1], [@pone.0032805-Seet1]. Naturally occurring PTMs can either result from enzyme modifications or arise spontaneously [@pone.0032805-Doyle1], [@pone.0032805-Uy1]. However, PTMs can also be used by bacterial and viral pathogens to compromise critical immune responses and host factors acting against infection [@pone.0032805-Ribet1]. The breakdown of immunological tolerance resulting from such modifications may render endogenous antigens immunogenic [@pone.0032805-Doyle1], [@pone.0032805-Utz1]. A large array of PTM peptides presented by major histocompatibility complex (MHC) class I and class II molecules have now been described [@pone.0032805-Engelhard1], [@pone.0032805-Petersen1], and it has been demonstrated that modifications such as cysteinylation [@pone.0032805-Meadows1], [@pone.0032805-Pierce1], glycosylation [@pone.0032805-Backlund1], [@pone.0032805-Haurum1] or phosphorylation [@pone.0032805-Andersen1], [@pone.0032805-Zarling1] may affect T cell immunoreactivity, resulting in immune escape and/or initiation of autoimmunity.
Protein tyrosine nitration is a hallmark of inflammation, and is associated with up-regulated expression of inducible nitric oxide synthase (iNOS) [@pone.0032805-Abello1], [@pone.0032805-Aulak1]. Nitrotyrosinated proteins (NT-proteins) are generated *in vivo* by nitration with peroxynitrite, itself derived from nitric oxide and superoxide which are both released from activated inflammatory cells [@pone.0032805-Ohmori1]. NT-proteins may accumulate in apoptotic or inflamed tissues [@pone.0032805-Burkhardt1]. Indeed, accumulation of the amino acid homolog 3′-nitrotyrosine (NY) and of NT-proteins has been observed at inflammatory sites in Alzheimer\'s disease [@pone.0032805-Drew1], arthritis [@pone.0032805-Cuzzocrea1], [@pone.0032805-Loeser1], [@pone.0032805-Beckmann1], [@pone.0032805-Shishehbor1], atherosclerosis [@pone.0032805-Beckmann1], [@pone.0032805-Shishehbor1], autoimmune diabetes [@pone.0032805-Ceriello1], autoimmune uveitis [@pone.0032805-Liversidge1], celiac disease [@pone.0032805-terSteege1], ischemia-reperfusion injury [@pone.0032805-Noiri1], multiple sclerosis [@pone.0032805-Smith1], Parkinson\'s disease [@pone.0032805-Giasson1], respiratory disease [@pone.0032805-vanderVliet1] and transplant rejection [@pone.0032805-Pieper1], as well as in various cancers and infectious diseases [@pone.0032805-Giorgio1]. NT self-proteins may thus be highly immunogenic, eliciting both humoral and cellular responses [@pone.0032805-Ohmori1], [@pone.0032805-Nakazawa1].
The potential immunogenicity of MHC class II-restricted NT autologous peptides has been previously investigated using the I-E^k^-restricted T cell pigeon/moth cytochrome c (PCC/MCC~88--103~) [@pone.0032805-Birnboim1] as well as the I-A^k^-restricted hen egg-white lysozyme (HEL) epitopes [@pone.0032805-Herzog1]. Both studies demonstrated unequivocally that conversion of tyrosine to nitrotyrosine resulted in dramatic consequences for T cell recognition. Indeed, processing of native proteins by activated antigen presenting cells (APCs) resulted in the presentation of MHC class II-restricted NT-epitopes in lymphoid tissues, significantly altering specific T cell responses and eliciting NT-specific CD4^+^ T cells that evaded negative selection in the thymus and thus central tolerance [@pone.0032805-Nakazawa1]. Likewise loss of tolerance by NT-specific CD4^+^ T cells has recently been shown to be critical for the production of autoreactive antibodies [@pone.0032805-Gauba1]. These studies indicate that NT-proteins generated during inflammation might constitute an important class of neoantigens that could promote autoimmune T cell responses.
The well-established lymphocytic choriomeningitis virus (LCMV) glycoprotein system was also used by Hardy *et al* to demonstrate that conversion of tyrosine to NY also profoundly affected T cell recognition of MHC class I-restricted epitopes [@pone.0032805-Hardy1]. A significant amount of the overall CD8^+^ T cell response to LCMV is dominated by very few viral epitopes, comprising the H-2D^b^-and H-2K^b^- restricted peptide gp33--41 (hereafter referred to as gp33; KAVYNFATC) and the H-2K^b^-restricted peptide gp34--41 (gp34; AVYNFATC) [@pone.0032805-Kotturi1], [@pone.0032805-Masopust1]. Both gp33 and gp34 contain a single tyrosine residue at positions 4 (p4Y) and 3 (p3Y), respectively. T cell populations, exclusively specific to the nitrotyrosinated MHC complexes H-2D^b^/NY-gp33, H-2K^b^/NY-gp33 and/or H-2K^b^/NY-gp34 [@pone.0032805-Hardy1], were elicited in C57/BL6 mice (H-2D^b+^/H-2K^b+^) following immunization with the nitrated peptide NY-gp33. Importantly, CD8^+^ T cell hybridomas specific for NY-gp33 comprised two distinct subsets recognizing either H-2D^b^/NY-gp33 or H-2K^b^/NY-gp33. While the T cell hybridoma 24H1 responded to stimulation with both H-2K^b^/NY-gp34 andH-2K^b^/NY-gp33, it did not recognize the unmodified wild-type MHC complex H-2K^b^/gp34 nor H-2K^b^/gp33 [@pone.0032805-Hardy1]. Similarly, the H-2D^b^/NY-gp33-specific T cell hybridoma 4C8 did not recognize H-2D^b^/gp33. In contrast, nitrotyrosination of the main T cell receptor (TCR)-interacting peptide tyrosine residue p4Y abrogated recognition of H-2D^b^/NY-gp33 MHC complexes by H-2D^b^/gp33-specific T cells from P14 transgenic mice [@pone.0032805-Hardy1].
The present study provides a comparative biochemical and structural analysis that explains how nitrotyrosination of the LCMV-associated immunodominant epitopes gp33 and gp34 can alter T cell recognition in the context of the two different MHC class I molecules H-2D^b^ and H-2K^b^. Nitrotyrosination of the MHC-restricted peptide impairs TCR recognition through reduced stability and alteration of the molecular surface of the MHC complex. The possible implications for the role of nitrotyrosination in the creation of modified neoantigens that allow for viral escape and/or breaking of immune tolerance that possibly results in autoimmune disorders are discussed.
Materials and Methods {#s2}
=====================
Preparation and crystallization of H-2K^b^in complex with gp34 and NY-gp34 {#s2a}
--------------------------------------------------------------------------
Peptides gp34 (AVYNFATM) and NY-gp34 (AV-p3NY-NFATM) were purchased from Genscript (Piscataway, NJ, USA). Refolding and purification of MHC/peptide complexes were followed according to previously published protocols [@pone.0032805-Achour1], [@pone.0032805-Achour2], [@pone.0032805-Sandalova1], [@pone.0032805-Sandalova2]. The best crystals for H-2K^b^/gp34 and H-2K^b^/NY-gp34 were obtained in hanging drops by vapor diffusion in 2.1 M NaH~2~PO~4~/K~2~HPO~4~ (pH 6.4), 1.5% MPD and 1.8 M NaH~2~PO~4~/K~2~HPO~4~ (pH 6.7), 1.5% MPD, respectively. Typically, 2 µl of 6 mg/ml protein were equilibrated against 2 µl of crystallization reservoir solution at 20°C.
Data collection and processing {#s2b}
------------------------------
Data collection was performed under cryogenic conditions (temperature 100 K) at beam lines ID14-2 and ID14-4 at the synchrotron radiation facility at ESRF (Grenoble, France) to a resolution of 2.0 and 2.6 Å for H-2K^b^/gp34 and H-2K^b^/NY-gp34, respectively. Crystals were soaked in a cryoprotectant solution containing 25% glycerol before data collection. A total of 180 and 360 images were collected with 0.5° oscillation per frame for both H-2K^b^/gp34 and H-2K^b^/NY-gp34. Data were processed with MOSFLM [@pone.0032805-Leslie1] and SCALA from the CCP4 suite [@pone.0032805-The1]. The space group was determined to be *P2~1~2~1~2~1~* for H-2K^b^/gp34 with unit-cell parameters *a* = 88.4, *b* = 92.6, *c* = 128.8 Å. The space group for H-2K^b^/NY-gp34 was*P2~1~* with unit-cell parameters *a* = 50.5, *b* = 88.5, *c* = 119 Å, *α* = *γ* = 90.0° and *β* = 94.7°. Data collection statistics are provided in [Table 1](#pone-0032805-t001){ref-type="table"}.
10.1371/journal.pone.0032805.t001
###### Data Collection and Refinement Statistics.
{#pone-0032805-t001-1}
H-2K^b^-gp34 H-2K^b^-NYgp34
--------------------------------------------------------- ------------------------------------------------- -------------------------------------------------------
**PDB code** 3ROO 3ROL
**Cell parameters (Å)** a = 88.4, b = 92.6, c = 128.8, α = β = γ = 90.0 a = 50.5, b = 88.5, c = 119.0, α = γ = 90.0, β = 94.7
**Data Collection**
**Space group** *P 2~1~ 2~1~ 2~1~* *P 1 2~1~ 2*
**Resolution range (Å)** 57.3 -- 2.0 50.3 -- 2.6
**Number of molecules/asymmetric unit** 2 2
**Number of reflections**
**Observed** 338476 (47869) 138558 (20164)
**Unique** 72101 (10411) 32286 (4682)
***I/σ (I)*** 11.6 (2.6) 12.9 (3.3)
**Completeness (%)** 99.9 (100.0) 100.0 (100.0)
[1](#nt102){ref-type="table-fn"} ***R~sym~ (%)*** 8.3 (67.0) 11.9 (39.8)
**Multiplicity** 4.7 (4.6) 4.3 (4.3)
**Refinement Statistics**
[2](#nt103){ref-type="table-fn"} ***R~cryst~*** **(%)** 22.5 24.7
[3](#nt104){ref-type="table-fn"} ***R~free~*** **(%)** 26.7 29.5
**Number of protein atoms** 6695 6391
**Water/other molecules** 396/4 283/4
**rmsd from ideal geometry**
**Bond length (Å)** 0.009 0.010
**Bond angle (°)** 1.136 1.285
**Ramachandran Plot (%)**
**Residues in preferred regions** 96.25 95.95
**Residues in allowed regions** 3.75 4.05
**Non-glycine residues in disallowed regions** 0 0
Values in parentheses are for the highest resolution shell.
*R~sym~* = Σ*~h~*Σ*~i~\|I~h,i~−I~h~\|*/Σ*~h~*Σ*~i~I~h,i~*, where *I~h~* is the mean intensity of the *i* observations of symmetry related reflections of *h*.
*R~cryst~* = Σ\|*F~obs~*−*F~calc~*\|/Σ*F~obs~*, where *F* ~obs~ and *F* ~calc~ are the observed and the calculated structural factors, respectively.
*R* ~free~ was calculated using 5% of the reflections.
Crystal structure determination and refinement {#s2c}
----------------------------------------------
The crystal structure of H-2K^b^/gp34 was solved by molecular replacement (MR) using Phaser [@pone.0032805-McCoy1] and the H-2K^b^/GNYSFYAL MHC complex (PDB code 1LK2) as a search model. Five percent of the total reflections were set aside for monitoring refinement by *R~free~*. The crystal structure of H-2K^b^/NY-gp34 was solved thereafter by MR using H-2K^b^/gp34 as a search model. Refinement of the two crystal structures was performed using REFMAC5 [@pone.0032805-Winn1]. After each round of refinement, missing residues were added in successive cycles of manual building followed by restrained refinement cycles in REFMAC5 [@pone.0032805-Winn1]. The final refinement parameters are presented in [Table 1](#pone-0032805-t001){ref-type="table"}. Figures were created using the program PyMOL [@pone.0032805-DeLano1].
Peptide-MHC binding affinity assays {#s2d}
-----------------------------------
Peptide-MHC binding affinity assays were performed using transporter associated with antigen processing (TAP)-deficient RMA-S cells as described previously [@pone.0032805-vanStipdonk1], by assessing the capacity of the different peptides to stabilize cell surface expression of H-2K^b^ complexes. Briefly, 5×10^5^ RMA-S cells were pulsed with different concentrations of indicated peptides in serum free AIM-V medium (Invitrogen, Carlsbad, CA, USA) at 26°C overnight in 5% CO~2~. Cells were subsequently washed and incubated in AIM-V medium at 37°C for 60 min in the absence of peptides. Cells were then washed twice with PBS before staining with anti-H-2K^b^ AF6-88.5 (BD PharMingen/BD Biosciences, MountainView, CA, USA). Following washing and fixation in 1% PFA, H-2K^b^ cell surface expression was detected by flow cytometry on BD FACSCalibur (BD Biosciences). Flow cytometry data was analyzed using Cell Quest Pro (BD Biosciences). The mean fluorescence intensity (MFI) of H-2K^b^ expression for the indicated peptide concentrations was divided by the observed MFI on cells without peptide as an estimate of peptide binding. The HIV-derived H-2D^d^-restricted epitope P18 (RGPGRAFVTI) was used as a negative control while the H-2K^b^-restricted Moloney murine leukemia virus peptide MULV (SSWDFITV) was used as a positive control.
Assessment of MHC complex stability using circular dichroism (CD) analysis {#s2e}
--------------------------------------------------------------------------
CD measurements were performed in 20 mM K~2~HPO~4~/KH~2~PO~4~ (pH 7.5) using protein concentrations from 0.15 to 0.25 mg/ml. Spectra were recorded with a JASCO J-810 spectropolarimeter (JASCO Analytical Instruments, Great Dunmow, UK) equipped with a thermoelectric temperature controller in a 2 mm cell. pMHC denaturation was measured between 30°C and 75°C at 218 nm with a gradient of 48°C/hour at 0.1°C increments and an averaging time of 8 seconds. The melting curves were scaled from 0% to 100% and the T~m~ values extracted as the temperature at 50% denaturation. Curves and T~m~-values are an average of at least two measurements from two independent refolding assays per MHC complex. Spectra were analyzed using GraphPad Prism 5 and T~m~ values compared using an unpaired, two-tailored T test.
Assessment of MHC complex stability using Thermofluor analysis {#s2f}
--------------------------------------------------------------
Differential scanning fluorimetry experiments were carried out using an iQ5 real-time PCR detection system (Bio-Rad). 96-well PCR plates were filled with 23 µl of MHC solution (0.25 mg/mL) in 10 mM Na^+^ HEPES buffer (pH 7.3), 150 mM NaCl per well. Finally, the fluorophore (2 µl of 125× SYPRO Orange (Sigma S5692)) was added to each well and the plate was sealed with optical sealing tape (Bio-Rad). An iQ5 Real-Time PCR Detection System, calibrated with External Well factor solution was used to monitor the changes in fluorescence intensity of the fluorophore. The temperature of the samples was changed from 20 to 95°C at a heating rate of 1°C/min and the fluorescence was recorded every 0.2°C. The melting temperature (T~m~) of each melting curve was calculated as the maximum of the first derivative of the curve. The average T~m~ value of each protein was determined from three distinct melting curves.
Molecular modeling of TCR in complex with wild-type and nitro-tyrosinated MHC complexes {#s2g}
---------------------------------------------------------------------------------------
Molecular models of the TCR P14 in complex with H-2D^b^/gp33 or H-2D^b^/NY-gp33 were created using previously described protocols [@pone.0032805-Achour3], [@pone.0032805-Velloso1]. Interactions between P14 and each MHC complex were analyzed visually for improper sterical contacts. Docking of P14 on its MHC/peptide ligand was also assessed according to contacts between evolutionary conserved amino acids suggested to be important for TCR/MHC interaction [@pone.0032805-Marrack1]. The molecular model was subjected to several rounds of energy minimization using the CNS suite of programs [@pone.0032805-Brunger1]. The coordinates of the TCR/MHC/peptide complexes will be provided upon request.
Results and Discussion {#s3}
======================
Overall structures of H-2K^b^ in complex with wild-type gp34 and nitrotyrosinated NY-gp34 {#s3a}
-----------------------------------------------------------------------------------------
The three dimensional structures of H-2K^b^ in complex with the wild-type gp34 (AVYNFATM) and with the nitrotyrosinated NY-gp34 (AV-p3NY-NFATM) were determined to 2.0 and 2.6 Å resolutions, respectively ([Table 1](#pone-0032805-t001){ref-type="table"}). Both crystal structures displayed good stereochemistry. The final electron density maps are of good quality with well-defined polypeptide chains ([Figures 1A and 1B](#pone-0032805-g001){ref-type="fig"}). In particular, the electron densities for all H-2K^b^ residues in contact with the peptides gp34 and NY-gp34 are clearly defined. The overall three-dimensional structures of H-2K^b^/gp34 and H-2K^b^/NY-gp34 are remarkably similar with a root mean square deviation (rmsd) of 0.37 Å^2^ for the entire MHC complexes (heavy chains in complex with peptides and β~2~-microglobulin (β~2~m)) and of 0.27 Å^2^ for heavy chain residues 1--176 corresponding to the α~1~ and α~2~ domains.
{#pone-0032805-g001}
Protrusion of residue p1K in H-2K^b^/gp33 does not alter the conformation of MHC residues within the N-terminal part of the peptide-binding cleft {#s3b}
-------------------------------------------------------------------------------------------------------------------------------------------------
The previously determined crystal structure of H-2K^b^ in complex with gp33 (KAVYNFATM) revealed that the nonameric peptide binds to H-2K^b^ as an octamer with the stretch of residues AVYNFATM filling the peptide-binding cleft, while the highly flexible residue p1K extended out of the binding groove [@pone.0032805-Achour3] ([Figure 2A](#pone-0032805-g002){ref-type="fig"}). As for p2A in H-2K^b^/gp33, the first peptide residue p1A in both H-2K^b^/gp34 (AVYNFATM) and H-2K^b^/NY-gp34 (AV-p3NY-NFATM) is surrounded by a cluster of tyrosine residues (Y7, Y159 and Y171) that forms a network of hydrogen bond interactions with the nitrogen atom of p1A. However, although the protrusion of p1K out of the peptide-binding cleft in H-2K^b^/gp33 results in a significant modification of the position of the nitrogen atom of residue p2A when compared to p1A in H-2K^b^/gp34 or H-2K^b^/NY-gp34, this does not affect the conformation of the cluster of tyrosine residues that surround the N-terminal part of the peptide ([Figure 2B](#pone-0032805-g002){ref-type="fig"}). Furthermore, the conformation of the stretch of peptide residues AVYNFATM bound within the peptide-binding cleft is very similar in both H-2K^b^/gp33 and H-2K^b^/gp34 ([Figure 2A](#pone-0032805-g002){ref-type="fig"}). Thus although the N- and C- peptide termini play crucial roles in binding of epitopes within the peptide-binding cleft formed by the α~1~α~2~ groove [@pone.0032805-Bouvier1], these are not always necessarily required. It has been previously demonstrated that longer peptides may protrude out of the groove and that modified and/or shorter peptides can still bind efficiently within the groove through the use of a network of water molecules within the peptide binding cleft [@pone.0032805-Achour3], [@pone.0032805-Bouvier2], [@pone.0032805-Collins1], [@pone.0032805-Yague1].
{#pone-0032805-g002}
In the prior study by Hardy *et al*, T cells could be readily elicited in C57/BL6 mice following immunization with NY-gp33 [@pone.0032805-Hardy1]. Interestingly, while the H-2K^b^/NY-gp33-specific CD8^+^ T cell hybridoma 24H1 recognized H-2K^b+^ target cells coated with both NY-gp33 and NY-gp34, it did not recognize H-2K^b^/gp34 MHC complexes. Furthermore, it should also be noted that 24H1 T cells recognized H-2K^b^/NY-gp34 to a higher level when compared to H-2K^b^/NY-gp33. The relatively lower recognition of H-2K^b^/NY-gp33 by 24H1 could be explained by the protrusion of the side chain of p1K from the N-terminal part of the peptide-binding cleft of H-2K^b^/NY-gp33, which could potentially limit optimal recognition by the TCR. However, the question remained as to why the H-2K^b^/NY-gp33 and H-2K^b^/NY-gp34-specific TCR of 24H1 did not recognize H-2K^b^/gp34?
Nitrotyrosination of peptide residue p3Y induces a profound conformational change of the H-2K^b^ residue E152, altering TCR recognition {#s3c}
---------------------------------------------------------------------------------------------------------------------------------------
The conformations of the peptides gp34 and NY-gp34 are very similar when bound to H-2K^b^, besides a lateral shift of 0.7 Å of the side chain of the nitrotyrosine p3NY in H-2K^b^/NY-gp34 when compared to the conformation of p3Y in H-2K^b^/gp34 ([Figure 3A](#pone-0032805-g003){ref-type="fig"}). In H-2K^b^/gp34 the side-chain of p3Y projects down into the D-pocket formed by residues Q114, E152, R155, L156 and Y159 ([Figures 3A and 3B](#pone-0032805-g003){ref-type="fig"}). Three hydrogen bond interactions are formed between the side chain of p3Y and the side chains of the H-2K^b^ residues E152 and R155 ([Figure 3B](#pone-0032805-g003){ref-type="fig"}). Nitrotyrosination of p3Y alters only the conformation of the H-2K^b^ heavy chain residue E152 which, in order to accommodate the side-chain of p3NY, undergoes a large conformational modification in H-2K^b^/NY-gp34 when compared to H-2K^b^/gp34 ([Figure 3C](#pone-0032805-g003){ref-type="fig"}). In contrast, the rest of the H-2K^b^ peptide-binding groove is not affected by the nitrotyrosination of the peptide in H-2K^b^/NY-gp34 when compared to H-2K^b^/gp34.
{#pone-0032805-g003}
In conclusion, substitution of the tyrosine residue at position 3 of the peptide gp34 to a nitrotyrosine exclusively affects the conformation of the side-chain of the H-2K^b^ residue E152. The specificity of the T cell hybridoma 24H1 to H-2K^b^/NY-gp34 thus seems to be dependent on the conformation of the MHC region surrounding the side-chains of p3NY and the H-2K^b^ residue E152. It should be noted that previous studies have demonstrated the importance of this region for recognition by H-2K^b^-specific TCRs [@pone.0032805-Garcia1], [@pone.0032805-Reiser1]. One may thus speculate that nitrotyrosination of the immunodominant peptide which alters considerably T cell recognition may be advantageous for the virus in order to escape immune recognition.
Nitrotyrosination of gp34 strains the overall conformation of the H-2K^b^ complex and significantly decreases its stability {#s3d}
---------------------------------------------------------------------------------------------------------------------------
Two additional interactions are formed between peptide residue p3NY and the heavy chain residues Q114 and Y116 in the H-2K^b^/NY-gp34 peptide when compared to H-2K^b^/gp34 ([Figures 3B and 3C](#pone-0032805-g003){ref-type="fig"}), suggesting that nitrotyrosination of the peptide may result in higher stabilization capacity of H-2K^b^ complexes by NY-gp34 when compared to gp34. However, binding affinity assays using TAP-deficient cells demonstrated that while both gp33 and gp34 displayed similar binding affinities to H-2K^b^, nitrotyrosination of peptide residue 3 significantly reduced binding affinity of NY-gp34 to H-2K^b^ ([Figure 4A](#pone-0032805-g004){ref-type="fig"}).
{#pone-0032805-g004}
Furthermore, the capacity of each peptide to stabilize H-2K^b^ complexes was also investigated using circular dichroism (CD) by assessing the thermostability of soluble H-2K^b^ molecules in complex with gp34 or NY-gp34. Melting temperatures (T~m~) were derived from changes in ellipticity at 218 nm, corresponding to loss of secondary structure during denaturation. Our analysis demonstrates that the T~m~ value for H-2K^b^/NY-gp34 (47.3°C) was nearly 10°C lower than for H-2K^b^/gp34 (56.7°C), reflecting the significantly reduced capacity of NY-gp34 to stabilize H-2K^b^ complexes ([Figure 4B](#pone-0032805-g004){ref-type="fig"}). Similar very significant shifts in thermal stability were also obtained for the two MHC class I complexes upon analyzing their stability with differential scanning fluorimetry (DSF, thermofluor) [@pone.0032805-Kopec1]. The melting temperatures T~m~ corresponding to the mid-point of the sharp transition from folded to unfolded state were 54.4±0.1 and 44.5±0.2 for H-2K^b^/gp34 and H-2K^b^/NY-gp34 ([Figure 4C](#pone-0032805-g004){ref-type="fig"}). These significant differences in stabilization capacity were at first surprising when considering that additional hydrogen bonds and long ionic interactions are formed between NY-gp34 and H-2K^b^ when compared to H-2K^b^/gp34 ([Figure 3C](#pone-0032805-g003){ref-type="fig"}). However, further comparative analysis of the two crystal structures provided an explanation to these unexpected results.
In H-2K^b^/NY-gp34, the side-chain of the nitrotyrosine p3NY projects within the D-pocket of H-2K^b^, resulting in structural over-packing ([Figures 3A and 3C](#pone-0032805-g003){ref-type="fig"}) and a 'strained' overall conformation of the MHC complex. Although this has not, to our knowledge, yet been reported for MHC class I molecules, strained conformations are relatively common and have been described for several proteins including e.g. native serine protease inhibitors [@pone.0032805-Huber1] and spectrin SH3 core-domains [@pone.0032805-Ventura1]. Induction of strained conformations is also important within several processes including catalysis in thymidylate synthase [@pone.0032805-Montfort1] and nuclear transport of exportins [@pone.0032805-Zachariae1]. Proteins with strained 'spring-loaded' conformations are usually intrinsically unstable. For example, the release of the course of the strained conformation in circularly permutated barnases resulted in a 20°C shift in melting temperature [@pone.0032805-Butler1] or in a 25-fold increase in stability for mutated serpins [@pone.0032805-Im1]. In the crystal structure of the H-2K^b^/NY-gp34 complex, introduction of the side chain of p3NY resulted in close contacts between the nitrotyrosinated peptide and several H-2K^b^ residues, forcing them away from the relaxed low-energy conformation observed in H-2K^b^/gp34 in order to accommodate the side chain of p3NY, thus resulting in unfavorable strained conformations. Besides straining the backbone of the heavy chain residues 124--126 and 156--157, the side chain of p3NY also incurred strained higher internal energy conformations for the side-chains of residues V97, I98 and Q114, when compared to H-2K^b^/gp34.
In conclusion, the potential gain in enthalpy resulting from the formation of two additional interactions between p3NY and the H-2K^b^ residues Q114 and Y116 may be outweighed by the loss of a hydrogen bond interaction formed with the side chain of E152 combined with the free energy cost resulting from the strained conformations of several MHC residues. Our results indicate that formation of additional interactions between peptides and MHC residues does not always necessarily increase the overall stabilization of MHC complexes. Furthermore, alteration of MHC complex stabilization through nitrotyrosination of the presented epitope may represent an additional alternative strategy used by the virus to prevent adequate presentation of the peptide at the surface of the infected cell.
Nitrotyrosination of p4Y in H-2D^b^/NY-gp33 directly affects recognition by H-2D^b^/gp33-specific TCRs {#s3e}
------------------------------------------------------------------------------------------------------
The previously determined crystal structures of H-2D^b^ and H-2K^b^ in complex with gp33 demonstrated that the peptide binds in two diametrically opposed manners to the two MHC class I molecules [@pone.0032805-Achour3]. Thus in contrast to H-2K^b^/gp33 and H-2K^b^/gp34, in which the side-chain of the tyrosine residue acts as a secondary anchor position, the side-chain of p4Y in H-2D^b^/gp33 protrudes out of the peptide-binding cleft, playing a key role in TCR recognition [@pone.0032805-Achour2], [@pone.0032805-Sandalova2], [@pone.0032805-Achour3]. Indeed, mutation of p4Y to a phenylalanine (Y4F) reduced the affinity of the H-2D^b^/gp33-specific TCR P14 by 100-fold [@pone.0032805-Velloso1], [@pone.0032805-Tissot1]. A molecular model of P14 in complex with the crystal structure of H-2D^b^/gp33 indicated that the side-chain of the protruding p4Y is perfectly positioned in the hotspot of the TCR composed of the CDR3 loops from both the Vα and Vβ domains. It forms three hydrogen bonds with the TCR residues Y36(Vα) and G102(Vβ), as well as with the side chain of the H-2D^b^ histidine residue H155, linking this domain of the heavy chain to the TCR ([Figure 5A](#pone-0032805-g005){ref-type="fig"}). In contrast, insertion of the negatively charged NO~2~ group in p4NY would result in deleterious sterical clashes with Y36(Vα) or G102(Vβ) ([Figures 5B and 5C](#pone-0032805-g005){ref-type="fig"}). The side chain of one of the possible p4NY rotamers would also repulse the negatively charged side-chain of the MHC residue E163, important for TCR recognition ([Figure 5B](#pone-0032805-g005){ref-type="fig"}).
{#pone-0032805-g005}
In conclusion, nitrotyrosination of p4Y in H-2D^b^/NY-gp33 is clearly deleterious for recognition by P14 as previously demonstrated [@pone.0032805-Hardy1]. Thus, one may here speculate again about the possibility that nitrotyrosination of the H-2D^b^-restricted peptide gp33 may represent an important strategy used by LCMV in order to avoid CD8^+^ T cell recognition and escape immune recognition. Furthermore, and as for H-2K^b^/NY-gp34, our structural analysis also strongly indicates that NY-gp33 may act as an H-2D^b^-restricted neo-epitope with the capacity to break tolerance, selecting and inducing inadequate T cell responses that may result in autoreactivity.
Concluding remarks {#s3f}
------------------
Our study provides a structural platform underlying the effects of peptide nitrotyrosination on initiation of TCR responses. Such PTMs can directly affect TCR recognition by modifying the properties of key TCR-interacting residues on the presented peptide or by altering the conformations of other MHC residues that are of importance for TCR recognition. Nitrotyrosination can also indirectly affect TCR recognition by severely destabilizing the MHC complex. Although additional interactions were formed between the PTM peptide and H-2K^b^, the conformation of a large amount of surrounding residues was strained, significantly reducing the overall stability of the MHC complex. We have previously demonstrated that subtle modifications in MHC-restricted peptides can result in significant alterations of MHC stabilization [@pone.0032805-Velloso1], a phenomenon that should be taken into account and measured upon trying to design altered peptide ligands for modulation of TCR response [@pone.0032805-vanStipdonk1], [@pone.0032805-Insaidoo1]. Several other amino acid modifications caused by oxidative stress could result in similar effects on TCR recognition [@pone.0032805-Ischiropoulos1], [@pone.0032805-Ischiropoulos2]. In conclusion, our structural study indicates that the impact of post-translational modifications could be dual, possibly allowing for viral immune escape from TCR recognition but also potentially inducing the expansion of subset of T cells that could induce autoreactivity. Likewise, nitrotyrosination of self-peptides during periods of inflammation or oxidative stress could lead to the formation of neo-epitopes that, through the same mechanisms described herein for gp33/34, could escape the constraints of central tolerance. The impact of these modified *de-novo* MHC complexes on the initiation of unwanted T cell responses that may result in autoreactivity remains to be studied.
We gratefully acknowledge access to synchrotron beam lines 14.2 and 14.4 at ESRF (Grenoble, France), MX-41 at BESSY (Berlin, Germany) and I911 at MAX II (Lund, Sweden).
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**This work was supported by grants from the Canadian Institute of Health Research to RJW, the Swedish Research Council to AA, the Swedish Cancer Society to AA, the Swedish Childhood Cancer Foundation to AA and the Swedish National Board of Health to AA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: CM ADD TS AA. Performed the experiments: CM ADD. Analyzed the data: CM ADD TS JRW AA. Contributed reagents/materials/analysis tools: JRW AA. Wrote the paper: CM TS JRW AA.
| {
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Background
==========
Type 2 diabetes and depression are two long course diseases with modifiable risk factors. Diabetes, the fourth leading cause of death has affected an estimated 246 million people in the world \[[@B1]\]. In 2007, the number of people affected by diabetes in Asia rose to 110 million, with different proportion for young and middle aged people \[[@B2]\]. Among type 2 diabetics, the prevalence of newly diagnosed diabetes in people aged 25 years or above is 5.1% males and 6.8% females in urban areas and 5% males and 4.8% females in rural areas of Pakistan \[[@B3]\]. Depression accounted for 10% prevalence in primary care settings at various developed as well as developing countries \[[@B4]\]. In Pakistan, mean overall prevalence of anxiety and depressive disorders is 34% \[[@B5]\].
Among patients with type 2 diabetes, depression is found to be strongly associated with increased morbidity and mortality \[[@B6],[@B7]\]. Multiple studies from developed countries have proved positive association of type 2 diabetes with depression \[[@B8],[@B9]\]. Similarly, in the developing countries of South Asia, the association was significant in rural areas of Bangladesh, predominantly among females \[[@B10]\]. Zhao et al found this association in young adults \[[@B11]\] and it has also been found to be significantly associated with physical inactivity \[[@B12]\]. Beside age and gender \[[@B13]\], multiple other risk factors and co-morbidities are associated with either one or both diseases. These include socioeconomic status \[[@B14]\], past history of (h/o) depression \[[@B15]\], family h/o diabetes \[[@B16]\], h/o gestational diabetes \[[@B17]\], family type \[[@B18]\], Body Mass Index (BMI) \[[@B19]-[@B21]\], h/o smoking \[[@B22]\], h/o high cholesterol \[[@B23],[@B24]\] and h/o high blood pressure (BP) \[[@B25]\].
Epidemiological transition and lifestyle modification in South Asian countries have increased the prevalence of type 2 diabetes and depression. The healthcare systems of these countries have adequate services for diagnosis and management of type 2 diabetes but depression is not considered to be a regular or essential part of health or social milieu. Going to a psychiatrist for any mental problem including depression is regarded as a taboo. Usually people follow cultural or religious traditions to address mental illnesses rather than getting professional help. Untreated depression might be responsible for increase in diabetic co-morbidities because of poor self care, non-compliance with diet and exercise guidelines or lapse in filling prescription for oral diabetes medication \[[@B19]\]. Eventually, positive association between diabetes and depression has been identified \[[@B10]\]. Exploring this association at the initial stage of type 2 diabetes would help clinicians and psychiatrists to find avenues for preventive strategies and advocacy could be done at policy making level for early reduction of morbidity and mortality.
Although developed countries have enough evidence on this fatal combination, developing countries still have insufficient data on association of depression with type 2 diabetes especially with newly diagnosed type 2 diabetes. We, therefore, explored the association of depression with newly diagnosed type 2 diabetes among adults aged between 25 to 60 years in Karachi, Pakistan.
Research design, patients and methods
=====================================
From July 2006 to September 2007, a matched case control study (n = 592) was conducted in the Out Patient Department (OPD) of Civil Hospital, Karachi. After being granted permission by competent authorities, incident cases of type 2 diabetes (n = 296) were identified opportunistically and recruited from diabetic OPD of Civil Hospital, Karachi. Cases were operationally defined according to American Diabetic Association guidelines \[[@B26]\] which requires the \"presence of any two of the following: Symptoms of polyuria (frequent urination) and polydispia (increased thirst and consequent increased fluid intake) plus random blood glucose level \> = 200 mg/dl (11.1 mmol/l), or elevated fasting blood glucose level \> = 126 mg/dl (7.0 mmol/l) or 2 hour post prandial glucose level (\> = 200 mg/dl (11.1 mmol/l) after 75 gm glucose load\". Newly diagnosed type 2 diabetics were incident cases of type 2 diabetes diagnosed within the last one month. They had been referred to the OPD from wards or doctors of the same hospital or other hospitals of the city. The diagnosis made one month earlier was confirmed by documented proof in the form of diabetic card or prescription of medication or referral form. All those who failed to show proof of diagnosis of diabetes within the previous one month or could not give valid signed consent or refused to participate in the study or pregnant women were excluded from the study.
One control (n = 296) per case was recruited from medical OPD adjacent to diabetic OPD of the same hospital. They were the attendants accompanying medical patients waiting in medical OPD for their turn of clinical care. Those attendants who did not have polyuria and polydispia and had random blood glucose level of \< 200 mg/dl (measured by glucometer) were selected as control. The attendants of cases under study, those having a blood relationship with respective cases or those who shared the same household environment with the case or had polyuria and polydispia or were detected as having random blood glucose level \> = 200 mg/dl were excluded from the study. Matching on age and sex was done to minimize variation among cases and controls. Initially, on first two days of week, a bulk of 30 cases were identified and listed. On 3^rd^day, eligible controls were matched on age and sex with the listed cases. From 4^th^day onwards, cases and controls were recruited on alternate days to complete the sample size. Separate informed consents were used for cases and controls and study subjects were interviewed by two trained data collectors through pre-tested structured questionnaire for exploring the association of depression (level) and other hypothesized confounding factors with newly diagnosed type 2 diabetes.
The main Independent Variable, \"depression\" was screened through Siddiqui Shah Depression Scale (SSDI) Questionnaire. It was validated in Pakistani population for three different categories of depression: mild, moderate and severe \[[@B27]\]. In this study three categories of depression was used: no depression (score 0-25), mild depression (score 26-36) and moderate to severe depression (score 37+). For obesity and overweight, Body Mass Index (BMI) at the Asian cut off was measured (underweight: \<18.50 kg/m^2^, healthy: 18.50 to 22.90 kg/m^2^, overweight: 23.00 to 24.90 kg/m^2^, obese: \>25.00 kg/m^2^) \[[@B28]\] by height scale & calibrated bathroom scales. Information about socio-demographic factors such as age, sex, marital status, socioeconomic status, educational status, occupation and house ownership was gathered. Past history of depression, family h/o diabetes, h/o gestational diabetes, nuclear family type, h/o smoking, h/o exercise, h/o high cholesterol and h/o high BP, past h/o depression was collected by means of verbal inquiry from the study subjects.
Participation in the study was voluntary and the confidentiality of the study subjects was maintained. Potential patients of newly diagnosed type 2 diabetes identified in the control group or of depression detected in either cases or controls were given an authentic referral letter and sent to the OPD clinics of Civil Hospital, Karachi for timely management.
This study is a product of thesis done for Master in Epidemiology and Biostatistics at Aga Khan University Hospital, Karachi. It was ethically approval by Ethics Review Committee of Aga Khan University Hospital, Karachi (575 CHS/ERC-06) and was funded by Aga Khan University Research Council Grant (Project ID: 06GS008 CHS).
Statistical analysis
--------------------
Taking 15% (approximately) \[[@B29],[@B30]\] as the prevalence of risk among controls (p~0~= 0.15, α = 5% and β = 0.2 at 95% confidence level with corresponding odds ratio of 2 and inflating to 10% for the non-response and errors), sample size calculated by NCSS Pass statistical software was 592. Data was entered into EpiInfo version 6.04 and subsequently converted to SAS version 9.1 (Statistical Analysis Software Inc. NC USA) for performing univariate and multivariate analyses. Frequencies and percentages were computed for all the categorical variables. Newly diagnosed type 2 diabetes (present or absent) was taken as dependent variable. The category of independent variables with minimum level of association with newly diagnosed type 2 diabetes was taken as reference. Conditional logistic regression was performed to evaluate the association of depression and other independent variables with newly diagnosed type 2 diabetes at 95% C.I. and P \< 0.05, controlling for potential confounders.
Results
=======
Table [1](#T1){ref-type="table"} shows that the study sample included 432(73%) male and 160(27%) females with a higher proportion of married ones (cases: 227(76.69%); controls: 251(84.80%). Although the proportion of working people in both cases and control was equal (cases: 59(19.93%); controls: 59(19.9%) but both mainly belonged to middle socioeconomic status (cases: 145(48.99%); controls: 165(55.74%). The proportion of past h/o depression in newly identified type 2 diabetics was higher in cases as compared to controls (cases: 49(16.55%); controls: 41(13.85%).
######
Baseline characteristics of study subjects in cases & controls.
Male to female ratio is 432(73%): 160(27%)
-------------------------------------------- --------------------- ------------------------
**Characteristics** **Cases (n = 296)** **Controls (n = 296)**
**n\* (%)** **n\* (%)**
**Marital status**
\- Married 227 (76.69%) 251 (84.80%)
**Family type**
\- Nuclear family 207 (70.61%) 175 (59.12%)
\- Extended family 87 (29.39%) 121 (40.88%)
**Socioeconomic status**
\- Lowest 91 (30.74%) 85 (28.72%)
\- Middle 145 (48.99%) 165 (55.74%)
\- Upper 60 (20.27%) 46 (15.54%)
**Occupation**
\- Salaried workers 29 (9.8%) 23 (7.77%)
\- Daily wages laborer 31 (10.47%) 36 (12.16%)
\- Not working 236 (79.73%) 237(80.07%)
**House ownership**
\- Shared accommodation 15 (5.07%) 11 (3.72%)
\- On rent 122 (41.21%) 114 (38.51%)
\- Own house 159 (53.72%) 171 (57.77%)
**Illiterate** 150 (50.68%) 160 (54.05%)
**H/o smoking** 21 (7.09%) 20 (6.76%)
**H/o high cholesterol** 16 (5.41%) 12 (4.05%)
**H/o exercise** 51 (17.23%) 35 (11.83%)
**Past h/o depression** 49 (16.55%) 41 (13.85%)
\* frequency
Univariate analyses calculated unadjusted odds ratio and 95% confidence interval at 25% significance level to include most of the biologically and statistically important independent variables (Table [2](#T2){ref-type="table"}). Conditional logistic regression was applied and significant association of depression (level), history of (h/o) gestational diabetes, nuclear family type, family history of diabetes and BMI (obese vs healthy to underweight and overweight vs healthy to underweight) with newly diagnosed type 2 diabetes were found, independent of confounding effect of age, sex, marital status, h/o smoking and h/o high BP (Table [3](#T3){ref-type="table"}). The odds of mild depression among cases were 3.86 times the odds among controls (95% CI: 2.22, 6.71) while odds of moderate to severe depression among cases were 3.41 times the odds among controls (95% CI: 2.07, 5.61). Cases were 2.83 times more likely to have history of gestational diabetes (95% CI: 1.05, 7.64), 1.79 times more likely to have nuclear family type (95% CI: 1.14, 2.69) and 1.59 times more likely to have family history of diabetes as compared to controls (95% CI: 1.04, 2.43). Also, BMI showed statistically significant association with newly diagnosed type 2 diabetes (obese: mOR = 1.62; 95% CI: 1.01, 2.60 and overweight: mOR = 2.12; 95% CI: 1.19, 3.79).
######
Univariate aanalyses of associated factors with newly diagnosed type 2 diabetes (n = 592)
--------------------------------------------------------------------------------------------------------------
Characteristics Cases (n = 296)\ Controls (n = 296)\ Unadjusted mOR^\#^(95% C.I.\*\*)
n(%) n(%)
---------------------------------- ------------------ --------------------- ----------------------------------
**Depression level^+1^**
\- Moderate to severe 103 (34.80%) 63 (21.28%) 2.79 (1.84, 4.32)^++^
\- Mild 73 (24.66%) 40 (13.51%) 3.13 (1.94, 5.06)^++^
**Marital status (single)^+2^** 69 (23.31%) 45 (15.20%) 1.77 (1.14, 2.76)^++^
**Nuclear family type^+3^** 209 (70.61%) 175 (59.12%) 1.71 (1.12, 2.44)^++^
**Family h/o diabetes^+4^** 106 (35.81%) 64 (21.62%) 1.93 (1.35, 2.77)^++^
**H/o high blood pressure^+5^** 195 (65.88%) 173 (58.45%) 1.42 (0.99, 2.03)^++^
**H/o gestational diabetes^+6^** 23 (7.77%) 6 (2.03%) 3.83 (1.56, 9.41)^++^
**BMI^+7^**
\- Obese 140 (47.30%) 150 (50.68%) 1.11 (0.76, 1.64)^++^
\- Overweight 77 (26.01%) 52 (17.57%) 1.81 (1.12, 2.92)^++^
--------------------------------------------------------------------------------------------------------------
\+ References: +1 no depression, +2 married, +3 extended family type, +4 no family history of diabetes, +5 no history of high BP, +6 no history of gestational diabetes, +7 healthy
++ Significant: at P \< .25 \# matched odds ratio \*\* Confidence Interval
######
Conditional logistic regression analyses for factors associated with newly diagnosed type 2 diabetes (n = 592)
Characteristics Adjusted mOR^\#^(95% C.I. \*\*)
---------------------------------- ---------------------------------
**Depression level^+1^**
\- Moderate to severe 3.41 (2.07, 5.61)^++^
\- Mild 3.86 (2.22, 6.71)^++^
**H/o gestational diabetes^+6^** 2.83 (1.05, 7.64)^++^
**Nuclear family type^+3^** 1.75 (1.14, 2.69)^++^
**BMI^+7^**
\- Obese 1.62 (1.01, 2.60)^++^
\- Overweight 2.12 (1.19, 3.79)^++^
**Family h/o diabetes^+4^** 1.59 (1.04, 2.43)^++^
\+ References: +1 no depression, +6 no history of gestational diabetes, +3 extended family type, +7 healthy, +4 no family history of diabetes
++ Significant: at P \< .05 \# matched odds ratio \*\* Confidence Interval
Our study sample had 68(23%) of controls with impaired glucose tolerance (having random blood glucose 140 mg/dl to 199 mg/dl). To check the validity of final model on the whole study sample 592(100%), two more additional analyses were conducted: by excluding those with impaired glucose tolerance 456(77%) and on those who had impaired glucose tolerance 136(23%). All three analyses showed almost similar results. Therefore, we can conclude that depression is independently associated with newly diagnosed type 2 diabetes adjusting for potential confounders.
Discussion
==========
The study findings revealed that depression is significantly associated with newly diagnosed type 2 diabetes among adults aged 25 to 60 years. Type 2 diabetes needs adherence to long term treatment regimens including regular medical examination, investigating blood sugar levels and subsequent dietary, oral or injectable treatment. During its course, fear of complications, feeling of helplessness and non-compliance to treatment may cause depression. Previously published literature proved that there is three times higher risk of depression among diabetics as compared to non-diabetics \[[@B11]\]. Their concomitant existence may adversely affect the working capacity of the individuals. Our results also show that mild depression has higher odds ratio as compared to moderate to severe depression (refer: Table [3](#T3){ref-type="table"}). Most probably, diagnosis of type 2 diabetes might have led to mild depression which but later on association decreased due to patient\'s acceptance of the disease. Therefore, it is a strong point for policy makers to formulate preventive strategies against this fatal mix in the initial stage of diagnosis.
Along with lifestyle medication, family structure in Pakistan has been changed from extended to nuclear family type in Pakistan. Most of the families are living as nuclear families these days. It means parents live with their children only. Homes without grandparents might have reduced the burden of responsibilities and cumulative support could be rendered for prevention and management of type 2 diabetes with co-morbidities and complications. Our findings strongly support the association of nuclear family with newly diagnosed type 2 diabetes. It is consistent with previous literature \[[@B31]\] but Irving et al. \[[@B32]\] showed insignificant association with family structure which is needed to be explored with longitudinal study design. Along with lifestyle modification, genetic factors have also been documented as an associated factor for type 2 diabetes. Framingham offspring study proved family history as a significant factor for type 2 diabetes \[[@B33]\]. Our study also reaffirmed the positive association of family history of diabetes \[[@B34]\]. Additionally, our results also re-confirmed the significant association of past history of gestational diabetes with type 2 diabetes \[[@B23],[@B35]\]. It emphasizes the importance of selective screening for gestational Diabetes.
Our study results show (refer to Table [1](#T1){ref-type="table"}) that cases of diabetes were exercising more than control but it might be due to the prompt compliance of the advice given by their doctors after diagnosis of diabetes. But with the passage of time and progression of diabetes, patients might have ignored the exercise routine. Supporting the fact, newly diagnosed type 2 Diabetics have higher BMI as overweight and obese; which might be due to non-exercise culture or ineffective health seeking behavior.
One of the major strength of the study is its matched case control design which controlled two fundamental confounders of age and sex at design stage. Selection timings of both cases and controls were the same and they were interviewed under strictly similar conditions using the same questionnaire. Recruitment of cases and controls was done on the basis of disease status which avoided selection biases from the study. Both cases and controls were selected from the same source population, so they were representative of the source population with respect to depression within age sex matched strata. It achieved the goal of unbiased control. The screening tool used for depression exclusively screened out depression, thus avoided false positive results due to anxiety and somatic symptoms. Finally, sensitivity analysis was done by analyzing data set in different subgroups to address the sample size issue for wide range of prevalence. All showed no major differences among themselves.
Since depression is taken as a stigma, subjects suffering from depression might have refused to acknowledge it. Another limitation of the study is that controls were selected on the basis of Self-reported absence of symptoms and screening of random blood glucose by glucometer with only one reading which is against the established standard of diagnosis of type 2 diabetes \[[@B26]\].
It could not be verified by laboratory investigation because the study was the course requirement of a Master of Epidemiology and Biostatistics and there were monetary constraints. Furthermore, the questions asked about \"h/o high BP\", \"past h/o gestational diabetes\", \"h/o high cholesterol\" and \"past h/o depression\" were subjectively asked and could not be verified with lab investigations.
Conclusions
===========
Our study concludes that depression is significantly associated with newly diagnosed type 2 diabetes. Therefore, patients, clinicians and psychiatrists should be informed and educated about the associated burden of depression with newly diagnosed type 2 diabetes. All diabetics within 25 to 60 years should be simultaneously screened for depression. Concomitant preventive strategies for gestational diabetes, nuclear family and high BMI should also be made to prevent mortality/morbidity in 25 to 60 years.
Abbreviations
=============
OPD: out patient department; BMI: body mass index; SAS: Statistical Analysis Software; h/o: history of; BP: blood pressure: mOR: matched odds ratio; C.I.: confidence interval.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
SP, MSO, MNS, GR and JH participated in conceptualization, study design and protocol preparation. SP performed data collection, its supervision and monitoring. SP and JH did data analysis and its interpretation. SP, MSO, MNS, JH and GR contributed in manuscript drafting and revising. All authors read and approved the final manuscript.
Acknowledgements
================
I acknowledge the guidance of Dr Masood Kadir and Dr Rehana Siddiqui for their scientific input, Akhtar and Mehwish for data collection and Ms Sumera Nasheed for reviewing the English language of the manuscript.
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1.. Introduction {#s1}
================
Discs of gas and dust surround essentially all young analogues of our Sun \[[@RSOS160652C1]\]. The lifetime of the gas in these discs is very short compared with the stellar lifetime, and within a few million years has accreted onto the star, been lost to space in photoevaporative flows, and contributed to building planets. The evolution of the dust during this phase is uncertain, but the existence of gas giant planets makes it clear that planetary building blocks, and of course some planets, form on a similar or shorter timescale.
Beyond the first few million years, a typical star hosts a planetary system, the components being the planets themselves and a residual disc of small bodies. These 'planetesimals'---the asteroids and comets---make up the 'debris disc', where the standard picture is that destructive collisions between them generate a size distribution of fragments that extends down to micrometre-sized dust \[[@RSOS160652C2]--[@RSOS160652C4]\].
The state of planetary systems as they emerge from the gas-rich phase is uncertain. Planets' locations are not finalized at this epoch, but may move by interacting with other stars, planets and/or planetesimals in the system \[[@RSOS160652C5]--[@RSOS160652C7]\]. Similarly, the state and origin of the debris disc is uncertain. At stellocentric distances near 1 AU, the region of interest in this article, it could be that dust observed at this time is related to the final stages of planet formation \[[@RSOS160652C8],[@RSOS160652C9]\], originates in young analogues of our asteroid belt \[[@RSOS160652C10]\], is a signature of comets scattered inwards from more distant regions \[[@RSOS160652C11]\] or is simply a remnant of the gas-rich disc that has yet to be dispersed \[[@RSOS160652C12]\]. In the absence of gas detections that argue for the latter scenario, discerning among these various scenarios, which are not mutually exclusive, is difficult.
A promising way to probe these inner regions is by observing temporal variability \[[@RSOS160652C13]\]. Optical and IR stellar variation has been studied for decades \[[@RSOS160652C14],[@RSOS160652C15]\], and has recently been reinvigorated by large-scale efforts \[[@RSOS160652C16],[@RSOS160652C17]\] and as a side effect of large-scale surveys for transiting planets \[[@RSOS160652C18],[@RSOS160652C19]\]. Of many different classes of variables, those of most interest and relevance here are the 'UXors', named for the prototypical system UX Orionis \[[@RSOS160652C15]\]. These are usually Herbig Ae and late-type Herbig Be stars \[[@RSOS160652C20]\], and typically show several magnitudes of extinction that is generally attributed to variable obscuration by circumstellar dust \[[@RSOS160652C15],[@RSOS160652C21],[@RSOS160652C22]\].^[1](#FN1){ref-type="fn"}^ Three related arguments that favour circumstellar dust as the cause are (i) a maximum depth of dimming events of roughly three magnitudes, suggesting that a few per cent of the visible flux is not directly from the star, but scattered off a disc that surrounds the star and remains visible even when the star itself is completely occulted, (ii) 'blueing', where the star is reddened for small ($\lesssim$1 mag) levels of dimming but returns to the stellar colour (i.e. becomes 'bluer') for the very deep ($\gtrsim$1 mag) events where the star is mostly occulted---the reddening indicates dimming by circumstellar dust, and a stellar colour is typical of light scattered off circumstellar dust \[[@RSOS160652C28]\], and (iii) increased polarization fraction during dimming events, caused by a greater fraction of the flux being contributed by dust-scattered light (e.g. \[[@RSOS160652C29]\], which also shows that the surrounding dust does not reside in a spherical shell). UXors therefore reveal information on the degree of non-axisymmetry, the 'clumpiness', of dust orbiting a star on spatial scales similar to the star itself. The observations can span multiple orbits to test for repeated dimming events \[[@RSOS160652C30]\], and by using different bandpasses and polarization can estimate dust grain sizes \[[@RSOS160652C28],[@RSOS160652C31]\].
In the majority of UXor-like cases (i.e. those related to obscuration by dust), including other classes such as 'dippers' \[[@RSOS160652C17],[@RSOS160652C32]\], the processes causing young stars to vary are attributed to gas-rich protoplanetary discs. For Herbig Ae/Be stars, the obscuration is thought to be caused by hydrodynamic turbulence that lifts dust above the puffed up inner rim of a self-shadowed disc \[[@RSOS160652C33]\]. For the dippers, which are observed around low-mass stars, the obscuration is attributed to dust in accretion streams that link the inner disc and the stellar surface, and/or to variations in the height of the inner disc edge \[[@RSOS160652C30],[@RSOS160652C32],[@RSOS160652C34]\]. The common theme is therefore that the location of the occulting dust is as close to the star as physically possible, being set by sublimation \[[@RSOS160652C35]\]. These systems tell us about the nature of turbulence and accretion in gas-dominated discs, but so far reveal little about how these discs transition to the debris phase and the subsequent evolution.
Here, we focus on RZ Psc, a star that shows UXor-like variability \[[@RSOS160652C36]--[@RSOS160652C38]\]. As a young K0V-type star with no evidence for gas accretion and a strong infrared (IR) excess, this system appears unique among UXors and may provide us new information on the structure of inner planetary systems during or following dispersal of the gas disc \[[@RSOS160652C39],[@RSOS160652C40]\]. However, the IR excess indicates the over 5% of the starlight is intercepted by the disc, which is a level more akin to gas-rich protoplanetary and transition discs than debris discs. Specifically, we use a decade of ground-based optical photometry of RZ Psc (§[3](#s3){ref-type="sec"}) to draw conclusions on dust location (§[4](#s4){ref-type="sec"}), and discuss the possible disc structure and evolutionary state in §[5](#s5){ref-type="sec"}. We conclude in §[6](#s6){ref-type="sec"}.
2.. A clumpy dust ring near 0.5 AU? {#s2}
===================================
There is significant evidence that the optical variations seen towards RZ Psc are caused by circumstellar dust: (i) during dimming events, the colour becomes redder \[[@RSOS160652C36],[@RSOS160652C41]\] in a way consistent with that expected for dust \[[@RSOS160652C42],[@RSOS160652C43]\], (ii) the maximum depth is about 2.5 mag and during these events, the colour returns to near stellar values, suggesting that the remaining emission is from light scattered off the circumstellar dust (i.e. the star is fully occulted \[[@RSOS160652C28],[@RSOS160652C42]\], and (iii) the polarization fraction increases during the transits, as expected if an increasing fraction of the light is scattered off a disc of circumstellar dust \[[@RSOS160652C28],[@RSOS160652C38],[@RSOS160652C44]\].
What separates RZ Psc from other UXors (and dippers) is (i) the spectral type is K0V rather than Herbig Ae/Be for UXors and late K to M type for dippers, (ii) the occulting dust lies well beyond the sublimation radius, and (iii) the star is not associated with a star-forming region so is inferred to be a few tens of millions of years old. The dust distance has been inferred from the speed of ingress of dimming events, which was previously estimated as about 0.6 AU (for circular orbits \[[@RSOS160652C40]\]). Corroborating evidence comes from approximately 500 K temperature of the dust seen in the mid-IR, which places it near 0.4--0.7 AU (depending on optical depth) and therefore at a location consistent with the occulting dust \[[@RSOS160652C40]\]. The distance to RZ Psc is unknown, but as an apparently isolated star that shows Li absorption the age has been estimated as a few tens of Myr, and therefore beyond the age at which a gas-rich disc would normally exist \[[@RSOS160652C39],[@RSOS160652C45]\]. Further distinguishing features are that the duration of the dimming events is consistently short compared with other UXors, a few days rather than days to a few weeks, and that no near-IR (i.e. K-band) excess or accretion signatures are seen \[[@RSOS160652C45]\], so interpretations related to accretion of disc material onto the star \[[@RSOS160652C21],[@RSOS160652C30],[@RSOS160652C34]\] are unlikely.
Thus, the potentially compelling and unique aspect for RZ Psc is that we are observing dimming events from dust in a main-sequence planetary system that resides at about 0.5 AU. This dust is also seen in thermal emission, so deriving joint constraints on the dust properties and structure may be possible. As argued by de Wit *et al.* \[[@RSOS160652C40]\], a picture is emerging in which RZ Psc is surrounded by a massive young version of our own asteroid belt, in which planetesimals are continually being destroyed. These collisions generate the large collective surface area of small dust that emits strongly in the mid-IR, and the system geometry means that this dust also sometimes passes in front of the star.
While this asteroid belt picture is intriguing, and makes RZ Psc a system that could be of great interest and worthy of detailed study, it is not the only possibility. Well over 1% of the starlight is reprocessed by the circumstellar disc, which is more typical of the primordial gas-rich discs seen around nearly all young stars. The discovery of systems such as HD 21997, that appear to be a few tens of Myr old and host gas-rich discs \[[@RSOS160652C46]\], shows that stellar age is not a perfect indicator of disc status. Thus, a considerable part of our analysis focuses on the question of the status of the disc around RZ Psc.
Given the proposed interpretation related to individual planetesimal disruptions, rather than hydrodynamics, it is perhaps surprising that to date the dimming events are not seen to be periodic \[[@RSOS160652C40],[@RSOS160652C47]\]. The only cyclical variation seen in light curves for RZ Psc is a 12.4 year variation with an amplitude of 0.5 mag, which is attributed to either a magnetic cycle or precession of an otherwise unseen outer disc owing to perturbations from an unseen companion \[[@RSOS160652C40]\].
3.. Time series photometry {#s3}
==========================
3.1.. Optical {#s3a}
-------------
To study the temporal variability of RZ Psc, we use two seasons of public data from the wide-angle search for planets (WASP \[[@RSOS160652C48],[@RSOS160652C49]\]), and nine seasons of data from the Kilodegree Extremely Little Telescope North (KELT-North \[[@RSOS160652C50]\]). We also collected, but ultimately did not use, photometric observations of RZ Psc from a wide variety of other sources (\[[@RSOS160652C15],[@RSOS160652C36],[@RSOS160652C41],[@RSOS160652C44],[@RSOS160652C51]--[@RSOS160652C53]\], the Catalina Sky Survey, the American Association of Variable Star Observers, the All-Sky Automated Survey). Aside from the Harvard plate photometry published by Gürtler *et al.* \[[@RSOS160652C37]\], we have not sought unpublished photometry, so the light curve remains incomplete.^[2](#FN2){ref-type="fn"}^
Here, we focus on the WASP and KELT-North data, as it has not been previously analysed and has considerably higher cadence (many measurements per night) and temporal coverage (nightly, weather permitting) than other datasets. The WASP data from 2004 and 2006 are public and were obtained from an online archive.^[3](#FN3){ref-type="fn"}^ These data were processed in a manner similar to that described by van Werkhoven *et al*. \[[@RSOS160652C54]\], where common-mode variations were removed using 50 quiet nearby stars. The WASP bandpass is broad, with roughly uniform transmission from 400 to 700 nm \[[@RSOS160652C48]\]. The KELT-North data, 2006--2014, were used in raw form, the only specific treatment being a 4% relative correction being made to ensure observations taken in the 'east' and 'west' telescope orientations have the same calibration. The bandpass is redder than for WASP, with most transmission between 500 and 800 nm \[[@RSOS160652C50]\]. For a full description of the KELT-North data reduction, see \[[@RSOS160652C55]\].
We normalized each year's data from each instrument separately by converting magnitudes to flux density and dividing out the sigma-clipped median, so the light curve has an out-of-occultation baseline of 1. In doing so, we are assuming that variations owing to the slightly different filter bandpasses are unimportant. Each row in [figure 1](#RSOS160652F1){ref-type="fig"} shows a season's data, starting on 1 May each year (JD also indicated). Most year's data therefore extend into the next year, so the '2006 data' refers to data from the 2006/2007 observing season. Figure 1.WASP and KELT-North data. Photometry is shown in dimensionless form, relative to a quiescent level of 1, and was converted from observed magnitudes as described in the text.
### 3.1.1.. Qualitative light curve overview {#s3a1}
It is clear from [figure 1](#RSOS160652F1){ref-type="fig"} that RZ Psc undergoes the very deep dimming events that are typical of UXors. These are seen a few times each observing season and vary in complexity, with a few extended events (e.g. 2006) and a greater number of 'neater' single events (e.g. 2010). In some years, there is also significant variability at shallower depths. Of particular note is the pair of deep events in 2006; these appear to be about 70 days apart, and given the suggestion that the putative asteroid belt analogue resides near 0.5 AU a natural inference is that these two events are related. If true, this repetition corresponds to a semi-major axis of about 0.3 AU, which given uncertainties in the true disc spectrum could be consistent with the location of the asteroid belt. In 2004, there are about 100 days of near-consecutive nights of data and no deep events, so either the true period is longer than 100 days or dust clumps can be created (and perhaps destroyed) on timescales of a year or so.
In [figure 2](#RSOS160652F2){ref-type="fig"}, we have selected most of the events from each year and shown them at a greater temporal resolution. The scale in each panel is the same, so wider boxes simply cover longer events. Most events appear to last at least a few days, suggesting that only having nightly coverage does not seriously hinder our ability to detect most events. However, the events are sufficiently short and irregular that the true shape of events remains uncertain. While it is likely that interpolation of the photometry for the fourth event in 2011 (i.e. the fourth box from the left in the row corresponding to 2011 in [figure 2](#RSOS160652F2){ref-type="fig"}) would resemble the true light curve, this assumption seems very unlikely to yield the true evolution of more complex events like those in 2006. Figure 2.WASP and KELT-North data, focusing on dimming events. The vertical and horizontal scales in each subpanel are the same.
Nevertheless, [figure 2](#RSOS160652F2){ref-type="fig"} shows an unprecedented view of dimming events seen towards RZ Psc, and that key information on the ingress and egress of dimming events is present. The dimming rate is such that it can be resolved temporally, and hence the velocity and radial location of the dust clumps estimated. While such estimates have been made in the past based on one or two individual events \[[@RSOS160652C36]\], these data make them possible for an ensemble of tens of events.
A fairly basic question is whether the light curve could result from objects that all have the same properties or whether a range is required. Given the existence of both long shallow events and short deep events, at a minimum, the clumps must vary in size and/or velocity across the face of the star, but probably also have different optical depths. The star can be completely occulted, so the clumps can be optically thick and star-sized, but it has already been shown that the events are not grey, so the clumps must have a density gradient rather than have sharp edges. Where sufficient data exist, it is clear that not all events have the same relative shape, so the shape of the clumps varies. Thus, the broad picture is of roughly star-sized clumps, whose shape and orbital elements vary. The fact that the dimming events can be shown in a series of panels with the same scale in [figure 2](#RSOS160652F2){ref-type="fig"} suggests that the range over which these properties vary is of order factors of a few, not many orders of magnitude.
3.2.. Infrared {#s3b}
--------------
While the optical photometry reveals information about how RZ Psc is itself dimmed, IR photometry beyond a few micrometres is dominated by emission from the circumstellar disc. Thus, IR variation reveals information about how the emitting surface area, temperature and perhaps composition, of the dust change with time. Such variation is indeed apparent, both from comparison of an AKARI 18 μm non-detection at a lower level than the WISE 22 μm detection, and from several individual WISE measurements taken at six month intervals.
Motivated by this variation, we obtained VLT/VISIR observations of RZ Psc; an N-band (10 μm) spectrum on 16 August 2016 and Q-band (18μm) photometry on 27 July 2016 (programme 097.C-0217). These data, and the related calibration observations, were reduced using the standard ESO `esorex` pipeline. The wall-clock integration time for the spectrum was 50 min at an airmass of 1.66, and was calibrated using an observation of HD 189831 taken immediately afterwards at an airmass of 1.63. The spectra in individual chop/nod cycles are consistent, so the shape of the spectrum is reliable. The absolute calibration is uncertain at approximately 10% levels \[[@RSOS160652C56]\], which is sufficiently precise for our purposes here. The spectrum was trimmed to mask highly uncertain regions shortward of 8 μm, and longward of 13 μm, and near the telluric absorption at 9.5 μm. The Q-band photometry took 45 min at an airmass of 1.65--1.7, and was calibrated against an observation of HD 2436 taken immediately afterwards at an airmass of 1.5. Photometry of RZ Psc and HD 2436 was done using a 0.9^′′^ radius aperture and a sky annulus from 1 to 2^′′^. In addition to the conversion from adu/s to Jansky using HD 2436, we applied an additional upward correction to account for the slightly lower airmass for the calibrator ($\exp(0.3\lbrack 1.675 - 1.5\rbrack \approx 1.05$, where an extinction of 0.3 per unit airmass was used, derived from archival calibration data using a method similar to that of Verhoeff *et al.* \[[@RSOS160652C57]\]). Uncertainties were estimated as the standard deviation of the flux density in 50 apertures around RZ Psc. The final calibrated flux is 86±10 mJy.
The VISIR spectrum and photometry are shown in [figure 3](#RSOS160652F3){ref-type="fig"}, which also shows the other available near- to far-IR photometry. The absolute level of the spectrum agrees well with the IRAS, WISE (from the ALLWISE catalogue) and AKARI observations; that four observations spanning over 30 years are consistent suggests that while this part of the spectrum may vary, the shape and level shown is typical. The same cannot be said near 18 μm, where the AKARI and VISIR (and perhaps WISE depending on the disc spectrum) flux densities are inconsistent. The flux near 10 μm being relatively constant, and the 18μm flux changing could be indicative of shadowing of an outer disc by an inner disc; that is, evidence that the disc around RZ Psc has significant radial extent. This type of behaviour is seen as 'see-saw' variability in IR spectra towards some transition discs \[[@RSOS160652C58],[@RSOS160652C59]\]. Figure 3.Flux density distribution of RZ Psc, including 2MASS, WISE, AKARI, VISIR and IRAS data and their (approximate) year of observation. The dark blue line shows a stellar photosphere model at the approximate stellar temperature of 5350 K, and the light blue line a 500 K blackbody. The latter is not a fit, but an approximate continuum level that illustrates that the WISE 3.4 and 22 μm photometry cannot both be accounted for with a single blackbody, if the silicate feature seen with VISIR was present in 2010.
The spectrum clearly shows solid-state emission (and the continuum level is not actually clear), which indicates (i) that the dust is of order micrometres in size and (ii) that the dust includes silicates. The smooth rise and fall suggests that the silicates are largely amorphous; crystalline silicates have sharper features, notably a depression between two peaks at 10 and 11 μm, rather than the flat top seen here. Other systems thought to host bright asteroid belt analogues (rather than gas-rich discs), such as HD 69830, BD+20 307 and HD 113766A, tend to show crystalline features \[[@RSOS160652C60]--[@RSOS160652C62]\], which may argue against such a scenario for RZ Psc. However, such comparisons are largely speculative as there is also a high degree of variation among silicate features, for both gas-rich and gas-poor discs.
To further explore this variability, we use the WISE 'epoch' photometry, for which the telescope scanning strategy results in clusters of measurements that are spaced six months apart. These data appear at approximately days 70 and 250 in the relevant years in [figure 1](#RSOS160652F1){ref-type="fig"}, but do not coincide with any optical dimming events. Photometry is not available in all four channels since launch in early 2010, owing to exhaustion of the coolant after 7.7 months and a 2.5-year hiatus from mid-2011 to 2014 (see \[[@RSOS160652C63],[@RSOS160652C64]\]). These data are shown in [figure 4](#RSOS160652F4){ref-type="fig"}, where [figure 4](#RSOS160652F4){ref-type="fig"}*a* shows the 3.4 μm magnitude as a function of time, and that there is significant variation on six month timescales, and an even greater variation overall. Inspection of the individual clusters, which are on hour-to-day timescales, shows no significant variation with time. The dashed lines have the slope expected for disc brightness variation that is independent of wavelength; the slopes are not exactly 1, because the total flux is not dominated by the disc near 3--5 μm, and hence the slopes are slightly flatter. Comparing the observed and expected correlations, we conclude that the data do not show significant evidence for changes in the spectral shape (i.e. changing temperature or composition). However, the ratios including 12 and 22 μm observations are most sensitive to these changes, but only include the first two sets of observations where the brightness changes were relatively small. Figure 4.WISE epoch photometry at 3.4, 4.6, 12 and 22 μm (W1, W2, W3 and W4, in magnitudes). Panel (*a*) shows the time variation in W1 over 5.5 years. Panels (*b*--*d*) show how W1 correlates with W2, W3 and W4, which do not have observations at all W1 epochs. The dashed lines show the slope expected for constant disc flux variation with wavelength (the variation is smaller in W1/2, because the total flux is not dominated by the disc).
Nevertheless, the amplitude of the change in 3.4 μm brightness over 5 years is about 30%. Considering that the disc flux density is only 45% of the total flux at this wavelength, the disc brightness increased from 2010 to 2015 by about a factor of two. A similar variation can be inferred by comparing the 18 μm upper limit from AKARI in 2007 and our VISIR measurement in 2016. Given these increases, it is surprising that the N-band spectrum does not appear much higher than the IRAS, AKARI and WISE photometry. A possible explanation would be that the increased emission originates in larger grains, which would result in greater continuum flux but similar levels in spectral features. However, without wider spectral and more frequent temporal coverage quantifying such effects is difficult. This level of IR variation is seen towards both protoplanetary \[[@RSOS160652C58],[@RSOS160652C65]\] and debris discs \[[@RSOS160652C13],[@RSOS160652C66],[@RSOS160652C67]\], so these data provide little means to distinguish between scenarios.
4.. Where are the occulting bodies? {#s4}
===================================
The main part of our analysis concerns attempts to extract information from the optical light curve, taking advantage of the great number of dimming events seen over 10 seasons. In this section, we focus on the radial location of the bodies ('clumps') that pass in front of RZ Psc, first searching for periodicity associated with repeat events, and then using the light curve gradients to constrain the projected velocities. This analysis primarily focuses on what can be gleaned from the light curves, and the implications of these results for different clump origins are then explored in §[5](#s5){ref-type="sec"}.
4.1.. Search for periodic dimming events {#s4a}
----------------------------------------
We begin by estimating the lifetime of an occulting clump as a check on the plausibility that dimming events should repeat. The angular rate at which clumps are sheared out is *R*d*Ω*/d*R*=−3*Ω*/2. Accounting for shear in forward (interior) and backward (exterior) directions, the shear velocity across a clump of radius *R*~cl~ is then $$v_{sh} = 3R_{cl}\Omega,$$so the clump expansion rate owing to shear in units of clump radii is only three times the orbital frequency. That is, after one orbit a clump will be stretched by a factor of 6*π*, and the radial and vertical optical depth will be roughly 6*π* lower (though it might still be optically thick). Thus, clumps that are not bound by their own self-gravity are expected to have a short lifetime at optical depths that are large enough to cause detectable dimming events, but could cause repeated dimming events if they are initially optically thick.
The temporal coverage of the observations in an individual season is 100--150 days. Thus, if the occulting material resides in an asteroid belt closer than approximately 0.5 AU, periodicity in the dimming events may be visible in a single season's data. Longer orbital periods may be visible across seasons, though the six month gap between seasons makes unambiguously linking events harder. Non-detection of periodicity would imply that the data are not sufficient or that strict periodicity does not exist. An intermediate possibility is that occultations happen with a range of periodicities, perhaps reflecting their origin in a radially broad region, and that discerning this scenario from randomly occurring occultations is not possible given the data.
In an attempt to find the expected periodicity, we tried several approaches. These are similar in that they aim to quantify whether some feature in the light curve is repeated again at a later time, but differ in how well they reveal evidence for a periodic signal. We found no evidence for events that are related from one year to another, so focus on statistics derived from individual seasons' data (though these are sometimes combined).
### 4.1.1. Autocorrelation {#s4a1}
We first used autocorrelation to search for periodicity rather than used methods related to Fourier transforms (e.g. periodograms). The motivation being that an individual transit event may be followed by another some number of days later, and perhaps repeat a few times, but other similarly (but not exactly) separated events may happen years later or earlier with a phase that is totally different. We therefore used the discrete autocorrelation function (DACF) proposed by Edelson & Krolik \[[@RSOS160652C68]\], though do not include uncertainties on individual measurements. For a time series with measurements *a*~*i*~ at times *t*~*i*~, the DACF first computes the mean $\overline{a}$ from the light curve (here we used the sigma-clipped mean to remove the dimming events), then, for each pair of points *a*~*i*~, *a*~*j*~ (with *i*≠*j*) computes $U_{ij} = ((a_{i} - \overline{a})(a_{j} - \overline{a})/\sigma_{a}$, with each *U*~*ij*~ associated with a time lag Δ*t*~*ij*~=*t*~*j*~−*t*~*i*~. A series of time lags centred at times *t*~lag~ with width Δ*t*~lag~ are then used as bins, and the average in each bin is the DACF. The DACF is not computed for lag bins with no data. The units of the DACF are standard deviations of the light curve *σ*~*a*~ (again calculated using sigma clipping).
The results are shown in [figure 5](#RSOS160652F5){ref-type="fig"} for time lags (i.e. trial periods) of 10--155 days in half-day bins. Comparison of these with the light curves shows that the DACF recovers most, but not all, events. Conversely, not all DACF peaks are necessarily associated with real repeat events, as there may of course be multiple distinct clumps orbiting the star at any given time. Not all pairs of events show a strong DACF signal, as they can comprise only a few measurements and the mean for that *t*~lag~ dominated instead by a much larger number of measurements elsewhere in the light curve closer to the quiescent level (i.e. near $\overline{a}$). Our attempts to avoid this issue by using autocorrelation on interpolated data yielded mixed results; heavy filtering, such as setting all data above a given level to 1, was needed for results similar to the DACF shown in [figure 5](#RSOS160652F5){ref-type="fig"}. Figure 5.Discrete autocorrelation function for yearly WASP and KELT-North data, computed for lags between 10 and 155 days. The second and third lines from the top show all data, and 2006-excluded data. The topmost line shows the number of years that show a peak more than 3*σ* above the clipped DACF mean within each 5 day bin. The peak at 70 days is 7 years, the dashed line is zero and the *y*-axis scale is shown to the right.
While several strong peaks appear in the DACF of all data, most of these arise from 2006, as can be seen in the DACF when these data are excluded. Some peaks remain near 70 days, as well as at 120 and 145 days, and the latter two could be aliases of periods near 60--70 days, arising simply because an event was missed. That is, the irregular sampling means that absence of evidence of power at some period in the DACF is not evidence of absence.
The pair of events separated by 70 days in 2006 provides the strongest signal, and most other years also show events near this period (2007, 2009, 2011--2014). To illustrate these numbers the topmost line in [figure 5](#RSOS160652F5){ref-type="fig"} quantifies the number of years that show a peak, in 10 day bins. The peak of 7 years is at 65--75 days, which is suggestive but not conclusive because a K--S test shows that this distribution is consistent with being uniform in period.
### 4.1.2.. Iterative event finding {#s4a2}
In an attempt to avoid some of the difficulties arising from the DACF, we tried a similar approach that first identifies individual occultation events and then computes the time delays between them. The main aim was to identify and use all events in a way that avoids biases related to the sampling of the data and the different relative depths of potentially repeated events. By repeating this prescription for synthetic data, we are able to test different scenarios for how the occultations do or do not repeat.
For this approach, an event is initially identified as the lowest point that is 6*σ* below the mean, where the mean and standard deviation are again estimated by sigma-clipping. This lowest point is noted, as are all immediately adjacent points that are also below the threshold noted. The points so included constitute a single dimming event. The points belonging to this event are removed from the light curve and the process repeated until no significant events remain. The time of the event is the time of the lowest point, and the duration the time between the two endpoints that are consistent with the quiescent level. Thus, if an event is in a region of sparse sampling, the duration can appear to be longer than it probably is, though we discard any events that occur at the beginning or end of an observing season to avoid unreasonably long events.
For a given set of event times and durations, the range of possible times between events is then calculated using the maximum allowed by the duration. This calculation is done for all combinations, yielding *N*(*N*−1)/2 inter-event time ranges. These ranges are then 'stacked' into a histogram (i.e. counting +1 for time differences within a given bin) that shows a measure of the power present at a given time difference.
The solid lines in [figure 6](#RSOS160652F6){ref-type="fig"} show this power in histogram form (the same in each panel), generated from 60 events that were identified in the light curve. The *y*-axis should be interpreted as the number of events that are consistent with that period in the *x*-axis. As before, there is evidence for a peak, now slightly shifted to near 65 days. In contrast to the DACF analysis, this power does not all arise from a single year. Most is contributed by 2004 and 2009, but exclusion of events from these years results in a similar (but noisier) histogram. Figure 6.Power at a given period from the iterative event finding. The solid line shows the power from the data, and is the same in each panel. The dashed lines show the mean and ±1*σ* power from simulated dimming events, which from top to bottom are: random, periodic between 64 and 75 days with three repeats, and periodic at 64 days with 10 repeats.
To test what could have been detected, and quantify the variation in power expected, we created synthetic light curves with the same temporal sampling as the WASP and KELT-North data. To do this, we set the flux to 1, and randomly injected a number of dimming events with a flux of 0.1. These events are therefore relatively easy for the algorithm to detect, but suffer the same sampling issues. We tried individual randomly occurring events, and periodic events that repeat a fixed number of times. All events have durations randomly distributed between 1 and 4 days, similar to the observed events. For random events, the remaining parameter in this model is simply the number of events---this is the total number over the 11 year period covered by the WASP and KELT-North data, including when measurements were not being taken. For repeating events, the range of periods and the number of repeats are additional parameters. To estimate the level of variation in the power spectrum, we repeated the process of injecting synthetic events 500 times, and in each bin estimate the mean and standard deviation of the power.
The results are shown in [figure 6](#RSOS160652F6){ref-type="fig"}, where the dashed lines show the mean and ±1*σ* deviations from the simulations (and solid lines show the data). Each panel shows a different scenario, from top to bottom these are: 295 random events, 100 events with periods between 64 and 75 days and three repeats, and 100 events with a period of 64 days and three repeats. The purely random events are marginally disfavoured; while the data lie outside the dashed lines, these are only 1*σ*. Nevertheless, the middle and bottom panels show that the possible peak near 65 days could be caused by events with a range of periods, but that only a single period of 64 days is actually needed and yields a slightly stronger peak. The remaining excess periodicity near 10 days is not accounted for by any of the models, but could be an indication that on this timescale events are related (e.g. a clump that has separated into several). That the real signal nearly lies within the 1*σ* variation expected for randomly occurring events shows that the evidence for periodicity is weak, but that as found by the autocorrelation analysis, this weak evidence points towards 64 days as a possible period.
A side effect of the simulations is an estimate of about 300 dimming events in total over the period between 11 June 2004 and 21 February 2015 (3851 days). On average, an event therefore occurs about once every 13 days, and there are 30 dimming events each year (and roughly 15 per observing season). If events repeat three times, then a *new* dimming event would appear every 39 days on average, but we would also expect to see two other unrelated events during this time.
The total is five times larger than the 60 events detected by the iterative search. Most of these events were therefore either missed by the WASP and KELT-North observations or not counted because they were misidentified as single events. From [figure 1](#RSOS160652F1){ref-type="fig"}, a rough estimate is that three in five events would have been missed owing to incomplete temporal coverage, meaning that about one in five simulated events were sufficiently close to other events that they were not separately identified. Not all events are deep, but as could be surmised from [figure 1](#RSOS160652F1){ref-type="fig"}, near continuous observation of RZ Psc would yield a rich light curve. If continuous coverage allowed closely occurring events to be separated, then the number identified in a given time period would approximately double.
### 4.1.3.. Summary of period search {#s4a3}
We found a weak periodic signal near 60--70 days, but neither of the methods described above show compelling evidence that the dimming events seen towards RZ Psc are periodic and not random. While we presented the results for single seasons' data, we saw no evidence for periodicity on longer periods. Aside from the 12 year variation, no periodicity has been seen in the past \[[@RSOS160652C40]\]. These searches used periodograms, which are sensitive to variations with fixed phase and poorly motivated, so we explored autocorrelation and a similar method. We found a possible signal that can be attributed to both a different method and the significantly better temporal coverage of the WASP and KELT-North data.
A lack of strong evidence for periodicity is perhaps surprising, because material that occults the star once and is on an unperturbed orbit must pass in front of it again. Not all material need return at the same time however, and the prediction of the shearing estimate made at the outset, that the visible lifetime of clumps when they are optically thin is similar to the orbital period, appears to be borne out. Of course, shearing is not the only possible explanation, as pressure effects in a hydrodynamic turbulence scenario might also disperse a clump (as the sound crossing time for a star-sized clump near 1 AU is of order or shorter than an orbit). The latter scenario relies on a significant gas reservoir, so the primary test to distinguish between different clump scenarios lies with the evolutionary status of the disc, which we explore in §5.3.
4.2.. Gradient analysis {#s4b}
-----------------------
Given the possibility of a 60--70 day periodicity, the location of the occulting bodies could be relatively close to the star, with semi-major axes of about 0.3 AU. This distance is comparable to the 0.4 AU estimated for optically thin dust at 500 K \[[@RSOS160652C40]\]. To further investigate the location, we turn to a different aspect of the light curves that provides information on the velocity of the occulting bodies: the gradients. To convert gradients measured in the light curves to velocity and orbital distance, we first outline a simple model, and then use this model to interpret the data.
### 4.2.1.. 'Curtain' model {#s4b1}
This section considers a simple one-dimensional model (along *x*) of a cloud that dims a star. The main assumption is that the cloud is larger than the star, so for a cloud that passes in front of the star from left to right, the vertical (*y*) size of the cloud can be ignored. The large cloud extent is not only suggested primarily by the large depths of the dimming events, but also because no flat-bottomed (i.e. planet transit-like) events are seen. It seems likely that not all clumps are this large, and that a variety of sizes (and impact parameters) exist, but for our purposes this simplification is sufficient. Thus, the cloud is modelled as a semi-opaque screen or 'curtain' that dims the star, as in previous analyses of related phenomena (e.g. KH-15D, J1407 \[[@RSOS160652C54],[@RSOS160652C69],[@RSOS160652C70]\]).
The star is dimmed by the passage of a cloud located at *x*~cl~ from the star centre. The one-dimensional geometrical optical depth structure of the cloud is given by some function centred at *x*~cl~ (e.g. a top hat or Gaussian) so is *τ*(*x*−*x*~cl~). The star has a surface brightness $I(\sqrt{x^{2} + y^{2}})$, which could allow for limb-darkening. The observed flux from the star is then $$F(x_{cl}) = \int_{- R_{\star}}^{R_{\star}}(1 - \tau(x - x_{cl}))\, dx\int_{- \sqrt{R_{\star}^{2} - x^{2}}}^{\sqrt{R_{\star}^{2} - x^{2}}}I\left( \sqrt{x^{2} + y^{2}} \right)dy,$$which first integrates vertically over the star at some *x* (i.e. independently of *τ*), and then along *x*, which includes the effect of the cloud. The light curve is therefore the convolution of the one-dimensional stellar brightness profile with the clump's optical 'thin-ness' profile (i.e. 1−*τ*). The flux profile (light curve) is a function of time, but the star and clump profiles are functions of *x*, and the conversion that links these is the cloud velocity.
The simplest case is a star of uniform surface brightness that is occulted by an optically thick screen that covers the star from *x*=−1 to *u* (i.e. the units of length are now *R*~⋆~). Then, *I*=1 and *τ*(*x*−*x*~cl~) is a step function at *u* and the fraction of the total stellar flux (*F*~⋆~=*π*) seen is \[[@RSOS160652C69]\] $$f = \frac{F}{F_{\star}} = \frac{\left( \cos^{- 1}\lbrack u\rbrack - u\sqrt{1 - u^{2}} \right)}{\pi},$$where *f* has the same units as our normalized light curve. If the curtain is not completely optically thick, then the fraction is instead $$f = \frac{F}{F_{\star}} + \left( 1 - \frac{F}{F_{\star}} \right)(1 - \tau).$$
The gradient of the normalized light curve as the curtain is pulled across is *df*/*du*, and therefore the sky-projected (i.e. minimum) velocity of a clump is $$\frac{du}{dt} = - \frac{\pi}{2\tau\sqrt{1 - u^{2}}}\,\frac{df}{dt}.$$
A cloud that is not completely optically thick has a shallower flux gradient because it reaches a shallower depth for the same velocity, and the factor 1/*τ* accounts for this effect. Stated another way, for this curtain model the optical depth and cloud velocity are degenerate in producing some flux gradient. However, this degeneracy can be partially broken, because some information on *τ* exists; *τ* must be greater than the depth of the dimming event (i.e. 1−*f*~min~, the minimum normalized flux). Objects somewhat smaller than the star are also accounted for; an optically thick clump that covers half the star produces approximately the same light curve as a *τ*=0.5 clump that covers the whole star.
These expressions can be further simplified by assuming that the maximum gradient occurs as the cloud 'edge' passes the centre of the stellar disc (i.e. *u*=0). By adopting a radius for the star, this velocity can be converted to physical units, and assuming a stellar mass and that the clump is on a circular orbit converted to a semi-major axis. If *df*/*dt* is in units of fractional stellar flux per day (i.e. the light curve is normalized and has time units of days) and *du*/*dt* in stellar radii per day (i.e. units of *u* are *R*~⋆~), then the numbers for these quantities can be used in the following equations $$v \approx 8050\frac{R_{\star}}{R_{\odot}}\,\frac{du}{dt}{m\, s}^{- 1}$$and $$a_{circ} = 14\frac{M_{\star}}{M_{\odot}}\left( \frac{R_{\odot}}{R_{\star}}\,\frac{dt}{du} \right)^{2}{AU} = 8.7\frac{M_{\star}}{M_{\odot}}\left( \tau\frac{R_{\odot}}{R_{\star}}\,\frac{dt}{df} \right)^{2}{AU}.$$While the assumption of *u*=0 yields a simple conversion between the light curve gradient and the velocity and semi-major axis, it is of course possible to measure gradients that are not at *u*=0. For example, the gradient when a dimming event reaches minimum is zero, which implies zero velocity and an infinite semi-major axis (e.g. the first minimum in 2006 in [figure 2](#RSOS160652F2){ref-type="fig"}). In addition, orbits may not be circular and thus the actual velocity is greater than the sky-projected velocity during a dimming event. Thus, the gradients and velocities must be taken as lower limits, and the semi-major axes as upper limits.
### 4.2.2.. Curtain model application {#s4b2}
Using the simple formalism described above, we can use gradients derived directly from the light curves to estimate the radial location of the occulting clumps. The gradients are estimated by least-squares fitting straight lines to each night's observations, for which only nights with six or more measurements are used. This procedure is possible, because in nearly all cases, an individual night's observations only cover ingress or egress, not both. These gradients are plotted against the minimum nightly flux *f*~min~ in [figure 7](#RSOS160652F7){ref-type="fig"}. We plot gradients whose uncertainty is less than 4× their value as open circles, with symbol sizes proportional to the inverse of this uncertainty. All other gradients are plotted as small dots, as a check that the gradients for unocculted fluxes are near zero. The horizontal scatter of these gradients near *f*~min~=1 provides a further estimate of the uncertainty of individual gradients. The solid lines show the gradients expected for circular orbits at a range of semi-major axes. Figure 7.Gradient and minimum flux measured from individual nights' observations. Open circles have gradients significantly different from zero, and a symbol size proportional to the inverse of the gradient uncertainty. Dots are consistent with zero slope. The lines show the gradients implied by the velocities for circular orbits at 0.3, 1, 10 and 100 AU.
An unusual feature in [figure 7](#RSOS160652F7){ref-type="fig"} is that the gradients may be biased towards negative values at low *f*~min~. For the 15 negative, and four positive gradients below *f*~min~=0.5, and approximately equal numbers of positive and negative gradients above, Fisher's exact test yields a *p*-value of 0.02. Thus, there is evidence that the distributions of gradients above and below minimum fluxes of 0.5 are different, with negative gradients more commonly seen for deep dimming events. In the range 0.5\<*f*~min~\<0.8, there are 15 and 25 negative and positive gradients, a reversal of the trend, but this difference only has a *p*-value of 0.1. Thus, there are about equal numbers of gradients measured with minimum fluxes below 0.8, but their distributions are different.
The bias to negative gradients below *f*~min~=0.5 suggests that ingress tends to be slower than egress; the egress is too quick to be caught. However, the equal numbers between 0.5\<*f*~min~\<0.8 suggest that the rapid egress does not return the light curve to quiescence, just to a level above *f*~min~=0.5. Thus, the statistics suggest that a typical deep dimming event has an ingress at a rate of −1 to −2 day^−1^, after which the flux rapidly rises to *f*~min~≈0.5, and then the remaining egress is at a rate similar to ingress.
Qualitatively, this inference is consistent with a scenario of a disrupted asteroid, whose structure is dictated by shear and radiation pressure. The fragment size distribution is such that at least half of the optical depth is contributed by grains large enough that their orbits relative to the original body are dominated by shear; forward shearing is more rapid than backward shearing, so the clump has a sharper rear edge than front edge, accounting for the different number of positive and negative gradients measured for *f*~min~\<0.5. The fragments also comprise small grains whose dynamics are dominated by radiation pressure, which form a 'tail' much like a comet's and account for the egress where *f*~min~\>0.5. We leave the development of a quantitative study of this scenario for the future, noting that tests of such a model would require photometry at multiple wavelengths.
A look at specific dimming events shows that such a simple scenario will face challenges, as shown by the first set of events in 2006 ([figure 2](#RSOS160652F2){ref-type="fig"}); following the *f*≈0.5 dip, there are two more nights of data that have higher average fluxes, but both nights actually have negative gradients. This evolution does not invalidate the above analysis, but shows that the temporal evolution is complex, and at any given time multiple clumps, which may or may not be related, could be occulting the star.
In [figure 8](#RSOS160652F8){ref-type="fig"}, we have converted the gradients into semi-major axes using equation ([4.7](#RSOS160652M4x7){ref-type="disp-formula"}). The dashed line shows the stellar radius, estimated to be approximately solar based on an age of 25 Myr \[[@RSOS160652C71]\] and an effective temperature of 5350 K \[[@RSOS160652C45]\], using Siess *et al.'s* \[[@RSOS160652C72]\] isochrones. [Figure 8](#RSOS160652F8){ref-type="fig"} also includes solid lines of constant gradient, computed using equation ([4.7](#RSOS160652M4x7){ref-type="disp-formula"}) and *f*~min~=1−*τ*. Figure 8.Semi-major axes estimated from the light curve gradients in [figure 7](#RSOS160652F7){ref-type="fig"}. Open circles have gradients significantly different from zero, and a symbol size proportional to the inverse of the gradient uncertainty. Dots are consistent with zero slope. The stellar radius (grey dashed line) has been estimated as solar. Because gradients may have lower minimum fluxes, they can move down along lines parallel to the solid lines. Points near or above the blue line are consistent with zero light curve gradient.
The blue line shows the semi-major axis implied by the scatter in gradients near *f*~min~=1 in [figure 7](#RSOS160652F7){ref-type="fig"}. Above this line, the gradients can be considered consistent with zero, and the inferred semi-major axes largely meaningless. A related point is that because the points near *f*~min~=1 have very small *τ*, the derived velocities and semi-major axes can be rather extreme and should be disregarded. An additional issue is highlighted by the two large circles near *f*~min~=0.5 and between *a*~*circ*~=30 and 100 AU, which are the first two nights of 2006 observations shown in [figure 2](#RSOS160652F2){ref-type="fig"}. The first is at the time of minimum flux, and the other also appears to be at a turning point, so the assumption behind equation ([4.7](#RSOS160652M4x7){ref-type="disp-formula"}) that *u*=0 (i.e. the cloud edge is passing the stellar disc centre) is clearly incorrect. These points should therefore be associated not with clumps at 30--100 AU, but with clumps that lie somewhere interior.
The green line shows the semi-major axis implied by a gradient of 1 per day. As the lowest flux measured on a given night is not necessarily the minimum flux for that dimming event, the inferred semi-major axis could be greater; in this case, points move downward parallel to this and similar lines. [Figure 8](#RSOS160652F8){ref-type="fig"} suggests that the clumps orbit with projected velocities that are consistent with circular orbits at or beyond about 1 AU. The conclusion from the IR excess, and possibly the periodicity analysis, is that they may lie closer, near 0.3--0.7 AU. Reconciling these differences requires that the clumps are on eccentric obits. For a semi-major axis of 0.3 AU, the minimum clump eccentricity (i.e. all clumps transit at apocentre) is about 0.6. Such an orientation is of course highly unlikely, so either the semi-major axis is larger, or the true eccentricities need to be higher. Perhaps coincidentally, an eccentricity of 0.6 yields a pericentre velocity of 120 km s^−1^, similar to the velocity shifts in sodium absorption lines seen towards RZ Psc, which are discussed further in §5.2 (noting, however, that the pericentre velocity is tangential, and the absorption lines show projected radial velocity \[[@RSOS160652C73]\]).
4.3.. Summary of light curve analysis {#s4c}
-------------------------------------
The aim of this section was to estimate the location of the bodies that cause the dimming events towards RZ Psc. Using 10 years of WASP and KELT-North data, we investigated the periodicity of possible repeat events and the light curve gradients. While we found evidence for a 60--70 day period, this signal is weak and not statistically significant. This period would place the clumps near 0.3 AU, similar to the distance inferred for the dust seen as an IR excess (though this location is also uncertain). The gradient analysis places loose constraints on the clump orbits (less than 10 AU), so is consistent with a scenario where the clumps have semi-major axes near 0.3 AU. Thus, based on the light curve analysis, there is no reason to disfavour the model proposed by de Wit *et al.* \[[@RSOS160652C40]\], that the dimming events are associated with clumps being created by planetesimal collisions within an asteroid belt analogue.
The primary uncertainty lies with the disc evolutionary state. If a significant gas reservoir remains, a turbulent inner rim scenario similar to that proposed for UXors might produce a similar light curve. The lack of accretion and a likely age beyond which gas-rich discs are typically seen, may argue against this scenario, though the disc may be in transition to the debris phase and retain some primordial gas. We revisit the disc status from the perspective of the flux distribution in §5.3.
An additional result from the gradient analysis concerns the structure of individual clumps. The non-uniform gradient distribution is qualitatively consistent with post-collisional asteroidal fragments being dispersed by a combination of shearing and radiation pressure. Assuming small dust well-coupled to gas in a hydrodynamic turbulence scenario, a uniform gradient distribution seems more likely, so we interpret the gradients as providing circumstantial evidence for the planetesimal fragment scenario.
5.. Disc structure and evolutionary state {#s5}
=========================================
Based on 10 years of relatively high-cadence photometry, RZ Psc is regularly occulted by what are almost certainly star-sized clumps of dust. These clumps can be optically thick, and previous measurements of colour variations show that at least some of the dust must be small \[[@RSOS160652C38]\], which may be supported by the light curve gradient statistics. The previous interpretation of this system was that the clumps are the fragments arising from planetesimal collisions within an asteroid belt analogue that is also detected in the mid-IR. While the results from the previous section are consistent with this scenario, the evidence is at best circumstantial as its does not rule out the alternative of a UXor-like hydrodynamic inner rim scenario.
We now address several open questions, each taking a slightly wider view. Primary among these is whether the dimming events and the IR excess are caused by the same dust, as suggested by de Wit *et al.* \[[@RSOS160652C40]\]. Two further aspects are then the implications for the origin of the clumps and the evolutionary status of the disc in which they reside. We finish by considering the proposed scenarios for dippers and UXors, and why RZ Psc appears to be a rare object that lies between these classes.
5.1.. Does the occulting dust account for the IR excess? {#s5a}
--------------------------------------------------------
One of the reasons that RZ Psc is worthy of detailed study is that circumstellar dust is inferred from both the dimming events and the IR excess. Different properties of the dust grains, and the larger structure in which they reside, are revealed by each method; the dimming events yield information on dust 'clumpiness' on a star-sized scale, whereas the IR excess provides evidence for a disc that captures approximately 7% of the starlight, and thus a measure of the total surface area of dust. The proposed interpretation is that the clumps orbit within an asteroid belt, and the dimming events therefore provide some information on the size distribution and collisional evolution within the belt \[[@RSOS160652C40]\]. This expectation relies on co-location of the clumps and the belt, for which circumstantial evidence is provided by the light curve gradients and perhaps the periodicity analysis (see also \[[@RSOS160652C39]\]).
The fraction of starlight intercepted by the dust is a variable common to the dimming events and the IR excess. For the former, we use the average extinction $\overline{E}$, which is simply taken from the normalized light curve, as 1 minus the average flux, yielding 0.05 (the light curve median is 0.995). This estimate assumes that all dimming events are independent, and the value would be smaller if not, because the dust in some clumps may be being counted two or more times. The lack of strong evidence for periodicity suggests that multiple counting is not a serious issue however. Another issue is that the star could be reddened, and therefore that the normalized light curve has already had some constant level of extinction removed. Based on photospheric colours, this unseen extinction is probably small, in the range of zero to a few per cent \[[@RSOS160652C74]\].
If we assume that this average extinction applies over a uniform sphere around the star, and that the dust has a low albedo (i.e. the dimming events are dominated by dust absorption, not scattering of light out of our line of sight), then the IR fractional luminosity is equal to the average extinction. That is, both are equal to the fraction of starlight intercepted by the dust.
To explore possible geometries we use a simple relation between fractional luminosity *L*~disc~/*L*~⋆~, (uniform) geometrical optical depth *τ*, and the disc opening angle *θ* \[[@RSOS160652C12]\], $$\frac{L_{disc}}{L_{\star}} = \tau\sin\left( \frac{\theta}{2} \right),$$which says that the fractional luminosity is the optical depth of the dust multiplied by the fraction of the sky covered as seen from the star. The dust belt must therefore have an opening angle of at least 8^°^ to capture 7% of the starlight. However, for this minimal estimate, the dust is optically thick, yet RZ Psc is not seen to be reddened. If we instead require $\tau \sim \overline{E}$, then as stated in the previous paragraph, the dust distribution must instead be near isotropic. Given the ubiquity of disc-like structures around young stars, such a spherical distribution seems physically unlikely. In addition, the increased polarization during deep dimming events argues against a spherical distribution.
Thus, the picture of RZ Psc as a star seen *through* a disc, where the clumps account for all of the dust and sample some representative part of an asteroid belt (e.g. the midplane), is untenable because that belt would cause much more reddening than is observed. These characteristics distinguish RZ Psc from heavily reddened objects, where the dimming events could be sampling a more representative section of the disc \[[@RSOS160652C75]\]. This issue can be avoided by invoking a spherical distribution of material, but the problem then shifts to whether such a distribution is physically plausible.
A more likely alternative, which we favour, is that most of the dust does not lie on orbits that pass in front of the star, and the occultations are caused by a small fraction of objects that have higher vertical locations (or greater orbital inclinations) than average. In this case, the component that causes most of the IR excess may or may not be clumpy, and could be radially optically thick (i.e. the opening angle could be as small as 8^°^). This picture unfortunately loses any strong connection between the occulting clumps and the IR excess, essentially adding a free parameter that is the fraction of material that is 'kicked' or resides above the disc, but seems to be the simplest and most probable scenario. Dullemond *et al.* \[[@RSOS160652C33]\] used essentially the same argument for UXors, so our picture is therefore inevitably similar to dust occultation models proposed for UXors \[[@RSOS160652C22],[@RSOS160652C33],[@RSOS160652C76]\]. As the disc is probably radially optically thick with a scale height similar to gas-rich protoplanetary discs, it could be that the scenario for RZ Psc is in fact the same as proposed for UXors. In this case, the IR excess would originate from the inner edge of a more extended disc, which is not detected at longer wavelengths for reasons discussed below.
We therefore conclude that while there is almost certainly some connection between the dimming events and the IR excess, it is at best indirect; we are not viewing RZ Psc through a representative part of an asteroid belt analogue. As with other UXors, a clear prediction is that the disc is not seen edge-on, but at an intermediate inclination.
5.2.. Origin of the occulting structures {#s5b}
----------------------------------------
One of the distinguishing characteristics for RZ Psc is the relatively short duration of the dimming events *t*~dim~, which are a few days compared with a few weeks for other UXors \[[@RSOS160652C21],[@RSOS160652C77]\]. If we assume near-circular orbits at speed *v*~kep~, *t*~dim~=2(*R*~cl~+*R*~⋆~)/*v*~kep~, where the clump has radius *R*~cl~. Solving for the clump radius yields the relation \[[@RSOS160652C32],[@RSOS160652C78]\]: $$R_{cl} \approx 1.85t_{\dim}\left( \frac{M_{\star}}{M_{\odot}}\frac{1{AU}}{a} \right)^{1/2} - R_{\star},$$where here *R*~cl~ and *R*~⋆~ are in units of *R*~⊙~ and *t*~dip~ is in days. Equation ([5.2](#RSOS160652M5x2){ref-type="disp-formula"}) says that dimming events of a given duration can, in general, be caused by larger clumps that orbit close to the star or smaller clumps that orbit farther out.
[Figure 9](#RSOS160652F9){ref-type="fig"} shows the radius that clumps must have to cause dimming events of different durations as a function of semi-major axis. For clumps much larger than the star the stellar radius is unimportant, and the stellar mass dependence is relatively weak, so this plot can be applied to RZ Psc and UXors. The radius is of course the sky-projected size of a clump along the orbit, so whether this scale also applies vertically and radially depends on the specific scenario. This plot assumes circular orbits, and that the star has solar radius and mass. The approximate locations of the RZ Psc belt (or the inner edge of a more extended disc) and the range of inner edge radii for Herbig Ae stars \[[@RSOS160652C35]\] are shown by the hatched regions. Figure 9.Clump properties assuming circular orbits for a range of dimming event durations (as labelled). The brown region marks the approximate location of the RZ Psc asteroid belt (or the inner edge of a more extended disc). The blue-shaded region shows the range of Herbig Ae inner disc edge radii. The dashed line shows where a clump has an azimuthal extent similar to the scale height of a typical gas-rich disc.
The dashed line shows where clumps extend a tenth of the semi-major axis---approximately, the scale height for a gas-rich disc. If the variability of both UXors and RZ Psc originates from dust structures arising from hydrodynamic turbulence at the disc inner edge, and these structures are related to the disc scale height, then a wide range of dimming event times is expected. These times should correlate strongly with the inner edge location, and because this location is set by sublimation \[[@RSOS160652C35]\] should correlate with the luminosity of the star. We did not find evidence for such a correlation in time-variability studies of UXors \[[@RSOS160652C79]\], suggesting that the clump radii do not vary strongly with the inner edge location. In any case, we have already noted that RZ Psc has shorter dimming events than 'typical' UXors, so while RZ Psc has dust at a radius that falls within the range of UXors, the occulting clumps are inferred to be several times smaller.
A further difference between RZ Psc and UXors is the origin of the dust location. For Herbig Ae/Be stars (and by extension, UXors), the dust inner radius is set by sublimation. However, the dust around RZ Psc is roughly 500 K, so much cooler than the approximately 1500 K sublimation temperature. That is, if the origin of RZ Psc's variability is interpreted as similar to other UXors and originates in a gas-rich disc, then it must host a transition disc rather than host a 'full' primordial disc. Therefore, while these comparisons show RZ Psc to be unusual compared with typical UXors, they do not argue strongly for or against a specific scenario.
A final aspect to discuss regarding the origin of the clumps, and their relation to the IR excess, is the transient absorption features. These are seen towards UXors, but also seen towards some main-sequence A stars \[[@RSOS160652C80]--[@RSOS160652C82]\], so are not exclusive to stars that host gas-rich discs. For A-type stars, these features are generally interpreted as Sun-grazing 'exocomets', and the same may apply to UXors and RZ Psc. A potential issue with this interpretation is that the absorption lines towards RZ Psc are so far blue-shifted, and may instead originate in an outflow \[[@RSOS160652C73]\]. However, blue-shifting could also occur if evaporation mostly occurs near periastron passage, which might be expected if the bodies originate in an asteroid belt and are thus more refractory than the exocomets seen towards other stars (i.e. an asteroid in the Solar System would need to pass very close to the Sun to have a tail). Such a scenario is also consistent with the conclusion of §[4](#s4){ref-type="sec"} that the occulting clumps could be on eccentric orbits with high velocities at periastron. Models of such low-periastron asteroids or comets invariably require a perturbing planet \[[@RSOS160652C83],[@RSOS160652C84]\]. Assuming the same 4 : 1 mean-motion resonance picture of Beust & Morbidelli \[[@RSOS160652C84]\] implies a planet about 2.5 times more distant than the source. That is, if the asteroid belt is at 0.3 AU, the planet is near 0.75 AU. It may be possible that this planet is inclined and causes the asteroid belt to precess, thus causing the 12.4 year modulation of the stellar flux seen by de Wit *et al*. \[[@RSOS160652C40]\].
Overall, the origin of the clumps remains unclear, and raises many further questions. A distinguishing feature of RZ Psc's light curve is that most of the time the star is near the quiescent level; if the dimming events are related to variable structure at the inner rim of a gas-rich disc, then why are the events so rare? Is the geometry so finely tuned that only the most extreme fluctuations are visible? In the asteroid belt scenario, the details are equally unclear; if the clumps are recently disrupted planetesimals, how do they appear above the bulk of the disc when collisions are most likely to occur at the midplane? Are there two populations of objects, one that forms the main disc, and another that causes the dimming events? Could such a scenario be reconciled with the transient absorption features? Answers to these questions will require further study, and will almost certainly require new observations.
5.3.. Disc evolutionary state {#s5c}
-----------------------------
Many young stars with gas-rich discs show broadly similar variability (UXors, AA Tau analogues, dippers). With sporadic and deep photometric minima, and transient absorption lines, RZ Psc is most similar to UXors and has often been associated with this class, albeit as an unusual member \[[@RSOS160652C39]\]. Initial distinctions were made based on the late spectral type (K0V), the relatively short dimming events, and a lack of near-IR excess and accretion signatures. The more recent findings that the stellar age is probably several tens of Myr, and that the IR excess is suggestive of an asteroid belt analogue, further distinguish RZ Psc as a potentially remarkable object where the early gas-poor stages of main-sequence debris disc evolution can be studied. There remain similarities between RZ Psc and UXors. Primarily, we concluded that the clumps that cause the dimming events are most likely seen when they are well above the densest regions of a disc, consistent with the turbulent inner rim scenario proposed by Dullemond *et al.* \[[@RSOS160652C33]\].
To consider RZ Psc within the context of other young disc-hosting systems, [figure 10](#RSOS160652F10){ref-type="fig"} shows the spectral energy distribution (SED) of RZ Psc, and several other systems that could be considered to be at a similar evolutionary stage (a similar plot appeared in \[[@RSOS160652C12]\]). GM Aur hosts a transition disc \[[@RSOS160652C85]\], the status of HD 166191's disc is ambiguous and may lie somewhere between the transition and debris phase \[[@RSOS160652C12],[@RSOS160652C86]\], and HD 113766A is generally considered to host a bright warm debris disc (based on a 10--16 Myr age and a lack of gas \[[@RSOS160652C87]\], but based on similarities with HD 166191 was also noted as potentially ambiguous \[[@RSOS160652C12]\]). Figure 10.The spectral energy distribution of RZ Psc in comparison with other young disc-hosting stars. All SEDs are normalized to a common flux density at *H* band. The triangles are AKARI and IRAS upper limits for RZ Psc. Measurements were made at different times, so the apparent discrepancy between the WISE detection at 22 μm and the AKARI upper limit at 18μm is an indicator of IR variability (see §3.2).
In contrast to UXors, there is no obvious reason for dust around RZ Psc to lie near 0.3 AU, as the dust sublimation distance is much closer. For RZ Psc to host a gas-rich disc, it would therefore probably need to be a transition object that has as-yet undetected far-IR emission from the outer disc. As illustrated by [figure 10](#RSOS160652F10){ref-type="fig"}, the limits set by IRAS are not particularly stringent, but in comparison with GM Aur the SED beyond 20 μm is a factor of two lower, despite the mid-IR SED being a factor of two brighter. Transition discs have a wide variety of spectra however \[[@RSOS160652C58],[@RSOS160652C88]\], so the main conclusion from this comparison is that if an outer disc exists, it is not very bright. This far-IR deficit is a signature of self-shadowing, which is of course a key characteristic of UXors. If such a picture were true for RZ Psc, a prediction is that the outer disc may still be detectable at millimetre wavelengths (roughly mJy levels), whereas an extrapolation based on the asteroid belt scenario would not (roughly μJy). Specifically, self-shadowing can be caused by settling of dust towards the disc midplane, which can occur with little loss of vertical optical depth \[[@RSOS160652C89]\]. Thus, the far-IR flux can be much lower than for a typical disc, whereas the mm-wave flux is not.
In [figure 10](#RSOS160652F10){ref-type="fig"}, RZ Psc looks more similar to HD 166191 and HD 113766A, thus falling in the category of systems whose interpretation in terms of disc status is ambiguous (the high fractional luminosity would also mark RZ Psc as unusually extreme for a debris disc). With the aid of mid-IR interferometry, the disc around HD 113766A has been shown to comprise two components, one at 0.6 and another at 9 AU \[[@RSOS160652C90]\]. This is by no means evidence that RZ Psc has a similar structure, but merely reinforces the fact that the SED does not rule out such possibilities and that the dust need not be confined to a single belt near 0.3 AU, and need not be interpreted as an asteroid belt. Similarly, the disc around HD 166191 was modelled as an optically thick transition disc extending from 1 to 25 AU \[[@RSOS160652C12]\]. Thus, as was concluded above by considering self-shadowing, it could be that RZ Psc hosts a disc that extends from 0.3 to a few tens of AU.
While the lack of a large near-IR excess for RZ Psc suggests that the disc is at least in the transition to a debris disc (i.e. has an inner hole), it does not preclude the possibility that gas resides in that hole and may still be accreting onto the star. No emission lines that would provide evidence of accretion have been seen \[[@RSOS160652C45],[@RSOS160652C73]\], but as a further test we reconsidered the spectral energy distribution. Specifically, we included photometry from the galaxy evolution explorer (GALEX \[[@RSOS160652C91]\]) to quantify the level of any ultraviolet (UV) excess, a complementary accretion indicator \[[@RSOS160652C92]\]. This exercise is hindered somewhat by the possibility that optical photometry was obtained when RZ Psc was not near the quiescent level. To circumvent this issue, we used just the 2 μm all-sky survey (2MASS \[[@RSOS160652C93]\]) and GALEX photometry, fitting a PHOENIX atmosphere model \[[@RSOS160652C94]\], finding a best-fit effective temperature of 5485 K (assuming no reddening, or 5600 K if some reddening is allowed to improve the fit slightly). These temperatures are consistent with that derived from a high-resolution spectrum; 5350±150 K \[[@RSOS160652C45]\]. Alternatively, fixing the temperature to the spectroscopic value yields a mild UV excess, less than a factor of two, that may be chromospheric. We therefore conclude that there is no evidence for accretion seen as a UV excess.
Finally, the mid-IR variability discussed in §3.2 provides a measure of the changing emitting area of the disc around RZ Psc, and thus potentially information about the disc structure and status. [Figure 4](#RSOS160652F4){ref-type="fig"} shows that for the epochs where contemporaneous 3--22 μm photometry exists there is no evidence of 'see-saw' variability over six months, but that strong conclusions are limited by a lack of data. Over 5 years, the 3--5 μm disc flux varied by about a factor of two, with no major changes in the behaviour of the optical light curve. However, given that the bulk of the disc emission probably originates from material that is not occulting the star, direct links between the optical and IR behaviour are not necessarily expected. Indeed, towards young stars hosting gas-rich discs, correlated and uncorrelated optical/IR variability is seen \[[@RSOS160652C17]\]. Similarly, the IR flux of bright warm debris discs has been seen to vary strongly, whereas the optical brightness remains constant \[[@RSOS160652C95]\]. The main benefit of more intensive IR monitoring would be to search for mid-IR 'see-saw' variability, because it would provide evidence that the disc around RZ Psc has a significant radial extent.
In summary, the status of the disc surrounding RZ Psc is unclear. Despite a reasonable near/mid-IR characterization, at longer wavelengths the SED is not detected. Comparison with the IR spectra of other discs suggests that the disc is at a minimum well evolved towards the debris phase, and may have reached it already. This possibility, and the rarity of objects such as RZ Psc, opens the possibility that it is being observed at a special time with a specific geometry, so may yield better insights than most systems.
5.4.. The rarity of Sun-like dippers/UXors {#s5d}
------------------------------------------
We finish by briefly discussing RZ Psc in the context of the general classes of dippers and UXors, considering why neither of these classes includes many young Sun-like stars such as RZ Psc. To aid this discussion, [figure 11](#RSOS160652F11){ref-type="fig"} shows simplified cartoons of the proposed scenarios for dippers and UXors, and possible reasons for a lack of significant dust-related dimming events towards Sun-like stars (see \[[@RSOS160652C34]\] for similar figures for dippers). Figure 11.Cartoon shows possible origins of dippers and UXors, and why Sun-like stars may only rarely show analogous behaviour. In each case the star, magnetic dipole and rotation axis are shown at the left (the stellar magnetic field is not necessarily always tilted with respect to the disc). Possible disc structures viewed edge-on to the right. (*a*) Low-mass stars (dippers) are occulted by co-rotating material that is accreting onto the star, and the dust sublimation radius is interior to co-rotation \[[@RSOS160652C34]\]. (*b*) Sun-like stars are rarely seen as dippers or UXors because (i) dust sublimates outside co-rotation (represented by the grey accretion column in the upper half \[[@RSOS160652C34]\]) or (ii) material lifted by turbulence is shadowed by the outer disc (spiral in the lower half). (*c*) Herbig Ae/Be stars (UXors) are occulted by turbulence that appears above self-shadowed discs \[[@RSOS160652C33]\].
From the perspective of dippers, which so far have spectral types later than K5, Bodman *et al.* \[[@RSOS160652C34]\] explain their tendency to be low-mass stars as a consequence of the relation between the magnetospheric truncation, co-rotation and sublimation radii. The periodicity of dippers suggests that the occulting material is near the co-rotation radius, and is therefore near the base of any accretion columns. For low-mass stars, the dust temperature at this distance is cool enough that the columns contain significant dust mass and hence the dipping phenomenon is seen ([figure 11](#RSOS160652F11){ref-type="fig"}*a*). For earlier type stars, the sublimation radius is outside the co-rotation radius, so any dust in the accretion columns has sublimated, and no dipping is seen (grey accretion column in [figure 11](#RSOS160652F11){ref-type="fig"}*b*).
An as-yet unexplored corollary of this scenario is that the outer discs in dipper systems must not be more flared than the height of the accretion columns (as seen from the star). This could be because discs around low-mass stars are simply less flared in general \[[@RSOS160652C96]\], because discs in dipper systems tend to be more evolved than average \[[@RSOS160652C32]\] or because there is less relation between the outer disc geometry, the inner disc and accretion columns than would be naively expected \[[@RSOS160652C97]\]. Of course, if discs around Sun-like stars are sufficiently flared that the inner regions are not visible, then whether the accretion streams are transparent or not is moot.
From the perspective of UXors, a possible explanation for their tendency to be late B- and A-type stars is that the specifics of self-shadowing are different for Sun-like stars \[[@RSOS160652C33]\]. There is little reason to believe that self-shadowing does not happen for young Sun-like stars \[[@RSOS160652C89],[@RSOS160652C98]\], so it seems either that self-shadowing is simply rarer, or that the nature is different in a way that affects whether the inner disc can occult the star (i.e. shadowing is by a larger portion of the inner disc regions rather than by a puffed-up inner rim \[[@RSOS160652C89],[@RSOS160652C99]\]).
Thus, it seems that the rarity of Sun-like stars among dipper and UXor populations may be understood as a result of the scenarios for both. Accretion columns are optically thin, because the dust has sublimated, and any turbulence that rises above the inner disc may not be seen as it is hidden behind a flaring outer disc.
In this context, the rarity of RZ Psc-like objects can be explained in two ways. The first simply sidesteps the above discussion by interpreting the disc as a gas-poor asteroid belt analogue. Examples of such bright discs at a few AU are rare \[[@RSOS160652C10]\], and only a subset of these will be oriented such that dimming events are seen above the disc midplane (recalling that the occulting bodies are at tens to hundreds of stellar radii). The second explanation relies on RZ Psc's age and SED, which suggest that it hosts a gas-rich transition disc that is well settled (i.e. not significantly flared), and hence turbulence above the disc inner edge is visible. The non-detection of RZ Psc in the far-IR ([figure 10](#RSOS160652F10){ref-type="fig"}) might also be the result of such settling, and suggests that the brightness at millimetre wavelengths might be brighter than expected given the mid/far-IR brightness \[[@RSOS160652C89],[@RSOS160652C99]\]. The rarity is again explained by an unlikely geometry, and perhaps that the period during which the inner disc can be seen above the outer disc as it settles is relatively short.
6.. Summary and conclusions {#s6}
===========================
Long considered a member of the UXor class of variables, RZ Psc is almost completely occulted by dust for several days, multiple times during each observing season. The 'typical' UXor (which is generally a Herbig Ae object) shows day to month long dimming events that are thought to be caused by hydrodynamic turbulence above the disc inner rim, and where the outer disc is self-shadowed \[[@RSOS160652C33]\]. Various anomalous characteristics distinguish RZ Psc from other UXors; the possible age of a few tens of Myr, the K0V spectral type, the few-day long dimming events and the location of the IR-excess emitting dust well beyond the sublimation radius. These characteristics have been used by de Wit *et al.* \[[@RSOS160652C40]\] to suggest that RZ Psc hosts a gas-poor asteroid belt analogue at 0.4--0.7 AU and that the dust clumps that occult the star are the dispersed fragments produced in destructive planetesimal collisions.
To take a critical look at this intriguing scenario, we have presented and analysed 10 years of WASP and KELT-North photometric monitoring of RZ Psc. We found circumstantial evidence that some dimming events repeat and have a semi-major axis consistent with that inferred from the IR excess, but the signal is not significant (1--2*σ*). The light curve gradients are consistent with this picture, but the constraints are poor. The statistics of the light curve gradients suggest that a typical dimming event has an egress rate that is initially faster, and then slower, than ingress. While this evolution seems qualitatively consistent with the structure expected from a planetesimal collision, quantitative models are needed.
By considering the joint constraints allowed by the light curve and the IR excess, we find that the objects causing the dimming events are unlikely to be representative of the structure causing the IR excess. The two can only be reconciled if the IR excess originates in a spherical shell of clumpy but on average optically thin dust, a scenario disfavoured by the increased polarization during deep dimming events. Assuming a disc-like structure, the belt is almost certainly optically thick with an opening angle of a few tens of degrees, with the system viewed at an inclination that allows clumps residing above this belt to pass in front of the star. While such a geometry is possible for both UXor-like and asteroid belt scenarios, the relatively cool temperature of the dust around RZ Psc means that if it hosts a gas-rich disc it must be a transition object. Indeed, comparison of RZ Psc's spectrum with other objects suggests that it is similar to objects whose status is ambiguous, and could be either debris or transition discs. The low far-IR disc luminosity could arise if the outer disc is shadowed (as suggested for UXors), or simply, because there is no outer disc. The spectrum is poorly sampled and would benefit from millimetre photometry, specifically to test whether there is a settled outer disc.
Overall, we conclude that the status of RZ Psc's disc is uncertain, and therefore that so is the origin of the clumps. The lack of near-IR excess shows that the disc is beyond the primordial phase, but could be in the final throes of dispersal and the occulting structures a related phenomenon. Several specific observations would help: (i) mid-IR spectral monitoring would allow comparisons with transition disc systems that show 'see-saw' variability, (ii) continuous photometry (ideally with multiple colours) would yield the detailed shape of individual dimming events and in some cases the distribution of dust size across the clump.
As a young Sun-like star showing disc-related stellar variability, RZ Psc is a rarity. The reason may be twofold: (i) the sublimation radius is greater than for low-mass stars, so any accretion streams are transparent and/or (ii) in contrast to more massive stars, turbulence above the inner rim may be shadowed by the outer disc. While the asteroid belt scenario avoids the need to consider primordial disc structure, in the context of such models, RZ Psc-like variability could be explained by the evolved state of the disc, which may have settled enough that the inner rim is visible.
We thank Joachim Gürtler for sharing photometry from the Sonneberg and Harvard Plates in a palatable form, AAVSO observers for monitoring RZ Psc, and Simon Hodgkin and Jim Pringle for useful discussions. This paper makes use of data from the DR1 of the WASP data \[[@RSOS160652C49]\] as provided by the WASP consortium, and the computing and storage facilities at the CERIT Scientific Cloud, reg. no. CZ.1.05/3.2.00/08.0144 which is operated by Masaryk University, Czech Republic.
Not all stars occulted by dust are UXors. Two other classes are: those occulted by circumstellar material beyond $\gtrsim$10 AU, such as AA Tau and V409 Tau which show $\gtrsim$year-long dimming events \[[@RSOS160652C23],[@RSOS160652C24]\], and systems such as *ϵ* Aur, J1407, EE Cep and OGLE-LMC-ECL-11893 where the occultations are attributed to circumsecondary discs \[[@RSOS160652C18],[@RSOS160652C25]--[@RSOS160652C27]\].
All normalized photometry is available at <https://github.com/drgmk/rzpsc>.
See <http://wasp.cerit-sc.cz>.
Data accessibility {#s7}
==================
Materials used in the preparation of this contribution can be found online at <https://github.com/drgmk/rzpsc>.
Authors\' contributions {#s8}
=======================
G.M.K. initiated the project, collated data, did most of the analysis and wrote the paper. M.A.K. contributed to the time-series analysis and conceived the iterative event finding method. K.G.S. provided independent SED models to test for a UV excess. As members of the KELT collaboration, J.P., J.E.R., R.J.S. and K.G.S. acquired the time-series photometry. M.C.W. was a co-I on the proposal to obtain the VISIR data. All co-authors provided input on the style and content of the manuscript.
Competing interests {#s9}
===================
We declare we have no competing interests.
Funding {#s10}
=======
G.M.K. is supported by the Royal Society as a Royal Society University Research Fellow. J.E.R. is supported as a Future Faculty Leaders Fellow at the Harvard-Smithsonian Center for Astrophysics. M.C.W. acknowledges support from the European Union through ERC grant no. 279973. Early work on KELT-North was supported by NASA grant no. NNG04GO70G.
| {
"pile_set_name": "PubMed Central"
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1. Introduction
===============
Silicosis, one of the earliest recognized occupational diseases, is a pathological condition of the lungs due to inhalation of particulate matter containing crystalline silica \[[@b1-ijerph-08-02923]\]. Silicosis may reflect a failure in adequate control of occupational dust exposure. Although advances in occupational safety and health make this disorder highly preventable, silicosis remains the most prevalent occupational disease worldwide \[[@b2-ijerph-08-02923]\]. The situation is particularly serious in developing countries, where millions of workers are at risk of developing silicosis. Since crystalline silica was classified as a human carcinogen by IARC in 1997 \[[@b3-ijerph-08-02923]\], a series of quantitative risk assessments has been conducted for respirable silica dust exposure, especially for low exposure levels \[[@b4-ijerph-08-02923]--[@b8-ijerph-08-02923]\].
Although the published exposure-response relationships and excess risk estimations have resulted in advanced risk communications, methodological limitations in published studies have stirred much debate within the scientific community. Besides the common problems in design, lack of adjustment of confounding \[[@b9-ijerph-08-02923]\], limited quality and poor comparability of exposure data between studies \[[@b10-ijerph-08-02923]\], inadequate consideration of exposure data or inappropriate biological assumptions of the exposure-response relationship in data analysis also seem to be important limitations.
Previously, the exposure-response relationship and excess risk estimations of silicosis have been based mainly on an assumption of a monotonic association between cumulative silica dust exposure and the incidence or prevalence of silicosis. The impact of exposure patterns (various combinations of long-term average respirable silica dust concentration, change of exposure concentration over time and latency) on risk estimations has, to our knowledge, never been considered. Previous analysis indicated that the lack of consideration of disease latency in data analysis may lead to a difference in the estimated health risks by as much as a factor of 10 \[[@b11-ijerph-08-02923]\]. In order to quantify the possible influence of exposure patterns on exposure-response relationships and long-term risk estimations of silicosis, an analysis was conducted on a cohort of Chinese pottery workers.
2. Methods
==========
2.1. Design and Study Population
--------------------------------
Details of the study design and methods, including case identification, diagnostic procedures, exposure monitoring methods, and follow-up information have been described previously \[[@b12-ijerph-08-02923]--[@b14-ijerph-08-02923]\]. Briefly, a cohort of silica exposed workers, established in the late 1980s, was followed for silicosis morbidity and all-cause mortality from Jan. 1, 1960 to Dec. 31, 1994 in 29 Chinese mines and factories (active employment between Jan. 1, 1960 and Dec. 31, 1974). An extended follow-up of cohort members in four pottery factories, six tungsten mines and four tin mines was conducted until Dec. 31, 2003. In this extended follow-up smoking habits were also assessed. The extended follow-up was conducted in a research cooperation between Tongji Medical College, China and the Institute for Occupational Safety and Health of German Social Accident Insurance.
The study population in this analysis are cohort members of the four pottery factories who attended the extended follow-up with the following inclusion criteria:
- Starting-date of employment after January 1, 1950 and aged over 15 years
- Minimum employment of 1 year
- Without unknown external silica dust exposure
In total, 7,373 employees joined the extended follow-up. Due to the inclusion criteria, 4,123 persons were excluded from the initial cohort. This resulted in a final cohort of 3,250 employees for analysis ([Figure 1](#f1-ijerph-08-02923){ref-type="fig"}).
2.2. Ascertainment of Incident Cases of Silicosis
-------------------------------------------------
A program for detection of silicosis by regular chest radiographs was launched in China in the early 1950s. Later, this program was continued in the form of a national law in 1963 \[[@b15-ijerph-08-02923]\]. According to this law, each company was and is responsible for providing yearly chest radiographs for dust-exposed workers, and for maintaining a register for employees with silicosis. This law also makes provision for a radiographic examination of workers exposed to dust every 2--3 years even after the cessation of dust exposure \[[@b15-ijerph-08-02923]\].
Radiographic reading and diagnosis of silicosis were performed in China in each province by specially trained medical teams. Diagnostic criteria were based on a standardized radiographic grading system usually applied in China, which classifies silicosis as stage 0 (suspected cases), I, II or III. If a worker has silicosis of stage I or above, he will be defined as having definite silicosis. In an inter-reader comparison study between the Chinese and Internatinoal Labor Organisation (ILO) classification system (revised ED 1980), a good agreement of 89.3% was found between the Chinese stage I and ILO profusion category 1/1 \[[@b15-ijerph-08-02923]\].
The onset of silicosis was defined in this study as the date of the radiograph leading for the first time to classification as stage I or higher. For workers without silicosis, the end of the follow-up-period was defined as the date of the last X-ray examination.
2.3. Occupational Exposure Assessment
-------------------------------------
More than 100,000 historical industrial hygiene data dating back to the 1950s are available for total dust, particle size and percentage of free silica in the pottery factories studied. These data were used in this study to create a job exposure matrix (JEM) for average total dust for each calendar year. Approximately 220 facility/job title combinations over 30 calendar periods, starting in 1950, were available. Over 60 percent of facility, job title, and calendar year data were estimated based on direct monitoring data. The remainder were estimated from monitoring data for similar jobs or data for the same job at different times, with adjustment for other historical exposure information and task descriptions for the job title. A detailed description of the JEM has been published elsewhere \[[@b14-ijerph-08-02923]\].
To estimate cumulative and long-term average dust exposures, complete individual work histories for each study subject were assessed using employment records in the personnel files of the mining companies and factories involved.
For estimation of the respirable silica dust exposures, special monitoring programs were designed to compare the Chinese total dust with the US respirable crystalline silica dust concentrations based on side-by-side measurements during 1988--1989. Conversion factors between Chinese total dust and US respirable crystalline silica dust exposure have been published in the past \[[@b16-ijerph-08-02923]\]. These conversion factors were updated in an analysis by the addition of recent measurements data from 2000 to 2006. The new analysis confirmed that the contents of free crystalline silica did not change substantially over time. However, respirable dust measurement values based on the German measurement strategy are about twice those based upon the US measurement strategy \[[@b17-ijerph-08-02923]\].
Respirable crystalline silica dust exposures were estimated by multiplication of the total dust exposures by the corresponding conversion factors (respirable crystalline silica concentration = total dust concentration × conversion factor).
2.4. Statistical Analysis
-------------------------
The statistical analysis was conducted in two steps. In the first step, exposure-response relationships between respirable crystalline silica dust exposure and the incidence of silicosis were quantified by Poisson regression analysis adjusted for age at first exposure (classified in three categories in the model), sex and smoking (ever *vs.* never). Exposure patterns were considered time-dependently in the model and presented as various combinations of long-term average exposure, highest exposure ever and time since the first exposure (time since first exposure is not identical with follow-up duration, since the starting date of employment is not the starting date of follow-up). Highest exposure ever was defined as the highest annual average concentration ever experienced during the working lifetime. Since the main purpose of this analysis is the quantification of absolute risk of silicosis at low exposure levels rather than the identification of possible threshold values of silica dust exposures, no thresholds were assessed in this study \[[@b18-ijerph-08-02923]\]. Calculation of time-dependent person-years was performed with the help of Wood's algorithm \[[@b19-ijerph-08-02923]\]. The effect estimates were calculated by PROC GENMOD (SAS 9.2) \[[@b20-ijerph-08-02923]\].
In the second step, the expected long-term risk of silicosis was quantified for a selected target population based on the estimated exposure-response relationships \[[@b21-ijerph-08-02923]\].
In this analysis, the German population was chosen as the target population. The silicosis risk was quantified based on a typical exposure situation in which workers begin employment aged 20 years and retire aged 65 years with a working life of total of 45 years. The risk of silicosis at each working year (or age) can be quantified by the Poisson regression model (depending on the exposure level, latency, smoking *etc.*). The long-term risk was estimated by cumulating the risks of silicosis over the whole working life between the ages of 20 years and 65 years, corrected for natural deaths within the target population (life table for male in Germany in 1995);
R
l
i
f
e
t
i
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i
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20
65
N
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=
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i
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where:
R~long-term~: Estimated long-term risk of silicosis over the whole working life between the ages of 20 years and 65 years;
N~i~ : Risk set at age i;
*Î~i~*: Incidence of silicosis at age i estimated by the Poisson regression model;
λ~i~: All-cause mortality of target population at age i;
x~j~: Other parameters considered in the Poisson regression model;
*β̂~j~*: Effect estimates of variables x~j~ in the Poisson regression model.
3. Results
==========
[Table 1](#t1-ijerph-08-02923){ref-type="table"} provides a description of the study population. In total, 3,250 workers were followed for silicosis morbidity for a median duration of 36.6 years. During the follow-up period, a silicosis cumulative incidence of 15.5% were identified. In addition to dust exposure, the smoking status was assessed almost completely. About 60% of cohort members had smoked at some point.
Information on both total dust and respirable crystalline silica dust exposure is presented in [Table 2](#t2-ijerph-08-02923){ref-type="table"}. On average, respirable crystalline silica dust accounted for around 4% of total dust exposure. The long-term average respirable crystalline silica dust exposure varied between 0 (below the detection limit) and 1.16 mg/m^3^, with a highest exposure ever up to 1.95 mg/m^3^. The average duration of respirable crystalline silica dust exposure was around 28 years, with a longest duration of more than 46 years.
[Tables 3](#t3-ijerph-08-02923){ref-type="table"} and [4](#t4-ijerph-08-02923){ref-type="table"} provide the results of the Poisson regression analysis for total dust and respirable crystalline silica dust exposure, respectively. The goodness-of-fit of the models was evaluated in a comparison between the observed and the estimated number of silicosis cases for each covariate in the model. They show excellent agreement (results not shown). The risk of silicosis increased consistently with increasing respirable crystalline silica dust exposure. Similar trends in exposure-response relationships can be observed for both total dust and respirable crystalline silica dust exposures. The estimated effects shown in [Table 4](#t4-ijerph-08-02923){ref-type="table"} were used for calculation of the incidence rate of silicosis and the exposure-response relationship as shown in [Figure 2](#f2-ijerph-08-02923){ref-type="fig"}.
[Figure 2a](#f2-ijerph-08-02923){ref-type="fig"} shows a dynamic change in silicosis incidence among workers exposed to dust over their whole working lives between the ages of 20 years and 65 years. Where workers begin employment aged 20 years, virtually no risk of silicosis is expected during the first 10 years of their working lives. Thereafter, the risk of silicosis increases rapidly, peaking around 35 years after the onset of exposure. After this point, the incidence decreases. This figure expresses the estimated exposure-response relationship between respirable crystalline silica dust exposure and silicosis over time.
The estimated exposure-response relationship between respirable crystalline silica dust exposure and silicosis was also presented by the cumulative risk estimator as shown in [Figure 2b](#f2-ijerph-08-02923){ref-type="fig"}. The cumulative risk curve shows a typical s-form. The peak of each curve represents the estimated long-term risk of silicosis.
The long-term risks of silicosis under various exposure patterns are summarized in [Table 5](#t5-ijerph-08-02923){ref-type="table"}. Under the same exposure conditions (long-term average exposure), workers having short-time higher exposure in their work history tend to have a higher risk of silicosis. In contrast, workers having constant lower exposure over a longer exposure duration have a much lower silicosis risk. A baseline risk of silicosis of 13.6/1,000 was identified in this analysis even among workers without dust exposure. Presumably, this finding indicates a false positive diagnosis in silicosis detection (e.g., due to smoking or other reasons). Given this baseline risk, an "excess" risk of silicosis of 0.9/1,000--1.9/1,000 was estimated among workers if all annual respirable crystalline silica dust concentrations are kept below 0.1 mg/m^3^ which implies that the long-term respirable crystalline silica dust exposure is limited at the same time to 0.1 mg/m^3^.
4. Discussion
=============
Previous analysis in the quantification of silicosis risk has often suffered from two types of limitation: limited historical exposure monitoring data; and poor comparability of exposure information reported in various studies or countries---differences in average crystalline silica dust exposure between industrialized countries of up to a factor of 12 were reported even in the same industrial sector and calendar periods \[[@b10-ijerph-08-02923]\]. This difference is more likely a reflection of the different exposure control measurement strategies used in various countries (e.g., selection of dust sampling site, compliance *vs.* non-compliance measurements) rather than of the true differences in their related working conditions. This problem often makes comparison of studies and a valid interpretation difficult.
Compared with most silicosis studies published to date, this study has advantages, including the ascertainment of incident cases of silicosis via well-established silicosis registries, clear definition of follow-up end-point for both silicotic and non-silicotic workers (via periodical X-ray examination), long-term follow-up, complete ascertainment of smoking habit, and in particular, the large quantity of well-documented historical exposure monitoring data.
The comprehensive historical measurement data permit, for the first time, a detailed quantification of the dynamic change in exposure-response relationship between various patterns of respirable crystalline silica dust exposure and the incidence of silicosis over time. The results of this analysis indicate that the risks of silicosis depend not only on respirable crystalline silica dust exposure levels, but also on their related exposure patterns and time. "Time" was presented in this analysis by two variables: "age at first exposure" and "time since the first exposure". Previous analysis indicates that, age as a parameter of biological time have an independent effect on silicosis appearance \[[@b21-ijerph-08-02923]\].The combination of age, exposure duration and latency allows flexibility in estimating the influence of time on the occurrence of silicosis. The peaks presented in [Figure 2a](#f2-ijerph-08-02923){ref-type="fig"} demonstrate the influence of latency on the occurrence of silicosis. The decreased incidences of silicosis after the peaks exhibit a typical healthy-survivor effect (risk decreases even though the residence time of silica dust in the lung continues to increase with time).
To ensure a valid exposure assessment in this study, workers with higher exposure uncertainties (unknown external dust exposure, employment before 1950 or below the age of 15 years) were excluded from the data analysis. The conversion factors between total dust and respirable dust exposures were compared by the use of different measurement strategies. The content of crystalline silica in the dust was also quantified by comparison between different laboratories. Our analysis demonstrates that comparable crystalline silica content in the dust was found in different laboratories in the USA and Germany. However, conversion factors estimated with use of the German measurement strategy were about twice as high as those estimated with use of the US measurement strategy. In this analysis, conversion factors between total dust and respirable dust exposures were quantified on the basis of the German measurement strategy.
To quantify the stability of the effect estimates in this study, we compared the results by using the updated conversion factor (based on measurement data conducted during the time 1988--1989 and 2000--2006) with previously published conversion factors (based only on the 1988--1989 measurements). Little change was found for the estimated long-term "excess" risk with use of the different conversion factors.
One main purpose of this analysis is the quantification of long-term risks of silicosis for a target population by use of the estimated effects of respirable crystalline silica dust exposures derived from a well-conducted epidemiological study. Since the impact of silica dust exposure on the occurrence of silicosis is unlikely be influenced by the population studied. Pooled analysis based on various type of population groups have been conducted so far to estimated the long-term risk of silica related health issues \[[@b6-ijerph-08-02923],[@b7-ijerph-08-02923]\]. In this analysis, we quantify the long-term risk of silicosis based on a cohort of Chinese pottery workers.
The long-term risk estimates developed in this study describe the probability of contracting silicosis due to a long-term exposure to respirable crystalline silica dust under special exposure levels or patterns. These risk estimates are accumulated over the whole working life. We applied the NIOSH approach \[[@b22-ijerph-08-02923]\], ignoring certain recommended refinements \[[@b23-ijerph-08-02923]\].
Previous estimations of the long-term risk of silicosis were based mainly on the assumption of a monotonic exposure-response relationship between cumulative crystalline silica dust exposure and the incidence or prevalence of silicosis. One important limitation of this assumption is that, time (especially latency) was not considered in the analysis. For a given cumulative exposure, exposure could be accumulated over 5 years (higher exposure) or 40 years (lower exposure). Depending on the time (especially latency) given in the data, the estimated risks may either be underestimated or overestimated. Risk quantifications based on this assumption may therefore be misleading.
In this analysis, the exposure-response relationship was presented by the incidence of silicosis (under a certain exposure pattern) over time. The long-term risk of silicosis was quantified by accumulation of the risks of the target population strictly "over time" from the ages of 20 years to 65 years, as shown in [Figure 2b](#f2-ijerph-08-02923){ref-type="fig"}. Since the risk quantification is corrected for competing deaths by application of the life table of the target population, this analysis actually gives the expected long-term risk of silicosis of the target population, given the population were exposed.
Overall, a baseline risk of silicosis of 13.6/1,000 was found in this study, even among workers not subject to dust exposure. Possible reasons for this finding may partly be associated with the common diagnostic uncertainty of early detection of silicosis \[[@b24-ijerph-08-02923],[@b25-ijerph-08-02923]\]. This is consistent with previous findings among workers not subject to dust exposure. These show that 0.2% to 11.7% of workers not subject to dust exposure have small lung opacities in radiographs which are likely to be diagnosed as silicosis or pneumoconiosis \[[@b26-ijerph-08-02923],[@b27-ijerph-08-02923]\]. In this study, one case of silicosis was also identified in an individual not subject to dust exposure (excluded from the data analysis) and in two cases, the 'highest annual exposure ever' was below 0.1 mg/m^3^.
5. Conclusions
==============
Compared to the baseline risk, a long-term "excess risk" of silicosis of 0.9/1,000 to 1.9/1,000 is expected among employees with a long-term respirable crystalline silica dust exposure below 0.1 mg/m^3^. This estimate is conservative, since it does not take potential threshold effects into account \[[@b17-ijerph-08-02923]\]. It is worth noting that, this estimate is quantified by using the German dust measurements strategy. Similar risk will be expected for an exposure level of 0.05 mg/m^3^ by using the US dust measurement strategy.
{#f1-ijerph-08-02923}
{#f2-ijerph-08-02923}
######
Description of the study population.
Potteries
----------------------------- ------------------------------- ------------
N 3,250
Age (years) Start of follow-up: mean (SD) 25.5 (6.5)
End of follow-up: mean (SD) 60.3 (10.6)
Sex (% female) 24.4
Follow-up-duration (years) Mean (SD) 34.8 (8.9)
Median (range) 36.6 (1.1--45)
Silicosis n (%) 504 (15.5)
Smoking Information available (%) 99.9
Non-smoker (%) 39.6
Ex-smoker (%) 28.6
Smoker (%) 31.8
######
Description of dust exposures.
Total dust Respirable crystalline silica dust
--------------------------------------------------------------------------------- ------------------ ------------------------------------
Long-term average exposure (mg/m^3^)
Mean (SD) 6.1 (4.8) 0.27 (0.19)
Median (min.--max.) 4.8 (0--36.7) 0.22 (0--1.16)
Highest exposure ever[\*](#tfn1-ijerph-08-02923){ref-type="table-fn"} (mg/m^3^)
Mean (SD) 23.5 (11.6) 0.73 (0.34)
Median (min.--max.) 23.8 (0--65.8) 0.74 (0--1.95)
Cumulative exposure (mg/m^3^-year)
Mean (SD) 226.4 (171.0) 7.32 (5.22)
Median (min.--max.) 183.7 (0--861.7) 6.20 (0--26.6)
Exposure duration (years)
Mean (SD) 27.8 (7.4)
Median (min.--max.) 27.8 (1.1--46.4)
Highest exposure ever was defined as the highest annual average concentration ever experienced during the working lifetime.
######
Results of Poisson-regression analysis based on total dust exposure.
No. of silicosis Person-years β 95% CI
-------------------------------------------------------------------------------------------- -------------- ------------------ -------------- ------------- ---------------
Intercept −11.58 −13.56, −9.58
Age at first exposure (years) ≤20 210 49,286 0 --
20--30 225 54,441 0.03 −0.16, 0.22
\>30 69 9,272 0.51 0.24, 0.78
Sex female 39 29,031 0 --
male 465 83,960 1.31 0.95, 1.66
Smoking never 135 47,839 0 --
ever 369 65,159 0.18 −0.04, 0.39
Highest total dust exposure ever (mg/m^3^)[\*](#tfn3-ijerph-08-02923){ref-type="table-fn"} 1\. tertile 166 46,256 0 --
2\. tertile 150 38,424 0.01 −0.22, 0.25
3\. tertile 188 28,319 0.37 0.01, 0.64
Time since the first exposure (years) 0--9 1 21,463 0 --
10--19 30 31,597 2.92 0.92, 4.91
20--29 208 29,510 4.90 2.93, 6.86
30--39 244 20,657 5.44 3.48, 7.41
≥40 21 9,771 3.84 1.83, 5.85
Long-term average total dust exposure [\*\*](#tfn4-ijerph-08-02923){ref-type="table-fn"} 1\. quintile 100 28,167 0 --
2\. quintile 100 17,902 0.37 0.09, 0.66
3\. quintile 102 19,089 0.48 0.19, 0.77
4\. quintile 101 15,132 0.66 0.34, 0.97
5\. quintile 101 32,709 0.05 −0.30, 0.40
Highest exposure ever was defined as the highest annual average concentration ever experienced during the working lifetime.
Highest exposure ever (mg/m^3^): 1. tertile: from 0 to \<19.6 (total dust); 2. tertile: from 19.6 to \<29.8 (total dust); 3. tertile: from 29.8 to 65.8 (total dust).
Long-term average exposure (mg/m^3^): 1. quintile: from 0 to \<3.94 (total dust); 2. quintile: from 3.94 to \<5.90 (total dust); 3. quintile: from 5.90 to \<8.67 (total dust); 4. quintile: from 8.67 to \<11.73 (total dust); 5. quintile: from 11.73 to 37.7 (total dust).
######
Results of Poisson-regression analysis based on respirable crystalline silica dust exposure.
No. of silicosis Person-years β 95% CI
---------------------------------------------------------------------------------------- -------- ------------------ -------------- ------------- ----------------
Intercept −13.99 −16.41, −11.56
Age at first exposure (years) ≤20 67 61,766 0 --
20--30 390 36,849 0.02 −0.17, 0.21
\>30 47 14,384 0.48 −0.21, 0.75
Sex female 39 29,031 0 --
male 465 83,960 1.40 1.04, 1.76
Smoking Never 135 47,839 0 --
ever 369 65,159 0.16 −0.06, 0.37
Highest silica exposure ever (mg/m^3^)[\*](#tfn5-ijerph-08-02923){ref-type="table-fn"} \<0.10 2 8,386 0 --
0.1--0.5 194 45,524 2.08 0.66, 3.50
\>0.5--1.0 283 50,849 2.30 0.87, 3.73
\>1.0 25 8,237 1.27 −0.22, 2.76
Time since the first exposure (years) 0--9 1 21,463 0 --
10--19 30 31,597 2.95 0.96, 4.94
20--29 208 29,510 4.99 3.02, 6.95
30--39 244 20,657 5.58 3.61, 7.55
≥40 21 9,771 3.96 1.95, 5.97
Long-term average silica exposure (mg/m^3^) \<0.05 18 10,657 0 --
0.05--\<0.10 35 9,811 0.24 −0.33, 0.82
0.10--\<0.15 63 11,500 0.40 −0.13, 0.93
0.15--\<0.20 85 14,459 0.59 0.07, 1.11
20--\<0.30 133 21,977 0.75 0.24, 1.27
0.30--\<0.40 95 16,155 0.84 0.29, 1.38
\>=0.40 75 28,439 0.50 −0.07, 1.07
0.05 mg/m^3^ increase 0.07 0.02, 0.11
Highest exposure ever was defined as the highest annual average concentration ever experienced during the working lifetime.
######
Estimated long-term risk of silicosis by respirable crystalline silica exposure patterns in the low exposure region.
Highest silica exposure ever (mg/m^3^)[\*](#tfn6-ijerph-08-02923){ref-type="table-fn"} Long-term average silica exposure (mg/m^3^)
---------------------------------------------------------------------------------------- --------------------------------------------- ------------ ------------ ----------- -----------
Long-term "excess" risk (compared to baseline)
\<0.1 0.9/1,000 1.9/1,000
0.1--0.5 138/1,000 147/1,000 157/1,000
\>0.5--1.0 179/1,000 191/1,000
Long-term risk
\<0.1 13.6/1,000 14.5/1,000 15.5/1,000
0.1--0.5 152/1,000 161/1,000 171/1,000
\>0.5--1.0 193/1,000 205/1,000
Highest exposure ever was defined as the highest annual average concentration ever experienced during the working lifetime
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Plant-pollinator interactions are complex. Pollinators visit flowers to obtain nectar and pollen as food; in turn, pollinators facilitate sexual reproduction in plants. While this relationship is mostly mutualistic, floral nectar and pollen sometimes contain toxins or other plant compounds^1-5^which can harm pollinators. The ecological rationale for the presence of such compounds in nectar and pollen is not clear in all settings. One outstanding question in this field is how pollinators such as bees can detect and avoid flowers with nectar containing toxins.
The bumble bee species, *Bombus terrestris*(Linnaeus, 1758), is a generalist pollinator that visits the flowers of many plant species including those producing nectar containing toxins^6^. Bumble bees have been shown to avoid consuming solutions containing high concentrations of toxins in a 24 hr two-choice assay^7^. This assay of food consumption described by Tiedeken *et al*.^7^ revealed that bees can detect bitter compounds in solutions. However, this assay was unable to distinguish taste from post-ingestive processes such as malaise that could also affect feeding behavior over this time interval^8-10^.
Bees possess gustatory sensilla on their antennae, mouthparts and tarsi to detect compounds^11-13^. The proboscis extension reflex (PER) experiments involve restraining individual bees in a harness and then stimulating the bee\'s antennal sensilla to produce the feeding reflex^14-17^. Bees can be restrained in individual harnesses and then stimulated to produce the feeding reflex as an assay of their ability to taste compounds^18,19^. Others have modified the PER assay to study the sensitivity of the antennae or mouthparts to toxins^9,20^. However, bees are subjected to stress during harnessing. This could affect how they respond to compounds^21^.
Here, a new assay is described to assess the behavioral taste response of freely-moving bumble bees to sucrose and quinine, an alkaloid that has previously been reported to be deterrent^9^ and toxic^10^ to honey bees (*Apis mellifera*) and bumble bees (*Bombus terrestris*)^7,22^. Although quinine has not been found in plant nectar, this alkaloid is often used as an aversive stimulus in behavioral and physiological studies in bees^7,9,12,13,22^. The method involves video recording the bumble bees\' mouthparts at great resolution during the initial proboscis contact with the test solutions. Specifically, the fine structure of the feeding response is examined by continuously scoring behavior over a 2 min interval. The volume of solution consumed is measured during the feeding period and so the amount of food eaten can be correlated with the microstructure of the feeding behavior. Also the speed of the proboscis retraction is measured, as an indicator of an active avoidance, and therefore pre-ingestive detection.
Protocol
========
1. Capturing Bees from the Colony and the Starvation Period
-----------------------------------------------------------
Note: Experiments described here were performed at Newcastle University, UK with *Bombus terrestris audax*. Multiple (2-3) commercially purchased colonies were used per treatment. The colonies have been maintained on a bench at laboratory conditions (25 ± 2 °C and 28 ± 2% RH) in constant darkness and were fed with honey bee collected pollen and sugar solutions *ad libitum*.
1. Collect individual worker bumble bees using a plastic vial (7 cm long, 2.8 cm inner diameter) with a perforated plastic stopper, after having opened the gate to the colony just long enough for one bee to exit and be trapped.
2. Prior to the experiment, individually starve all bumble bees for 2-4 hr in the plastic vials and keep at room temperature in complete darkness.
2. Transferring Bees into the Holding Tubes and the Habituation Phase
---------------------------------------------------------------------
1. After the starvation period, transfer a bumble bee directly from the plastic vial into a holding tube. The holding tube is a modified 15 ml centrifuge tube (length: 119 mm; diameter 17 mm), with a 4 mm hole drilled at the tip and a piece of steel mesh (base: 8 mm; height 30 mm) fixed inside by melting the plastic of the tube with a heated dissecting steel needle.
2. Fix the holding tube containing the bumble bee onto a polystyrene holder with dental wax. Fix two pieces of cardboard on either side of the holding tube. This is to shield the bee from visual stimuli that might interfere with the experiment.
3. Position a digital microscopic camera 5 cm above the tip of the holding tube and connect the camera to a compatible laptop.
4. Adjust the holding tube so that at least the first 18 mm of the holding tube tip is within the video frame. Prior to the experiment, commence the 3 min habituation period.
3. Pre-test Phase: Presenting a Drop of Sucrose
-----------------------------------------------
1. Connect a syringe to a female adapter containing a droplet of sucrose solution (\~3.5 µl, 500 mM sucrose dissolved in deionized water). Present the sucrose inside the holding tube tip to motivate the extension of the proboscis.
2. Give the bumble bee up to 5 min to consume the sucrose droplet. If the droplet is not consumed, remove the bumble bee from the experiment.
3. Begin video recording after the habituation period. In this study, the proboscis activity was recorded at 26.7 frames/sec^-1^ with a 25X magnification rate.
4. Test Phase: Presenting the Test Solution
-------------------------------------------
1. Fill a 100 µl microcapillary tube with the test solution. Connect it to a piece of silicone tubing (6 cm length, 1 mm inside diameter) and fix it to a micro manipulator. Connect the tubing via a male adapter to another silicone tubing (6 cm length, 4 mm inside diameter), which acts as a pipette bulb.Position the microcapillary tube 5-10 mm away from the holding tube tip. Gently squeeze the tubing to maintain the feeding solution at the tip of the microcapillary tube.
2. After the bumble bee consumes the sucrose droplet, immediately remove the syringe containing the 500 mM sucrose solution.
3. Begin the 2 min test phase when the bumble bee\'s proboscis contacts the solution inside the microcapillary tube. To control for possible evaporation, fill two additional microcapillary tubes with sucrose or water and manipulate it exactly as during the test phase.
4. Before and after each trial scan the liquid levels inside the microcapillary tube using a scanner at 600 dpi to measure the amount of food consumed (**Figure 4A**).
5. Image Analyses
-----------------
1. Determine the volume of solution consumption using ImageJ (version 1.48), an image processing software. Upload the image file and zoom into the image (\~400%). To set the reference scale, select the straight line tool and draw a line between the two ends of the microcapillary tube. Select \'Analyze\' then \'Set Scale\'. Input the total length of the tube under \'Known distance\' and the corresponding unit under \'Unit of length\'.Select the straight line tool again and draw a line between the two ends of the liquid level. Select \'Analyze\' then \'Measure\'. In the results window the length of the liquid is given under the \'Length\' column.
2. Calculate the volume of solution consumption by using the formula: where is the length of the microcapillary tube and and are the measured lengths of the liquid inside the microcapillary tube before and after the test phase, respectively.
6. Video Analyses
-----------------
1. Score the feeding behaviors during the 2 min testing phase of each video using an event logging software (See **Materials Table**). At first, define the feeding behaviors (*i.e*. the elements) in the behavioral classes menu of the recording software. The feeding behaviors are as followed:(1) proboscis out/contact: the proboscis extends and is in contact with the solution inside the microcapillary tube (2) proboscis out/no contact: the proboscis extends and is not in contact with the solution inside the microcapillary tube, (3) proboscis stowed: the proboscis is not extended but instead stowed under the head and (4) out of sight: the bumble bee is out of the video frame.Set each behavior as a \'state\' and \'mutually exclusive\' in the properties menu and make continuous recordings for a 2 min interval. Replay the videos in slow-motion mode (2 times slower) for more precision.
2. Measure the speed of proboscis retraction from the test solution after the first contact between two consecutive frames (separated by 37.5 msec in the video recordings shown here) using a motion tracking video software (See **Materials Table**). Upload the video file and skip to the frame where the proboscis first contacts the solution.To set the reference scale, select the line tool and draw a line on the width of the microcapillary tube in the video frame. Right click on the line and select \'Calibrate measure\'. Input the width of the capillary tube and the corresponding unit.Select \'Image\' then \'Coordinate system origin\'. On the new window click on the tip of the proboscis and select \'Apply\'.Select the hand move tool, right click on the tip of the proboscis on the video frame and select \'Track Path\'. Move to the next frame and readjust the tracking point to the tip of the proboscis.
3. Right click on the tracking point and select \'Configuration\'. Select \'Complete Path\' and select \'Speed\' under measurement. Select \'Apply\'. The speed is then displayed.
Representative Results
======================
The novel assay is used to test the feeding responses to 1 M sucrose, 1 M sucrose solution plus 1 mM quinine and deionized water alone. The immediate feeding responses to each treatment are determined by quantifying the duration of proboscis contacts with the test solution, the frequency of the feeding bouts and the speed of the proboscis retracting away from the test solution after the first contact during the 2 min test phase. The volume of solution consumed is also measured after the test phase. In this study, we have chosen a bout criterion interval of 5 sec (**Figure 1,**see **Supplemental File**) based on previous work by French *et al*.^25^ who used a 5 sec threshold to characterize the proboscis retraction behavior by *Drosophila* in response to deterrent compounds^25^. Thus, we defined a feeding bout as a contact between the extended proboscis and the solution not interrupted by an absence of contacts of 5 sec or more.
In comparison to sucrose and deionized water alone, adding quinine to sucrose solution evidently deters feeding by bumble bees as they will rapidly move away if they detect an aversive substance (**Video Figure 1**).
In this experiment, the treatments have a significant effect on the cumulative duration of proboscis contacts during the test phase (ANOVA on the log-transformed data, F~2,31~= 41, *p*\<0.001). The cumulative duration of contact time with sucrose containing quinine is significantly reduced in comparison to sucrose alone (*p*\<0.001) but not to deionized water alone (*p*= 0.219) (**Figure 2**). Similarly, the treatments have a significant effect on the cumulative duration of feeding bouts (ANOVA on the log-transformed data, F~2,31~= 27.95, *p* \<0.001, **Figure 3A**). The cumulative duration of feeding bouts with sucrose containing quinine is significantly reduced in comparison to sucrose alone (p \<0.001) but not compared to deionized water alone (p = 0.41). The treatments have also a significant effect on the frequency of feeding bouts (Poisson GLM with a log link function, change in deviance compared to the *c*^2^ distribution: *p*\<0.050), whereby the number of bouts with sucrose containing quinine is significantly higher in comparison to sucrose (*p*\<0.01) but marginally significantly different to the deionized water treatment (*p*= 0.055, due to one bumblebee displaying seven feeding bouts on water, **Figure 3B**). Likewise, the speed of proboscis retraction differs significantly between treatments (ANOVA on the log-transformed data, F~2,31~= 5.12, *p*\<0.050). Bumble bees retract the proboscis away from the test solution significantly faster after the first contact with sucrose containing quinine than with sucrose or deionized water alone (*p*\<0.050, **Figure 3C**). These results suggest that quinine triggers an active avoidance behavior in bumble bees. The treatments also have a significant effect on the total volume of solution consumed (ANOVA on the log-transformed data, F~2,32~= 62.5, p \<0.001), whereby the consumption of sucrose containing quinine is reduced in comparison to sucrose (*p* \<0.001) but not to deionized water (*p* = 0.457) (**Figure 4B**). The volume of solution evaporated from the capillary during the test period is negligible. At laboratory conditions (25 ±2°C and 28 ±2% RH), the evaporation varies between 0.033 to 0.883 µl with an average of 0.276 µl and 0.171 µl for deionized water and 1 M sucrose respectively.
In this assay contacts between the antenna and the test solution cannot be prevented. Nonetheless, the percentage of bumble bees using their antennae to taste the feeding solution during the test phase (sucrose: 46.1%, sucrose plus quinine: 60.0% and deionized water: 33.3%) is not significantly different between the treatments (binomial GLM, change in deviance compared to the *c*^2^ distribution: *p*= 0.450*)*. No effect of the treatments is found on the latency between the first antennal contacts and the test solution and the first contacts of the proboscis (median: 2.67 sec for sucrose; 1.10 sec for sucrose plus quinine; 0.80 sec for deionized water, ANOVA on the log-transformed data, F~2,13~= 0.620, *p*= 0.550). In addition, the percentage of bumble bees extending the proboscis to taste the test solution remains constant across the treatments (sucrose: 66.7%; sucrose plus quinine: 50.0%; deionized water: 52.2%; binomial GLM, change in deviance compared to the *c*^2^ distribution: *p*= 0.840). Together these results suggest that the antennae play a minor role in the detection of the toxins in this assay.
A separate experiment examines whether it is necessary to test bees for a period of time longer than 2 min. The amount of food consumed by bees is tested with the 1 M sucrose or 1 mM quinine in 1 M sucrose solutions in two conditions: a 2 min test period and a 10 min test period. For both treatments, total food consumption does not differ for the test periods and no significant interactions occur between the test period and the treatment (N = 6 - 13, ANOVA on the log transformed data; effect of the treatments:*F*~1,31 ~= 54.8, *p*\<0.001; effect of the test period: F~1,31~= 0, *p*= 0.979; effect of the interaction: F = 0.1, *p*= 0.457). In summary, a 2 min test period is sufficient to assess the effect of the solution on the total amount of food consumed by bumblebees and the deterrent effects of toxic or repellent substances in this assay. Thus, by measuring food consumption and assaying feeding behavior, it is possible to correlate total food consumption to the fine structure of feeding during the assay.
**Figure 1: Latency Periods between the Proboscis Contacts during the First 2 Min of the Feeding Assay.** Density plots of the time latency periods separating each proboscis contact with the 1 M sucrose solution, the 1 M sucrose + 1 mM quinine solution and water. The cumulative data from 13, 10 and 11 bees are represented respectively. [Please click here to view a larger version of this figure.](https://www.jove.com/files/ftp_upload/54233/54233fig1large.jpg)
**Figure 2:Proboscis Contact Durations during the First 2 Min of the Feeding Assay.** Density plots of the proboscis contact durations by bumblebees feeding on 1 M sucrose, 1 M sucrose + 1 mM quinine or water. Sample size as in Figure 1. [Please click here to view a larger version of this figure.](https://www.jove.com/files/ftp_upload/54233/54233fig2large.jpg)
**Figure 3: Proboscis Activity of Bumblebees Feeding on 1 M Sucrose, 1 M Sucrose + 1 mM Quinine or Water.**(**A**) The cumulative duration of feeding bouts during the test phase (**B**) the frequency of feeding bouts and (**C**) the speed of the proboscis retraction after first contact. Lettering indicates a significant difference: treatments with different letters indicate P \<0.05. Box plots represent the median (black bars), the lowest and the highest data points still within 1.5 of the interquartile range (whiskers) and the outliers (circles). Sample size as in Figure 1. [Please click here to view a larger version of this figure.](https://www.jove.com/files/ftp_upload/54233/54233fig3large.jpg)
**Figure 4: Quinine supresses feeding by bumble bees.**(**A**) Scanned images of the microcapillary tubes showing the level of the 1 M sucrose or the 1 M sucrose plus 1 mM quinine solution (indicated by a black line) before and after the test phase respectively. (**B**) Consumption of 1 M sucrose, 1 M sucrose plus 1 mM quinine or deionized water alone by bumble bees after the test phase. Lettering indicates a significant difference: treatments with different letters indicate P \<0.001. Box plots represent the median (black bars), the lowest and the highest data points still within 1.5 of the interquartile range (whiskers) and the outliers (circles). Sample size as in Figure 1. [Please click here to view a larger version of this figure.](https://www.jove.com/files/ftp_upload/54233/54233fig4large.jpg)
**Video Figure 1:**Video Recordings of the Proboscis Activity toward (**A**) [1 M Sucrose](https://www.jove.com/files/ftp_upload/54233/Video_figure_1A_1M_Sucrose.m4v), (**B**) [1 M Sucrose Plus 1 mM Quinine](https://www.jove.com/files/ftp_upload/54233/Video_figure_1B_Quinine.m4v) and (**C**) [Deionized Water during the Test Phase](https://www.jove.com/files/ftp_upload/54233/Video_figure_1C_Water.m4v).
Discussion
==========
With this novel behavioral assay, quinine is shown to deter feeding of the buff-tailed bumble bee. The reduced proboscis contact time and feeding bout frequency with water or the sucrose solution laced with quinine is interpreted here as a refusal to initiate further feeding on non-nutritive or potentially toxic solutions. When quinine is added to 1 M sucrose solution, bumble bees not only reduce the volume of solution they consume, they also retract the proboscis faster, thus reducing contact time between the mouthparts and the solution containing a toxin. Together, these results suggest that quinine is perceived by the gustatory receptor cells on the mouthparts of the bumble bee, as already previously identified in the honey bee^9^. Quinine is a toxin for insects that induces malaise-like behavior in the honey bee^10^ and knockdown in the malaria mosquito (*Anopheles gambiae*)^23^. This assay could well lead to the identification of some deterrent and potentially toxic compounds that are perceived by the taste receptor cells on the mouthparts in the bumble bees.
It is crucial for the microcapillary tube to be filled with a sufficient volume of test solution to last throughout the test phase. It is recommended that at least around three quarters of the microcapillary tube (*e.g.* 70-80 µl) is filled. However, care should be taken to not completely fill the microcapillary tube to reduce the risk of spillage during the process of scanning and attaching the microcapillary tube to the experimental apparatus. Care should also be taken when presenting the 500 mM sucrose droplet to the bumble bee, so that the experimenter avoids leaking the droplet into the holding tube.
The 4 mm hole at the tip of the holding tube is large enough for an adult worker bumble bee to naturally extend its proboscis toward the test solution. However it is possible that bumble bees can taste the solution with their antennae before extending their proboscises. This might affect the probability of proboscis extension as PER could be elicited in bumble bees by stimulating their antennae with a sugar solution^15^. In fact the antennae of Hymenoptera like the parasitoid wasp (*Trissolcus brochymenae*)^24^ or the honey bee^13^ are equipped with taste sensilla, allowing them to taste sugars and toxins like quinine. Consequently, initial antennal contacts with solutions containing highly deterrent compounds like quinine could also reduce the motivation of a bumble bee to extend its proboscis and therefore affect the experimental success rate. Although antennal contact with the test solution cannot be controlled, in the present study we did not find any significant effect of antennal contact on proboscis extension toward the test solution. In this assay, immediately setting up the microcapillary tube after the pre-test phase when the bumble bees\' antennae are still within the holding tube can reduce the opportunity for the bumble bees to taste the test solution with their antennae.
The main limitation of this assay arises when tracking the proboscis retraction away from the test solution after the first proboscis contact using the motion tracking video software. The video footage only displays 2D movement of the proboscis, so the given output of the speed measurement can be under or over estimated. However with some modifications, this aspect of the assay could be improved.
This assay can be used to observe natural feeding responses toward solutions containing different compounds including natural-occurring plant secondary metabolites. Observing the immediate feeding responses with this assay gives detailed information about how bumble bees detect these compounds. This is advantageous over existing \'go-no go\' methods like PER^18,19^ and over two-choice assays^7^ because this method produces several behavioral response measures including food consumption during a discrete feeding bout.
Measuring several parameters simultaneously allows a better evaluation of the palatability of a compound. For example in our assay, bumble bees avoid consuming water or the sucrose solution laced with quinine. Retraction of the proboscis could be caused by a change in the responses of the sugar receptor cells^12,13^. Our assay shows that bumble bees retract the proboscis faster after contacting the sucrose plus quinine solution than water alone; this could suggest that quinine affects a distinct set of neurons in addition to inhibiting sugar sensing neurons^9,12,13,25^.
Our assay permits the analysis of the temporal pattern of behavioral responses during feeding. A similar protocol where the consumption time and the number of bouts is measured has already been implemented to evaluate the feeding response of *Drosophila* to nutritive and non-nutritive sugars^26^. We predict that bees will exhibit a more reliable response to feeding stimulants in our assay than in other methods such as PER because the bees are free to move in the holding tube^21^. This technique will permit an exhaustive analysis of the taste thresholds for nutrients and toxins to illuminate the mechanisms of feeding in bumble bees and potentially other bee species.
Disclosures
===========
The authors declare no conflict of interest.
This work was funded by the Leverhulme Trust grant (RPG-2012-708) and a BBSRC grant (BB/M00709X/1) to GAW.
[^1]: Correspondence to: Geraldine Wright at <jeri.wright@newcastle.ac.uk>
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Echocardiography is a useful tool for risk stratification and prognosis assessment following acute myocardial infarction (AMI). Several echocardiographic parameters, such as left ventricular (LV) volume, ejection fraction (EF), wall motion score index (WMSI), presence of mitral regurgitation, and left atrial volume, have been shown to provide prognostic information [@pone.0086959-Gibbons1]--[@pone.0086959-Mollema1]. LV volume and EF are the primary means for assessing myocardial systolic function and myocardial damage after AMI. Nevertheless, it must be taken into account that these indices are global and load-dependent. The development of cardiac magnetic resonance imaging (CMR) with the tagging approach and echocardiography with the speckle-tracking strain imaging has provided additional tools to assess global and regional functions according to myocardial fiber orientation and position within the myocardial thickness [@pone.0086959-Vartdal1]--[@pone.0086959-Hung1]. As a result, longitudinal, radial, and circumferential functions can be distinctively assessed. Using speckle-tracking imaging, several studies have demonstrated the usefulness of longitudinal and circumferential strains in differentiating between sub-endocardial and transmural AMI, and assessing post-AMI prognosis [@pone.0086959-Hung1]--[@pone.0086959-Chan1].
CMR is currently considered to be the most reliable method for determining microvascular obstruction (MVO) in the first days after reperfusion [@pone.0086959-Wu1] and for measuring accurately infarct size a few weeks later [@pone.0086959-Gerber1], [@pone.0086959-Wu2], both parameters being well-established prognosticators [@pone.0086959-Gerber1], [@pone.0086959-Wu3], [@pone.0086959-Gerber2]. However, CMR accessibility is limited, whereas echocardiography is readily available.
In the present study, we sought to prospectively assess the value of speckle tracking imaging performed within the first days after successful reperfusion in ST-segment elevation myocardial infarction (STEMI) patients in order to predict initial microvascular obstruction (MVO) and infarct size at a later time point.
Method {#s2}
======
Patients with STEMI admitted to the Angers university hospital were prospectively enrolled. Inclusion criteria were as follows: primary or rescue percutaneous coronary intervention (PCI) for first STEMI within 12 hours of symptom onset; age above 18 years; culprit coronary artery with proximal occlusion, *i.e.,* proximal or mid-left anterior descending coronary artery, proximal dominant circumflex coronary artery, or proximal right coronary artery; thrombosis in myocardial infarction (TIMI)-flow Grade 0 or 1 prior to PCI, and successful revascularisation with TIMI-flow Grade 2 or 3 after stenting. Diagnosis of STEMI was defined by chest pain for at least 30 minutes, ST-segment elevation ≥0.1 mV in at least two or more limb leads, or ST-segment elevation ≥0.2 mV in two or more contiguous precordial leads. Exclusion criteria were cardiogenic shock, history of myocardial infarction or aorto-coronary bypass surgery, contraindication to CMR and cardiac arrest before PCI.
Baseline echocardiography was performed within 5 days after myocardial reperfusion. CMR was performed at baseline, within 10 days after myocardial reperfusion in order to assess MVO, with the examination repeated at 3-month follow-up in order to quantify infarct size and infarct transmurality.
The protocol was approved by the Institutional ethics committee at the University Hospital of Angers (France), and the study was conducted in accordance with the Declaration of Helsinki and French regulatory requirements. Prior to being included into the study, the patients gave their written informed consent.
Echocardiography {#s2a}
----------------
Images were obtained in the left lateral decubitus position with a commercially available VIVID 7 system (GE Healthcare, Horten, Norway) using a 2.5 MHz transducer at a depth of 14 to 16 cm. Standard data on bi-dimensional echocardiography was collected according to American Society of Echocardiography (ASE) guidelines [@pone.0086959-Hanekom1], with LV size evaluated by M-mode on a parasternal long axis view, and wall motion scored using a 16-segment LV model as follows: 1 = normokinetic, 2 = hypokinetic, 3 = akinetic, and 4 = dyskinetic. LV and left atrial volumes were estimated using the biplane Simpson's method from apical four-chamber and two-chamber views. Aortic stenosis and mitral regurgitation were quantified [@pone.0086959-Hanekom1].
For 2D-strain analysis, apical four-, three-, and two-chamber views, and parasternal short axis view at papillary muscle level were stored in cine-loop format, triggered to the QRS complex during one heart cycle with frame rates between 55 and 70 frames/sec. These cine loops were then analyzed off-line on the EchoPAC (GE healthcare, Horten, Norway) system, based on frame-by-frame tracking of natural acoustic markers [@pone.0086959-Leitman1]. Peak systolic longitudinal strain (LS) was determined for each of the 16 segments, and longitudinal global strain was then calculated. Peak radial and circumferential systolic strains were determined from papillary muscle level short axis view, with mean values computed. All acquisitions and analyses were carried out by a single experienced cardiologist blinded to the CMR results ([Figures 1](#pone-0086959-g001){ref-type="fig"} & [2](#pone-0086959-g002){ref-type="fig"}).
{#pone-0086959-g001}
{#pone-0086959-g002}
For 2D-strain analysis, inter-observer and intra-observer agreements were determined based on recordings obtained from twenty random patients.
Cardiac Magnetic Resonance Imaging {#s2b}
----------------------------------
CMR scans were performed with 1.5-T imager (Avanto, Siemens, Erlangen, Germany) using an 8-element phased-array cardiac receiver coil. Localization was carried out using breath-hold real-time and steady-state free precession images of true anatomical axes of the heart.
LV function was assessed by means of cine imaging using a segmented steady-state free precession pulse sequence in multiple short-axis, long-axis, and four-chamber views covering the entire LV. Typical in-plane resolution was 1.6×1.9 mm, with a 7.0 mm section thickness and the following parameters: repetition time (TR)/echo time (TE): 2.6 ms/1.3 ms; flip angle: 80°; matrix: 256×208; temporal resolution: 35--50 ms. Receiver bandwidth was set to 930 Hz/px and adapted to the shorter echo spacing (3.1 ms).
Late gadolinium enhancement (LGE) was performed 10--12 minutes following initiation of gadoterate meglumine (DOTAREM, Guerbet, Aulnay-sous-Bois, France), administered in cumulative dose calculated at 0.2 mmol/kg of body weight, along with a two-dimensional segmented inversion-recovery gradient-echo pulse sequence. Typical in-plane resolution was 1.68×1.68 mm, with a 7.0 mm section thickness and the following parameters: TE: 4.66 ms; flip angle: 30°; image acquisition triggered at every other heartbeat; matrix: 256×208. Inversion time was set to null the normal viable myocardium signal and typically ranged from 240 ms to 300 ms.
All deidentified images were analyzed at the central core laboratory by two experienced observers who were blinded to the patient data. Commercially available software (QmassMR 7.1, the Netherlands) was used for the analysis. Endocardial and epicardial borders were outlined manually on all end-diastolic and end-systolic short-axis cine slices. LV end-systolic and end-diastolic volumes, EF, and LV myocardial mass were then calculated in a standard manner.
The recommended 17-segment system was employed [@pone.0086959-Cerqueira1], with the two blinded observers' consensus required.
LV segmental motion was assessed visually using a three-grade scale: 0 = normal; 1 = hypokinesia; 2 = akinesia, dyskinesia, or systolic expansion.
Transmural myocardial LGE extension was assessed visually. The segmental extent of LGE was assessed as follows: 0 = 0%; 1 = 1%--25%; 2 = 26%--50%; 3 = 51%--75%; 4 = 76%--100% hyperenhancement. We considered myocardium non viable if LGE exceeded 75% of mural extent. LGE area was manually defined on all segments, and infarct size was defined as the total LGE mass. Microvascular obstruction (MVO) was identified by the presence of a central hypoenhancement within the bright signal [@pone.0086959-Wu1], and the MVO area was manually traced on all segments. In cases where no MVO was present, MVO mass was set at zero. For CMR parameter analysis, including LV volumes, EF, mass, LGE mass, and MVO mass, inter-observer agreements were determined based on data gathered from 20 random patients.
Infarcted Area Analysis {#s2c}
-----------------------
A segment was considered as part of the infarcted area if LGE was present in more than 50% of the circumferential extent for that segment [@pone.0086959-OrtizPerez1]. Mean LGE segmental extent in the infarcted area was calculated, with a value \>75% required for the diagnosis of transmural infarction.
The mean value of longitudinal strain was calculated within the infarcted area (infarct LS) so as in the remote myocardium area.
Statistical Analysis {#s2d}
--------------------
Data was presented as mean±standard deviation or median (25^th^;75^th^ percentiles) in cases of non-normal distribution, with categorical data expressed as frequencies and percentages. We compared baseline with 3-month CMR data using McNemar test or a Chi-squared test, as appropriate. Baseline echocardiographic and CMR data obtained at 3-month follow-up was compared using Pearson's correlation test. P-values \<0.05 were considered statistically significant.
For multivariate analyses, echocardiographic data was tested by a descending step-by-step linear regression analysis, including variables with p values \<0.05. The linear relation between dependent and independent variables was tested, as was the normal distribution of the residuals on PP plots. Analysis was performed using SPSS version 15.0 for Windows (SPPS, Inc, Chicago, IL).
We calculated the specificity, sensitivity, as well as positive and negative values of different parameters for predicting myocardial contractility recovery at 3-months (absence of recovery was defined by ≥3 akinetic segments at 3 month). ROC curves were generated.
Inter-observer agreement was studied in a blinded fashion using echocardiographic and CMR scans randomly selected from 20 patients, which were analyzed on EchoPac and QmassMR software by two different observers. Intra-observer agreement was also tested in a blinded fashion using 20 speckle tracking examinations, which were analyzed at two different times on EchoPac. Inter-and intra-observer agreements were assessed using the Bland and Altman method [@pone.0086959-Bland1], and intraclass correlation coefficients (ICC) [@pone.0086959-Landis1] were calculated. For radial and circumferential strain reproducibility analysis, two examinations were not evaluable due to lack of echogenicity on parasternal view.
Results {#s3}
=======
In total, 44 patients were included in the study between February 2009 and March 2010. Of these, three were excluded from analysis, one because of surgically treated significant ischemic mitral regurgitation at 1-month follow-up, another because of significant aortic stenosis, and the remaining patient because of pace-maker implantation prior to the second CMR. Characteristics of the remaining 41 patients are summarized in [Table 1](#pone-0086959-t001){ref-type="table"}. Of note, two patients with acute anterior STEMI presented small inferobasal, previously unknown infarctions. Echocardiographies were conducted 3.9±1.2 days and CMR 5.6±2.5 days and 99.4±4.6 days following myocardial reperfusion, with echocardiographic and CMR data summarized in [Table 2](#pone-0086959-t002){ref-type="table"}.
10.1371/journal.pone.0086959.t001
###### Characteristics of the patient population (n = 41).
{#pone-0086959-t001-1}
Variable
--------------------------------------------------------------------------------------------------------------------------- -------------------
Age , y 57±12
Male, % (n) 82 (34)
Smoking, % (n) 56 (23)
Diabetes, % (n) 19 (8)
Hypertension, % (n) 39(16)
Family history of CAD, % (n) 41 (17)
Dyslipidemia, % (n) 56 (23)
Primary PCI, % (n) 80 (33)
Time onset of symptoms to PCI (min) 226 (176.5;303.7)
Culprit coronary artery: LAD/LCx/RCA (%) 66/17/17
Peak CPK, UI/L 1845 (1196;3149)
Medications at 3 months including aspirin, statins, clopidogrel, angiotensin-renin systeminhibitors, and beta-blockers, % 95
CAD: coronary artery disease; PCI: percutaneous coronary intervention; LAD: left anterior descending coronary artery; LCx: left circumflex coronary artery; RCA: right coronary artery; CPK: creatinin phosphokinase.
10.1371/journal.pone.0086959.t002
###### Echocardiography and CMR data.
{#pone-0086959-t002-2}
Baseline 3-monthsfollow-up
---------------------------- -------------------------------------------------- ---------------------------------------------------
**Echocardiographic data**
LVEDV index (mL/m^2^) 75.3 (60.7;90.4)[†](#nt104){ref-type="table-fn"}
LVESV index (mL/m^2^) 34.9 (24.9;46.4)[†](#nt104){ref-type="table-fn"}
WMSI 1.4±0.2
LVEF (%) 51.2±7.3
E/E' 10 (8;13)
Deceleration time ofE (ms) 207±43
LA volume (mL) 40 (26.6;49.8)
Sphericity index 1.6±0.2
MR grade 1/ 2 /3/4 (%) 24/2/0/0
GLS (%) −13.9±3.4
CGS (%) −14.5±3.6
RGS (%) 32.7±11.7
**CMR data**
LGE mass (g) 17.5 (12.0;35.0) 14.0 (7.5;24.5)[\*](#nt103){ref-type="table-fn"}
TransmuralInfarction % (n) 63 (26) 34 (14)[\*](#nt103){ref-type="table-fn"}
LVEDV index (mL/m^2^) 83.4 (72.9;96.3) 80.6 (69.2;93.9)
LVESV index (mL/m^2^) 38.6 (34.1;54.5) 34.0 (27.0;51.0)[\*](#nt103){ref-type="table-fn"}
LVEF (%) 50 (45.0;55.5) 57.0 (47.5;62.0)[\*](#nt103){ref-type="table-fn"}
LVM index (gr/m^2^) 62.9±10.9 56.0±11.0[\*](#nt103){ref-type="table-fn"}
MVO mass (g) 2.4±2.5
LV: left ventricular; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; WMSI: wall motion score index; LVEF: LV ejection fraction; LVM: LV mass; E: E peak velocity on trans-mitral Doppler; E': E peak velocity on tissue Doppler at the mitral annulus; LA: left atrium; MR: mitral regurgitation; GLS: longitudinal global strain; CGS: circumferential global strain; RGS: radial global strain; LGE: late gadolinium enhancement.
signifies p\<0.05 when comparing baseline and 3-month CMR results (McNemar or Wilcoxon test).
signifies p\<0.05 when comparing baseline global functional parameters as assessed by TTE and CMR (Wilcoxon test).
Feasibility and Reproducibility of 2D Strain Analysis {#s3a}
-----------------------------------------------------
No patient was excluded from analysis due to insufficient apical acoustic windows. Longitudinal global strain data was assessed in 40 (97%) patients, and radial and circumferencial strain data in 35 (85%).
Bland and Altman tests for longitudinal, radial, and circumferential global strains and LVEF are illustrated in [Figures 3](#pone-0086959-g003){ref-type="fig"} and [4](#pone-0086959-g004){ref-type="fig"}. While longitudinal strain was the most reproducible method (ICC 0.97 \[0.93;0.99\]), circumferential (ICC 0.92 \[0.78;0.97\]), and (ICC 0.90 \[0.71;0.96\]) radial strains were sufficiently reproducible to be integrated in the afterward analysis. Excellent inter-observer agreements for CMR parameters were achieved with the corresponding ICC coefficients as follows: LVEF (0.99 \[0.98;1.00\]), LVEDV index (0.99 \[0.99;1.00\]), LVESV index (0.99 \[0.98;1.00\]), LGE mass (0.99 \[0.98;1.00\]), and MVO mass (0.99 \[0.98;1.00\]).
{#pone-0086959-g003}
{#pone-0086959-g004}
Correlations between Early Echocardiographic Assessment and Infarct Size as Measured by CMR at 3-month Follow-up {#s3b}
----------------------------------------------------------------------------------------------------------------
LGE mass evaluated at 3 months significantly correlated with the following echocardiographic data: longitudinal global strain (GLS) (R = 0.601, p\<0.001), radial global strain (RGS) (R = −0.405, p = 0.010), circumferential global strain (CGS) (R = 0.526, p = 0.001), LV EF (R = −0.699, p\<0.001), WMSI (R = 0.539, p = 0.001), and left atrial volume (R = 0.510, p\<0.001).
In multivariate analyses, as illustrated in [Table 3](#pone-0086959-t003){ref-type="table"}, baseline LVEF and GLS were independent predictors of LGE mass at 3 months (respectively, p = 0.004 and p = 0.033).
10.1371/journal.pone.0086959.t003
###### Multivariate analysis of echographic data for predicting CMR-derived infarct size (n = 35).
{#pone-0086959-t003-3}
variable Linear correlationcoefficient P value Linear regressioncoefficient ß Confidence Intervals P value
----------- ------------------------------- --------- -------------------------------- ---------------------- ---------
GLS 0.601 \<0.001 0.877 0.078 −1.679 0.033
RGS −0.405 0.010
CGS 0.526 0.001
LVEF echo −0.699 \<0.001 −0.653 −1.077 - −0.229 0.004
WMSI 0.539 0.001
LA volume 0.510 0.001 0.195 −0.015--0.405 0.068
GLS: global longitudinal strain; RGS: radial global strain; CGS: circumferential global strain; LVEF echo: LVEF obtained with echocardiography; WMSI: wall motion score; LA: left atrium.
When studying transmural extension of LGE at 3 months, LVEF and CGS were related to LGE above 75% of transmural extension (respectively, p = 0.002 and p = 0.037).
Echocardiographic Assessment of MVO Measured by CMR at Baseline {#s3c}
---------------------------------------------------------------
MVO was reported in 24 patients (58.5%), and MVO mass significantly correlated with the following echocardiographic data: GLS (R = 0.376, p = 0.010), WMSI (R = 0.387, p = 0.011), and EF (R = −0.389, p = 0.011).
In multivariate analyses, GLS was the only independent predictor of MVO mass ([Table 4](#pone-0086959-t004){ref-type="table"}).
10.1371/journal.pone.0086959.t004
###### Multivariate analysis for predicting MVO mass (n = 35).
{#pone-0086959-t004-4}
variable Linear correlationcoefficient p value Linear regressioncoefficient ß Confidence Interval P value
----------- ------------------------------- --------- -------------------------------- --------------------- ---------
GLS 0.376 0.01 0.403 0.071--0.605 0.015
RGS −0.259 0.06
WMSI 0.387 0.011
LVEF echo −0.389 0.011
GLS: global longitudinal strain; RGS: radial global strain; WMSI: wall motion score; LVEF echo: LVEF obtained with echocardiography.
Segmental Analysis and Follow-up Functional Prognosis {#s3d}
-----------------------------------------------------
Mean GLS was −13.9±3.4%. Mean LS in the remote and infarcted segments was −15.7±3.7% and −10.5±4.6%, respectively. Concerning the infarcted myocardium, mean LS of viable and non viable segments was −14.8±4.6% and −9.1±4.7%, respectively.
Overall, 23 patients presented an akinesia affecting at least three segments at baseline, 10 having recovered at 3-month follow-up.
ROC curves for predicting akinesia ≥3 segments at follow-up are displayed in [Figure 5](#pone-0086959-g005){ref-type="fig"}. A cut-off of infarct LS \>−6.0% exhibited a 96% specificity for this prediction ([Figure 5](#pone-0086959-g005){ref-type="fig"} & [Table 5](#pone-0086959-t005){ref-type="table"}). The association of infarct LS \>−6.0% with a MVO extending over two segments increased sensitivity and specificity ([Table 5](#pone-0086959-t005){ref-type="table"}).
{#pone-0086959-g005}
10.1371/journal.pone.0086959.t005
###### Imaging parameters for the prediction of an akinesia affecting at least 3 segments at follow-up.
{#pone-0086959-t005-5}
Sensitivity (%) Specificity (%) PPV (%) NPV (%)
---------------------------------------------- ----------------- ----------------- --------- ---------
Infarct LS\>−6.0% 61 96 89 84
MVO 57 85 48 89
MVO over 2 segments 54 100 100 82
Infarct LS\>−6.0% AND/OR MVO over 2 segments 85 96 92 93
LS: longitudinal strain; MVO: microvascular obstruction.
Discussion {#s4}
==========
In the present study, 2D-strain echocardiography conducted within the first days after successful reperfusion in STEMI patients was feasible and provided relevant prognostic information. GLS was a marker of 1) MVO mass assessed a few days after reperfusion and 2) infarct size at 3-month follow-up, while 3) infarct LS \>−6.0% measured at the acute phase showed good specificity for diagnosis of a persistent akinesia (≥3 segments) at 3-month follow-up but a poor sensitivity (61%).
Cardiac imaging techniques at the acute myocardial infarction phase mainly aim to provide prognostic factors. Infarct size is well-recognised as a strong outcome predictor following myocardial infarction [@pone.0086959-Kan1], [@pone.0086959-Pfeffer1], while the evaluation of transmural myocardial infarct extension is predictive of myocardial viability [@pone.0086959-Kim1]. CMR performed a few weeks after AMI occurrence allows for quantifying these two prognostic factors using LGE [@pone.0086959-Wu2], [@pone.0086959-Gerber3]. When LGE-CMR is performed earlier, *i.e.,* a few days after reperfusion, the examination is instrumental in detecting and quantifying MVO [@pone.0086959-Wu1], [@pone.0086959-Gerber2]. All these factors are predictive of LV remodeling, which is associated with poorer outcome [@pone.0086959-Bolognese1]. Echocardiography, as a bedside tool, is able to provide prognostic information at the acute myocardial infarction phase [@pone.0086959-Moller1], [@pone.0086959-Burns1], and LVEF and WMSI are the main echocardiographic parameters strongly predictive of all-cause mortality following AMI [@pone.0086959-Savoye1], [@pone.0086959-Sutton1]. In the majority of cases, LVEF is assessed using the Simpson's biplane method [@pone.0086959-Lang1]. However, this measurement is a global LV function evaluation using a volumetric approach, and its reproducibility requires training and experience. In contrast, WMSI is rather a regional function parameter, and its evaluation is subjective. Cardiac mechanics are complex, and these two parameters do not allow us to study the spatial organization of myocardial fibers [@pone.0086959-TorrentGuasp1]. While longitudinal or long-axis function is mainly accounted for by sub-endocardial layers organized in an oblique clockwise orientation, short-axis function, reflecting the thickening of the myocardium visible in 2D-echocardiography, is mainly due to circumferentially oriented mid-layers and an outer layer arranged in an oblique anticlockwise direction.
New echocardiographic modalities, such as strain imaging, provide new insights into cardiac mechanics, with 2D-strain echocardiography being a highly promising tool [@pone.0086959-Leitman1]. This angle-independent echocardiographic method has been validated against sonomicrometry in animals and tagged-CMR in humans for measuring myocardial deformation [@pone.0086959-Amundsen1]. Longitudinal, radial, and circumferential strain can be quantified, reflecting these different deformations. Longitudinal strain has been shown to be the first altered function in AMI due to early necrosis of sub-endocardial layers of myocardial fibers [@pone.0086959-Reant1], while radial function is still preserved. It has been demonstrated that circumferential strain allows investigators to differentiate between sub-endocardial and trans-mural infarction [@pone.0086959-Chan1], [@pone.0086959-Zhang1]. Thus, though load-dependent, these parameters provide a more accurate insight into regional myocardial function and its impact on global function.
GLS has recently been studied in AMI, being shown to exhibit a predictive value for infarct size and LVEF recovery [@pone.0086959-Park1], [@pone.0086959-Antoni1]--[@pone.0086959-Munk1]. Several studies have demonstrated the feasibility of measuring these parameters in the AMI setting [@pone.0086959-Vartdal1], [@pone.0086959-Sjoli1]. Our study confirmed GLS's value as a marker of infarct size in AMI patients, as evaluated by CMR at 3 months. We studied strain parameters in the three main axes (radial, longitudinal, and circumferential), whereas most other published studies were chiefly focused on longitudinal strain alone [@pone.0086959-Grabka1]. Reproducibility and feasibility of GLS have been well-established in the AMI setting [@pone.0086959-Vartdal1], [@pone.0086959-Park1], [@pone.0086959-Antoni1], [@pone.0086959-Mollema2], [@pone.0086959-Sjoli1], which underlines the parameter's value as a prognostic tool. However, it appears crucial to also assess radial and circumferential strains on account of the complex organisation and interaction between these deformation parameters [@pone.0086959-Hung1]. Therefore, in our prospective study comparing CMR and echocardiography, baseline circumferential strain provided information on the transmural extension of infarction. This clinically relevant observation is concordant with results obtained in chronic ischemic cardiomyopathy [@pone.0086959-Chan1], [@pone.0086959-Hanekom1], [@pone.0086959-Zhang1]. In the infarction setting, radial strain is conserved and has thus less usefullness. In the recent VALIANT echocardiographic sub-study, both longitudinal and circumferential strains were predictors of outcomes, whereas only circumferential strain was predictive of remodeling, suggesting that preserved circumferential function might help restrain ventricular enlargement after AMI. [@pone.0086959-Hung1].
Our CMR study provided additive information on MVO, another post-MI prognostic factor, as GLS was shown to significantly correlate with MVO mass. To the best of our knowledge, MVO's influence on myocardial strain is still unclear, although MVO was shown to exhibit a significantly negative impact on regional function at the segmental level [@pone.0086959-Bergerot1].
Limitations {#s5}
===========
This was a prospective, monocentric study, with echocardiography and CMR required to be performed within the first days after reperfusion. Therefore, including a large patient number proved difficult, and the main limitation of our study is its small sample size, essentially due to limited CMR accessibility. As a consequence, we were not able to assess initial strain imaging values so as to predict later LV remodeling, although encouraging results were obtained with surrogate markers, notably myocardial infarct size and MVO.
Our study was exclusively based on 2D strain analysis, itself limited to the quality of view acquisition and to its 2D nature. In-plane and out-of-plane motion may, in fact, pose a problem for strain analysis, which not encountered when using the 3D or MR-tagging approach [@pone.0086959-Reant2].
It is worth mentioning that although the cut-offs and corresponding sensitivity, specificity, and predictive negative and positive values were specific to our dataset, they were nevertheless of particular interest in terms of defining how GLS relates to akinetic territory. However, when computing the limits of agreements for GLS (see [figure 3](#pone-0086959-g003){ref-type="fig"}) and the differences in GLS between viable and not viable segments in infracted myocardium, it should be cautioned that this thresholds may be insufficiently precise for an individual assessment.
Conclusion {#s6}
==========
2D-strain echocardiography carried out as a bedside test within a few days after reperfusion in STEMI patients provides indices similar to those obtained using CMR. Further studies involving larger patient series and registries are needed to confirm the parameters' prognostic value in terms of LV remodeling and clinical patient outcome.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: LB ED FP. Performed the experiments: LB ED GK GT SW. Analyzed the data: LB ED SW AF FP. Contributed reagents/materials/analysis tools: SW AF. Wrote the paper: LB ED.
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Key Teaching Points•The use of synthetic cannabinoids is on a rise in the United States, more so in the younger population.•Synthetic cannabinoids are not detected by routine toxicology screens.•Synthetic cannabinoids may have cardiovascular side effects including cardiac arrhythmias and even cardiac arrest.•Patients admitted with nonarrhythmic side effects of synthetic cannabinoid use should still be monitored for potential sudden cardiac arrest (SCA).•Drug-related SCA should not always be considered reversible until a thorough evaluation to determine if other sudden cardiac death (SCD) predispositions are present that may impact decisions regarding secondary prevention of SCD with implantable cardioverter-defibrillator therapy.
Introduction {#sec1}
============
Synthetic cannabinoids (SC), often referred to as K2 or spice, comprise a significant burden of overall illicit drug use in the United States. Contributing to its popularity may be the longer-lasting marijuana-like effects and lack of detection by standard toxicology screens, compared to natural cannabinoids. There is mounting evidence that SC may be responsible for potentially fatal cardiac arrhythmias. How to manage long-term risk of sudden cardiac death (SCD) in such patients has not been studied. Patients with drug use--related cardiac arrest are considered to have a reversible cause of the arrest and thus do not fit the class I indication for receiving an implantable cardioverter-defibrillator (ICD). However, the degree of reversibility is poorly defined when genetic predisposition for SCD may be unmasked rather than caused by SC use. Concern for continued drug use could also challenge the label of reversibility.
Case report {#sec2}
===========
A 52-year-old woman with no significant past medical history was found unresponsive shortly after consuming K2-laced cigarettes. Cardiopulmonary resuscitation was started and Emergency Medical Service (EMS) was called. Return of spontaneous circulation was attained in the field by EMS after 15 minutes of cardiopulmonary resuscitation. Her initial rhythm was ventricular fibrillation (v-fib), for which she required 2 shocks and in addition 1 dose of epinephrine and lidocaine each. Therapeutic hypothermia was initiated in the Emergency Department. Initial electrocardiogram (ECG) 1 hour after the sudden cardiac arrest (SCA) was notable for normal sinus rhythm, prolonged QTc of 534 ms, and lateral lead T-wave inversion in V~2~--V~6~ ([Figure 1a](#fig1){ref-type="fig"}). Her history did not reveal any home medications, nor did she receive any QT interval--prolonging medications by EMS or the hospital. Initial serum electrolyte levels revealed potassium of 3.6 mmol/L, magnesium of 1.6 mg/dL, and calcium of 8.6 mg/dL. The patient had another SCA with torsades de pointes degenerating to v-fib after admission to the Cardiac Care Unit, with successful resuscitation. Immediately after return of spontaneous circulation was achieved, a temporary transvenous pacemaker was inserted owing to significant bradycardia requiring transcutaneous pacing, atropine boluses, and dopamine and epinephrine infusions. ECG 20 minutes after initial arrest revealed a prolonged QTc of 634 ms ([Figure 1b](#fig1){ref-type="fig"}). Serum electrolyte levels checked immediately after the second arrest showed a potassium level of 4.3 mmol/L, magnesium of 2.6 mg/dL, and calcium of 8.1 mg/dL. Cardiac troponin levels were unremarkable. Urine toxicology screen was negative. Blood alcohol level was undetectable. Echocardiogram showed a depressed left ventricle ejection fraction (LVEF) of 20% with global hypokinesis. Cardiac magnetic resonance imaging 3 days later showed improving LVEF of 42% with no evidence of infiltrative disease or evidence of an ischemic insult. A dual-chamber, transvenous ICD (TV-ICD) was implanted owing to recurrent SCA associated with QTc prolongation and reduced LVEF with atrial pacing support to allow for beta-blocker titration and atrial overdrive pacing, as durable future resolution of QTc prolongation and cardiomyopathy were still uncertain. Device settings included the following: 2 tachycardia zones with a ventricular tachycardia (VT) zone between 188 and 230 beats per minute (bpm) detected for 24 consecutive beats, treated with 3 decremental scanning bursts of antitachycardia pacing prior to cardioversion shocks; a v-fib zone \> 231 bpm detected for 30 of 40 intervals with antitachycardia pacing during charging prior to defibrillation shocks; and bradycardia support at DDD 40--130 bpm. ECG done on day 4 of the presentation before discharge and at 2 years follow-up showed normalization of QTc interval ([Figure 2a](#fig2){ref-type="fig"} and [b](#fig2){ref-type="fig"}).Figure 1**a:** Electrocardiogram after return of spontaneous circulation post first cardiac arrest showing prolonged QTc and lateral lead (V~2~--V~6~) T-wave inversions (*blue arrows*). **b:** Electrocardiogram done in Intensive Care Unit after return of spontaneous circulation post second cardiac arrest, again showing prolonged QTc and lateral lead (V~3~--V~6~) T-wave inversions (*blue arrows*).Figure 2Electrocardiograms done **a:** on day 4 of admission and **b:** at 2 years follow-up after discharge, showing normalization of QTc interval with inverted T waves in leads V~3~--V~6~ at day 4 and upright T waves in lateral leads at 2 years (*blue arrows*).
The patient was seen at a follow-up visit 8 months after initial presentation and was found to have an improved LVEF of 55% on guideline-directed medical therapy including angiotensin-converting enzyme inhibitors and low-dose beta blockers. Her ECG showed a normal QTc interval at the visit. Eighteen months following ICD implant, she suffered both inappropriate ICD shocks for supraventricular tachycardia and less frequent appropriate shocks for VT in the setting of continued nonsynthetic marijuana use. The dose of beta blockers was up-titrated to limit the recurrence of episodes of supraventricular tachycardia.
Discussion {#sec3}
==========
Cannabis is the most prevalent illicit drug used in the United States, with legalization in some states. It is estimated that 89.5% and 10.5% of cannabis use is recreational and medicinal, respectively.[@bib1] SC, commonly known as synthetic marijuana, spice, K2, black magic, or crazy clown, are being increasingly abused, especially among high school students.[@bib2] They have marijuana-like effects and are not detectable by routine urine drug screen. SC chemical composition differ, as they lack delta-9-tetrahydrocannabinol (THC), the key component of cannabinoids. They stimulate the human cannabinoid receptors (CB1) with a greater binding affinity, as they are full agonists rather than partial agonists. SC are rapidly absorbed into the blood and have longer half-lives, resulting in more rapid and durable cannabinoid effects compared to other cannabinoids (usually 3--5 days).[@bib3]^,^[@bib4] With an increase in the use of these drugs, an increasing number of patients are being seen in facilities for various complications including rhabdomyolysis, seizures, acute kidney injury, and arrhythmias leading to SCA. Establishing causality of these adverse effects is difficult owing to the lack of available drug assays, and associations have been based on patient- or witness-reported drug use preceding hospital presentation. SCD is the leading cause of death in the United States, with estimates of 350,000--450,000 cases per year. As substance abuse prevalence rises, rates of death including SCD is expected to increase proportionately.[@bib5]^,^[@bib6]
To date, there have been few cases describing SCA related to reported SC use. We systematically searched the published literature for reports of SC-induced out-of-hospital and in-hospital SCA. The articles were collected as of September 2019 using PubMed and Google Scholar. After redundant articles were removed, 5 papers were included in the present study for the final review and analysis. A thorough reading of these articles yielded a total of 6 cases of SC-associated SCA.[@bib6], [@bib7], [@bib8], [@bib9], [@bib10] The data of the patients, including demographics, clinical features, comorbid conditions, diagnostic tools, prognosis, and outcomes, are summarized ([Supplementary Table S1](#appsec1){ref-type="sec"}).
A comprehensive review of these 6 cases revealed male predominance (men, n = 6) and ages from 15 to 56 years (mean = 32 years). This is consistent with results from studies that have reported SC use in male more than in female subjects, specifically in college students.[@bib3] Three patients suffered out-of-hospital and 3 patients in-hospital SCA. Two patients had pulseless electrical activity arrest, 2 had asystole (both in-hospital), and 2 pediatric patients had out-of-hospital v-fib arrest. SC use was reported in all of the cases, either by the patients or by any witness immediately preceding the event. Only 1 patient had a positive urine toxicology screen (cocaine and cannabinoids), while the screen was negative in other patients. ECG findings post--cardiac arrest were variable and included sinus tachycardia, atrial fibrillation, sinus bradycardia with asystole, and prolonged QTc. On further cardiac testing including echocardiogram, stress testing, and left heart catheterization, no other cause of arrhythmias was identified, including native coronary artery disease. One patient with known coronary artery disease but with near-normal ejection fraction received an ICD for secondary prevention of SCD. One of the pediatric patients who received an ICD was found to have appropriate therapy for VT. The other pediatric patient underwent electrophysiology study and ablation of an accessory pathway combined with medical management. In the pediatric patient with bradycardia and asystole, who did not require resuscitation, no intervention was done. One patient died and could not be resuscitated, while 1 had a complicated hospital course that led to change in code status and discharge to a long-term care facility.
There is a paucity of data owing to the rarity of this presentation and follow-up regarding the use of secondary-prevention ICD in patients who have SC-associated SCA. Two of 6 patients had an ICD placed and device interrogation data were reported for 1 of them, revealing a potentially appropriate shock. In our patient who received an ICD, follow-up device interrogation revealed multiple appropriate therapies for VT.
Placement of a subcutaneous ICD (S-ICD) or TV-ICD requires individualized shared decision-making. Factors that may favor S-ICD vs TV-ICD include patient age, battery longevity, pacing or antitachycardia pacing indications, detection algorithm--associated risks of inappropriate ICD therapy, risks of endovascular infection, device position, incision size and number, and personal choice based on review of these factors. S-ICD may be a better option in patients with previous infections or those who are at risk of infections, such as intravenous drug users, because they lack the transvenous lead and hence possess lower chances of lead-related infections or lead malfunction.[@bib11] The rationale for choosing a TV-ICD in our patient was her young age, the potential need for pacing therapy to allow for optimal therapy of cardiomyopathy and prolonged QTc, and lack of intravenous drug use history.
Appropriate use criteria for ICD implantation lists drug-induced arrhythmias as "rarely appropriate" indication for secondary prophylaxis. The panel consisted of experts in electrophysiology, heart failure, and cardiology.[@bib12] However, drug-related arrhythmias may unmask other predispositions to SCD that may not be reversible. In our case, the QTc interval returned to normal after the patient quit SC use; however, appropriate shocks for VT were still observed. We suggest that cases of drug-induced cardiac arrest should not be placed into "reversible causes of cardiac arrest" by default until other SCD susceptibilities have been fully evaluated.[@bib13]
There is ample data regarding arrhythmogenic potential of nonsynthetic marijuana. While adrenergic stimulation and atrial ischemia from cannabis can lead to atrial arrhythmias, catecholamine surges have been linked with v-fib. The postulated mechanism of long QTc seen in cannabis users involves blocking of the hERG channels. In 1 retrospective analysis of a nationwide pediatric database, QTc prolongation was the most common arrhythmia reported in cannabis users (353 patients, or 513.1 per 100,000 cases of cannabis users).[@bib14] Our patient, who initially presented for cardiac arrest from prolonged QTc owing to SC use, later continued to use nonsynthetic marijuana and experienced appropriate shocks for VT.[@bib14]
Conclusion {#sec4}
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This case demonstrates that the reversibility of drug use--related cardiac arrhythmias may uncover other arrhythmic predispositions such as genetic risks or accessory pathways, as described in our literature review. There remains a potential of continued drug use and genetic predispositions may exist in patients that make them susceptible to fatal arrhythmias that are unmasked by drug use. We conclude that in these patients, the reversibility of illicit drug-related SCA should be scrutinized more to identify other predispositions to SCD that would prompt placement of secondary-prevention ICD.
Appendix. Supplementary data {#appsec1}
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Supplementary Table S1
Supplementary data associated with this article can be found in the online version at <https://doi.org/10.1016/j.hrcr.2020.02.002>
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Background {#Sec1}
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Gram-positive bacteria, such as *Staphylococcus aureus, Enterococcus faecalis,* and *Enterococcus faecium* are dangerous and challenging agents of infection due to their increasing resistance to antibiotics \[[@CR1]\]. Among these, *S. aureus* are the most frequently isolated, making up 29.1% of the isolated Gram-positive bacterial populations, and infections caused by *S. aureus* (both community-associated or nosocomial-associated) are reported all over the world \[[@CR2], [@CR3]\]. *Enterococci* (including *E. faecalis* and *E. faecium*) are the second frequently isolated Gram-positive bacteria at 19.5%. Past generations of *enterococci* were mainly associated to urinary-tract infection, but recently more and more *enterococci* are isolated from other infections \[[@CR4]\], and an outbreak of vancomycin resistant *enterococci* (VRE) in a solid organ transplant unit was reported in 2018 \[[@CR5]\].
The epidemiology of resistance in Gram-positive bacteria has undergone major changes in recent decades, with methicillin-resistant *S. aureus* (MRSA) and VRE now being of international concern \[[@CR6]\]. Antimicrobial resistances of Gram-positive bacteria have been reported in many countries \[[@CR7]--[@CR9]\]; however, it was only in recent years that *vanM*-carrying *E. faecalis* strains were isolated from patients and the clinical environment in the Second Affiliated Hospital of Zhejiang University School of Medicine \[[@CR10]\], we speculate that if *vanM* infection has been spreading in the Zhejiang province, it may lead to an increase of VRE overall. In China, although several local studies have examined the prevalence of resistance in Gram-positive bacteria \[[@CR11], [@CR12]\], the studies usually only cover the tertiary hospitals. Therefore, the present study is a retrospective surveillance covering a wider range of hospitals, including tertiary and secondary hospitals. Our aim is to seek trends in antimicrobial resistance among clinical isolates of important Gram-positive bacteria in the Zhejiang province of China.
Materials and methods {#Sec2}
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Bacterial isolates {#Sec3}
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We extracted data of *S. aureus*, *E. faecalis* and *E. faecium* infections from the Zhejiang surveillance system's outpatient and inpatient records from January 1, 2015 to December 31, 2017. The number of surveyed hospitals for each city was as follows: Hangzhou (*n* = 24), Huzhou (*n* = 4), Jiaxing (*n* = 11), Jinhua (*n* = 9), Lishui (*n* = 6), Ningbo (*n* = 10), Quzhou (*n* = 5), Shaoxing (*n* = 6), Taizhou (*n* = 5), Wenzhou (*n* = 5), and Zhoushan (*n* = 1). Sample collection was in accordance with the clinical microbiology manual \[[@CR13]\]. To avoid duplicate isolates, only one isolate from the same species was included per patient, as determined by the personal identifying code and hospital name. Species identification of the isolates was performed by standard biochemical methods, automated system (the Vitek 2 compact, BD Phoenix-100, MicroScan WalkAway-96) or Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS).
Antimicrobial susceptibility testing {#Sec4}
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Standard operation procedures were established according to the Clinical and Laboratory Standards Institute's (CLSI) criteria, M100-S24 \[[@CR14]\]. Every participating laboratory conducted the antibiotic susceptibility testing for the clinical isolates using the Kirby-Bauer disk diffusion method or the commercialized automated system following the instrument specifications. *S. aureus* (ATCC 25923, ATCC 43300) and *E. faecalis* (ATCC 29212, ATCC 51299) were used as quality control strains for the testing.
Statistical analysis {#Sec5}
--------------------
We analysed the risk factors for MRSA and VRE proportions, and for the multivariable model, year, region, hospital level, patient age, specimen type and patient category were considered. The lowest detection rates of MRSA and VRE were set as contrasts. The risk factors and the differences in MRSA and VRE detection rates were compared using odds ratio (OR) with 95% confidence interval (95% CI) and Chi-square test. Chi-square values were corrected when the quantities of VRE isolates were less than forty. Results with *P*-value \< 0.05 were considered statistically significant. SPSS 19.0 (IBM Company, Chicago, IL) and WHONET 5.5 (WHO) software were used for all statistical analyses.
Results {#Sec6}
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Distribution of MRSA, *Enterococci* and VRE isolates {#Sec7}
----------------------------------------------------
During the years 2015--2017, the total number of MRSA isolates increased from 9292 to 10,237 cases; in contrast, VRE isolates decreased from 173 to 137 cases (data not shown). Among the 11 cities that participated in the survey, Hangzhou contributed 37.6% of all MRSA isolates (29,866 cases); and 41.0% of all *Enterococci* isolates (58,329 cases), although the population of Hangzhou is only about 9.5 millon (16.5% of the Zhejiang province population). This high number of isolates may be due to Hangzhou having more IIIA hospitals, which were the contributors of 67% of all MRSA and *enterococci* isolates. However, Hangzhou also contributed 245 VRE isolates, which was 54.2% of all of the VRE isolates in the study, and a much higher percentage than the province's MRSA and *enterococci* contributions. MRSA were most frequently isolated from respiratory samples (14,339 or 48.0% of all samples); VRE were dominantly isolated from urine samples (313 or 69.2% of all samples) (data not shown). The number of samples based on age of patient and inpatient vs. out-patient were also categorized. Patients older than 75 and patients who were hospitalized (especially those in the ICU) had dominantly higher MRSA, *enterococci* and VRE isolation rates compared to other patient groups (data not shown).
Resistance rates of MRSA, *E. faecalis* and *E. faecium* to antimicrobial agents from 2015 to 2017 {#Sec8}
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During the period of 2015 to 2017, the resistance rates of MRSA to trimethoprim-sulfamethoxazole and nitrofurantoin increased from 9.66 to 14.94% (*P* \< 0.001) and from 0.51 to 0.95% (*P* \< 0.001) respectively. The resistance rates of MRSA to erythromycin, tetracycline, tigecycline and levofloxacin decreased from 90.32 to 88.24% (*P* = 0.003), from 35.58 to 29.05% (*P* \< 0.001), from 0.14% to 0, and from 53.01 to 40.77% (*P* \< 0.001) respectively (Fig. [1](#Fig1){ref-type="fig"}). MRSA showed 100% susceptibility to vancomycin and teicoplanin, however, a few linezolid-resistant strains were found in 2016. The resistance rates of *E. faecium* to nitrofurantoin and gentamicin-high level increased from 51.90 to 55.47% (*P* \< 0.001) and from 34.15 to 41.67% (*P* \< 0.001) increased respectively. The resistance rates of *E. faecium* to linezolid, tetracycline, teicoplanin and vancomycin decreased from 1.15 to 0.67% (*p* = 0.168), from 38.33 to 30.77% (*p* = 0.243), from 0.51 to 0.16% (*P* \< 0.001), and from 1.69 to 0.90% (*p* = 0.006) respectively (Table [1](#Tab1){ref-type="table"}). The resistance rate of *E. faecalis* to gentamicin-high level increased from 7.32 to 41.67% (*P* \< 0.001). The resistance rates of *E. faecalis* to ampicillin, nitrofurantoin, linezolid, tetracycline, and levofloxacin decreased from 5.72 to 3.04% (*p* = 0.021), from 3.95 to 2.56% (*P* \< 0.001), from 3.17 to 2.73% (*p* = 0.002), from 77.29 to 76.32% (*P* \< 0.001), and from 20.78 to 19.79% (*p* = 0.009) respectively (Table [1](#Tab1){ref-type="table"}). Tigecycline showed 100% susceptibility against *E. faecalis* and *E. faecium*. The resistance rates of *E. faecium* to ampicillin, nitrofurantoin, ciprofloxacin, gentamicin-high level, levofloxacin, teicoplanin and vancomycin were higher than that of *E. faecalis*, while lower resistance rates to tetracycline and linezolid were seen in *E. faecium* in comparison to *E. faecalis*.Fig. 1Resistance rates of MRSA to antimicrobial agents during 2015 to 2017. / No comparison when appearing resistance rate was 0%. Results of nitrofurantoin were only from urine isolates. Abbreviations: F nitrofurantoin, SXT trimethoprim-sulfamethoxazole, E erythromycin, DA clindamycin, RD rifampicin, LZD linezolid, CN gentamicin, TE tetracycline, TGC tigecycline, TEC teicoplanin, VA vancomycin, LEV levofloxacinTable 1Resistance rates (%) of *E. faecalis* (E.fa) and *E. faecium* (E.fm) to antimicrobial agents during 2015 to 2017Antimicrobial agent201520162017*P* valueE.fa(*n* = 9109)E.fm (*n* = 7106)E.fa(*n* = 10,740)E.fm(*n* = 9135)E.fa (*n* = 11,860)E.fm (*n* = 10,379)E.faE.fmAmpicillin5.7288.224.6689.583.0489.130.021\< 0.001Nitrofurantoin3.9551.903.1954.962.5655.47\< 0.001\< 0.001Gentamicin-High7.3234.1518.0641.5741.6741.67\< 0.001\< 0.001Ciprofloxacin22.5489.1323.1490.4722.0590.320.0090.147Linezolid3.171.152.971.002.730.670.0020.168Tetracycline77.2938.3376.9033.7576.3230.77\< 0.0010.243Tigecycline000000//Teicoplanin00.5100.4500.16/\< 0.001Vancomycin0.671.690.371.220.420.90\< 0.0010.006Levofloxacin20.7887.4920.4389.0419.7988.470.0090.195/ No comparison when appearing resistance rate was 0%
Resistance rates of *S. aureus* and *Enterococci* to antimicrobial agents among different patients {#Sec9}
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For isolates collected from the outpatients, non-ICU inpatients and ICU inpatients, resistance rates of *S. aureus* to all the antimicrobial agents increased gradually except for clindamycin and trimethoprim-sulfamethoxazole (Fig. [2](#Fig2){ref-type="fig"}). Among which, the resistance rates to oxacillin, gentamicin and levofloxacin increased more dramatically. Resistance rates of *enterococcus* (including *E. faecalis* and *E. faecium*) to all the antimicrobial agents increased gradually except for linezolid and tetracycline (Fig. [3](#Fig3){ref-type="fig"}).Fig. 2Resistance rates of *S. aureus* to antimicrobial agents among different patients. / No comparison when appearing resistance rate was 0%. Results of nitrofurantoin were only from urine isolates. Abbreviations: OX oxacillin, F nitrofurantoin, SXT trimethoprim-sulfamethoxazole, E erythromycin, DA clindamycin, RD rifampicin, LZD linezolid, P penicillin, CN gentamicin, TE tetracycline, TGC tigecycline, TEC teicoplanin, VA vancomycin, LEV levofloxacinFig. 3Resistance rates of *enterococci* (*E. faecalis* and *E. faecium*) to antimicrobial agents among different patients. / No comparison when appearing resistance rate was 0%. Results of nitrofurantoin, tetracycline, ciprofloxacin and levofloxacin were only from urine isolates. Abbreviations: AMP ampicillin, F nitrofurantoin, CN gentamicin-high level, CIP ciprofloxacin, LZD linezolid, TE tetracycline, TGC tigecycline, TEC teicoplanin, LEV levofloxacin, VA vancomycin
Analysis of risk factors associated with MRSA and VRE strains {#Sec10}
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In multivariable analysis for risk factors associated with MRSA and VRE (Table [2](#Tab2){ref-type="table"}), we observed that the 2015 isolation rates of MRSA (OR = 1.022, 95% CI 1.010--1.035, *p* \< 0.001) and of VRE (OR = 1.005, 95% CI 1.003--1.006, *p* \< 0.001) were higher than those in 2017. Compared to Huzhou, Zhoushan was the most likely city to have MRSA isolates (OR = 1.775, 95% CI 1.676--1.880, *p* \< 0.001). Compared to Lishui, Huzhou was the most likely city to have VRE isolates (OR = 1.025, 95% CI 1.017--1.033, *p* \< 0.001). The cities with high detection rates of MRSA were distributed in the east and west of the Zhejiang province, and the cities with high detection rates of VRE were distributed in the west of the Zhejiang province (Fig. [4](#Fig4){ref-type="fig"}). Isolates from tertiary hospitals were more likely to be MRSA and VRE than isolates from secondary hospitals. Analysis based on age groups revealed that isolates derived from patients older than 75 years had the highest proportion of MRSA (OR = 1.443, 95% CI 1.409--1.478, *p* \< 0.001) and VRE (OR = 1.011, 95% CI 1.009--1.013, *p* \< 0.001). Isolates from bile had the highest proportion of MRSA (OR = 1.385, 95% CI 1.151--1.665, *p* \< 0.001) and isolates from blood had the highest proportion of VRE (OR = 1.007, 95% CI 1.003--1.010, *p* \< 0.001). Patients who were in ICU had the highest proportion of MRSA (OR = 1.439, 95% CI 1.407--1.473, *p* \< 0.001) and VRE (OR = 1.019, 95% CI 1.013--1.025, *p* \< 0.001).Table 2Analysis of risk factors associated with MRSA and VRE strainsMRSAVREOR (95%CI)*P* valueOR (95%CI)*P* valueYear 20151.022 (1.010--1.035)\< 0.0011.005 (1.003--1.006)\< 0.001 20161.018 (1.006--1.030)0.0021.001 (0.999--1.003)0.214 20171--1--Region Hangzhou1.329 (1.295--1.363)\< 0.0011.010 (1.008--1.012)\< 0.001 Huzhou1--1.025 (1.017--1.033)\< 0.001 Jiaxing1.109 (1.080--1.139)\< 0.0011.006 (1.003--1.009)\< 0.001 Jinhua1.229 (1.195--1.265)\< 0.0011.005 (1.002--1.008)0.001 Lishui1.030 (1.002--1.059)0.0451 (0.997--1.003)1 Ningbo1.406 (1.365--1.447)\< 0.0011.002 (1--1.004)0.054 Quzhou1.453 (1.398--1.511)\< 0.0011.017 (1.011--1.024)\< 0.001 Shaoxing1.189 (1.156--1.224)\< 0.0011.003 (1.001--1.006)0.015 Taizhou1.197 (1.163--1.231)\< 0.0011.005 (1.002--1.009)0.001 Wenzhou1.282 (1.245--1.319)\< 0.0011-- Zhoushan1.775 (1.676--1.880)\< 0.0011.004 (0.999--1.010)0.079Hospital Level IIIA (*n* = 46)1.267 (1.213--1.322)\< 0.0011.016 (1.009--1.024)0.031 IIIB (*n* = 24)1.189 (1.138--1.242)\< 0.0011.010 (1.002--1.019)0.143 IIA (*n* = 15)1.139 (1.089--1.190)\< 0.0011.016 (1.006--1.025)0.045 IIB (*n* = 1)1--1--Age \< =7d1.130 (1.063--1.201)\< 0.001\*\* 8d-28d1.085 (1.043--1.129)\< 0.001\*\* 1 m-1y1.054 (1.030--1.079)\< 0.001\*\* 2y-3y1--1.008 (0.998--1.019)0.015 4y-19y1.015 (0.992--1.037)0.2031-- 20y-43y1.057 (1.035--1.080)\< 0.0011.004 (1.002--1.005)0.044 44y-59y1.104 (1.080--1.128)\< 0.0011.007 (1.005--1.009)0.002 60y-74y1.190 (1.164--1.217)\< 0.0011.006 (1.004--1.008)0.004 75 + y1.443 (1.409--1.478)\< 0.0011.011 (1.009--1.013)\< 0.001Specimen type Blood1.142 (1.101--1.185)\< 0.0011.007 (1.003--1.010)\< 0.001 Bile1.385 (1.151--1.665)\< 0.0011.001 (0.999--1.004)0.403 Respiratory1.318 (1.278--1.360)\< 0.0011.003 (0.995--1.011)0.515 Urine1.186 (1.139--1.235)\< 0.0011.006 (1.004--1.008)\< 0.001 Secretion1.115 (1.081--1.150)\< 0.0011-- Puncture fluid1--1.004 (1--1.009)0.012 stool1.342 (1.262--1.426)\< 0.0011.001 (0.997--1.005)0.839 Other1.137 (1.102--1.173)0.0021 (0.998--1.002)1Patient category Outpatient1--1.004 (0.995--1.014)0.4 Inpatient-non ICU1.132 (1.118--1.147)\< 0.0011-- Inpatient-ICU1.439 (1.407--1.473)\< 0.0011.019 (1.013--1.025)\< 0.001OR odds ratio, CI confidence intervalIIIA number of bed more than 500, comprehensive examination score more than 900 pointsIIIB number of bed more than 500, comprehensive examination score between 750 and 899 pointsIIA number of bed between 100 and 499, comprehensive examination score more than 900 pointsIIB number of bed between 100 and 499, comprehensive examination score between 750 and 899 pointsComprehensive examination including departments, staffing, management level, technical level, work quality and technical facilitiesd day old, m month old, y year oldRespiratory containing sputum and bronchoalveolar lavage fluidSecretion containing pus and wound swabPuncture fluid containing hydrothorax, ascites, articular cavity fluid, pericardial fluid and Cerebrospinal fluid\* No VRE isolateFig. 4Distribution of MRSA (**a**) and VRE (**b**) by geographic area
Discussion {#Sec11}
==========
It is a global trend that the drug resistance of Gram-positive bacteria decreases gradually. In our study, there is a slight decline in the isolation rates of MRSA (from 34.98 to 33.53%). The downward trend is also observed in France, Germany and the UK \[[@CR15], [@CR16]\], but it is not seen in Saudi Arabia, where MRSA is maintaining its high level \[[@CR17]\]. The use of alcohol-based hand-rub and decolonization with antimicrobial agents may have helped to reduce MRSA transmission. As with other studies, linezolid, tigecycline and vancomycin are the most active agents against MRSA \[[@CR18]\]. There were no MRSA isolates that were resistant to vancomycin and teicoplanin. Unlike other reports, the minimum inhibitory concentration (MIC) of MRSA to vancomycin was distributed around 0.5 and 1 mg/L, and was stable during the three years study \[[@CR19]\]. These results may be attributed to vancomycin being an unlikely empirical therapy due to its narrow-spectrum and its injection-only administration, thus resistances may not have had a chance to form. Meanwhile, vancomycin belongs to the highest limit level in the classification management system of antibiotics. Linezolid resistances in coagulase negative *staphylococci* are greater than resistance in *S. aureus* \[[@CR20], [@CR21]\]. *Staphylococcus capitis* isolates were the highest in Zhejiang province, and the resistance rates to linezolid increased from 1.8% in 2014 \[[@CR20]\] to 3.5% in 2016 \[[@CR21]\]. This is due to an outbreak of linezolid-resistant *S. capitis* infection in Zhejiang. Since the first report of linezolid resistance in methicillin-resistant coagulase negative *staphylococci* in the Second Affiliated Hospital of Zhejiang University School of Medicine in 2011, linezolid resistance in human clinical isolates has become an increasing problem in China \[[@CR22], [@CR23]\], and the *cfr*-carrying plasmid has appeared in *S. aureus* \[[@CR24]\]. We isolated several linezolid-resistant *S. aureus* strains in 2016, but not in 2015 and 2017. No outbreaks of linezolid resistance were seen in *S. aureus* isolates*.*
The results of the present study show that the resistance rates of *E. faecium* are greater than the resistance rates of *E. faecalis,* and the resistance rates of *E. faecium* to ampicillin and quinolones are more than 80%. Therefore infections caused by *E. faecium* present a serious clinical challenge for physicians \[[@CR25]\], and treatment options for these infections are limited. Vancomycin is one treatment option that could be considered. In the last ten years, a weak downward trend for VRE cases was found worldwide and may be seen in the Zhejiang province. We also confirmed that the resistance rate of *enterococcus* to vancomycin remains at a low level, and showed downward trends, similar to other reports in China and lower than that in Ireland \[[@CR26]\]. We found that the resistance rates of *E. faecalis* and *E. faecium* to vancomycin are higher than the resistance rates to teicoplanin. We speculate that strains with the dominant gene *vanB* may have the highest resistance to vancomycin \[[@CR27]\]. These may have a different resistance than strains with the *vanM* gene, seen in China \[[@CR28]\] or in strains with *van*A dominance, seen in Poland \[[@CR29]\]. In our study, resistance rate of *E. faecalis* to linezolid increased from 1.6% in 2008 to 2.97% in 2016 \[[@CR21]\] and linezolid resistance was higher in *E. faecalis* than in *E. faecium.* In the past, mutations in the central loop of domain V of the 23S rRNA represented the most common mechanism of oxazolidinone resistance in *enterococci*, with G2576 T (*Escherichia coli* numbering) as the predominant mutation \[[@CR30]\]. Increasingly, transferable oxazolidinone resistance from the multi-resistance genes *cfr* and *optrA* are being reported all over the world \[[@CR31]--[@CR33]\]. Furthermore, it was reported that the *optrA* gene was detected more frequently from food-producing animals than from humans \[[@CR34]\].
In the multivariable analysis for risk factors associated with MRSA and VRE, we found that Hangzhou and Quzhou simultaneously had high MRSA and VRE detection rates. As the provincial capital of Zhejiang, Hangzhou has more tertiary hospitals and receives a greater number of critically ill patients from other cities. As a relatively under-developed city, Quzhou may have a poorer sanitary arrangement, and the compliance with antibiotics may be worse. Meanwhile, we found that the cities of relatively high MRSA and VRE detection rates tend to neighbour one another. This phenomenon may be caused by the increased likelihood of interaction between the populations and patients of these adjacent cities, allowing greater dissemination of MRSA and VRE isolates. To be a patient in a IIIA hospital (the highest classified and possibly largest hospital type in China) and a patient in the ICU ward are the greatest risk factors associated with MRSA and VRE infection. These findings are in accordance with the literature \[[@CR35], [@CR36]\] and can be attributed to patients with severe co-morbidities. Stratifying the data by patient age, it is observed that the proportion of MRSA and VRE was the highest in isolates from elderly patients older than 75 years, but was the lowest from children aged 2 years - 3 years and infants younger than 1 year. The high numbers of MRSA and VRE isolates from elderly patients may be due to these patients having more underlying diseases and a greater history of antibiotic use than the child and the infant group. As well, decreased nutrition and immune function, often seen in the elderly, may also be contributing elements. Other identified risk factors associated with MRSA and VRE include having a source of isolates. We found that puncture fluid (containing hydrothorax, ascites, articular cavity fluid, pericardial fluid and cerebrospinal fluid) has the lowest proportion (21.32%) of MRSA, though the underlying reason for this phenomenon needs to be further studied. In terms of treatment, we should be wary of methicillin-sensitive *S. aureus* when *S. aureus* is isolated from puncture fluid in Zhejiang province. With the noted exception of blood and urine, there is no difference in the specimen types in proportions of VRE isolates.
These findings will provide valuable information for infection control practices. Although many surveillance projects of antibiotic resistance have been carried out in China, they always cover only the tertiary hospitals. In our study, a wider range of scenarios were seen where resistant strains could occur, indicating the importance of performing regional antibiotic resistance surveillance. The current study had some limitations: we had a relatively short span of time (3 years) for the data collection, we had a limited number of IIB hospitals to contribute data to the study, and because ours was a retrospective analysis, we had an inability to obtain the original strains. In the future, we would like to expand this surveillance to cover more IIB and rural area clinics and also collect the original strains for intensive study.
Conclusion {#Sec12}
==========
The detection rates of MRSA stayed at moderate levels, and VRE stayed at low levels during the last three years, and local dissemination was found in MRSA and VRE isolates. The highest risk factors for MRSA and VRE infection were patient status in a IIIA hospital, age older than 75 years and hospitalization in the ICU ward. As a result of our findings, we suggest that sustained surveillance is necessary to prevent the spread or clonal dissemination of drug-resistant strains in Zhejiang China.
CI
: Confidence interval
ICU
: Intensive care unit
MALDI-TOF-MS
: Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry
MIC
: Minimum inhibitory concentration
MRSA
: Methicillin-resistant *Staphylococcus aureus*
OR
: Odds ratio
VRE
: Vancomycin-resistant *enterococci*
We want to thank the participating hospitals for their work and cooperation. We also want to thank the drug resistance monitoring group of the Zhejiang Province.
Funding {#FPar1}
=======
This study was supported by grants from the National Natural Science Foundation of China (No. 81871705 from Gongxiang Chen).
Availability of data and materials {#FPar2}
==================================
All data generated or analysed during this study are included in this manuscript.
HL, CJM, LHY, CS, DSB and ZR contributed to the acquisition and analysis of the data. HL wrote the initial draft of this paper. HYY and ZHW performed the statistical analyses. CGX contributed to the concept of the study, the revision of this paper, and the final approval of the version to be published. All authors have read and approved the final manuscript.
Not applicable
Not applicable
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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INTRODUCTION {#S1}
============
Identification and training of struggling learners have become one of the most challenging aspects for graduate medical education (GME) training programmes.^[@R1]^ Competencies are commonly measured but may not identify specific deficits in troubled learners.^[@R2]^ Changes in GME common programme requirements and the introduction of Next Accreditation System allow for earlier identification of struggling house staff, providing earlier opportunities for intervention. When troubled learners are identified, much of the process of establishing their learning deficit is inferential rather than pursuant to direct, systematic observation.^[@R2]^ It is particularly difficult to define the reasons learners suffer from poor clinical decision making.^[@R3]^ While in some cases signs may point towards poor knowledge acquisition skills (such as poor standardised examination scores), in reality, the issue may be due to complex and subtle deficits that revolve around the learner's inability to acquire and integrate pertinent information. This may have patient safety implications: one recent study suggested that selective data gathering or interpretation may significantly increase medical errors and harm.^[@R4]^
These issues are compounded by the use of electronic health records (EHRs). Our group and others have documented the negative impact of EHRs on efficiency, and demonstrated that providers at all training levels have significant difficulty in finding and/or recognising important trends in clinical data.^[@R5][@R6]^ This is exacerbated by the fact that GME programmes recruit trainees with prior exposure to, and different levels of experience with, multiple disparate EHRs that invariably affects their information retrieval skills and subsequently their overall clinical competency.
We previously described the use of high-fidelity simulation activities integrated with screen capture and eye tracking to study resident--EHR interactions,^[@R5][@R7]^ which led us to hypothesise that we could employ our EHR simulation to identify and assist learners struggling with clinical efficiency and decision making, and specifically assist in delineating whether the learners' issues lay in suboptimal data retrieval/interpretation and/or clinical knowledge deficits.
METHOD {#S2}
======
The study was approved by the Oregon Health & Science University Institutional Review Board. All data were de-identified and stored securely.
Troubled learners were identified by their Programme Director (PD) and enrolled in this study in a purposive fashion to participate in a high-fidelity EHR use simulation. All simulations were conducted by a member of our study team on a dedicated mobile desktop computer with an attached Tobii Pro X120 eye tracker affixed to the bottom of the monitor (Tobii Systems, Danderyd Municipality, Sweden). This system does not require subjects to wear specialised glasses and is reliable in a variety of lighting conditions with an optimal sampling rate at a user distance of 65 cm. At this distance, the viewing angle is up to 30° with an accuracy of 0.3°.
In addition to the dedicated eye-tracking and screen-tracking software (Tobii Studio), the computer contained the same software package as clinical workstations including access to a dedicated simulation instance of our institution's EHR (Epic-Care, Epic Systems, Madison, Wisconsin, USA) which featured full EHR functionality and all user-specific customisations. The simulation instance was populated with validated medical intensive care unit (ICU) charts that featured rich multiday clinical data, including multiprofessional documentation. Cases were designed to assess both efficient and effective data extraction from the EHR, with effective use being assessed by recognition of a series of built-in patient safety issues and dangerous trends in the patient's condition, medication errors and failure to adhere to best practices, and a full detail of these safety issues and their performance characteristics have been previously published.^[@R5][@R7]^ Learners were provided a written sign-out on the patient and then given 10--15 min to review the EHR in the context of their assuming care for the patient in preparation for daily rounds. Screen capture and eye tracking of the subjects' navigation were recorded using Tobii Studio (Tobii Technologies) to document their EHR usability patterns and guide debriefing.^[@R8]^ After review of the chart, the learner presented the case and was assessed for the number of safety issues identified. Subsequently, we reviewed screen use and eye-tracking patterns with the learners using visualisation maps showing where specific gaze fixations occurred on the EHR screen in a 'talk aloud' session to qualify the learners' thought process regarding data visualisation, interpretation and EHR use. Learners completed a brief survey (see online supplementary table 1) after the sessions to gather feedback, which was analysed to identify themes using a constant comparative method. Exemplar quotes were identified, which are highlighted in the results section below. After the talk aloud session and data analysis, a detailed report was provided to the PD to assist in the development of a personalised learning plan, with primary input from the study authors, that emphasised EHR best use practices specific to the deficiencies identified during the simulation.
RESULTS {#S3}
=======
Subject \#1 {#S4}
-----------
This subject was nearing completion of his Intern year in internal medicine (IM). His PD stated: "This is an intern who is struggling with appropriate collection of data from his chart reviews."
During the simulation, the subject only identified 14% of relevant safety items. Analysis of EHR navigation patterns revealed that while the subject used screens appropriate for data gathering, there was failure to identify the significance of the data observed on the screen. In essence, the subject would preferentially navigate to screens and hope that relevant data would 'stand out' as opposed to having a dedicated plan for searching for specific clinical data items. However, once the information was highlighted, the subject made appropriate decisions, implying adequate knowledge. Subsequent to the simulation debriefing, the learner stated: "I think the biggest gap in my current system is that, as we saw, I tend to review data without much of a critical eye, and as a result I fail to recognize when there are data points of concern. If I could somehow come up with a system that forces stopping points to integrate & interpret data I think I'd keep a much better handle on patients' active issues..."
As part of their personalised learning plan, the subject was instructed to create specific templates to facilitate a systematic approach to data collection, focusing on pursing specific data elements within the EHR. Currently, the subject is performing well with no further problems.
Subject \#2 {#S5}
-----------
Subject was a neurology intern in his preliminary year, with extensive prior exposure to IM services, including the ICU. The PD was concerned about problems with basic medical knowledge and medical decision making.
This subject recognised nearly 78% of the safety items during the simulation. However, while the subject could recognise important trends, this learner's differential diagnosis was extremely limited, suggesting deficits in medical knowledge.
In this scenario, the personalised learning plan focused on improving general clinical knowledge and recommended participating in additional case-based simulation focused on critical decision making. Unfortunately, the learner continued to struggle with cognitive reasoning despite remediation and resigned from the programme shortly after. The PD noted: "He could find data OK \[but\] he could not integrate it and that he never developed the core clinical skills in medical school he needed to function as an intern."
This example underscores the premise that some learners may struggle with issues linked to cognitive concerns as opposed to issues with the EHR itself.
Subject \#3 {#S6}
-----------
This subject was halfway through his preliminary IM internship year. The PD stated his primary issue was that he frequently missed significant and relevant data elements, despite spending an inordinate amount of time in data collection.
During the simulation, the subject recognised 42% of safety issues. However, on review of his navigation, it became apparent that his use pattern did not include viewing EHR screens used by the majority of his house staff peers (as identified by analysing over 200 recorded simulations with peer users); instead, the subject used screens seldom viewed by his peers which also contained significant blind spots in the presentation of critical data elements.
This suggests that prior EHR use patterns (in this case, the learner had used a different EHR in medical school) can result in information retrieval habits that may be detrimental when applied to another EHR. As part of this subject's personalised learning plan, we provided a script that encouraged the use of high-yield screens for data collection. The subject has since adopted this script and stated: "I found the EMR simulation very helpful, and I'm grateful for the opportunity to partake in it. It was particularly constructive to receive feedback on how my practices are discordant with standard/optimal use."
DISCUSSION {#S7}
==========
In this report, we demonstrate the use of EHR-based simulation to diagnose the aetiology of perceived cognitive issues and deficient medical decision making in identified troubled learners. Interestingly, we discovered distinct phenotypes of learning deficits using this technique.
First, the simulation allowed us to distinguish between learners with difficulty in data processing as opposed to data acquisition. Second, simulation allows us to distinguish troubled learners who engaged in two distinctly different workflows of selective data gathering that resulted in poor clinical decision making.
Of these, our ability to identify learners who struggle to recognise basic trends in data highlights an extreme example of our prior work.^[@R5]^ Such learning deficits may be resolved by prescribing a structured approach to EHR use facilitating data acquisition with the overall aim of the learner successfully adapting a new information retrieval strategy.
In contrast, selective data gathering can also be pursuant to using EHR screens that only partially display pertinent data, resulting in blind spots in data collection. Learners demonstrating this EHR use pattern require in a different remediation strategy focused on best practices for EHR navigation. Interestingly, the ability of one subject to self-identify their learning issue after participating in the simulation suggests that learners may possess latent insight into their deficits that can be metacognitively self-identified as they participate in EHR simulations.
The model of EHR-centric high-fidelity simulation that we have described may assist GME programmes in effectively diagnosing learner deficits rapidly and facilitate the development of personalised, effective solutions that the learners themselves find useful. We are now working on integrating this model into the global GME assessment process to assist struggling learners in our institution. We plan on using the data gathered from additional struggling learners to in turn inform changes in initial EHR training and system redesign, as well as the creation of electronic tools to better identify struggling learners.
**Contributors** VM and JAG helped design, interpret and conduct the study. GS created the simulations. GS, VM and JAG all contributed to the writing of the manuscript.
**Competing interests** None declared.
**Patient consent** No consent required for this study.
**Ethics approval** OHSU IRB.
**Provenance and peer review** Not commissioned; externally peer reviewed.
######
Qualitative results from post intervention survey
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Question Learner 1 Learner 3
--------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Was the simulation effective?** Effective in that it posed common clinical challenges in a realistic environment Yes; it was very helpful for identifying systematic weaknesses in my approach to chart review in Epic.
**What did you learn from the simulation?** Degree of inefficiency, difficulty processing information in Epic, unfamiliarity with the clinical scenario presented in the simulation I suboptimally utilised several summary views, particularly the medication, labs, and vitals accordions. I often searched for isolated lab results that did not display data in context. I also had a tendency to miss vent settings and drips (which are not consistently visible in different Epic activities).
**Did your use of the EHR change in any way after the simulation? If so, How?** Made me actively seek out further epic training to increase my efficiency I developed a habit of more frequently referring to the data views in ICU accordion, which are great for systems- focused review.
**Have you noticed a difference in patient outcomes after the simulation?** As a result of the above, I'm able to more effectively comb through chart history, assess trends in clinical data. Unclear if this has changed outcomes as there is no metric to evaluate avg LOS, hospital mortality, readmission, etc of patients with whose care I've been associated Yes\
-fewer misses in changes of titratable infusions\
-greater appreciation of vitals and lab trends over time\
-faster and more systematic prerounding and preadmitting in general
**Have you noticed a change in your performance and/or evaluation of your performance since the simulation?** Meteoric rise in efficiency stemming from further training and \~2 years of extra experience. Lower stress, markedly less frequent 'misses', increased overall awareness of patients' history and current trajectory. Subjectively, yes. No formal feedback yet available.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Note: learner 2 left the institution before the survey could be administered.
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Introduction
============
Few published studies have comprehensively explored the epidemiology of depression among truck drivers and none, to our knowledge, have done so in sub-Saharan Africa. Long-distance truck drivers may have a high prevalence of depression (and possibly other mental health disorders) due to occupational related stressors ([@ref-27]; [@ref-29]; [@ref-47]; [@ref-50]), such as road hazards, fixed delivery deadlines, and irregular sleeping schedules ([@ref-39]). Globally, several studies have estimated the prevalence of depression among truck drivers, but these estimates have varied by country, ranging from 14% to 70% ([@ref-15]; [@ref-16]; [@ref-38]; [@ref-35]; [@ref-40]; [@ref-44]).
Truck drivers are also at high-risk of HIV infection because of the high rates of sexual risk behavior, including engaging in condomless sex and having multiple partners ([@ref-14]; [@ref-28]; [@ref-51]; [@ref-12]). A small number of studies in sub-Saharan Africa have explored the relationship between depression and sexual risk, including in a general population at risk for HIV infection in South Africa ([@ref-41]; [@ref-31]) and among social networks of young men in Tanzania ([@ref-19]). Although this relationship is complex, one possible explanation may be that maladaptive thought patterns arising from depression, such as those related to impulsivity and hopelessness ([@ref-48]), might lead to the adoption of risky behavior and increase the propensity for self-harm. In other words, depression might lead to increased sexual risk behavior. General self-efficacy, which can be defined as "people's belief in their ability to influence events that affect their lives" ([@ref-8]), might mediate this association, as suggested by previous studies in sub-Saharan Africa ([@ref-46]; [@ref-4]; [@ref-43]).
In this study, we assessed the prevalence of depression and explored its association with sexual risk behavior in a sample of long-distance truck drivers seeking services at two roadside wellness clinics in Kenya. Additionally, we explored whether general self-efficacy mediated the association between depression and sexual risk behavior.
Materials and Methods
=====================
Study procedures
----------------
We used cross-sectional data collected as part of a randomized controlled trial evaluating whether offering HIV testing options (i.e., provider-administered blood-based (finger prick) rapid HIV test or a supervised oral self-administered rapid HIV test (OraQuick In-Home HIV Test) in the clinic or a self-test kit for home use) compared with the standard of care (i.e., only offering the provider-administered blood test) would increase HIV testing rates among truck drivers in Kenya. The methods and results of this trial are described in detail elsewhere ([@ref-23]).
Participants were recruited from two North Star Alliance roadside wellness clinics located along the northern transport corridor in Nakuru County, Kenya, one of the highest HIV prevalence areas in the country ([@ref-1]; [@ref-2]). The North Star Alliance provides healthcare services, including sexually transmitted infection and HIV testing and treatment ([@ref-32]), to key populations in transport corridor communities, including truck drivers and sex workers. At the time of this study, the North Star Alliance had 36 clinics in Africa, eight of which were in Kenya. Together, the two clinics included in this study served about 400 clients weekly, 30% of whom were long-distance truck drivers. Any male truck driver who visited either of the two clinics from October to December 2015 for services other than HIV treatment, was informed about the research study and, if interested, was referred to a fieldworker for information and eligibility screening. Eligibility criteria were: (1) at least 18 years old, (2) male (based on observation), (3) employed as a long-distance truck driver (anyone who works to make deliveries locally would be classified as a community resident and not eligible for our study), (4) primary residence in Kenya, (5) able to speak English or Kiswahili, (6) self-reported HIV-negative or unknown HIV status, (7) able to sign the consent form, and (8) willing to receive payment for participation via MPesa, a cell phone-based money transfer system widely used in Kenya. Overall, 319 men were referred for eligibility screening, of whom 305 were enrolled in the study. Informed consent was obtained from all participants.
Fieldworkers administered a baseline interview to truck drivers who met the eligibility criteria and consented to participate in the study. The baseline interview included questions about demographic characteristics and risk behavior, and several psychometric scales, including general self-efficacy. Following the baseline interview, participants were offered HIV testing with options dependent on randomization arm (i.e., choice of an alternative HIV testing modality vs. standard provider-administered blood test). After undergoing or refusing HIV testing, appointments were made for a follow-up interview at 6 months. At the 6-month interview, which was completed over the phone in most cases, research staff administered the Patient Health Questionnaire (PHQ)-9 ([@ref-25]) and asked about HIV testing in the past 6 months. The questionnaires were developed in English and Kiswahili, and the Kiswahili translation was translated and back-translated to English to ensure accuracy. Interviews were conducted in Kiswahili, English, or both, depending on the participant's preference. The study procedures were approved by the City University of New York Institutional Review Board (\#2015-0645), the Kenya Medical Research Institute Ethics Committee (\#541), and the University of KwaZulu-Natal Biomedical Research Ethics Committee (BFC025/15), and were in accordance with the 1964 Helsinki declaration and its later amendments.
Measures
--------
### Sexual risk behavior (main outcome of interest)
Number of condomless sex partners was used as a measure of sexual risk behavior and was determined by asking participants how many female and male condomless sex partners they had in the past 6 months. Participants who did not have sex in the past 6 months (*n* = 6) were categorized as having zero condomless sex partners. Only one reported ever having anal sex with another man. We retained this variable as a numeric, but also categorized responses (none, one, two or more) in bivariate analyses.
### Depression (main exposure of interest)
Depression was assessed with the PHQ-9, a widely used and validated nine-item questionnaire asking about each Diagnostic and Statistical Manual of Mental Disorders-IV depressive symptom within the last 2 weeks ([@ref-25]). Each item is scored based on symptom frequency, from 0 ("not at all"), 1 ("several days"), 2 ("more than half the days"), or 3 ("nearly every day"), with scores can ranging from 0 to 27. The PHQ-9 has been formally validated among general adult populations using an acceptable gold standard (i.e., psychiatric clinician diagnosis using Schedules for Clinical Assessment in Neuropsychiatry or Mini International Neuropsychiatric Interview) in East African primary healthcare settings ([@ref-17]; [@ref-18]). We constructed a summary score by summing responses to the nine questions allowing up to one item to be left unanswered; only two participants had any missing responses, and for these two participants, only one item was missing, so no one was excluded. The standardized Cronbach's alpha for the PHQ-9 in our study sample was 0.88, indicating good to excellent internal consistency. Scores were categorized as no/minimal depression (score 0--4), mild depression (score 5--9), and moderate-severe depression (score ≥10). A score of ≥10 has 88% sensitivity and specificity for major depressive disorder (MDD) ([@ref-25]).
### Demographic characteristics, self-efficacy, and other risk behavior
In the baseline interview, participants were asked their age, religion, educational attainment, and marital status. Age was based on reported age at their last birthday (in years). Religion was categorized as Christian (i.e., Protestant, Catholic) or non-Christian (i.e., Muslim, Hindu, traditional African, and no religion). Educational attainment was categorized as having completed at least secondary school vs. less than a secondary school diploma (i.e., no education, some primary school, completed primary school only, or some secondary school). Marital status was categorized as currently married (either legal or common-law) vs. unmarried (including divorced/separated, widowed, or single).
Average monthly income from truck driving was determined based on responses to the question "about how much money do you earn in an average month driving a truck?" and those who were unable or unwilling to specify their income were then asked "could you tell me if your income is less than 8,000 Kenyan shillings (Ks), 8,000--16,000, 16,001--24,000, 24,001--50,000, or \>50,001 Ks?" At the time of the study, 100 Ks was worth approximately 1.00 US dollar (USD). Income was dichotomized at about the first quartile into mid-high income (≥240 USD) vs. low income (\<240 USD). Length of time worked as a truck driver was determined by asking participants how many months and years they had worked as a truck driver. Driving accompanied or not was determined by asking participants if they usually drive alone or if someone else rides with them (e.g., assistant, main partner, non-main partner, family member, etc.). Days away from home in the past month was determined by asking participants how many nights they were away from home traveling for work in the past month.
General self-efficacy was measured using a 10-item scale ([@ref-36]), previously validated in a general non-white South African population ([@ref-24]), which presents statements related to belief in one's confidence to cope with a broad range of stressful or challenging demands. Response options were on a four-point Likert scale from "not at all true" to "exactly true," with a possible total score range of 10--40. We constructed a summary score by summing responses to the 10 questions allowing for one of the items to be left unanswered; only three participants had any missing responses and they were missing only one item. Cronbach's alpha for this scale in our sample was 0.89, indicating good to excellent internal consistency.
Number of sex partners was determined by asking how many people they had sexual intercourse with in the past 6 months, asking separately for female (vaginal/anal intercourse) and male (anal intercourse) partners. Alcohol use was determined by asking how often the participant had drinks containing alcohol in the past year. Similarly, illicit drug use was determined by asking how often the participant used drugs in the past year, with marijuana and cocaine listed as examples. Response options for both questions were "at least once a day," "a few times a week but not every day," "a few times a month but not every week," "a few times a year but not every month," and "never." We recoded these variables into two separate indicators for having consumed any alcohol or drugs in the past year (both versus none) for bivariate analyses. However, as there were very few reports of drug use and all participants who reported drug use also reported alcohol use, only the alcohol use variable was included in the regression models.
Statistical analysis
--------------------
We first described the sample overall and by depression category. We then used bivariate statistical tests (Pearson's Chi-square, Fisher's exact, and Kruskal--Wallis) to identify significant differences in the distribution of variables by depression category. We then ran Poisson regression models with number of condomless sex partners as the dependent variable. We ran crude models, then a multivariable model that included all variables explored in bivariate analyses (with the addition of clinic of enrollment). Next, we ran a multivariable model with imputed data to address potential bias due to missing data. The multivariable model had about 25% missing data, so we ran 25 imputations to achieve reliable *p*-values and standard errors ([@ref-11]).
We used fully conditional specification for the imputation method (logistic for classification variables and predictive mean matching for numeric variables) ([@ref-10]) with all variables in the model used to impute missing values. We then re-ran our multivariable model with the imputed data.
Finally, we assessed whether general self-efficacy mediated the association between MDD (PHQ-9 score ≥10) and number of condomless sex partners. This analysis was conducted using a SAS Macro developed by [@ref-45], with mediation methodology from [@ref-34]. The Macro used a bootstrapping approach with 100 samples to generate crude prevalence ratios (cPRs) and 95% confidence intervals (CIs) for the natural direct effect and natural indirect effect of the association between MDD and number of condomless sex partners ([@ref-45]). Presence of a statistically significant natural indirect effect was evidence of mediation.
All analyses were conducted in SAS 9.4 (SAS Institute Inc., Cary, NC, USA) and the significance level was set at a two-sided α of 0.05.
Results
=======
Description of the sample
-------------------------
The mean age of the overall sample was 36.9 years (standard deviation (SD) = 7.9). Most participants were married (83.0%), about one-third (37.0%) had a secondary school education or greater, about three-quarters (77.8%) were Christian, and nearly three-quarters (73.5%) earned ≥240 USD monthly. Participants had worked as a truck driver on average for 8.8 years (SD = 7.0), more than half (58.1%) usually drove alone, and in the previous 30 days, participants spent a mean of 21.6 days (SD = 23.0) on the road. Mean general self-efficacy score was 36.5 (SD = 4.6). In terms of risk behaviors, 53.5% had consumed alcohol in the past year and 3.2% reported drug use in the past year. The mean number of sex partners in the past 6 months was 2.8 (SD = 4.1); 58.2% reported having at least one condomless sex partner in the past 6 months and 27.3% reported having two or more ([Table 1](#table-1){ref-type="table"}).
10.7717/peerj.7253/table-1
###### Demographic variables, risk behaviors, and other characteristics overall and by depression category.
All participants (with depression data) None/minimal depression Mild depression Moderate-severe depression *p*-Value
----------------------------------------------------------- ----------------------------------------- ------------------------- ----------------------- ---------------------------- ------------------------------------------
*n* (%) 284 (100%) 157 (55.3%) 59 (20.8%) 68 (23.9%) NA
Age (years) *N* = 284
Mean (SD)/Median (IQR) 36.9 (7.9)/36.0 (10.0) 36.9 (8.0)/37.0 (10.0) 37.0 (7.0)/37.0 (9.0) 36.8 (8.5)/35.0 (11.5) 0.859[^a^](#table-1fn3){ref-type="fn"}
Married *N* = 282
Yes, *n* (%) 234 (83.0%) 130 (55.6%) 50 (21.4%) 54 (23.1%) 0.660[^b^](#table-1fn4){ref-type="fn"}
No, *n* (%) 48 (17.0%) 25 (52.1%) 9 (18.8%) 14 (29.2%)
Secondary school education or higher *N* = 284
Yes, *n* (%) 105 (37.0%) 57 (54.3%) 25 (23.8%) 23 (21.9%) 0.589[^b^](#table-1fn4){ref-type="fn"}
No, *n* (%) 179 (63.0%) 100 (55.9%) 34 (19.0%) 45 (25.1%)
Religion *N* = 279
Christian, *n* (%) 217 (77.8%) 117 (53.9%) 47 (21.7%) 53 (24.4%) 0.436[^b^](#table-1fn4){ref-type="fn"}
Non-Christian, *n* (%) 62 (22.2%) 39 (62.9%) 10 (16.1%) 13 (21.0%)
Monthly income *N* = 268
\<240 USD, *n* (%) 71 (26.5%) 34 (47.9%) 10 (14.1%) 27 (38.0%) 0.010[^b^](#table-1fn4){ref-type="fn"}
≥240 USD, *n* (%) 197 (73.5%) 112 (56.9%) 45 (22.8%) 40 (20.3%)
Years worked as a truck driver *N* = 284
Mean (SD)/Median (IQR) 8.8 (7.1)/7.0 (7.0) 9.2 (6.5)/8.0 (8.0) 9.3 (8.0)/6.7 (6.8) 7.6 (7.4)/4.2 (6.3) 0.016[^a^](#table-1fn3){ref-type="fn"}
Usually drives alone *N* = 284
Yes, *n* (%) 165 (58.1%) 97 (58.8%) 32 (19.4%) 36 (21.8%) 0.372[^b^](#table-1fn4){ref-type="fn"}
No, *n* (%) 119 (41.9%) 60 (50.4%) 27 (22.7%) 32 (26.9%)
Days away on road in past 30 days *N* = 277
Mean (SD)/Median (IQR) 21.6 (5.5)/23.0 (5.0) 21.5 (5.6)/22.0 (5.0) 21.7 (5.4)/22.0 (5.0) 21.9 (5.5)/24.0 (5.0) 0.766[^a^](#table-1fn3){ref-type="fn"}
General self-efficacy (score) *N* = 284
Mean (SD)/Median (IQR) 36.5 (4.6)/39.0 (5.0) 38.2 (2.9)/40.0 (3.0) 35.3 (5.3)/37.0 (8.0) 33.7 (5.6)/36.0 (10.5) \<0.001[^a^](#table-1fn3){ref-type="fn"}
Alcohol use in past year[^d^](#table-1fn6){ref-type="fn"} *N* = 284
Yes, *n* (%) 152 (53.5%) 79 (52.0%) 38 (25.0%) 35 (23.0%) 0.168[^b^](#table-1fn4){ref-type="fn"}
No, *n* (%) 132 (46.5%) 78 (59.1%) 21 (15.9%) 33 (25.0%)
Drug use in past year[^d^](#table-1fn6){ref-type="fn"} *N* = 283
Yes, *n* (%) 9 (3.2%) 0 (0.0%) 1 (11.1%) 8 (88.9%) \<0.001[^c^](#table-1fn5){ref-type="fn"}
No, *n* (%) 274 (96.8%) 129 (47.1%) 86 (31.4%) 59 (21.5%)
Number of sex partners in past 6 months *N* = 268
Mean (SD)/Median (IQR) 2.8 (4.1)/2.0 (3.0) 2.6 (5.4)/2.0 (2.0) 2.4 (1.7)/2.0 (2.0) 3.5 (3.5)/2.0 (4.0) 0.085[^a^](#table-1fn3){ref-type="fn"}
Number of condomless sex partners in past 6 months *N* = 275
Mean (SD)/Median (IQR) 1.5 (1.6)/1.0 (1.0) 1.2 (1.1)/1.0 (0.0) 1.3 (1.1)/1.0 (0.0) 2.2 (2.6)/1.0 (2.0) 0.003[^a^](#table-1fn3){ref-type="fn"}
None, *n* (%) 40 (14.6%) 23 (57.5%) 11 (27.5%) 6 (15.0%) 0.038[^b^](#table-1fn4){ref-type="fn"}
One, *n* (%) 160 (58.2%) 73 (45.6%) 54 (33.8%) 33 (20.6%)
Two or more, *n* (%) 75 (27.3%) 27 (36.0%) 21 (28.0%) 27 (36.0%)
**Notes:**
Some percentages may not add up to 100% due to rounding.
IQR, interquartile range; SD, standard deviation; USD, US dollars.
Kruskall--Wallis test.
Pearson's Chi-square test.
Fisher's exact test.
All participants who reported drug use also reported alcohol use. Drug use was not included in the regression models because of small numbers of participants in categories.
The mean and median scores on the PHQ-9 were 5.2 (SD = 5.0) and 4.0 (interquartile range = 9.0), respectively. Upon categorizing participants, 157 (55.3%) had none/minimal depression, 59 (22.8%) had mild depression, and 68 (24.0%) had moderate-severe depression (probable MDD) ([Table 1](#table-1){ref-type="table"}). The prevalence of any symptom frequency in the past 2 weeks based on the PHQ-9 was: low energy (50.7%), trouble concentrating (50.4%), anhedonia (little interest or pleasure in doing things) (48.8%), appetite problems (44.2%), feeling depressed (40.8%), sleep problems (41.2%), low self-esteem (38.3%), psychomotor problems (30.6%), and suicide/self-harm ideation (19.4%).
Bivariate analysis results
--------------------------
In bivariate analyses, there were significant differences in the distribution of monthly income, years worked as a truck driver, general self-efficacy, drug use, and number of condomless sex partners by depression category. Participants with lower monthly trucking income (\<240 USD) vs. higher monthly trucking income (≥240 USD) more often had moderate-severe depression (38.0% vs. 20.3%; *p* = 0.010). Truck drivers with moderate-severe depression vs. no/minimal depression tended to have been in the occupation for a shorter period of time (mean years: 7.6 vs. 9.2; *p* = 0.016). General self-efficacy scores were lower for participants with depression (mean score 38.2 (none/minimal depression), 35.3 (mild depression), 33.7 (moderate-severe depression); *p* \< 0.001). Participants who reported drug use were more likely to have depression (0.0% (no/minimal depression), 11.1% (mild depression), and 88.9% (moderate-severe depression)). Participants with moderate-severe depression were also more likely to have more condomless sex partners vs. participants with no/minimal depression (mean number of partners: 2.2 vs. 1.2; *p* = 0.003) ([Table 1](#table-1){ref-type="table"}).
Poisson regression results
--------------------------
In the crude Poisson regression model, moderate-severe depression (vs. none/minimal depression) was significantly associated with the number of condomless sex partners (cPR 1.80, *p* \< 0.001). This remained significant in the multivariable Poisson regression model (adjusted prevalence ratio (aPR) 1.63, *p* \< 0.001). For this model, the Pearson Chi-square value divided by the degrees of freedom was 0.92, indicating that under- and over-dispersion were not present. A goodness-of-fit Chi-squared test was not significant (*p* = 0.814), indicating reasonable model fit. In the multivariable model with imputed data, the associations between depression and condomless sex partners remained similar (mild depression: aPR 1.22, *p* = 0.124; moderate-severe depression: aPR 1.62, *p* \< 0.001) ([Table 2](#table-2){ref-type="table"}).
10.7717/peerj.7253/table-2
###### Poisson regression models examining the association of depression and other variables with number of condomless sex partners.
Crude models[^a^](#table-2fn2){ref-type="fn"} Multivariable model[^b^](#table-2fn3){ref-type="fn"} Multivariable model with imputed data[^b^](#table-2fn3){ref-type="fn"}
--------------------------------------------- ----------------------------------------------- ------------------------------------------------------ ------------------------------------------------------------------------
cPR (95% CI); *p*-value aPR (95% CI); *p*-value aPR (95% CI); *p*-value
*N* = 236 *N* = 305
Depression *N* = 275
None/minimal Reference Reference Reference
Mild 1.08 (0.84--1.37); *p* = 0.563 1.16 (0.88--1.52); *p* = 0.290 1.22 (0.95--1.56); *p* = 0.124
Moderate-severe 1.80 (1.43--2.26); *p* \< 0.001 1.63 (1.25--2.12); *p* \< 0.001 1.62 (1.26--2.08); *p* \< 0.001
Age (years) *N* = 296
1.00 (0.99--1.01); *p* = 0.817 1.01 (0.99--1.03); *p* = 0.720 1.01 (0.99--1.02); *p* = 0.360
Married *N* = 293
Yes 1.58 (1.17--2.12); *p* = 0.003 2.12 (1.41--3.19); *p* \< 0.001 1.67 (1.20--2.32); *p* = 0.003
No Reference Reference Reference
Secondary school education or higher *N* = 296
Yes 0.69 (0.57--0.85); *p* \< 0.001 0.79 (0.61--1.02); *p* = 0.065 0.73 (0.58--0.92); *p* = 0.007
No Reference Reference Reference
Religion *N* = 290
Christian Reference Reference Reference
Non-Christian 1.26 (1.02--1.56); *p* = 0.032 1.37 (1.02--1.82); *p* = 0.034 1.34 (1.03--1.73); *p* = 0.029
Monthly trucking income *N* = 279
\<240 USD Reference Reference Reference
≥240 USD 0.64 (0.52--0.77); *p* \< 0.001 0.76 (0.59--0.97); *p* = 0.029 0.72 (0.58--0.90); *p* = 0.003
Years worked as a truck driver *N* = 294
0.98 (0.97--1.00); *p* = 0.010 0.98 (0.96--1.00); *p* = 0.055 0.98 (0.96--1.00); *p* = 0.017
Usually drives alone *N* = 296
Yes 1.43 (1.19--1.72); *p* \< 0.001 1.16 (0.92--1.47); *p* = 0.220 1.10 (0.89--1.34); *p* = 0.383
No Reference Reference Reference
Nights away on road in past 30 days *N* = 288
0.99 (0.97--1.01); *p* = 0.183 0.98 (0.96--1.00); *p* = 0.106 0.99 (0.98--1.01); *p* = 0.475
Alcohol use in past year *N* = 296
Yes 1.14 (0.95--1.38); *p* = 0.158 1.11 (0.87--1.42); *p* = 0.400 1.06 (0.86--1.31); *p* = 0.593
No Reference Reference Reference
Number of sex partners in the past 6 months *N* = 288
1.04 (1.03--1.05); *p* \< 0.001 1.04 (1.03--1.05); *p* \< 0.001 1.04 (1.03--1.06); *p* \< 0.001
**Notes:**
aPR, adjusted prevalence ratio; cPR, crude prevalence ratio; USD, US dollars.
Not adjusted for any other variables.
Adjusted for clinic location in addition to the other variables listed.
Mediation analysis results
--------------------------
In the mediation analysis for general self-efficacy, the natural direct effect was not statistically significant (cPR 1.40; 95% CI \[0.94--1.90\]) and the natural indirect effect was significant (cPR 1.24; 95% CI \[1.14--1.44\]) for the association between moderate-severe depression and number of condomless sex partners.
Discussion
==========
We found high prevalence of probable MDD (24%) in this sample of truck drivers. When comparing across studies that also used the PHQ-9 from non-community-based samples, this estimate was higher than a sample of HIV-positive adult men attending clinics in Uganda (12%) ([@ref-46]), but lower than in a study among adult men who have sex with men (MSM) in coastal Kenya enrolled in an HIV research study (42%) ([@ref-37]). Nationally representative data on depression in the general population in Kenya are scant. According to the 2003 World Health Survey in Kenya, 5.5% of men reported having been diagnosed with depression in the past 12 months ([@ref-49]). Although we did not evaluate occupational stressors in this study, they may help explain this high prevalence of depression and might have a similar effect on mental health as the stressors reported by other high-risk groups, such as stigma-related abuse reported by MSM ([@ref-5]). Currently, the North Star Alliance does not offer mental health screening, diagnosis, and treatment at their roadside wellness clinics in East Africa; however, the results of this study suggest a need for such services. A next step that should be considered is piloting mental healthcare services integrated with existing HIV prevention and treatment services. Furthermore, it will be important to identify other co-morbidities that can be addressed so as to provide more holistic care to this population.
Truck drivers with moderate-severe depression had roughly 60% more condomless sex partners compared with those with no/minimal depression, after adjusting for demographic variables and number of sex partners. Although we cannot determine whether depression led to an increase in sexual risk behavior because our study was cross-sectional, this finding is consistent with a prospective study among men and women in South Africa, which found that baseline depressive symptomatology was associated with failure to use a condom at last sex during the follow-up interview ([@ref-31]). These findings are supported by Beck's cognitive theory of depression whereby negative feelings associated with depression can ultimately lead to negative behavioral reactions to situations ([@ref-9]), such as not using a condom when presented with the opportunity for sexual intercourse.
In our mediation analysis, the indirect effect (i.e., the effect of depression on sexual risk behavior that acts through self-efficacy) was statistically significant and the natural direct effect (i.e., the effect of depression on sexual risk behavior unexplained by general self-efficacy) was not statistically significant, thus suggesting self-efficacy as a mediator. In other words, depression might negatively affect an individual's confidence to cope with stressful situations and perform tasks, and consequently, this lower self-efficacy encourages an individual to engage in higher sexual risk behavior. Self-efficacy is a broad construct, so there may be multiple explanatory pathways between self-efficacy and sexual risk behavior, for example, pathways related to self-esteem, optimism about the future, or belief in the ability to prevent sexually transmitted infections ([@ref-22]). Other studies from sub-Saharan Africa have also found that self-efficacy was a mediator of this association ([@ref-46]; [@ref-4]; [@ref-43]), but have also been cross-sectional. Bandura's Social Cognitive Theory ([@ref-7]) might suggest that low self-efficacy could be a cause of depression implicating it as a confounder. A recent study among adolescents examining the bidirectional association found that depression resulted in negative changes in self-efficacy, rather than the other way around ([@ref-42]). Thus, further research is needed to understand the role of self-efficacy in this association. If self-efficacy is indeed a mediator, it could also be a target to reduce high sexual risk behavior, especially among individuals with depression, through interventions such as behavioral activation, problem-solving, and exploration of past successes and perceived competencies ([@ref-22]).
There are some limitations to consider when interpreting these data. First, clinic-based sampling alone, as done in our study, may result in higher prevalence estimates for mental health disorders than if non-clinic based sampling was also done ([@ref-33]). However, if the prevalence of depression is higher among those who seek care than those who do not, it would mean that the population with the greatest need for mental healthcare services is perhaps the easiest to reach because they are already in care. Roadside wellness clinics will be an important entry point to initiate the provision of these services to truck drivers. Another limitation related to sampling is that our study participants were recruited from two clinics in one geographical area, so we do not know how similar or different these truck drivers are compared with truck drivers who attend clinics in other areas of Kenya or East Africa. Second, our measure of sexual risk behavior was the number of condomless sex partners, which is not inclusive of all aspects of sexual risk ([@ref-6]). Although condomless sex with only a main partner would be considered lower sexual risk behavior compared with condomless sex with multiple partners, most men in our study had more than one sexual partner, even though most were married, and we adjusted for marital status and number of sex partners in the multivariable model. Third, some variables may have been subject to reporting bias. Because homosexuality is illegal in Kenya, it is understandable why study participants may have been reluctant to discuss same-sex partners. In fact, there was only one participant who reported previously having anal sex with another man. Similarly, participants may have also been reluctant to fully disclose drug and alcohol use because of potential consequences to their livelihood. Third, we did not have data on occupational stressors, which may have acted as confounders or common causes of depression and sexual risk behavior. Finally, as our study was cross-sectional, we do not know the direction of causality for the association between depression and sexual risk behavior. Although the data were interpreted as cross-sectional, measures were taken from two interviews that were 6 months apart, as this was a secondary analysis and the original trial was not specifically designed to answer the research questions in this paper. Because of the limited amount of time truck drivers had for interviews, the PHQ-9 depression scale was administered 6 months later than the questionnaires assessing demographic characteristics, HIV risk behavior, and self-efficacy, so measurement of our exposure (depression) occurred after measurement of our outcome (sexual risk). Although depression status may have changed over the 6 months between the baseline and follow-up interviews, we would not anticipate the association with sexual risk behavior to also change. It is possible that an HIV diagnosis caused depression during follow-up, but only two participants tested positive ([@ref-23]) and neither of these participants had a PHQ-9 score ≥10. Nevertheless, a longitudinal study is needed to establish the temporal order of events.
Beyond the truck driver population, there are major barriers to accessing mental health services, particularly social stigma and dearth of mental health clinicians ([@ref-3]) in Kenya. Although most stigma research in Kenya and sub-Saharan Africa has focused on HIV/AIDS, social stigma regarding mental health disorders appears to be pervasive ([@ref-30]), but a neglected topic. Individuals might be afraid to seek mental healthcare due to concerns about being labeled and stigmatized by the community, co-workers, or healthcare providers. Because there are a critically low number of mental healthcare providers in Kenya, integrating aspects of mental healthcare in services provided by primary care clinicians and lay health workers must be considered ([@ref-20]; [@ref-26]). This remains complicated considering the contradictory findings about the impact of providers' intervention on reducing symptoms of mental health disorders. For example, a trial in Zimbabwe found that lay health workers had a positive impact on reducing symptoms of common mental health disorders through a brief cognitive-behavioral intervention ([@ref-13]), whereas a trial in Kenya found that a mental health training program for primary care clinicians failed to improve diagnosis of mental health disorders ([@ref-21]).
Conclusions
===========
The results of this study suggest an association between depression and sexual risk behavior, and that this association might be mediated by general self-efficacy. Future research is needed to clarify the directionality of these associations. Despite frequent HIV risk-reduction counseling at the roadside clinics included in this study, the prevalence of sexual risk behavior in our participant population was high, emphasizing the importance of bolstering risk-reduction strategies. The prevalence of depression was also high, which emphasizes the importance of developing strategies to improve mental health diagnosis and treatment in this setting. For long-distance truck drivers specifically, future research in sub-Saharan Africa should identify occupational risk factors for depression and other mental health conditions to ensure that evidence-based interventions can be better tailored to this population.
Supplemental Information
========================
10.7717/peerj.7253/supp-1
###### SAS code to recreate analysis.
######
Click here for additional data file.
10.7717/peerj.7253/supp-2
###### Study questionnaire.
######
Click here for additional data file.
We thank the fieldworkers, Alice Opiyo, Deborah Ochungo, Lucy Wanjiru, and Oliver Simiyu, who recruited the study participants and conducted the interviews, the staff at the two participating North Star Alliance clinics who helped with recruitment, and the staff at the North Star Alliance Nairobi office who helped with administrative requirements, such as ethics committee applications. We also are grateful to the study participants who made this study possible.
Additional Information and Declarations
=======================================
Eva Mwai, Eston Nyaga, and Jacob O. Odhiambo are employees of the North Star Alliance. The remaining authors declare that they have no competing interests.
[Matthew L. Romo](#author-1){ref-type="contrib"} conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
[Gavin George](#author-2){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Joanne E. Mantell](#author-3){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Eva Mwai](#author-4){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Eston Nyaga](#author-5){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Michael Strauss](#author-6){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Jacob O. Odhiambo](#author-7){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Kaymarlin Govender](#author-8){ref-type="contrib"} performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
[Elizabeth A. Kelvin](#author-9){ref-type="contrib"} conceived and designed the experiments, performed the experiments, authored or reviewed drafts of the paper, approved the final draft.
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):
The City University of New York Institutional Review Board (\#2015-0645), the Kenya Medical Research Institute Ethics Committee (\#541), and the University of KwaZulu-Natal Biomedical Research Ethics Committee (BFC025/15) granted ethical approval to carry out this study.
The following information was supplied regarding data availability:
Data are available at Harvard Dataverse: Kelvin, Elizabeth A; Mantell, Joanne E, 2018, "Increasing HIV testing demand among Kenyan truck drivers and female sex workers", [DOI 10.7910/DVN/8GVXJY](DOI 10.7910/DVN/8GVXJY), Harvard Dataverse, V2, UNF:6:vT8Ct3ODFigMm1/pWnQzCw== \[fileUNF\].
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
Public health questionnaire surveys are the foundation of modern epidemiology and play an important role in devising policies and programs for the promotion of public health and prevention of diseases \[[@b1-epih-41-e2019012]\]. These surveys are conducted periodically, and accurate health survey data are highly useful for understanding the scope and trends of health problems \[[@b2-epih-41-e2019012]\]. In particular, researchers and policymakers are able to understand and predict current and future health problems and facilitate effective use of limited resources based on these data \[[@b3-epih-41-e2019012]\].
The two classic public health surveys conducted in South Korea (hereafter Korea) are Korea Community Health Survey (KCHS) and Korea National Health and Nutrition Examination Survey (KNHANES) controlled by the Korea Centers for Disease Control and Prevention (KCDC). KCHS presents health statistics in units of city, *gun*, and *gu* required for establishing community healthcare plans, thereby enabling interregional comparisons and serving as indices of community health projects \[[@b4-epih-41-e2019012]\]. KNHANES computes national statistics for people's health, health-related awareness and behavior, and food and nutrition intake and is used for goal-setting and assessment of the Health Plan. Further, it provides national statistical data requested by the World Health Organization (WHO) and the Organization for Economic Cooperation and Development, such as smoking, drinking, physical activity, and obesity data \[[@b5-epih-41-e2019012]\]. The target population for both surveys is Korean citizens, and they both include the entire region of Korea. Furthermore, they share many survey items although the two surveys serve different purposes.
In foreign countries, the estimates of surveys with different purposes but having duplicate items are continually compared to assess the validity of the surveys. The most accurate method to validate survey estimates is to examine the entire population, but this is practically impossible; therefore, these estimates can be compared with those of other surveys \[[@b6-epih-41-e2019012]\]. In the USA, studies have compared self-rated health estimates among four national health surveys, namely Behavioral Risk Factor Surveillance System (BRFSS), Current Population Survey, National Health and Nutrition Examination Survey (NHNES), and National Health Interview Survey \[[@b7-epih-41-e2019012]\], binge drinking rate estimates between BRFSS and National Survey on Drug Use and Health \[[@b8-epih-41-e2019012]\], and obesity estimates between BRFSS and NHNES \[[@b9-epih-41-e2019012]\].
However, there is a lack of studies comparing the estimates between different national surveys in Korea. This is because KCHS and KNHANES are virtually the only two national health surveys and because of their different fundamental purposes, assessing the validity of one survey with reference to the other survey is controversial. However, both surveys are actively utilized for policymaking and research and for diverse analyses in deviation from their purposes \[[@b10-epih-41-e2019012]-[@b13-epih-41-e2019012]\]. Identifying the similarities and disparities between the estimates of two surveys would promote appropriate use of the data.
Policymakers and researchers would be able to understand current health problems more accurately if they select the relevant data or appropriately utilize both data based on data features and the purpose of their analysis, as opposed to their convenience.
Therefore, this study aimed to compare estimates between KCHS and KNHANES, two surveys that serve as the foundation for computing important national statistics. To this end, we compared the estimates for six questionnaire variables in KCHS and eight questionnaire and physical examination variables in KNHANES over a six-year period and analyzed the time series stability of estimates of city-specific and sex- and age-specific subgroups.
MATERIALS AND METHODS
=====================
Study data
----------
Data from KCHS and KNHANES between 2010 and 2015 were used. Both surveys were conducted by the KCDC. KCHS were collected via an interview with all adult members (aged≥ 19 years) of the sample households. Nine hundred people per city, *gun*, and *gu* are surveyed, for a total of 220,000 people every year. Sampling is performed to ensure proportional sampling probability in consideration of household sizes based on the number of households by home type within *tong*, *ban*, and *ri*, and secondary sample households are selected via systematic sampling \[[@b4-epih-41-e2019012]\]. KNHANES is conducted on the members (≥ 1 year) of sample households, with about 10,000 people surveyed every year. Similar to KCHS, sampling is performed with a complex sample design with city/province, *dong/eup/myeon*, and home type as the stratification variables. A total of 3,840 households in 192 districts are chosen every year, and the members are classified as children (aged 1-11 years), adolescents (aged 12-18 years), and adults (aged ≥ 19 years). Age-specific items are used for each group, and health questionnaire, physical examination, and nutritional survey are administered for all participants \[[@b5-epih-41-e2019012]\].
In this study, participants were limited to adults aged 19 years or older, and the number of participants is shown in [Table 1](#t1-epih-41-e2019012){ref-type="table"}. Although the number of subjects to be surveyed shows a difference of about 40 times every year between KCHS and KNHANES, the weighted number of subjects is similar between the two surveys after applying weighted values to represent the target population ---the Korean population---considering that a complex sample design was used for both surveys. The weighted number of subjects differed by about 2% between 2010 and 2013 and by 1% from 2014 to 2015, an average of 2% difference over six years.
When the weighted subjects are divided into subgroups by city, as well as sex and age, there is an average of 9% and 5% difference with reference to the 2015 current smoking rate. The differences vary across years and variables, and arise from non-responses. In the present study, we considered these differences as a feature of the data and thus analyzed the data as is without age standardization.
Definition of variables
-----------------------
To minimize bias, we selected the same variables that the question can correspond to the two surveys. From KCHS, the following six variables were analyzed: self-rated health, current smoking rate, monthly drinking rate, hypertension diagnosis rate, diabetes diagnosis rate, and obesity prevalence. From KNHANES, the following eight variables were analyzed: self-rated health, current smoking rate, monthly drinking rate, hypertension diagnosis rate, diabetes diagnosis rate, obesity prevalence, hypertension prevalence, and diabetes prevalence. Obesity prevalence, hypertension prevalence, and diabetes prevalence in KNHANES were analyzed using physical examination data, and the remaining variables were analyzed based on questionnaire data.
Self-rated health was defined as the percentage of participants who perceived their health to be very good or good, and current smoking rate was defined as the percentage of participants who claimed to have smoked at least five packs (100 cigarettes) in their lifetime and currently smoke every day or occasionally. Monthly drinking rate referred to the percentage of participants who have drank at least one shot of drink in their lifetime and currently drink at least once a month. In KCHS, hypertension diagnosis rate and diabetes rate referred to the percentage of participants who had been diagnosed with hypertension and diabetes, respectively, by a physician. In KNHANES, hypertension diagnosis rate and diabetes diagnosis rate referred to the percentage of participants who responded "yes" to the question asking whether they have been diagnosed with hypertension and diabetes, respectively, by a physician.
KCHS measured body mass index (BMI) using self-reported height and weight, while KNHANES measured BMI using height and weight measured during physical examination. Obesity was defined as a BMI of 25 kg/m^2^ or greater based on the WHO AsiaPacific criteria for obesity \[[@b14-epih-41-e2019012]\], and obesity rates were compared between the two sets of data. Hypertension prevalence in KNHANES was defined as a systolic blood pressure (BP) of 140 mmHg or higher or diastolic BP of 90 mmHg higher for three repeated BP measurements in a physical examinations or the use of hypertension drugs. The hypertension prevalence was compared with hypertension diagnosis rate in KCHS. Diabetes prevalence in KNHANES was defined as a fasting (≥ 8 hours) blood glucose level of 126 mg/dL or higher measured in a physical examination, diagnosis by a physician, use of hypoglycemic agent, or use of insulin injections, and diabetes prevalence was compared with the diabetes diagnosis rate in KCHS. Because hypertension and diabetes are chronic diseases that can be controlled, as opposed to being cured, we deemed it appropriate to conclude individuals to have the disease if they had ever been diagnosed with it in their lifetime. Also, it would be appropriate to compare this estimate with an objective assessment of the disease based on health examination data.
Statistical analysis
--------------------
Because both surveys used a complex sample design, we considered weight, stratification and clustering for the computation of the estimates. When comparing the estimates, the absolute difference, 95% confidence interval (CI) of the difference, and relative difference (ratio of absolute difference to mean KNHANES estimate) were analyzed for the annual estimates for each variable. The absolute difference is the absolute value of the difference between estimates, and relative difference is the proportion of absolute difference in the mean estimate. Variables with low estimates tend to have low absolute differences, so examining relative difference more clearly shows the difference between estimates regardless of the size of the estimates.
Time series stability was compared among 16 city subgroups, excluding the city of Sejong, which was not separately surveyed until the sixth KNHANES (2013-2015), and among 14 sex and age subgroups (19-88 years divided into 10-year units for males and females). A simple linear regression line was computed using six years of estimates by subgroup, and the variability of the estimates were assessed using mean square error (MSE) of the estimates to the line. For example, if there is low variability in the estimates over six years, the MSE would be lower, and this would indicate high time series stability. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) and R version 3.4.4 (<https://cran.r-project.org/bin/windows/base/old/3.4.4/>).
Ethical statement
-----------------
This study was waived for review by the Institutional Review Board (IRB) at Seoul National University (IRB No. E1711/003-002).
RESULTS
=======
Comparison of estimates by variable between KCHS and KNHANES
------------------------------------------------------------
[Figure 1](#f1-epih-41-e2019012){ref-type="fig"} and [Table 2](#t2-epih-41-e2019012){ref-type="table"} show the comparison of estimates by variable between both surveys over six years from 2010 to 2015.
The mean absolute difference of self-rated health over six years was 10.8%p, with 10.5%p in 2010, 9.2%p in 2011, 11.4%p in 2012, 9.6%p in 2013, 9.9%p in 2014, and 14.0%p in 2015. The mean relative difference was 33.0%p. The differences were greater than those for other variables.
The mean absolute difference of current smoking rate over six years was 1.2%p, with 2.3%p in 2010, 2.3%p in 2011, 1.3%p in 2012, 0.1%p in 2013, 0.6%p in 2014, and 0.6%p in 2015. The mean relative difference was 4.9%p.
The mean absolute difference of monthly drinking rate over six years was 1.1%p, with 2.2%p in 2010, 0.7%p in 2011, 1.1%p in 2012, 0.4%p in 2013, 1.4%p in 2014, and 1.0%p in 2015. The mean relative difference was 1.9%p.
The mean absolute difference of hypertension diagnosis rate over six years was 0.8%p, with 0.8%p in 2010, 0.3%p in 2011, 0.3%p in 2012, 1.1%p in 2013, 2.1%p in 2014, and 0.0%p in 2015. The mean relative difference was 4.6%p.
The mean absolute difference of diabetes diagnosis rate over six years was 0.6%p, with 0.1%p in 2010, 0.2%p in 2011, 0.6%p in 2012, 0.8%p in 2013, 1.0%p in 2014, and 0.7%p in 2015. The mean relative difference was 9.0%p.
The mean absolute difference between obesity prevalence by self-reported height and weight in KCHS and by actually measured height and weight in KNHANES over six years was 8.3%p, with 8.9%p in 2010, 8.7%p in 2011, 8.8%p in 2012, 8.3%p in 2013, 6.6%p in 2014, and 8.2%p in 2015. The mean relative difference was 25.6%p.
The mean absolute difference between hypertension diagnosis rate by interview in KCHS and prevalence of hypertension by physical examination in KNHANES over six years was 8.4%p, with 12.9%p in 2010, 9.1%p in 2011, 8.8%p in 2012, 7.3%p in 2013, 5.1%p in 2014, and 7.4%p in 2015. The mean relative difference was 32.0%p.
The mean absolute difference between diabetes diagnosis rate by interview in KCHS and prevalence of diabetes by physical examination in KNHANES over six years was 1.9%p, with 2.1%p in 2010, 2.2%p in 2011, 1.5%p in 2012, 3.0%p in 2013, 1.5%p in 2014, and 1.1%p in 2015. The mean relative difference was 21.6%p.
Comparison of time series stability of subgroup estimates between KCHS and KNHANES
----------------------------------------------------------------------------------
[Figure 2](#f2-epih-41-e2019012){ref-type="fig"} shows the graph of time series trends by city as well as sex and age for current smoking rate, hypertension diagnosis rate and prevalence of obesity from 2010 to 2015. When divided by city, changes in current smoking rate estimates in KCHS were smaller than those in KNHANES in nearly all regions. However, when divided by sex and age, changes in estimates were similar between the two surveys. The trends for hypertension diagnosis rate were nearly identical to that of current smoking rate for both city as well as sex and age graphs. Regarding prevalence of obesity, KCHS computes the prevalence using self-reported height and weight while the KNHANES computes the prevalence using actually measured height and weight. For this reason, the difference of estimates is larger than that for current smoking rate or hypertension diagnosis rate. Similar to other variables, time series trends varied greatly for KNHANES in the city graph, but the variability was smaller in the sex and age graph.
[Tables 3](#t3-epih-41-e2019012){ref-type="table"} shows the MSE values for each variable by city as well as by sex and age. The MSE values for city subgroups were 0.5, 0.3, and 0.4 in KCHS and 22.6, 13.0, and 16.4 in KNHANES, showing a higher time series stability in KCHS. The MSE values for sex and age subgroups were 0.6, 0.4, and 0.3 in KCHS and 7.0, 13.6, and 9.2 in KNHANES, also showing a higher time series stability in KCHS.
DISCUSSION
==========
This study compared the estimates for six variables measured based on a questionnaire in KCHS and eight variables measured based on a questionnaire or physical examination in KNHANES from 2010 to 2015 and divided them by city as well as sex and age to compare time series stability. With the exception of self-rated health, all estimates measured based on questionnaires, namely current smoking rate, monthly drinking rate, hypertension diagnosis rate and diabetes diagnosis rate, showed an absolute difference of less than 1.0%p and relative difference ranging from 1.99.0%p. For prevalence of obesity, hypertension diagnosis rate and prevalence of hypertension, diabetes diagnosis rate and prevalence of diabetes using questionnaire data in KCHS and physical examinations data in KNHANES, the absolute difference ranged from 1.9-8.4%p and relative difference ranged from 21.6-32.0%p, showing greater differences in the estimates compared to those measured based on questionnaire data. Time series stability by subgroup was higher for KCHS than KNHANES, and in KNHANES, time series stability for sex and age subgroups was greater than that for regional subgroups.
In both surveys, the difference of estimates for variables measured using questionnaire data was small, but there was a large difference of the estimates in self-rated health. Although both KCHS and KNHANES showed a declining trend in self-rated health, the difference between the two surveys was relatively large despite the fact that both surveys used the same question "how would you rate your health?" and both surveys collected this data using a questionnaire. Differences also varied across variables in the study conducted by Fahimi et al. \[[@b3-epih-41-e2019012]\] comparing BRFSS and other national surveys, where there were relatively small differences of current smoking rate and influenza vaccination rate in the past year in various subgroups but larger differences in prevalence of asthma and self-rated health. Particularly, the absolute difference of the percentage of "fair" or "poor" responses regarding self-rated health between surveys was greater (4.2%; relative difference 33.9%) than that for other variables \[[@b3-epih-41-e2019012]\]. Although the absolute difference of self-rated health between surveys in the present study (10.8%) was greater than that found by Fahimi et al. \[[@b3-epih-41-e2019012]\], the relative difference was similar at about 33.0%. Salomon et al. \[[@b7-epih-41-e2019012]\] compared self-rated health data among various national surveys from 1971 to 2007. In the said study, the estimates of self-rated health were compared among four surveys by dividing the participants by sex, age, race, and education. In general, the estimates differed greatly and showed inconsistent trends across surveys. In contrast, variables such as diabetes and BMI differed less and showed consistent time series trends across surveys. Based on these results, Salomon et al. \[[@b7-epih-41-e2019012]\] suggested that it is difficult to provide a simple explanation of the differences in the estimates of selfrated health across surveys and that self-rated health is not an appropriate variable for monitoring the health of different groups over time. In a study comparing the estimates among three national surveys by Li et al. \[[@b15-epih-41-e2019012]\], the absolute difference of current smoking rate, prevalence of obesity, prevalence of hypertension, and no health insurance rate ranged from 0.7-3.9%p. Particularly, the absolute difference of self-rated health ranged from 0.4-3.1%p, which suggests similar estimates across surveys, but the trends were inconsistent. As shown here, multiple studies report that self-rated health estimates differ greatly across surveys.
The difference of prevalence between data measured using questionnaires and physical examinations was greater than that between data measured using questionnaires in other studies as well. In a study that compared the prevalence of obesity between that measured using self-reported height and weight in KCHS and that computed using actually measured height and weight in KNHANES in 2010, the prevalence of obesity differed by 8.6%p and prevalence of overweight differed by 7.8%p. This was because overestimation of height increased with age while weight was underestimated in males in their 20s and 30s and females in their 20s to 40s in KCHS \[[@b16-epih-41-e2019012]\]. In a study investigating the effects of prevalence of obesity on diabetes according to method of survey in adults aged 45 years or older, the difference of obesity prevalence by questionnaire in KCHS and that by physical examination in KNHANES was 6.4%p and the difference of diabetes prevalence was 2.6%p \[[@b17-epih-41-e2019012]\]. Other study also reported that self-reported data and actually measured data differ particularly according to sex \[[@b18-epih-41-e2019012]\].
Time series trend by subgroup was more stable in KCHS than in KNHANES. This may be attributable to the fact that KCHS has about 40 times more subjects than KNHANES. Variability of estimates in the regional subgroups was high in KNHANES, particularly in regions with a small number of subjects (≤200). There was a tendency of higher variability of estimates in the 79-88 years group for males and females compared to other age groups in KNHANES, but this age group had fewer subjects (≤150) than other age groups. This suggests that time series stability of regional estimates is not ensured in KNHANES, so extra precaution should be taken when using regional analyses as evidence of policies or research.
Nevertheless, this study has a few limitations. We were able to compare the estimates of only a few items, so the findings cannot represent the overall differences of estimates between the two surveys. Furthermore, although both KCHS and KNHANES use complex samples, they differ in the number of subjects and sampling method. KCHS surveys about 220,000 people every year with 900 individuals in each of the 251 public health centers in 16 cities nationwide, which enables even distribution of samples throughout all regions in Korea \[[@b19-epih-41-e2019012]\]. In contrast, KNHANES has a relatively smaller sample of 10,000 people in 3,840 households in 192 districts every year using sex, age, living space, and education of head of household as implicit stratification standards, and it aims to compute yearly national statistics, which may cause uneven distribution of subjects across regions. Such differences of features limits direct comparison of the two surveys. Furthermore, KNHANES is an annual survey, whereas KCHS conducts a survey for three months from August to October, so there is a chance that the estimates may differ due to the difference in survey periods. In addition, KCHS collects data through in-person interviews, while KNHANES collects data either through interviews or selfreported questionnaire depending on the survey item. Quality management of interviewers also differs between the two surveys. For KCHS, interviewers are selected for each region in June, and they undergo short-term training \[[@b20-epih-41-e2019012]\]. For KNHANES on the other hand, eight specialists comprise a team for interview survey and physical examinations survey, two of whom take charge of health interview, and a total of four professional survey teams travel around the country for the survey \[[@b21-epih-41-e2019012]\]. As shown here, the two surveys differ in several aspects, and estimates can differ not only due to the differences in the number of subjects, sampling method, and data collection method but also due to subtle differences, such as those in the nature of health parameters, phrasing of questions, and order of questions \[[@b22-epih-41-e2019012],[@b23-epih-41-e2019012]\]. Therefore, such minor differences should be meticulously reviewed when comparing different surveys \[[@b15-epih-41-e2019012]\]. When using KCHS and KNHANES data, the differences between the two surveys should be noted and either data should be selected or both should be used in supplementation depending on the purpose of analysis or policy.
Despite these limitations, this study clearly has strengths as well. Compared to similar Korean studies \[[@b16-epih-41-e2019012],[@b17-epih-41-e2019012]\], we performed a more comprehensive comparison using six and eight variables in KCHS and KNHANES, respectively, over six years, and also analyzed the differences of estimates according to method of survey by comparing estimates taken from interviews and estimates taken from physical examinations data. Furthermore, we analyzed the time series stability of subgroup estimates and demonstrated that subgroup estimates might differ between the two surveys even with little differences in yearly estimates overall.
This study proposed the similarities and disparities between two major health surveys in Korea that are utilized in policies and research, and our findings would contribute to preventing errors that may occur by using only one set of data as the basis of policies or research. Recent inter-survey comparison studies have expanded the scope of comparison to surveys across countries \[[@b24-epih-41-e2019012]\]. In the future, studies should compare Korean surveys with foreign surveys to lay a foundation to compare and share international policies.
The authors have no conflicts of interest to declare for this study.
SUPPLEMENTARY MATERIALS
=======================
Korean version is available at <http://www.e-epih.org/>).
{#f1-epih-41-e2019012}
{#f2-epih-41-e2019012}
######
Participants aged 19 years or older in the 2010-2015 Korea Community Health Survey (KCHS) and Korea National Health and Nutrition Examination Survey (KNHANES)
Year KCHS KNHANES Ratio (KCHS/ KNHANES)
------ --------- ------------ ----------------------- ------------ ------
2010 229,126 38,999,317 6,254 38,226,440 1.02
2011 229,186 39,904,832 6,027 39,050,481 1.02
2012 228,899 40,363,395 5,611 39,674,018 1.02
2013 228,764 40,781,906 5,362 40,164,541 1.02
2014 228,695 41,143,286 5,040 40,767,231 1.01
2015 228,558 41,554,658 5,571 41,176,539 1.01
######
Range of difference, mean of difference and relative difference between the estimates of Korea Community Health Survey (KCHS) and Korea National Health and Nutrition Examination Survey (KNHANES), 2010-2015
Variables Range of difference Mean difference^[1](#tfn1-epih-41-e2019012){ref-type="table-fn"}^ 95% confidence interval Relative difference^[2](#tfn2-epih-41-e2019012){ref-type="table-fn"}^
------------------------------------------------------------------------------------------------------------------------------ --------------------- ------------------------------------------------------------------- ------------------------- -----------------------------------------------------------------------
Self-rated health 9.2-1.4 10.8 9.1, 12.5 33.0
Current smoking rate 0.1-2.3 1.2 -0.3, 2.8 4.9
Monthly drinking rate 0.4-2.2 1.1 -0.6, 3.0 1.9
Hypertension diagnosis rate 0.0-2.1 0.8 -0.6, 2.2 4.6
Diabetes diagnosis rate 0.1-1.0 0.6 -0.2, 1.3 9.0
Obesity prevalence (self-reported height & weight, KCHS) vs. obesity prevalence (actually measured height & weight, KNHANES) 6.6-8.9 8.3 6.7, 9.9 25.6
Hypertension diagnosis rate (interview, KCHS) vs. prevalence of hypertension (physical exam, KNHANES) 5.1-12.9 8.4 6.7, 10.2 32.0
Diabetes diagnosis rate (interview, KCHS) vs. prevalence of diabetes (physical exam, KNHANES) 1.1-3.0 1.9 0.9, 2.9 21.6
Values are presented as %.
Mean difference (absolute difference) is the average estimate of the differences for each year.
Relative difference = mean difference (%, absolute difference) divided by average estimate(%) of 6-year of KNHANES.
######
Mean square error by city, sex and age in Korea Community Health Survey (KCHS) and Korea National Health and Nutrition Examination Survey (KNHANES), 2010-2015
Variables KCHS KNHANES
------------ ------ --------- ----- ------ ------ ------
City
Busan 0.2 0.0 0.1 4.4 4.0 13.7
Chungbuk 1.0 0.1 0.9 25.8 34.4 15.8
Chungnam 0.4 0.6 0.3 39.2 15.1 40.6
Daegu 0.7 0.3 0.1 1.0 6.3 17.6
Daejeon 0.9 0.2 0.5 38.7 6.1 16.5
Gangwon 0.2 0.2 0.3 46.7 35.5 32.2
Gwangju 1.6 0.5 0.6 18.6 8.8 12.5
Gyeongbuk 0.1 0.2 0.1 20.3 13.4 10.6
Gyeonggi 0.2 0.1 0.1 2.4 0.2 0.6
Gyeongnam 0.6 0.1 0.3 11.3 1.9 8.5
Incheon 0.4 0.1 0.1 20.3 2.1 7.2
Jeju 1.0 1.0 1.6 46.7 3.7 27.1
Jeonbuk 0.4 0.2 0.5 2.8 19.5 17.0
Jeonnam 0.2 0.0 0.1 20.0 21.4 25.3
Seoul 0.1 0.1 0.1 1.8 3.1 1.2
Ulsan 0.1 0.5 0.8 61.1 33.0 15.6
Mean 0.5 0.3 0.4 22.6 13.0 16.4
Sex
Female
19-28 0.0 0.0 0.1 5.9 0.0 3.8
29-38 0.0 0.0 0.1 2.1 0.3 4.4
39-48 0.0 0.1 0.2 1.0 2.4 13.5
49-58 0.1 0.1 0.1 3.1 5.8 5.8
59-68 0.0 0.1 0.1 1.1 9.7 11.1
69-78 0.0 0.5 0.1 0.4 8.7 3.1
79-88 0.1 2.1 1.2 3.7 30.8 17.9
Male
19-28 0.3 0.3 0.3 9.4 9.4 1.8
29-38 1.8 0.1 0.1 8.1 1.8 5.8
39-48 2.0 0.2 0.3 8.8 4.4 6.6
49-58 0.9 0.1 0.3 0.9 19.6 12.1
59-68 0.9 0.5 0.6 10.7 2.5 10.8
69-78 0.8 1.1 0.7 7.5 18.9 16.1
79-88 0.8 1.0 0.8 41.7 89.7 25.6
Mean 0.6 0.4 0.3 7.0 13.6 9.2
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#h0.0}
============
The ubiquitous predation of bacteria by bacteriophages (phages) has resulted in the evolution of numerous bacterial mechanisms that protect against phage attack ([@B1]). One of the most widespread is the CRISPR-Cas (CRISPR stands for clustered regularly interspaced short palindromic repeat) system. This system utilizes small RNA molecules that act as sequence-specific guides for nuclease activity ([@B2]). Different types (i.e., I, II, and III) and subtypes (i.e., I-A, I-B, etc.) of CRISPR-Cas systems exist across bacterial and archaeal species. Most of these systems, including all type I systems described here, target DNA, while some type III systems target RNA ([@B11]). The sophisticated functional mechanisms of CRISPR-Cas systems combined with their ability to gain immunity to newly encountered phages has led to intensive study of these systems in recent years.
CRISPR loci consist of multiple semipalindromic DNA repeats of 21 to 48 nucleotides, interspersed with variable "spacer" sequences of similar length. The spacers comprise sequences that are complementary to mobile genetic elements, including phages and plasmids ([@B3]). In type I CRISPR-Cas systems, the CRISPR locus is transcribed as a precursor RNA molecule that is processed into single repeat-spacer units (mature CRISPR RNA \[crRNA\]) ([@B4], [@B5]). The mature crRNA is then bound by a complex of CRISPR-associated (Cas) proteins ([@B2], [@B6]). The CRISPR-Cas complex is able to recognize DNA sequences that are complementary to the crRNA and direct the destruction of the foreign DNA ([@B7], [@B8]).
Since CRISPR-Cas systems provide a potent defense mechanism against phage infection, one might expect phages to possess means of inhibiting these systems. We recently identified five different anti-CRISPR proteins that inhibit the type I-F CRISPR-Cas system of *Pseudomonas aeruginosa*. The genes encoding these proteins are located at the same genomic position within a group of closely related *Pseudomonas* phages ([Fig. 1](#fig1){ref-type="fig"}); they appear to be contained within a unique operon inserted between two highly conserved head morphogenetic genes. Intriguingly, greater than half of the genes within these putative anti-CRISPR operons did not mediate anti-CRISPR activity against the type I-F CRISPR-Cas system reported in our previous work ([@B9]). In the present study, we demonstrate that many of these previously uncharacterized genes mediate anti-CRISPR activity directed against a different subtype of CRISPR-Cas system, the type I-E system of *P. aeruginosa*. This work reveals a second group of anti-CRISPR proteins comprising four distinct families and provides the first *in vivo* characterization of a naturally active type I-E CRISPR-Cas system.
{#fig1}
RESULTS {#h1}
=======
Two *P. aeruginosa* strains possess active type I-E CRISPR-Cas systems. {#h1.1}
-----------------------------------------------------------------------
To determine whether phage genes could mediate anti-CRISPR activity against the type I-E system of *P. aeruginosa*, it was imperative to identify strains in which this type of CRISPR-Cas system was active. A previous study identified several strains of *P. aeruginosa* that likely encode intact type I-E CRISPR-Cas systems; however, the activity of these systems was not tested ([@B10]). To determine whether these *P. aeruginosa* strains contain active type I-E systems, we used a plasmid transformation efficiency assay described in our previous work ([@B9]). In this assay, the transformation efficiency of a plasmid containing a "protospacer" sequence complementary to a CRISPR spacer within a given strain is compared to an empty-vector control. We synthesized and cloned three protospacer sequences, protospacer sequence 1 (PS1), PS2, and PS3, that were complementary to CRISPR spacers found within five type I-E-containing strains ([@B10]) ([Fig. 2A](#fig2){ref-type="fig"}; see [Fig. S1](#figS1){ref-type="supplementary-material"} in the supplemental material). We flanked each of these protospacer sequences with a protospacer adjacent motif (PAM), which is required for targeting ([@B11], [@B12]). Since PAM sequences for the *P. aeruginosa* type I-E system had not been experimentally determined, we used the PAM sequence 5′-CAT-3′, an experimentally validated PAM for the *Escherichia coli* type I-E system ([@B7]), or the sequence 5′-CTT-3′, a consensus PAM based on the alignment of putative type I-E protospacers found in *P. aeruginosa* phage genomes ([@B12]) ([Fig. 2B](#fig2){ref-type="fig"}).
{#fig2}
By testing strains using the plasmid transformation assay, we ultimately identified two strains, *P. aeruginosa* SMC4386 and SMC4389, that displayed 100-fold to 2,000-fold reductions in transformation efficiency of protospacer-containing plasmids ([Fig. 2C](#fig2){ref-type="fig"}). These results implied that these strains possess active type I-E systems. The plasmids that contained the PS1 and PS2 protospacers flanked by the PAM sequence 5′-CTT-3′ transformed both strains poorly, which is expected because both strains possess corresponding CRISPR spacers. Since only strain SMC4386 possessed a CRISPR spacer complementary to PS3, the plasmid bearing the PS3 sequence displayed reduced transformation efficiency only in this strain. We sequenced the type I-E CRISPR-associated (*cas*) genes of strain SMC4386 and found that all of the type I-E *cas* genes were present and that the Cas protein sequences were greater than 93% identical to the homologous proteins encoded in the genome of PA2192, a fully sequenced *P. aeruginosa* strain that contains a complete type I-E system ([@B10]).
Interestingly, both strain SMC4386 and strain SMC4389 targeted plasmids containing the PAM sequence 5′-CTT-3′, but not 5′-CAT-3′ ([Fig. 2C](#fig2){ref-type="fig"}), which is the most favored PAM sequence for the *E. coli* type I-E system. Therefore, the *P. aeruginosa* and *E. coli* type I-E systems clearly possess different PAM specificities ([@B7], [@B13]). To further define this specificity, we determined the transformation efficiencies of plasmids containing the PS2 protospacer flanked by a panel of different 3-nucleotide PAM sequences ([Fig. 2D](#fig2){ref-type="fig"}). Although 5′-CTT-3′ elicited the strongest inhibition of transformation, any PS2 construct with a PAM sequence of 5′-NTT-3′ resulted in at least a 100-fold decrease in transformation efficiency, while 5′-CCT-3′ exhibited a 10-fold reduction. Plasmids bearing other PAM sequences retained transformation efficiencies equal to the empty-vector control.
Some anti-CRISPR phages possess genes that inactivate the type I-E system. {#h1.2}
--------------------------------------------------------------------------
In our previous work, we described eight phages bearing genes that inhibit the *P. aeruginosa* type I-F CRISPR-Cas system ([Fig. 1](#fig1){ref-type="fig"}). These phages are temperate, so lysogenic strains can be isolated in which the phage genome is integrated into the host genome in the form of a prophage. The anti-CRISPR genes are transcribed from the prophage, thereby inactivating the CRISPR-Cas systems in lysogenic strains containing these phage genomes ([@B9]). To determine whether these anti-CRISPR phages encode genes able to inhibit the type I-E system, we created five *P. aeruginosa* SMC4386 lysogens that each contained a different anti-CRISPR prophage. We tested these lysogens using the transformation efficiency assay with the PS2 protospacer coupled with the 5′-CTT-3′ PAM. In contrast to SMC4386 lacking a prophage, the lysogens of phages JBD5, JBD24, DMS3, and JBD26 could be transformed with \~100% efficiency compared to the empty vector. The MP29 lysogen was transformed inefficiently at a level similar to that of the unlysogenized strain ([Fig. 3A](#fig3){ref-type="fig"}). These data indicate that each of these phages except MP29 possesses genes that inhibit the type I-E CRISPR-Cas system.
{#fig3}
Each of these phage genomes possesses a gene at the 3′ end of the putative anti-CRISPR operon encoding a protein with greater than 94% identity to JBD30-*36* ([Fig. 1](#fig1){ref-type="fig"}). We previously determined that these genes do not confer anti-CRISPR activity against the I-F system ([@B9]). All five of the lysogenic strains of SMC4386 tested here encode a JBD30-*36* homolog irrespective of their ability to inhibit the type I-E system. These findings suggest that the JBD30-*36* protein does not mediate anti-type I-E activity. Phage MP29, which did not mediate anti-type I-E activity, encodes only a JBD30-*36* homolog and a type I-F anti-CRISPR protein in this region. However, the four phages exhibiting type I-E anti-CRISPR activity (JBD5, JBD24, DMS3, and JBD26) all possess genes in their anti-CRISPR regions encoding proteins of unassigned function in addition to the JBD30-*36* homolog and any type I-F anti-CRISPR genes ([Fig. 1](#fig1){ref-type="fig"}). We postulated that these additional genes were the source of the observed type I-E anti-CRISPR activity.
Plasmid-based expression of four distinct genes found in anti-CRISPR phages mediates inhibition of the *P. aeruginosa* type I-E system. {#h1.3}
---------------------------------------------------------------------------------------------------------------------------------------
To aid in identifying the genes responsible for the anti-CRISPR effect observed in lysogenic strains, we searched for a phage that was inhibited by the type I-E system of *P. aeruginosa* SMC4386. We examined the genome sequences of *P. aeruginosa* phages in our collection to identify putative protospacers. Phage JBD8 has a protospacer sequence identical to a spacer in one of the type I-E CRISPR loci of strain SMC4386 flanked by a CTT PAM sequence. We tested the ability of JBD8 to form plaques on wild-type SMC4386 and the lysogenic strains of SMC4386 described above with an inactivated type I-E system ([Fig. 3A](#fig3){ref-type="fig"}). Phage JBD8 was not able to form plaques on wild-type SMC4386, but it was able to form plaques robustly when this strain was lysogenized by anti-CRISPR phage JBD5 ([Fig. 3B](#fig3){ref-type="fig"}). These data indicate that phage JBD8 is targeted by the type I-E system, and the ability of this phage to form plaques was restored in the presence of the JBD5 anti-CRISPR activity.
Once a phage that was targeted by the type I-E CRISPR-Cas system of strain SMC4386 was isolated, we were able to test directly the type I-E anti-CRISPR activity of individual genes from phage anti-CRISPR regions by expressing them from plasmids. Assays were performed utilizing plasmids expressing representatives of each unique family of genes of unassigned function within the phage anti-CRISPR regions ([Fig. 1](#fig1){ref-type="fig"}). As shown in [Fig. 3C](#fig3){ref-type="fig"}, expression of putative anti-CRISPR genes from phages JBD88a, DMS3, D3112, and JBD5 allowed CRISPR-sensitive phage JBD8 to form plaques with 10^3^- to 10^5^-fold-higher efficiency than on cells containing empty vector. In contrast, plasmids expressing any one of five different type I-F anti-CRISPR genes, or gene JBD30-*36* (homologs of which are found at the 3′ ends of all anti-CRISPR regions) had no effect on JBD8 plaquing. These results demonstrate that four distinct families of genes mediate anti-CRISPR activity against the type I-E CRISPR-Cas system of *P. aeruginosa*. We designate the proteins encoded by these anti-CRISPR genes as ACR88a-32, ACR3-30, ACR3112-31, and ACR5-34
Although the type I-E anti-CRISPR genes did not inhibit the *P. aeruginosa* type I-F system as assayed in our previous study ([@B9]), we tested whether they might inhibit the distantly related type I-E CRISPR system of *E. coli* (see [Fig. S3A](#figS3){ref-type="supplementary-material"} in the supplemental material). For this purpose, we used an inducible type I-E CRISPR-Cas system in *E. coli* strain K-12, engineered to target phage M13 ([@B14]). The system consists of a targeting strain expressing CRISPR-Cas machinery and crRNA targeting M13 and an isogenic nontargeting strain that lacks the locus encoding crRNA. We expressed each of the type I-E anti-CRISPR proteins from a plasmid in these strains and examined the ability of phage M13 to form plaques. The targeting strain reduced the efficiency of plating of M13 by \~10^5^-fold compared to the efficiency of plating of the nontargeting strain; we observed no change in the efficiency of plating upon expression of any anti-CRISPR gene ([Fig. S3B](#figS3){ref-type="supplementary-material"}). This result was not due to lack of protein expression in *E. coli*, since we examined induced lysates using SDS-PAGE followed by Coomassie staining and confirmed that all four anti-CRISPR proteins were robustly expressed ([Fig. S3C](#figS3){ref-type="supplementary-material"}). These data show that the anti-CRISPR proteins encoded by *P. aeruginosa* phages are not active against the type I-E system in *E. coli*.
Type I-E anti-CRISPRs function during the infection process to protect phage from CRISPR-Cas-mediated inhibition. {#h1.4}
-----------------------------------------------------------------------------------------------------------------
We used derivatives of phage DMS3m ([@B9]) to assess the requirement of a type I-E anti-CRISPR gene for lytic phage replication. Phage DMS3m possesses a protospacer matching *P. aeruginosa* SMC4386 CRISPR 1 spacer 3 that is flanked by the PAM CTT; thus, it should be targeted by the type I-E CRISPR-Cas system of strain SMC4386. However, phage DMS3m also has an active type I-E anti-CRISPR gene (gene *30*), encoding ACR3-30 ([Fig. 3C](#fig3){ref-type="fig"}), that is expected to protect it from inhibition by the CRISPR-Cas system. As shown in [Fig. 4](#fig4){ref-type="fig"}, wild-type DMS3m is able to form plaques robustly on strain SMC4386. In contrast, a mutant phage in which the DMS3m type I-E anti-CRISPR gene was replaced with a type I-F anti-CRISPR gene (DMS3m^30-*35*^ \[[@B9]\]) was not able to form plaques on SMC4386. A version of DMS3m containing a frameshifted version of the type I-F anti-CRISPR gene (DMS3m^30-35fs^) was also unable to form plaques on SMC4386. All three phages infected an *P. aeruginosa* strain lacking CRISPR-Cas activity with equal efficiency ([Fig. 4](#fig4){ref-type="fig"}). These results demonstrate that gene *30*, the type I-E anti-CRISPR gene of phage DMS3m, functions during lytic infection to inhibit the type I-E CRISPR-Cas system of SMC4386 and, thus, allow robust phage replication within this strain.
{#fig4}
Anti-CRISPR genes are widespread in Mu-like *P. aeruginosa* phages. {#h1.5}
-------------------------------------------------------------------
We detected both type I-E and I-F anti-CRISPR genes at a single defined genomic location in a number of closely related Mu-like *P. aeruginosa* phages. To extend these findings, we assessed the prevalence and diversity of anti-CRISPR genes in a larger collection of related phages. By sequencing the regions of these phages where anti-CRISPR genes were expected to be located, we identified 15 additional phages that possess an anti-CRISPR region at the same genomic position as the characterized anti-CRISPR phages ([Fig. 1](#fig1){ref-type="fig"}). Of these newly identified phages, nine had type I-E and I-F anti-CRISPR genes and six had only type I-F anti-CRISPR genes. Combining these data with our previous anti-CRISPR phage analyses, we have identified a total of 15 phages with both type I-E and I-F anti-CRISPRs, eight with type I-F anti-CRISPR genes, and one phage with only a type I-E anti-CRISPR gene ([Fig. 5A and B](#fig5){ref-type="fig"}). We constructed lysogenic strains using many of these phages in *P. aeruginosa* strain SMC4386 (to assay for type I-E anti-CRISPR activity) or strain PA14 (to assay for type I-F anti-CRISPR activity). Utilizing the plasmid transformation efficiency assay to test lysogens of the anti-CRISPR phages, we were able to detect *in vivo* type I-E anti-CRISPR activity for eight phages ([Fig. 3A, 5A, and C](#fig3 fig5){ref-type="fig"}), and type I-F anti-CRISPR activity for a total of 22 phages ([Fig. 5A](#fig5){ref-type="fig"}) ([@B9]). It should be noted that this was not an exhaustive study, so the relative proportions of the anti-CRISPR activities against each subtype may not be indicative of how widespread each is in nature.
![Anti-CRISPRs are widespread in *P. aeruginosa* Mu-like phages. (A) Transformation efficiency assays using PS2 with the CTT PAM construct are shown for a variety of *P. aeruginosa* SMC4386 lysogens. The anti-CRISPR gene regions of these phages have been sequenced, and their sequence groups are indicated. Prophages tested for type I-E anti-CRISPR activity as prophages in *P. aeruginosa* SMC4386 are indicated by an ampersand, and prophages tested for type I-F anti-CRISPR activity as prophages in *P. aeruginosa* PA14 are indicated by an asterisk. (B) The anti-CRISPR region sequence groups are defined. The boxes are drawn in proportion to the scale bar shown. Colored boxes indicate high sequence identity to one of the four type I-E anti-CRISPR genes (indicated by a white E on a black circle) or one of the five type I-F anti-CRISPR genes (indicated by a black F on a white circle). All anti-CRISPR-encoding phages have a conserved gene shown in blue, which does not have anti-CRISPR activity against the type I-E or I-F systems ([@B3]). (C) The phages belonging to each of the anti-CRISPR region sequence groups defined in panel B are listed.](mbo0021418030005){#fig5}
Although the experiments described above identified 15 new phages bearing anti-CRISPR genes, no novel anti-CRISPR gene families were discovered. Each newly sequenced anti-CRISPR region possessed closely related homologs (greater than 90% protein sequence identity) of one or more of the type I-F and type I-E anti-CRISPR proteins previously identified ([Fig. 5B](#fig5){ref-type="fig"}). Remarkably, homologs of the nine different families of type I-E and I-F anti-CRISPR genes appeared in nine different combinations in various phages ([Fig. 5A and B](#fig5){ref-type="fig"}), indicating that these genes have been "mixed and matched" during the evolution of this family of phages. All of the phages possessed a gene homologous to JBD30-*36* downstream of their anti-CRISPR genes, and all possessed a very similar DNA sequence upstream of their anti-CRISPR genes that appears to be a promoter, as judged by the presence of regions matching −10 and −35 transcription initiation sequences.
Active anti-CRISPR genes are found in *P. aeruginosa* mobile elements. {#h1.6}
----------------------------------------------------------------------
Searches for homologs of the proteins encoded by the type I-E anti-CRISPR genes mostly yielded closely related sequences encoded in phages or prophages similar to the other anti-CRISPR phages. However, homologs of type I-E anti-CRISPR ACR5-34 were found in several *P. aeruginosa* strains in nonprophage regions. Bioinformatic analysis of proteins encoded in the vicinity of these putative bacterial anti-CRISPR genes indicated that these regions encompass mobile DNA elements (i.e., several genes encode homologs of proteins involved in DNA transfer and conjugation \[see [Fig. S4A](#figS4){ref-type="supplementary-material"} in the supplemental material\]). Interestingly, one of these regions encodes homologs of both ACR5-34 and ACR5-35, a type I-F anti-CRISPR. These findings coupled with our previous discovery of type I-F anti-CRISPR genes in other *P. aeruginosa* mobile elements ([@B9]) suggests that anti-CRISPR genes may often protect other mobile DNA elements besides phages from destruction by CRISPR-Cas systems. To confirm that non-phage-encoded anti-CRISPR genes are functional, we demonstrated that two homologs of phage-encoded type I-F anti-CRISPRs (ACR30-35 and ACR5-35) and one homolog of a type I-E anti-CRISPR (ACR5-34) possess anti-CRISPR activity using a standard phage plating assay ([Fig. S4C](#figS4){ref-type="supplementary-material"} and [S4D](#figS4){ref-type="supplementary-material"}).
DISCUSSION {#h2}
==========
Phages and their bacterial hosts exert profound influences on the environment, the human microbiome, and the spread of disease. Thus, understanding the systems that affect the "arms race" between bacteria and phages is of crucial importance ([@B15]). Since CRISPR-Cas systems are key players in the phage-bacterium battle, inhibitors of these systems, if widespread, could have a large impact on bacterial and phage populations. Our recent discovery of type I-F anti-CRISPR genes in *P. aeruginosa* phages ([@B9]) raised new questions, such as whether anti-CRISPR genes are prevalent in many genomes and whether anti-CRISPR genes may inhibit other types of CRISPR-Cas systems. In this paper, we describe the discovery of an additional group of four distinct phage-borne anti-CRISPR genes that inhibit the type I-E CRISPR-Cas system of *P. aeruginosa*. As was observed for the type I-F anti-CRISPRs, these genes encode unique proteins with homologs in other closely related *P. aeruginosa* phages and in *Pseudomonas* mobile genomic elements.
The type I-E anti-CRISPR genes were almost always found adjacent to type I-F anti-CRISPR genes in closely related phages. These anti-CRISPR gene clusters all contain a conserved putative promoter region at their 5′ end and a conserved gene at their 3′ end. The latter gene encodes a protein that does not possess anti-CRISPR activity ([Fig. 3C)](#fig3){ref-type="fig"} ([@B9]) and contains a helix-turn-helix DNA-binding domain (Pfam accession no. [PF13412](http://pfam.sanger.ac.uk/family/PF13412)). This protein may be a regulator of this putative anti-CRISPR operon. A surprising aspect of the anti-CRISPR gene clusters is that we have identified nine distinct arrangements of various type I-E and type I-F anti-CRISPR genes, suggesting a process by which these genes have reassorted multiple times through horizontal gene transfer. Since the anti-CRISPR proteins lack sequence similarity to any other protein families, it is difficult to trace their evolutionary origin. However, the occurrence of these genes in a variety of arrangements in both *P. aeruginosa* phages and mobile DNA elements implies that they provide a meaningful fitness advantage.
Another important contribution of this work is identifying the first examples of bacterial strains with naturally active type I-E CRISPR-Cas systems. Although the type I-E system of *E. coli* has been intensively studied ([@B6], [@B13], [@B16][@B17][@B19]), it is not active in wild-type *E. coli* strains. The type I-E CRISPR-Cas systems of all *E. coli* strains investigated are transcriptionally repressed and do not encode crRNAs with complementarity to characterized phages or plasmids. For these and other reasons, it has been estimated that the *E. coli* type I-E system likely has not been functional for greater than 250,000 years ([@B20], [@B21]). The *P. aeruginosa* type I-E system is highly divergent from the *E. coli* system (see [Fig. S3A](#figS3){ref-type="supplementary-material"} in the supplemental material). One result of this divergence is that the PAM specificity of the two systems is distinct; the PAM sequence 5′-CAT-3′ of the *E. coli* system is not recognized by the *P. aeruginosa* system, and the *E. coli* 5′-CCT-3′ and 5′-CTC-3′ PAM sequences ([@B7], [@B13]) are only weakly targeted by the *P. aeruginosa* system. Our characterization of the distinctive *P. aeruginosa* type I-E system will open new avenues for comparative structural and functional studies. In addition, directed engineering or experimental evolution may allow the development of anti-CRISPRs to inhibit the *E. coli* system using the *P. aeruginosa* anti-CRISPRs as starting material.
The apparent fitness advantage provided by type I-E and I-F anti-CRISPR genes indicates that both of these CRISPR-Cas systems are naturally active in many strains of *P. aeruginosa*. The common occurrence of anti-CRISPR genes in prophages found in a diverse collection of *P. aeruginosa* isolates further suggests that phage-borne anti-CRISPR genes often suppress the activity of these systems in natural settings. Since anti-CRISPR genes protect not only the phage that encodes them but also any incoming foreign DNA that might be naturally targeted by the CRISPR-Cas system, undiscovered anti-CRISPR genes may explain difficulties in demonstrating natural *in vivo* CRISPR-Cas function in various organisms and system subtypes. Our discovery of anti-CRISPR genes in mobile genetic elements other than phages suggests that anti-CRISPR genes may play a significant general role in lateral gene transfer events by allowing incoming foreign DNA to bypass CRISPR-Cas systems.
In summary, we have shown that phage-encoded anti-CRISPRs are an important mechanism by which *P. aeruginosa* phages evade both type I-E and type I-F CRISPR-Cas activities encoded by their bacterial host. Similar phenomena may be at play in a wide variety of organisms and types of CRISPR-Cas systems.
MATERIALS AND METHODS {#h3}
=====================
Bacterial and phage growth. {#h3.1}
---------------------------
All bacterial and phage growth was performed as previously described ([@B9]). Phage lysogens in *P. aeruginosa* strain SMC4386 were created by streaking cells from the center of a phage plaque to single colonies. The presence of a prophage was confirmed by the development of resistance to superinfection by the phage in question and the production of that phage from the lysogenic strain.
Transformation efficiency assay. {#h3.2}
--------------------------------
Oligonucleotides encoding 32-nucleotide CRISPR spacers with five nucleotides upstream and downstream (encompassing the PAM sequence), flanked by NcoI and HindIII sites were synthesized and cloned into pHERD30T ([@B22]). Known concentrations of each of the protospacer-containing plasmids were used to transform *P. aeruginosa* via electroporation. One milliliter of overnight culture was collected by centrifugation, washed twice with 300 mM sucrose, and resuspended in 100 µl of sucrose. Aliquots of cells (100 µl) were transformed with 15 to 50 ng of plasmid DNA, diluted in 1 ml Luria broth, and incubated for 40 min at 37°C to allow for recovery. Serial dilutions of each transformation reaction were plated on LB agar containing gentamicin (50 µg/ml) and incubated overnight at 37°C. The following day, the colonies were counted and normalized according to the dilution factor and amount of plasmid DNA introduced during electroporation. The data were expressed as a percentage of colonies transformed with the empty-vector pHERD30T in the same experiment.
*P. aeruginosa* plaque assays. {#h3.3}
------------------------------
One hundred fifty microliters of overnight culture grown in LB was added to 3 ml LB agar containing 10 mM MgSO~4~, 1.5% agar, and top plated on LB agar plates containing 50 µg/ml gentamicin, 10 mM MgSO~4~, and 0.7% agar. Tenfold serial dilutions of phage lysates were spotted on the surface. The plates were incubated overnight at 30°C, and the numbers of plaques were counted.
*E. coli* M13 plaque assays. {#h3.4}
----------------------------
*E. coli* strains BW40114 and BW40119 contain an isopropyl-β-[d]{.smallcaps}-thiogalactopyranoside (IPTG)- and arabinose-inducible type I-E CRISPR-Cas system, and strain BW40119 contains a crRNA spacer that targets phage M13 ([@B14]). These cells were transformed with pHERD30T plasmids expressing each anti-CRISPR gene. Overnight cultures were diluted 1:100 into LB with 15 µg/ml gentamicin, grown to an optical density at 600 nm (OD~600~) of 0.6, and then induced with both 1 mM arabinose and 1 mM IPTG. After 3-h induction, cells were pelleted by centrifugation, resuspended in 3 ml soft agar, and then poured onto thick LB agar plates containing 1 mM arabinose and 1 mM IPTG. Serial dilutions of M13 phage were spotted on the surface. After incubation overnight at 30°C, plaques visible at the lowest dilution were counted, and the efficiency of plating (EOP) was calculated for each anti-CRISPR construct and for the pHERD30T empty vector in both strains.
Confirmation of anti-CRISPR expression in *E. coli*. {#h3.5}
----------------------------------------------------
*E. coli* strain BW40119 ([@B14]) transformed with various anti-CRISPR constructs in pHERD30T were subcultured 1:100 into LB containing the appropriate antibiotic, grown for 4 h, and then either induced with 3 mM arabinose or inhibited with 0.2% glucose. After 2 h of induction, 1 ml of culture was collected by centrifugation, and cells were resuspended in 100 µl SDS running buffer and analyzed by SDS-PAGE on a 15% Tris-Tricine gel, followed by Coomassie staining.
SUPPLEMENTAL MATERIAL {#h4}
=====================
######
Two of five *P. aeruginosa* isolates display type I-E CRISPR-Cas activity. (A) The type I-E CRISPR loci of the five strains tested are shown. CRISPR repeat units (black diamonds) and CRISPR spacer sequences (white boxes) are indicated. The direction of transcription of CRISPR loci is from left to right. The spacers in each strain targeting the plasmid-borne protospacer constructs PS1, PS2, and PS3 are shown. Note that strain SMC4389 does not encode a spacer match to PS3 and strain SMC4503 does not contain a match to PS1. (B) The protospacer sequences cloned into pHERD30T for the plasmid transformation efficiency assay are shown. The 32-nucleotide protospacers shown here in light blue were flanked by a PAM sequence highlighted in red (here, 5′-CTT-3′, although variant PAM sequences were used as indicated in the text). Download
######
Figure S1, JPG file, 0.1 MB
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Gene and protein sequences of type I-E anti-CRISPRs. Download
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Figure S2, JPG file, 0.2 MB
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The type I-E anti-CRISPRs do not inhibit the type I-E system of *E. coli* strain K-12. (A) The type I-E *cas* genes of *E. coli* strain K-12 and *P. aeruginosa* SMC4386 are compared. Pairwise amino acid sequence identities are indicated. Note that we were unable to obtain the sequence of the SMC4386 *cas3* gene by sequencing PCR products (although its presence was confirmed), so there is no protein identity calculated for Cas3 between SMC4386 and *E. coli* K-12. (B) The efficiency of plating (EOP) of phage M13 on either the nontargeting (BW40114) or targeting (BW40119) strain of *E. coli* ([@B16]) is shown normalized to the EOP of M13 on BW40114 cells containing empty vector. The values shown are averages of at least two replicates. The EOP is reduced 10^4^- to 10^5^-fold in the targeting strain, and no change was observed upon expression of plasmid-encoded type I-E anti-CRISPR genes as indicated. (C) Plasmid-based expression of type I-E anti-CRISPR proteins was confirmed by SDS-PAGE. Uninduced (− lanes; 0.2% glucose) and induced (+ lanes; 3 mM arabinose) samples are shown. Two irrelevant lanes were removed from the gel image between the empty vector and ACR3-*30* lanes. Download
######
Figure S3, JPG file, 0.1 MB
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Type I-E and type I-F anti-CRISPR homologs found in non-phage-related elements are functional. (A) Schematic diagram of part of a putative conjugative element from *P. aeruginosa* PA21_ST175 contig 00001 (GenBank accession no. AOIH01000001), highlighting the genes around a homolog of the type I-E anti-CRISPR, ACR5-34 (GI:453048050), and a homolog of the type I-F anti-CRISPR, ACR5-35 (GI:453048051). ACR5-34 homologs are present in a variety of *P. aeruginosa* genomes in regions closely resembling the element depicted here; however, in all other cases, it is not accompanied by a type I-F anti-CRISPR gene. (B) ClustalW protein sequence alignment of ACR5-34 and its homolog from PA21_ST175 (ACR5-34_PA21). The protein sequence identity is 62%. (C) ACR5-34_PA21 possesses anti-type I-E activity against the CRISPR-Cas system of *P. aeruginosa* strain SMC4386. Tenfold dilutions of phage lysate of either JBD8, a CRISPR-sensitive phage, or JBD93a, a control phage, were spotted on lawns of strain SMC4386 with the empty vector (e.v.) or the ACR5-*34*\_PA21 gene. (D) Tenfold dilutions of CRISPR-sensitive phage DMS3m or control phage DMS3 were spotted on *P. aeruginosa* strain PA14 lawns expressing type I-F anti-CRISPR homologs from *Pseudomonas stutzeri* strain TS44 YO5_18187 (ACR30-35 homolog; 73% protein sequence identity; GenBank accession no. [EIK54721.1](EIK54721.1)) or *P. aeruginosa* genomic island PAGI-5 (ACR5-34 homolog; 43% protein sequence identity; GenBank accession no. [ABR13384.1](ABR13384.1)) in the presence (+) or absence (−) of the arabinose (ara) inducer. These type I-F anti-CRISPR genes are functional. Notably, the ACR5-35 homolog from PAGI-5 tested here is 99% identical to the ACR5-35 homolog encoded in the PA21_ST175 locus depicted in panel A possessing only one single amino acid change. Download
######
Figure S4, JPG file, 0.1 MB
**Citation** Pawluk A, Bondy-Denomy J, Cheung VHW, Maxwell KL, Davidson AR. 2014. A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR-Cas system of *Pseudomonas aeruginosa*. mBio 5(2):e00896-14. doi:10.1128/mBio.00896-14.
We thank G. O'Toole and K. Cady for providing *P. aeruginosa* strains and K. Severinov for providing *E. coli* strains BW40114 and BW40119. We thank D. Bona for technical assistance and P. Sadowski for reading the manuscript.
This work was supported by Canadian Institutes of Health Research operating grants to A.R.D. (MOP-130482) and K.L.M. (MOP-62796) and an Emerging Team Grant to A.R.D. and K.L.M. (XNE86943).
[^1]: **Editor** Roger Hendrix, University of Pittsburgh
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Colorectal cancer incidence and mortality rates vary around the world. Globally, colorectal cancer is the third most commonly diagnosed cancer in men and the second in women \[[@CR1]\]. Surgical treatment of rectal cancer has changed radically in recent years. The introduction of total mesorectal excision \[[@CR2]\], neo-adjuvant therapy protocols \[[@CR3]\], and the laparoscopic approach \[[@CR4]\] has made rectal cancer treatment a multidisciplinary management.
While laparoscopic resection of colon cancer has slowly gained acceptance worldwide, the role of laparoscopy in the treatment of rectal cancer is still controversial \[[@CR4],[@CR5]\]. The laparoscopic treatment of rectal cancer raises specific issues related to its anatomical location: difficult exposure in a narrow pelvis, challenging nerve-sparing techniques, low intestinal transection, and total mesorectal excision \[[@CR6],[@CR7]\]. Ligation of the inferior mesenteric artery at the origin and mobilization of the splenic flexure are not routinely done worldwide \[[@CR8]-[@CR11]\]. The need for well-designed studies to compare the different vascular approaches in rectal surgery has been clearly expressed \[[@CR12]-[@CR14]\].
The level of arterial ligation can affect genito-urinary function (injury to the superior hypogastric plexus), the extent (and yield) of lymphadenectomy, and distal colonic arterial perfusion (especially in older people, where distal colonic arterial perfusion could be deficient due to degenerative disease), and causesympathetic nerve injures \[[@CR15]-[@CR19]\]. Moreover, colonic stump blood supply and anastomosis tension are the main factors in developing leaks in rectal surgery \[[@CR20]-[@CR26]\] and are dependent on the level of ligation.
The aim of this study is to compare the incidence of genito-urinary dysfunction and, secondarily, the incidence of anastomotic leakage and the oncological outcomes in laparoscopic anterior rectal resection with total mesorectal excision with high or low ligation of the inferior mesenteric artery in a controlled randomized trial.
Methods/design {#Sec2}
==============
Study design {#Sec3}
------------
The HIGHLOW study is a multicenter controlled trial. Patients are randomly assigned to high or low inferior mesenteric artery during laparoscopy once the surgeon has confirmed intraoperatively, that all inclusion criteria are fulfilled and that the procedure is feasible. All participating surgeons have performed at least 20 laparoscopic procedures per year during the past 5 years. Randomization is performed using sealed envelopes (Figure [1](#Fig1){ref-type="fig"}).Figure 1**Study flowchart.** LAR: Laparoscopic Anterior Resection. TME: Total Mesorectal Excision.
Primary and secondary endpoint measures {#Sec4}
---------------------------------------
The primary end-point measure is the incidence of post-operative genito-urinary dysfunction compared with a preoperative base-line assessment in both groups. The secondary endpoint measures are the incidence of clinical or subclinical anastomotic leakage in the two groups and the oncological outcomes in both groups.
The incidence of clinical or subclinical anastomotic leaks will be determined either as the presence of a radiologically, endoscopically, or surgically proved fistula or as the presence of a subclinical leak observed by endoscopic examination 30 days after surgery in both groups.
Oncological outcomes will be assessed in terms of retrieved lymph nodes, number of positive lymph nodes on the root of the inferior mesenteric artery total number of lymph nodes of the mesorectum, and positive lymph nodes of the mesorectum, disease-free survival, overall survival, local recurrence, distant metastasis in both groups.
Participating centers and {#Sec5}
-------------------------
Five non-academic public Italian hospitals will enroll patients.
Study population {#Sec6}
----------------
The study population consists of patients with cancer of the lower two-thirds of the rectum, who are eligible for laparoscopic low rectal resection with total mesorectal excision.
Inclusion criteria are middle or low rectal cancer (from 0 to 12 cm from the dental line), an American Society of Anesthesiologists score of I, II, or III, and a body mass index less than 30. The study will include both preoperatively irradiated and non-irradiated patients.
Exclusion criteria are prior surgery on the abdominal aorta and proven arteriosclerosis of the inferior mesenteric artery and its branches.
The study will include both pre operatively irradiated and non-irradiated patients.
Exclusion criteria: prior surgery on the abdominal aorta, proven arterosclerosis of IMA and IMA branches.
Ethical considerations {#Sec7}
----------------------
This study is conducted in accordance with the principles of the Declaration of Helsinki and "good clinical practice" guidelines. The study was approved by a Central Ethics Committee (list of names of the Ethical Body in Additional file [1](#MOESM1){ref-type="media"}). Prior to randomization written informed consent will be obtained from all patients.
Preoperative evaluation {#Sec8}
-----------------------
Patients with a preoperative diagnosis of adenocarcinoma of the middle or lower rectum and who satisfy the inclusion criteria will be asked to participate in the HIGHLOW trial. Once informed consent is obtained, investigations will include questionnaires for the International Index of Erectile Function \[[@CR27]\], International Consultation on Incontinence Questionnaire \[[@CR28]\], International Prostatic Symptoms Score, Female Sexual Function Inde \[[@CR29]\] Uroflowmetry and ultrasound measurement of post-void bladder volume will be performed pre-operatively for all patients.
Surgery {#Sec9}
-------
The following steps are required in all cases, independently of randomization.
### Laparoscopic anterior rectal resection with total mesorectal excision (without pelvic cylindrical excixion) {#Sec10}
The first step consist in the opening of the left part of the gastrocolic ligament and the division of the left part of transverse mesocolon. The splenocolic and phrenocolic attachments are then divided, achieving complete dissection of the left colonic angle. The pelvic peritoneum is opened below the sacral promontory and the hypogastric nerves are identified and preserved. The common iliac veins, the genitofemoral nerve, the gonadic vessels, and the left ureter are successively identified and preserved.
### For high ligation {#Sec11}
The opening of the peritoneum proceeds cephalad, towards the duodenojejunal angle of Treitz, and the mesenteric root is incised 1 cm below the inferior margin of the pancreas. The aortomesenteric window is opened wide and the inferior mesenteric vessels are exposed. The inferior mesenteric artery is ligated and divided at 2 cm from its origin. The inferior mesenteric vein is ligated and divided below the pancreatic margin
### For low ligation {#Sec12}
The opening of the peritoneum proceeds upward and then laterally towards the sigmoid colon. The leftcolic artery is identified and preserved while low ligation of the inferior mesenteric artery (the superior hemorrhoidal artery) is performed. Lymphadenectomy is performed medially along the inferior mesenteric artery as far as 2 cm from the aorta.
For both groups, dissection is then continued windowing Toldt's and Gerota's fascias up to the parietocolic gutter. Intra-pelvic dissection is carried out through standardized planes. Dissection of the rectum starts by incision of the peritoneal fold in the pelvis. Mesorectal excision starts posteriorly by dissection through Heald's "holy plane", it carries on towards the lateral region of the rectum, sparing the lateral part of the lateral rectal ligaments, and extends on the anterior side in front of Denonvilliers' fascia.
Post-operative evaluation {#Sec13}
-------------------------
Colonoscopy will be performed 30 days after surgery to evaluate anastomosis (leakage, signs of ischemia) \[[@CR26]\]. Accurate description and pictures of the anastomosis will be produced. Questionnaires for the Internation Index of Erectile Function \[[@CR27]\], International Consultation on Incontinence Questionnaire \[[@CR28]\], International Prostatic Symptoms Score, and Female Sexual Function Index \[[@CR29]\] will be administrated, and uroflowmetry and ultrasound measurement of post-void bladder volume will be performed 1 and 9 months post-operatively. Retrieved lymph nodes will be collected from the histopathological examination, and the number of positive lymph nodes on the root of the inferior mesenteric artery, the total number of lymph nodes on the mesorectum, and the number of positive lymph nodes on the mesorectum will be recorded Oncological follow up will be carried out for 5 years, according to National Comprehensive Cancer Network Guidelines \[[@CR30]\] Version 1.2015 Rectal Cancer/Surveillance ([www.nccn.org](http://www.nccn.org)).
Sample size calculation {#Sec14}
-----------------------
A two tail Fischer exact test applied to two cohorts of 100 patients each will have 84.45 power in estimating a 20% difference in the incidence of genitor-urinary dysfunctions. With a group size of 100 patients per arm it is possible to find a significant difference (α= 0.05, β= 0.1555).
With a drop-out estimated rate of 5%, the required sample size is 212 patients. If the number of drop-outs exceed 5%, we plan to ask the ethics committee to enroll more patients to be able to keep the power of analysis well above 80%.
Statistical analysis {#Sec15}
--------------------
The Primary end-point measure will be evaluated using Fisher's exact test (one-sided). The different incidences of genitor-urinary dysfunction according to sex and the results of each questionnaire will be evaluated using the Mann--Whitney *U test* and the *t-*test. Mc Nemar's test will be used to evaluate changes in questionnaires results overtime. Statistical analysis will be performed in accordance with the intention-to-treat principle.
Data collection and monitoring {#Sec16}
------------------------------
Data will be collected daily using an Access database by one physician for each hospital and referred to a research fellow who will monitor the included data for all institutions. Patients will fill out questionnaires during pre and post-operative physical examinations. There will be regular contact between the study coordinators and the participating centers through scheduled meetings every 3 months. Uroflowmetry and ultrasound measurement of post-void bladder volume will be performed by the urologists of each institution, colonoscopies will be performed by endoscopists of each institution.
Discussion {#Sec17}
==========
The discussion of the benefits or disadvantages between high tie and low tie in rectal surgery continues to be debated. Retrospective evaluations \[[@CR9],[@CR31]\] have not produced a high level of significance regarding this issue. On the one hand, the risk of poor blood supply of the anastomosis could outweigh the oncological benefits of performing high ligation of the inferior mesenteric artery routinely, on the other hand a more powerful disease staging achieved with high ligation of the inferior mesenteric artery is found to be associated with an acceptable anastomotic leak rate. There is an increased risk of poor colonic stump blood supply when relying on the marginal artery alone, therefore, if this artery is not adequate, a more extended intestinal resection has to be performed, even if it is oncologically unnecessary. Performing lymphadenectomy extending to the origin of the inferior mesenteric artery even for low ligation provides data on the disease involvement of apical nodes. A significantly lower incidence of anastomotic leak in one or other group could indicate a technical policy for patients for which the vascular component might play a key role. Reduction of fistula in laparoscopic anterior rectal resection will produce a significant improvement in quality of life for these patients. Investigating pre and post-operatibegenitor-urinary function will provide a more complete assessment of the impact of laparoscopic anterior rectal resection on functional outcomes. Arterial ligation far from the hypogastric plexus could help in preserving pelvic autonomic functions, giving a better quality of life to patients.
We expect the low tie group to present a lower rate of genito-urinary function depression and post-operative fistula because of the better blood supply provided and better nerve sparing achieved.cpr
Trial status {#Sec18}
------------
The study is not yet open for participant recruitment.
Additional file {#Sec19}
===============
Additional file 1:**Title and legend section.**
**Competing interests**
The authors declare that they have no competing interests.
**Authors' contributions**
All authors have made substantial contributions to either conception and design, acquisition of data, or analysis and interpretation of data. All authors have read and approved the final manuscript. MG: conceived the study and carries out the direction of the study. MD: conceived the study, performs the surgical procedure. CA: performs the surgical procedure. MA: carries out the enrollment and the follow up. RL: carries out the enrollment and the follow up. CJ: carries out the enrollment and the follow up. MC: carries out the enrollment and the follow up. DLS: carries out the enrollment and the follow up. FA: carries out the enrollment and the follow up. CP: carries out the enrollment and the follow up. NM: carries uut the statistical analysis. PC: performs the surgical procedure. VF: carries out the enrollment and the follow up. RM: carries out the enrollment and the follow up. BM: carries out the enrollment and the follow up. CE: performs the surgical procedure. LL: performs the surgical procedure. OM: carries out the enrollment and the follow up. SI: performs the surgical procedure. RF: carries out the enrollment and the follow up. DLA: carries out the enrollment and the follow up. FG: performs the surgical procedure. PR: carries out the enrollment and the follow up. All authors read and approved the final manuscript.
No sponsorship was received for this study. All work was up to the authors of the paper.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
The development of nanomedicine arose from a combination of nanotechnology and medicine, and due to the special properties of nanosized particles, nanomedicine provides potential encapsulation systems for medical agents used in both diagnostic and therapeutic applications.[@b1-ijn-10-3031],[@b2-ijn-10-3031] In the field of therapeutic applications, nanomedicines offer advantages over conventional medicines, including improvement of poor drug solubility, increased bioavailability,[@b3-ijn-10-3031],[@b4-ijn-10-3031] and targeted delivery of chemotherapeutic drugs.[@b5-ijn-10-3031],[@b6-ijn-10-3031] Application of nanotechnology to imaging methodologies is paving the way for diagnostic medicine. Nanoparticles have been designed and used as detectors for diagnostic application in clinical and basic medical research, including magnetic resonance imaging,[@b7-ijn-10-3031] positron emission tomography,[@b8-ijn-10-3031] IVIS^®^ spectrum imaging,[@b9-ijn-10-3031] and ultrasonic imaging.[@b10-ijn-10-3031],[@b11-ijn-10-3031] Ultrasonic imaging, a noninvasive and real-time procedure, is often used. Compared with other imaging techniques, ultrasound systems are cost-effective, portable, easy to use, and safe for both clinical staff and patients over repeated use due to lack of ionizing radiation.[@b12-ijn-10-3031]
Bubble formulations represent a new class of encapsulation systems with both diagnostic and therapeutic applications.[@b11-ijn-10-3031] They can transport drugs or genes to a specific location, at which point ultrasound is applied, causing the formulations to burst and leading to site-specific delivery of bioactive materials.[@b13-ijn-10-3031] In addition to targeted drug delivery, bubble formulations have also received attention as ultrasonic imaging agents. Micron-scale (μm) bubbles (microbubbles) can become trapped in the blood pool after intravenous injection. The reported use of microbubbles has largely been limited to research involving cardiovascular diseases, including inflammation, arteriosclerosis, and thrombus formation.[@b14-ijn-10-3031],[@b15-ijn-10-3031] In addition, microbubbles are often restricted in their utility for tissue-targeted imaging due to their large diameters.[@b15-ijn-10-3031],[@b16-ijn-10-3031] Therefore, nanoscale bubbles (nanobubbles) and nanodroplets have been developed and studied as contrast agents and drug carriers for diagnostic and therapeutic application due to their particle size.[@b11-ijn-10-3031],[@b15-ijn-10-3031],[@b17-ijn-10-3031]
A large number of polymeric nanoparticles have been evaluated when applied in basic and clinical medical studies. Many of these particles were able to alter the distribution profiles of encapsulated drugs.[@b1-ijn-10-3031],[@b18-ijn-10-3031] However, previous literature reports have indicated that these nanobubbles/nanodroplets are still in the early stages of development,[@b19-ijn-10-3031],[@b20-ijn-10-3031] whether in ultrasonic imaging or therapeutic applications.
Generally, the shells of nanobubbles are composed of nontoxic biodegradable materials (such as albumin or lipids), and encapsulated with poorly soluble and nonreactive gases (such as inert air or perfluorocarbon).[@b21-ijn-10-3031] Perfluoropentane (C~5~F~12~) is a liquid at room temperature, boils at 28.5°C, and is generally used to generate nanobubbles as gas. When these shells were filled with perfluoropentane to form nanodroplets,[@b22-ijn-10-3031] they can be activated by heat or other energy to form nanobubbles.[@b23-ijn-10-3031]
To our knowledge, there are no studies that have investigated the pharmacokinetics of drug-loaded nanobubbles/nanodroplets with ultrasound treatment. Additionally, the applications of nanobubble/nanodroplet ultrasound imaging are currently limited to observing tumor tissue.[@b15-ijn-10-3031],[@b24-ijn-10-3031] In this study, we designed perfluoropentane nanodroplets to investigate ultrasound-induced pharmacokinetics and development of novel diagnostic applications to improve our understanding of and ability to accurately predict the clinical applications and efficacy of echogenic bubbles.
Quercetin is a plant-derived bioflavonoid that is found in naturally occurring products. Pharmacological reports have demonstrated that quercetin has pronounced pharmaceutical effects, including anti-inflammatory, antioxidant, and antiviral activity.[@b25-ijn-10-3031] In addition, quercetin is included in a novel class of chemotherapeutic drugs for the treatment of various cancers[@b26-ijn-10-3031] and can also be combined with ultrasonic pretreatment to increase the concentration of quercetin to inhibit the growth of the prostate and skin cancer cell lines.[@b27-ijn-10-3031] However, quercetin has low aqueous solubility, poor absorption, and rapid metabolism (bioavailability approximately 1%--5%),[@b28-ijn-10-3031] all of which can generate in vivo results that differ from the powerful in vitro efficacy of quercetin. Thus, encapsulation of quercetin in nanodroplets may improve its pharmacokinetic profile, and with the use of ultrasound-triggered rupturing, enable effective drug delivery to provide anticarcinogenic effects.[@b29-ijn-10-3031],[@b30-ijn-10-3031]
The current study aimed to: design lipid-coated nanodroplets containing quercetin; evaluate the functions of the formulations in terms of their physicochemical characteristics after in vitro release of quercetin; assess their potential diagnostic applications by in vivo ultrasonic imaging of topical tissue (bladder) and the vascular system; and investigate the pharmacokinetic profile of quercetin loaded into nanodroplets and of ultrasound-triggered quercetin-loaded nanodroplets.
An improved understanding of the effect of combining quercetin-loaded nanodroplets with ultrasonic treatments on bioimaging and pharmacokinetics may help to better predict the clinical applications of nanodroplets and lead to more effective design preparation.
Materials and methods
=====================
Materials and experimental animals
----------------------------------
Perfluoropentane (96%, boiling point 28.5°C) was purchased from Strem Chemicals (Newburyport, MA, USA). Cholesterol, coconut oil, quercetin, heparin, α-chloralose urethane, and polyethylene glycol (PEG) 400 were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Hydrogenated soybean phosphatidylcholine (Phospholipon^®^ 80H) was supplied by American Lecithin Company (Oxford, CT, USA). Distearoyl phosphatidyl ethanolamine-PEG with a mean molecular weight of 5,000 (DSPE-PEG 5000) was obtained from Nippon Oil & Fats Co., Ltd (Tokyo, Japan). The cellulose membrane (Cellu-Sep^®^ T2, molecular weight cut-off 6,000--8,000 Da) was obtained from Membrane Filtration Products Inc. (Seguin, TX, USA). Ethanol, chloroform, ethyl acetate, methanol, and acetonitrile were purchased from EMD Millipore (Billerica, MA, USA). Triply deionized water from EMD Millipore) was used in the preparation of all aqueous solutions.
Adult male Sprague-Dawley rats (200±30 g body weight) were obtained from the National Yang-Ming University Animal Center, Taipei, Taiwan. The animals were specifically pathogen-free and had free access to food (Laboratory Rodent Diet 5001, PMI Nutrition International LLC, St Louis, MO, USA) and water. All experimental protocols involving animals were performed and approved by the Institutional Animal Care and Use Committee (1010613) of National Yang-Ming University.
HPLC analysis of quercetin
--------------------------
The high-performance liquid chromatography (HPLC) system was composed of a chromatographic pump (LC-20AT, Shimadzu, Kyoto, Japan), an autosampler (SIL-20AT, Shimadzu), a diode array detector (SPD-M20A, Shimadzu), and a degasser (DGU-20A5). A reversed-phase C18 column (250×4.6 mm, particle size 4 μm, Synergi Fusion RP 80, Phenomenex, Torrance, CA, USA) was used. The mobile phases consisted of acetonitrile 10 mM KH~2~PO~4~ (47:53, v/v) at pH 3.0, and the flow rate was 1 mL per minute. The ultraviolet wavelength was set to 370 nm and the sample injection volume was 20 μL. The limits of detection and quantification were defined as a signal-to-noise ratio of 3 and the lowest concentration from the linear regression, respectively.
Preparation of the nanodroplet-encapsulated quercetin formulation
-----------------------------------------------------------------
Soybean phosphatidylcholine (4.0%, w/v of the final product), cholesterol (1%, w/v), and DSPE-PEG 5000 (1%, w/v) were dispersed in 5 mL of a chloroform-methanol (2:1) solution. The organic solvent was evaporated in a rotary evaporator at 50°C, and the solvent traces were removed by maintaining the lipid film under a vacuum overnight. The film was hydrated with triply deionized water containing quercetin (solubility approximately 60 mg/L) using a probe-type sonicator (VCX600, Sonics and Materials, Newtown, CT, USA) at 35 W for 10 minutes at 60°C. Coconut oil 3.0% was then added to the mixture, followed by high-shear homogenization (Pro250, Pro Scientific, Monroe, CT, USA) for 5 minutes. The semi-finished product was cooled to 0°C. Finally, perfluoropentane (10%, w/v) was incorporated into the system, followed by homogenization (for 5 minutes at 0°C) and sonication (35 W for 5 minutes at 0°C). The total volume of the resulting product was set at 5 mL. After the formulations were prepared, they were left to stand for 1 day at 0°C degassed.
Determination of particle size and zeta potential
-------------------------------------------------
The mean particle size and polydispersity index of the formulations were measured by dynamic light scattering (90 Plus, Brookhaven Instruments Corporation, Holtsville, NY, USA). The zeta potential of the formulations was determined using a zeta potential analyzer (90 Plus, Brookhaven Instruments Corporation). The dispersion of the prepared formulations was diluted tenfold with triply deionized water for measurement of both size and surface charge. Particle characterizations were repeated three times per sample for three independent batches.
Encapsulation of quercetin in nanodroplets
------------------------------------------
Entrapment capacity was used to determine the amount of drug in the nanodroplet-encapsulated quercetin (NQ) formulation. The NQ formulations were centrifuged at 48,000× *g* at 4°C for 30 minutes in an Optima Max^®^ ultracentrifuge (Beckman Coulter, Brea, CA, USA) to separate the incorporated drug from the free form. The samples were collected from the supernatant at top of the tube. The amount of quercetin in the formulation was analyzed using the HPLC system. The entrapment capacity of NQ was calculated as follows: (\[total amount of drug − amount of drug detected only in the supernatant\]/total amount of drug) ×100.[@b31-ijn-10-3031] Three formulations was tested, and the data are expressed as the mean ± standard deviation (n=3).
Morphological observations by TEM
---------------------------------
The characteristic microstructure of NQ was assessed by transmission electron microscopy (TEM) as follows. First, a drop of a formulation (the formulation was diluted tenfold by triply deionized water) was applied to a 400-mesh carbon film-covered copper grid to form a thin film specimen, which was stained with 2% phosphotungstic acid (40 seconds). After the stained samples were allowed to dry in air, TEM samples were obtained. The samples were prepared at room temperature (about 22°C--24°C). The sample was then examined and photographed by TEM (JEM-1400EXII, JEOL, Tokyo, Japan).
In vitro drug release study
---------------------------
The drug release profile for the NQ formulation was measured using a Franz diffusion cell. A cellulose membrane (molecular weight cut-off 6,000--8,000 Da) was mounted between the donor and acceptor compartments, acting as a barrier in the experiments to examine drug release from the NQ. The donor medium consisted of 0.5 mL of vehicle containing the drug and a 0.5 mL sample of rat plasma and was added (or not added) to the donor compartment. The receptor medium consisted of 5.5 mL of ethanol with pH 7.4 buffer (3:7 ratio) to maintain physiological conditions. The practical diffusion area between the cells was 0.785 cm^2^. The stirring rate and temperature of the receptor were maintained at 600 rpm and 37°C, respectively. At each time point, 0.3 mL aliquots of the receptor medium were removed and immediately replaced by equal volumes of blank buffer. The concentration of quercetin released was determined using the HPLC system. In the study exploring the release of drug from NQ with or without ultrasound, the donor phase was exposed to ultrasound using a 1 MHz probe (Rich-Mar Sonitron 2000, Inola, OK, USA) with a 2.0 W/cm^2^ intensity and a 20% duty cycle.
In vivo ultrasonic imaging
--------------------------
To evaluate in vivo ultrasonic imaging of nanodroplets, the femoral vein was catheterized with polyethylene tubing for intravenous administration under anesthesia (urethane 1.0 g/kg and α-chloralose 0.1 g/kg). Ultrasonic scanning was performed by means of an Acuson S2000TM ultrasound system (Siemens, Washington, DC, USA) with a 9 MHz linear array probe. An initial baseline B-mode survey and a color/power Doppler analysis were performed. Ultrasound scan parameters, such as focal zone, persistence, linear gray-scale map, time-gain compensation curve, and focal settings, were fixed throughout the experiment. Next, the ultrasound device was set to cadence contrast pulse sequencing mode with a transmission frequency of 7 MHz and a mechanical index of 0.18 to avoid destruction of the formulation. About 1 minute after injection, the mechanical indices (MI) was manually adjusted and fixed to 1.0 for destruction of the formulation. Each examination lasted approximately 6 minutes after the bolus injection.
Surgical procedures
-------------------
The rats were initially anesthetized using a mixture of urethane 1.0 g/kg and α-chloralose 0.1 g/kg. A polyethylene tube was implanted into the right external jugular vein and femoral vein to collect blood and for drug administration, respectively. Subsequently, for the groups receiving ultrasound treatment, the hilum of the liver was gently exposed through a midline incision so that the hepatic portal vein could be accessed. Three minutes after drug administration then treated with ultrasound for 1 minute using a 1 MHz probe with a 2.0 W/cm^2^ intensity and a 20% duty cycle.
Drug administration and sampling
--------------------------------
Quercetin (dissolved in PEG 400 with 20% ethanol) or an equal quercetin dose in NQ (3 mg/kg) was intravenously injected via the femoral vein. A 500 μL aliquot of the blood sample was withdrawn from the jugular vein into a heparin-rinsed vial using a fraction collector set to a programmed schedule at 5, 10, 20, 30, 45, 60, 90, and 120 minutes.
Sample preparation
------------------
The blood samples were centrifuged at 16,000× *g* for 10 minutes at 4°C for plasma preparation. The supernatant was collected for plasma samples and preserved at −20°C before further sample analysis. A 50 μL aliquot of the plasma sample, 5 μL of internal standard apigenin (10 μg/mL), and 1,000 μL of ethyl acetate were added, and the mixture was vortexed for 30 seconds. The mixture was centrifuged at 16,000× *g* for 10 minutes at 4°C. The supernatant (950 μL) was transferred to tubes and dried under vacuum at room temperature. Finally, the samples were reconstituted in 50 μL of 50% acetonitrile, and 20 μL of the mixture was injected into the HPLC system.
Statistical analysis
--------------------
All pharmacokinetic analyses were carried out using WinNonlin Standard Edition version 1.1 (Scientific Consulting Inc, Apex, NC, USA). A noncompartmental model was utilized for the data fitting and parameter estimation. The pharmacokinetic results are represented as the mean ± standard error of the mean. Statistical significance was determined using one-way analysis of variance, followed by a Schiff post-test using Statistical Package for the Social Sciences version 10 software (SPSS Inc., Chicago, IL, USA), with *P*\<0.05 accepted as the minimal level of significance.
Results
=======
Physicochemical characteristics of nanodroplets and NQ
------------------------------------------------------
These formulations were prepared using the conventional thin film hydration technique, and the lipid film was prepared by modifying previous experimental methods, including modifying the evaporation temperature and vacuum time.[@b20-ijn-10-3031],[@b32-ijn-10-3031]--[@b34-ijn-10-3031] Therefore, the organic solvents could be completely removed under these conditions, and even if there were slight solvent residues, they did not affect the subsequent experiments.
Because perfluorocarbon-loaded nanodroplets are satisfactorily stable when circulating in the vasculature as blood pool agents, they act as carriers of these agents until the site of interest is reached.[@b13-ijn-10-3031] In this study, the formulation system containing perfluoropentane (with a boiling point of 28.5°C) was designed with soybean phosphatidylcholine, cholesterol, and DSPE-PEG 5000. The use of DSPE-PEG 5000 in nanodroplets can aid in increasing the circulation time in vivo and for sustained drug release.[@b35-ijn-10-3031] Coconut oil is extracted from coconut (kernel or meat), and was used as the oil phase due to its superiority in enhancing flavonoid solubility compared with other oils.[@b36-ijn-10-3031],[@b37-ijn-10-3031] Perfluoropentane is a nonpolar and hydrophobic substance, which can disperse in aqueous solution by surfactants.[@b38-ijn-10-3031] Cholesterol is an amphipathic compound containing both a water-soluble region and a fat-soluble region, regulating the lipid structure to improve the stability of formulations.[@b39-ijn-10-3031] Therefore, the core of the formulation was loaded with perfluoropentane, which was surrounded by coconut oil. Soybean phosphatidylcholine, DSPE-PEG 5000, and cholesterol, which functioned as emulsifiers, were located at the oil/water interface as a phospholipid membrane surrounding the quercetin-containing core.
To evaluate the physicochemical properties of the formulations, the particle size and zeta potential were examined. [Table 1](#t1-ijn-10-3031){ref-type="table"} summarizes the physicochemical characteristics of the nanodroplets and the NQ formulation. Dynamic light scattering indicated that the size of the nanodroplets and the NQ formulation was approximately 280 nm with a polydispersity index \<0.35, suggesting that the particle formulations were quite homogeneous. The zeta potentials of the nanodroplets and NQ were −42.18 and −34.66 mV, respectively. To calculate the loading of quercetin in the nanocomplex, nanoparticle counting instruments (such as Q-Nano) may be used to estimate the number of NQ particles. However, this method provides only a rough estimate due to the potential for errors associated with sample preparation (filtering of the sample may cause errors in number of particles counted). Therefore, we used encapsulation efficiency as an acceptable alternative. The entrapment capacity of NQ was calculated as follows: (\[total amount of drug − amount of drug detected only in the supernatant\]/total amount of drug) ×100.[@b31-ijn-10-3031] The encapsulation efficiency of NQ was 99% (about 2.97×10^19^ molecules of quercetin were encapsulated).
Stability of NQ formulation
---------------------------
Loss of gas is the main reason for destruction of bubble formulations.[@b40-ijn-10-3031] To assess the stability of NQ in the body and during storage in a freezer, the NQ formulation was evaluated using a 37°C oven and a 4°C freezer. As shown in [Figure 1](#f1-ijn-10-3031){ref-type="fig"}, NQ evaporated rapidly within 10 hours at 37°C and completely evaporated after 24 hours; however, the weight of NQ did not change significantly over 0--24 hours at 4°C. The size and zeta potential of the NQ formulation was also measured at 4°C after 24 hours, and the results indicated no significant differences from values recorded at 0 hours.
TEM of NQ formulation
---------------------
TEM with a multifunctional nanosensor was used to image and quantify the mechanical properties of the NQ formulation.[@b41-ijn-10-3031] [Figure 2](#f2-ijn-10-3031){ref-type="fig"} shows the microscopic structures of the spherical NQ formulation. A narrow size distribution was observed for the NQ groups, indicating the presence of a quite homogeneous particle population. The polydispersity index also exhibited a low value of 0.34, as determined by laser scattering. These results demonstrate good agreement between the laser scattering method and TEM.
In vitro drug release study
---------------------------
To examine the performance of the nanodroplets loaded with quercetin, an in vitro drug release test was performed. The in vitro release profiles of quercetin alone and NQ are presented in [Figure 3](#f3-ijn-10-3031){ref-type="fig"}. Release of quercetin from PEG 400 with 20% ethanol was used as the control. As shown in [Figure 3](#f3-ijn-10-3031){ref-type="fig"}, free quercetin in solution showed the highest release, and the NQ formulation showed suppressed and sustained release of quercetin compared with the control group.
Furthermore, to evaluate the release profile after injection, plasma was added to the donor phase and mixed with the test substances to simulate in vivo conditions. The amount of quercetin released from the NQ formulation (in the absence of plasma) showed no significant difference (*P*\>0.05) in comparison with NQ mixed with plasma ([Figures 3](#f3-ijn-10-3031){ref-type="fig"} and [4](#f4-ijn-10-3031){ref-type="fig"}). To investigate the influence of this novel use of ultrasound on drug delivery, the amount of quercetin released under ultrasound treatment was examined. [Figure 4](#f4-ijn-10-3031){ref-type="fig"} shows that ultrasound treatment of 1 MHz (1 minute) produced an increase in drug release from the NQ formulation (*P*\<0.05). The percentage of drug released after 24 hours from the NQ formulation with insonation showed a 2.19-fold and 9.07-fold increase in comparison with the nonultrasound group in the absence and presence of plasma, respectively.
In vivo ultrasonic imaging
--------------------------
To investigate the potential use of nanodroplets for diagnostic application, ultrasonic imaging was used to monitor the appearance of particles in topical (bladder) tissue and the vasculature in rats. No significant difference in the ultrasonographic images was observed between the preinjection time point and injection of double-distilled H~2~O, but many bubbles appeared in the bladder tissue after injection of the NQ formulation ([Figure 5](#f5-ijn-10-3031){ref-type="fig"}). The boiling point of perfluoropentane is 28.5°C. The increase in the temperature-induced droplet-to-bubble conversion is impeded by the high Laplace pressure inside the nanodroplets. However, nanodroplets are easily converted into bubbles under ultrasonication.[@b42-ijn-10-3031]--[@b44-ijn-10-3031] The in vivo ultrasonic imaging data provided additional confirmation of droplet-to-bubble conversion.
[Figure 6](#f6-ijn-10-3031){ref-type="fig"} illustrates the effect of ultrasound-targeted nanodroplets. [Figure 6A](#f6-ijn-10-3031){ref-type="fig"} represents the abdominal vein before NQ injection, which was a blank area. For NQ administration, the femoral vein was catheterized with polyethylene tubing for intravenous administration under anesthesia. [Figure 6B](#f6-ijn-10-3031){ref-type="fig"} shows that some bubbles appeared in the abdominal vein 30 seconds after intravenous administration of nanodroplets via the femoral vein. Ideally, the NQ formulation should be applied for site-specific delivery to areas where selective enhancement of activity by ultrasound is required.[@b45-ijn-10-3031] For the next step, to assess the ability of ultrasound-controlled targeted drug delivery, ultrasound-triggered drug-loaded nanodroplets were used. [Figure 6C](#f6-ijn-10-3031){ref-type="fig"} demonstrates that the NQ could be readily directed to specific sites for ultrasonic treatment. After ultrasound triggering, the number of bubbles appeared to decrease, which was confirmed in [Figure 6D](#f6-ijn-10-3031){ref-type="fig"}. These results suggest that the NQ formulation could potentially be used for clinical application to simultaneously enable ultrasound diagnostic imaging and drug targeted delivery.
Pharmacokinetics
----------------
To investigate the effects of ultrasound and nanodroplets with regard to the pharmacokinetics of quercetin, the portal vein was used as a topical target for ultrasonic treatment. Four parallel experimental groups were evaluated, ie, quercetin alone, quercetin triggered with ultrasound (quercetin + U), NQ, and NQ triggered with ultrasound (NQ + U). The quercetin concentration-time curves after intravenous administration in the four groups are shown in [Figure 7](#f7-ijn-10-3031){ref-type="fig"}. Quercetin could be detected in plasma up to 45 minutes after administration (3 mg/kg, intravenously), while this time was prolonged to 120 minutes for the NQ groups. The pharmacokinetic parameters were calculated using WinNonlin software version 1.1, and the comparative pharmacokinetic parameters for the four groups are reported in [Table 2](#t2-ijn-10-3031){ref-type="table"}.
[Figure 7](#f7-ijn-10-3031){ref-type="fig"} demonstrates that the quercetin concentration-time curves for the free drug and NQ were not significantly affected by ultrasonic treatment. The initial serum concentration (C~5\ min~) was significantly lower (*P*\<0.05) for the quercetin group than for the nanodroplet formulations (NQ and NQ + U) following intravenous administration.
[Table 2](#t2-ijn-10-3031){ref-type="table"} shows a significant increase (*P*\<0.05) in the area under the concentration--time curve (AUC) for the NQ and NQ + U groups (60.5±5.9 μg/mL per minute and 67.6±6.8 μg/mL per minute, respectively) compared with the quercetin alone group (18.4±7.0 μg/mL per minute). These results indicate that the absorption of quercetin increased when the drug was encapsulated in the formulation, regardless of whether NQ was combined with ultrasonic treatment. These data suggest that the quercetin group was rapidly eliminated from the plasma, whereas the nanodroplet formulations were able to significantly improve the pharmacokinetics of the drug by increasing the AUC, elimination half-life, and C~5\ min~, and by reducing (*P*\<0.05) clearance.
Discussion
==========
Physical and chemical properties, such as particle size and zeta potential, can affect the pharmacokinetic profile and ultrasonic imaging of nanodroplets.[@b11-ijn-10-3031],[@b46-ijn-10-3031] First, we designed and modified quercetin-loaded nanodroplets, which included an evaluation of factors such as dosage, encapsulation, and pharmacokinetic profile. For the conventional mode of injection, the typical injection volume is 1 mL/kg, with a dosage of 1 mg/kg, 3 mg/kg, 5 mg/kg, or 10 mg/kg. We therefore selected 1 mg/mL, 3 mg/mL, and 5 mg/mL quercetin loaded in nanodroplets to assess the feasibility of our experiments. The results showed that 1 mg/kg (1 mg/mL, injection volume 1 mL/kg) of quercetin could not be detected. We ultimately decided that quercetin 3 mg/mL loaded into nanodroplets was suitable for our in vitro and vivo studies.
As shown in [Table 1](#t1-ijn-10-3031){ref-type="table"}, the particle sizes of the nanodroplets and the NQ formulation were approximately 280 nm, with a low polydispersity index. Small size is an important characteristic for nanodroplets and increases the efficacy of intravenous administration. For example, Ferrara et al reported that the resonance frequency is strongly dependent on the diameter of the formulation, and specific ultrasound frequencies require a narrow size distribution.[@b21-ijn-10-3031] Quercetin is a lipophilic molecule (logP 2.16), and may be encapsulated in a matrix of nanodroplets; however, the structure of quercetin contains many OH groups, so may also be encapsulated within the surface of the shell. As shown in [Table 1](#t1-ijn-10-3031){ref-type="table"}, the zeta potential decreased from −42.18±7.64 mV to −34.66±2.18 mV when quercetin was loaded into nanodroplets. In theory, quercetin was neutral in the NQ formulation (the pKa of quercetin was 6.44, and the pH value of NQ was 5.5); therefore, some quercetin molecules interacting with the surface of NQ may shield some negative charges, resulting in reduced surface charges being present on NQ.[@b34-ijn-10-3031],[@b47-ijn-10-3031]
The uniformity of the particles was confirmed based on the polydispersity index. Our results show that the particle formulations were quite homogeneous (polydispersity index \<0.35), and TEM showed that the NQ groups had a narrow size distribution. Previous studies also used similar materials (coconut oil, perfluoropentane, and cholesterol) to produce nanodroplets, and the physical and chemical properties of the nanodroplets are consistent with our data.[@b20-ijn-10-3031],[@b36-ijn-10-3031] The suitability of a formulation for clinical application depends on several conditions, and the development of formulations capable of efficiently carrying a payload is one of the most important goals of research.[@b48-ijn-10-3031] Our data demonstrate that the NQ formulation achieved a near complete encapsulation efficiency of 99%. The small size and high encapsulation efficiency of this formulation may have a positive effect with regard to pharmacokinetic information.
The morphological characterization of NQ was done using SEM. However, NQ may have undergone structural degradation during preparation of samples for SEM, so disintegration was observed in the images of the NQ formulation. Atomic force microscopy was also used to assess the appearance of NQ, but the apparent dimensions of the NQ were altered during preparation of the sample for processing by atomic force microscopy.[@b49-ijn-10-3031] Therefore, we used TEM to confirm the morphology of NQ. We optimized the method used for TEM sample preparation, including the dye concentration, formulation concentration, and dye settling time, and obtained a clean TEM image ([Figure 2](#f2-ijn-10-3031){ref-type="fig"}).
Release of NQ was preliminarily evaluated in the in vitro study under different conditions and including various factors that may affect the release profiles in this model. The in vitro release rate of quercetin over 24 hours from the control solution of PEG 400+20% ethanol was rapid but could not achieve complete release. This limited amount released may be due to use of an in vitro Franz cell. Because the drug is released into the restricted space of the receptor chamber (5.5 mL) and because of the diffusion area is small (0.785 cm^2^), the amount of the drug that can be loaded into the receptor compartment is limited, so the concentration gradient between the donor and receptor may have been lost. Nevertheless, this experimental design is still useful for assessing drug release capabilities under different conditions.[@b50-ijn-10-3031] An incorporated drug appears to be released in a sustained manner, which is an important characteristic that is often correlated with improved pharmacokinetics and efficacy.[@b51-ijn-10-3031] Our results indicate that quercetin loaded into nanodroplets was released more slowly than free quercetin, suggesting that release of quercetin can be effectively controlled by use of nanodroplets.
Plasma is a viscous fluid composed of approximately 91% water and 9% other substances, such as proteins, ions, nutrients, and waste products. In this study, plasma was added to the formulation of the NQ to mimic actual in vivo conditions for the release test with ultrasonic treatment. According to the release profiles in the presence of plasma, drug release might be decreased due to increased viscosity and path lengths for drug diffusion.[@b36-ijn-10-3031] Another explanation may be that quercetin binds strongly to albumin and other proteins in plasma.[@b52-ijn-10-3031] Previously, ultrasound has been used as an external trigger to induce drug release from drug-loaded carriers. When treated with ultrasound pressure waves, bubble formulations start to break down and provide a surge in drug release.[@b53-ijn-10-3031] An increase in the amount of quercetin released after ultrasound exposure was observed for the NQ group. When quercetin was encapsulated in nanodroplets, a change in the amount of quercetin released was observed after ultrasound exposure.
[Figure 6](#f6-ijn-10-3031){ref-type="fig"} confirms that the nanodroplets have features similar to those of microbubbles and have potential for use as vascular contrast agents. Intravesical chemotherapy is a conventional treatment for nonmuscle-invasive urinary bladder cancer.[@b54-ijn-10-3031] However, intravesically administered drugs can be eliminated by voiding, and are unable to target certain areas of the bladder. In such situations, use of drug-loaded nanodroplets with ultrasound-triggered disruption is a promising strategy to increase local drug concentrations at the site of pathology while reducing systemic toxicity. Therefore, the results demonstrate the potential applications of nanodroplets for diagnostic imaging and therapy, including the treatment of bladder cancer and imaging of the vasculature.
Although some studies have explored the pharmacological activity of quercetin metabolites (such as the ability of quercetin-3-*O*-β-glucuronide and quercetin-3-*O*-β-glucoside to prevent the damage to red blood cell membranes done by smoking),[@b55-ijn-10-3031] most studies have focused on the substantial pharmaceutical effects of quercetin. In this experiment, we focused on improving the therapeutic effect and applications of quercetin via encapsulation into nanodroplets and ultrasound activation.
To investigate the pharmacokinetics of ultrasound- activated, quercetin-encapsulated nanodroplets, a suitable assay system for preparation of samples needed to be developed and optimized. Several methods for sample preparation, such as protein precipitation and liquid--liquid extraction, have been examined. Background interference was observed in the blank plasma extract generated using protein precipitation, whereas no background was observed with the liquid--liquid extraction process. Therefore, ethyl acetate was selected as the liquid solvent for liquid--liquid extraction.
To assess the effects of formulation used and ultrasonic treatment on the pharmacokinetics of quercetin, the pharmacokinetics of four parallel groups (quercetin, quercetin + U, NQ, and NQ + U) were evaluated. Pharmacokinetic studies must take into account the physical welfare of animal subjects. Previous reports have indicated that repeated blood sampling influences the normal physiological conditions of rats and determination of pharmacokinetic profiles.[@b56-ijn-10-3031] Thus, the volume of the blood sample and frequency of sampling must be selected carefully, and consequently the time points of the in vivo test can be used as a reference for the in vitro test.
The results indicate that nanodroplets could prolong the elimination time to achieve a sustained-release effect, which increased both the AUC and elimination half-life and thus reduced drug clearance. Liposomes are composed of phosphatidylcholine-enriched phospholipids and contain mixed lipid chains with surfactant.[@b57-ijn-10-3031] The characteristics of the outer shells of our lipid nanodroplets were similar to those of liposomes. Our data are consistent with previous reports,[@b5-ijn-10-3031],[@b58-ijn-10-3031] which also found an increased AUC and elimination half-life, as well as reduced clearance for drug-loaded liposomes compared with free drugs. This finding can be attributed to the protection provided by the lipid membranes and slow drug release from the formulation.[@b59-ijn-10-3031]
A previous in vitro report by Paliwal et al indicated that pretreatment of cells with ultrasound may change the concentration of quercetin in treated cell lines.[@b27-ijn-10-3031] Our in vivo experiment demonstrated that the C~5\ min~ increased from 0.5±0.1 μg/mL to 0.9±0.2 μg/mL after treatment with ultrasound ([Table 2](#t2-ijn-10-3031){ref-type="table"}). This observation suggests that treatment with ultrasound may cause the concentration of analyte to increase over a short period of time and have an acute effect. In recent years, many studies have confirmed the efficacy of ultrasound-triggered destruction of bubble formulations for drug delivery, both in vitro and in vivo.[@b13-ijn-10-3031],[@b60-ijn-10-3031] However, inconsistencies between in vitro and in vivo tests are not unexpected because pharmacokinetic profiles are affected by many factors, including route of administration, the macroenvironment or microenvironment in vivo, and protein binding.
Although no statistically significant difference was observed between the pharmacokinetic parameters of NQ and NQ + U, the initial (C~5\ min~) concentration for NQ + U was slightly higher than that in the NQ group. The elimination half-life of NQ + U was 72±10 minutes, which was between that of the quercetin alone (32±19 minutes) and NQ (135±43 minutes) groups, indicating that destruction of NQ during exposure to ultrasound may have caused some drug to be released from the nanodroplets,[@b13-ijn-10-3031],[@b61-ijn-10-3031] which slightly affected the pharmacokinetic profile. However, while ultrasound may induce a transient change in pharmacokinetics, encapsulation of quercetin in nanodroplets significantly altered the pharmacokinetic profile of quercetin.
In summary, the results of the in vitro tests provided an initial demonstration of the release capability of NQ in the absence of interfering factors, while the in vivo tests enabled a complete evaluation of the pharmacokinetic profile of NQ with ultrasound activation, providing a foundation for future clinical application.
Conclusion
==========
In conclusion, we developed quercetin-loaded, lipid-coated nanodroplets and investigated the efficacy of these particles for ultrasound diagnostic imaging of the bladder and vasculature, and ultrasound-triggered drug delivery in rats. To our knowledge, this is the first study of nanodroplets loaded with quercetin that has evaluated their ultrasound-induced pharmacokinetics and potential bioimaging applications. The in vitro and in vivo tests demonstrated that nanodroplets have potential application in simultaneous ultrasonic imaging and drug delivery, and the results and evaluation process could be useful for future preclinical studies.
The authors appreciate the assistance of Anthony Venuti in editing the manuscript for language and grammar. Funding for this study was provided in part by research grants from the National Science Council (NSC102-2113-M-010-001-MY3) of Taiwan and the Taipei City Hospital, Taipei, Taiwan (TCH 10401-62-004).
**Disclosure**
The authors report no conflicts of interest in this work.
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{#f3-ijn-10-3031}
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{#f6-ijn-10-3031}
{#f7-ijn-10-3031}
######
Characterization of nanodroplets without drug loading and the nanodroplet-encapsulated quercetin formulation by particle size, zeta potential, polydispersity index, and drug encapsulation
Size (nm) Zeta potential (mV) PDI Encapsulation (%)
------------------------ ------------- --------------------- ----------- -------------------
Nanodroplets (no drug) 270.1±24.01 −42.18±7.64 0.21±0.01
NQ 278.6±10.18 −34.66±2.18 0.34±0.01 99.41±0.20
**Note:** The data are expressed as the mean ± standard deviation (n=3).
**Abbreviations:** NQ, nanodroplet-encapsulated quercetin; PDI, polydispersity index.
######
Pharmacokinetic parameters of quercetin 3 mg/kg and the equivalent quercetin dose for the nanodroplet-encapsulated quercetin formulation in rat plasma after administration of an intravenous bolus
Pharmacokinetic parameters Quercetin Quercetin + U (3 min) NQ NQ + U (3 min)
--------------------------------- ----------- ----------------------- ------------------------------------------------------ ------------------------------------------------------
C~5\ min~ (μg/mL) 0.5±0.1 0.9±0.2 2.3±0.3[\*](#tfn4-ijn-10-3031){ref-type="table-fn"} 2.8±0.3[\*](#tfn4-ijn-10-3031){ref-type="table-fn"}
AUC (μg/mL per minute) 18.4±7.0 37.0±10.5 60.5±5.9[\*](#tfn4-ijn-10-3031){ref-type="table-fn"} 67.6±6.8[\*](#tfn4-ijn-10-3031){ref-type="table-fn"}
Elimination half-life (minutes) 32±19 29±18 135±43[\*](#tfn4-ijn-10-3031){ref-type="table-fn"} 72±10[\*](#tfn4-ijn-10-3031){ref-type="table-fn"}
Clearance (mL/minute per kg) 281±81 183±96 51.6±4.2[\*](#tfn4-ijn-10-3031){ref-type="table-fn"} 46.1±3.5[\*](#tfn4-ijn-10-3031){ref-type="table-fn"}
**Notes:** The data are expressed as the mean ± standard error of the mean (n=6).
*P*\<0.05 versus the quercetin group. C~5\ min~ is 5-minute serum concentration after quercetin administration; Quercetin + U (3 min) is ultrasound treatment after 3 minutes quercetin administration; NQ + U (3 min) is ultrasound treatment after 3 minutes NQ administration.
**Abbreviations:** AUC, area under the concentration--time curve; U, treatment with ultrasound; NQ, nanodroplet-encapsulated quercetin.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Porcine species have a great impact on studies of biomaterial production, organ transplantation, and specific biomodel development \[[@B2],[@B7]\]. Establishment of porcine parthenogenetic embryonic stem cells (ppESCs) is expected to make significant contributions to therapeutic applications. The advantage of parthenogenetic stem cells is that they can be banked and distributed as \"off-the-shelf\" embryonic stem cells (ESCs) with less ethical concerns than *in vitro* or *in vivo* fertilized oocyte-derived stem cells \[[@B9]\]. While many researchers have attempted to establish immune-matched parthenogenetic ESCs to increase the clinical application of ESCs \[[@B28]\], the low efficiency of producing viable embryos from which to derive the ESCs has made achieving this goal a challenge. In particular, embryo quality decreases after parthenogenetic activation \[[@B4],[@B18],[@B22]\]. Although several culture systems have been suggested for improving the establishment of ESCs \[[@B3],[@B25],[@B33],[@B34]\], previous studies have failed to show sufficient correlation between different culture conditions and improved establishment of ESCs. Therefore, more efficient culture and isolation methods are needed, especially for parthenogenetic embryos that are more sensitive to the culture environment than normally fertilized embryos \[[@B20],[@B29]\]. In the present study, we evaluated the effects of serum-rich and cytokine-supplemented culture conditions on the establishment of ppESCs.
Materials and Methods
=====================
Animal care and experiment materials
------------------------------------
Ovaries were collected from crossbred (Duroc × Landrace × Yorkshire) prepubertal gilts at a local slaughterhouse and transported to the laboratory in Dulbecco\'s phosphate-buffered saline (PBS; Invitrogen, USA) supplemented with 1× Pen Strep (penicillin 100 unit/mL streptomycin 100 µg/mL, Gibco-BRL, USA ). All procedures for animal management and surgery followed the standard protocols of Seoul National University (Korea). The Institutional Animal Care and Use Committee Review Board at Seoul National University approved the research proposal (SNU-200908-13). All chemicals were purchased from Sigma Chemical Company (USA) unless otherwise stated.
Oocyte collection and *in vitro* maturation
-------------------------------------------
Cumulus-oocyte complexes (COCs) were aspirated from antral follicles with a diameter of 3\~8 mm using an 18 G needle attached to a 10-mL syringe. Only COCs with multiple layers of compacted cumulus cells were selected. These complexes were then washed three times and transferred to a 4-well dish (ThermoFisher Scientific, USA) containing 500 µL of an *in vitro* maturation (IVM) medium with gonadotropin including 10 IU/mL pregnant mare serum gonadotropin (PMSG; Intervet, The Netherlands) and 10 IU/mL human chorionic gonadotropin (hCG; Intervet). The cells were incubated at 39℃ with 5% CO~2~ in a humidified atmosphere \[[@B31]\]. After maturation for 22 h, the COCs were incubated in IVM medium without PMSG or hCG for an additional 22 h.
Parthenogenesis, embryo culture, and blastocyst manipulation
------------------------------------------------------------
Oocytes that reached the MII stage after 44 h of culturing in IVM medium were activated with two pulses of 120 V/mm DC for 60 µsec in a 280-mM mannitol solution containing 0.01 mM CaCl~2~·2H~2~O and 0.05 mM MgCl~2~·6H~2~O. Following electrical activation, the oocytes were treated with 5 µg/mL cytochalasin B for 4 h. The activated oocytes were washed three times with fresh *in vitro* culture medium \[IVC medium; porcine zygote medium-3 (PZM-3) supplemented with 0.3% (w/v) fatty acid-free bovine serum albumin (BSA; Bioworld, Korea), 2.77 mM myoinositol, 0.34 mM trisodium citrate (Wako, Japan), and 10 µM β-mercaptoethanol\], which was designated as FBS (-) medium. The oocytes were cultured in 30-µL droplets of IVC medium under mineral oil and subsequently cultured at 39℃ in a humidified atmosphere of 5% CO~2~, 5% O~2~, and 90% N~2~ for 7 days. To culture with FBS (Hyclone, USA), the embryos were transferred into freshly prepared IVC medium containing 10% (v/v) heat inactivated FBS (Hyclone), which was designated as FBS (+) medium, on the 3rd day of IVC culturing using a micropipette tip. To artificially eliminate the zona pellucida (ZP), unhatched blastocysts on the 5th and 6th day of IVC culturing were treated with 50 µL of acidic Tyrode\'s solution (0.24 mg/mL CaCl~2~·2H~2~O, 0.1 mg/mL MgCl~2~·6H~2~O, 0.2 mg/mL KCl, 8 mg/mL NaCl, 1 mg/mL d-glucose, and 4 mg/mL polyvinylpyrolidone; pH 2.5 ± 0.3) for 5 min.
ICM implantation onto feeder cells and ppESC culturing
------------------------------------------------------
ZP-free blastocysts obtained by both spontaneous and artificial hatching processes were washed three times. The ICMs were then separated from the trophectoderm by gently pipetting with fine-pulled glass capillary pipettes in embryonic stem cell (ES)-basal media \[1:1 mixture of low-glucose Dulbecco\'s Modified Eagle Medium (DMEM) (Invitrogen) and Ham\'s F-10 medium (Invitrogen), 2 mM l-glutamine, 1% (v/v) non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and 1% (v/v) penicillin-streptomycin (Invitrogen)\] containing 15% (v/v) FBS (HyClone) supplemented with 20 ng/mL recombinant human basic fibroblast growth factor (bFGF; Invitrogen), 20 ng/mL recombinant human stem cell factor (SCF; R&D Systems, USA), and 20 ng/mL recombinant human leukemia inhibitory factor (LIF) before use. From this point, the cells were cultured at 37℃ with 5% CO~2~ in a humidified atmosphere.
Isolated ICMs were implanted on a monolayer of mitomycin C-treated mouse embryonic fibroblasts (MEFs) that were isolated using general methods \[[@B21]\]. After implantation, the culture medium was refreshed every other day. Attachment efficiencies of the ICMs were measured on the second day after implantation. Eight to 12 days after the first implantation, ICM-derived colony-forming cells (ppESCs) were mechanically removed from the MEFs and reseeded onto new MEFs. Next, the ppESCs were sub-cultured every 4 days and the culture medium was changed every day.
Alkaline phosphatase (AP) activity assay
----------------------------------------
Cells were fixed with 4% formaldehyde for 30 min and washed with PBS. After incubation with staining solution provided in a BCIP/NBT Alkaline Phosphatase Substrate Detection Kit IV (Vector Lab, USA) for 1 h at room temperature, the cells were washed with PBS and examined under an Eclipse Ti-U inverted microscope (Nikon, Japan).
Immunofluorescence (IF) staining
--------------------------------
ICM-derived colony-forming cells (ppESCs) were sub-cultured into new MEFs on glass cover slips (Marlenfeld, Germany) in 24-well culture plates (TPP, USA). After 4 days, the cells were fixed with 4% formaldehyde for 20 min at room temperature, permeabilized with 0.4% Triton X-100 in PBS for 30 min, and washed with PBS-T buffer (PBS containing 0.05% Tween-20). After incubating with blocking buffer (PBS-T buffer containing 3% BSA) for 1 h at room temperature, the cells were incubated overnight at 4℃ with each primary antibody diluted 1:100 in blocking buffer. Species reactivity of the primary antibodies had been verified in previous reports and the following primary antibodies were used: rabbit anti-octamer-binding transcription factor 3/4 (OCT3/4; Santa Cruz, USA) \[[@B19]\], goat anti-Nanog (Abcam, UK), mouse anti-stage-specific embryonic antigens (SSEA4; Millipore), mouse anti-cytokeratin-17 (CK17; Millipore) \[[@B8],[@B24]\], mouse anti-vimentin (Millipore), mouse anti-desmin (Millipore) \[[@B3]\], mouse anti-cytokeratin 8-18-19 (CK8-18-19; Abcam), mouse anti-α smooth muscle actin (SMA; Abcam), and mouse anti-microtubule-associated protein 2 (MAP2; Millipore). The cells were washed three times with PBS-T buffer and then incubated with the following secondary antibodies diluted 1:100 in blocking buffer for 1 h at room temperature: Alexa fluor 488-conjugated goat anti-rabbit IgG (Invitrogen), rhodamine-conjugated rabbit anti-goat IgG (Invitrogen), Alexa fluor 647-conjugated goat anti-mouse IgG (Invitrogen), or Oregon green 514-conjugated goat anti-mouse IgG (Invitrogen). After washing three times with PBS-T buffer, mounting solution with Hoechst 33258 (Invitrogen) was used for nuclear staining. Negative controls were produced by incubation with secondary antibody alone. The stained cells were observed under a Carl Zeiss Axiovert 200M inverted fluorescence microscope (Carl Zeiss, Germany).
Reverse transcription (RT)-PCR
------------------------------
Total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen, Germany). cDNA synthesis was performed with an RT kit (Promega, USA). DNA amplification was conducted with forward and reverse primers specific for the following factors using a Go-script PCR kit (Promega): porcine OCT3/4 (forward): 5\'-AG GTGTTCAGCCAAACGACC-3\', (reverse): 5\'-TGATCG TTTGCCCTTCTGGC-3\', porcine Nanog (forward): 5\'-ATCCAGCTTGTCCCCAAAG-3\', (reverse): 5\'-ATTTC ATTCGCTGGTTCTGG-3\', porcine GATA-binding factor-6 (GATA-6; forward): 5\'-CAGGAAACGAAAACCTAAGA GCAT-3\', (reverse): 5\'-TTCTCGGGATTAGCGCTCTC-3\', porcine neurofilament-H (NF-H; forward): 5\'-AGAGCT GGAGGCACTGAAAA-3\', (reverse): 5\'-TCCGACACT CTTCACCTTCC-3\', porcine bone morphogenetic protein-4 (BMP-4; forward): 5\'-TGAGCCTTTCCAGCAAGTTT-3\', (reverse): 5\'-CAACGCACAGATCAGGAAGA-3\', porcine glyceraldehyde 3-phosphate dehydrogenase (GAPDH; forward): 5\'-TCGGAGTGAACGGATTTG-3\', (reverse): 5\'-CCTGGAAGATGGTGATGG-3\'. PCR was performed at 94℃ for 5 min for initial denaturation followed by 27 cycles of 60℃ for 40 sec and 72℃ for 1 min before a final extension at 72℃ for 5 min. *GAPDH* was used as a reference gene. The amplified products were analyzed on 2% agarose gels and product size was confirmed with a DNA ladder.
Telomerase activity assessment
------------------------------
Telomerase activity of undifferentiated or differentiated ppESCs (ppESC1 and ppESC2) was measured using a TeloTAGGG PCR ELISA kit (Roche, USA) according to the manufacturer\'s protocol. The result was analyzed with positive control (Human embryonic kidney \[HEK\] 293 cells) and negative control (heat treated HEK 293 cells, lysis reagent and synthetic oligonucleotide).
Measurement of embryoid body (EB) formation and further differentiation
-----------------------------------------------------------------------
ppESCs were detached from the MEFs and further dissociated by incubation with 0.125% trypsin/EDTA for 5 min at 37℃. For EB formation, the single cells (approximately 3,000 cells/EB) were transferred to AggreWell400 plates (Stem Cell Technologies, USA) and then cultured for 4 days in ES-basal medium containing 10% (v/v) FBS (Hyclone) without bFGF, SCF, or LIF. Next, the EBs were transferred to culture dishes coated with 0.1% gelatin for further differentiation, and maintained in ES-basal medium including 10% (v/v) FBS without bFGF, SCF, or LIF that was refreshed every other day. After 4\~5 days of culturing, differentiated cells were used for RT-PCR analysis and IF staining.
Teratoma formation and histological analysis
--------------------------------------------
ppESCs were treated with a 10% (v/v) accutase solution (Invitrogen) in PBS (Invitrogen) for 2 min at 37℃. The cells were the separated by gently pipetting up and down five to six times. Dissociated ppESCs were neutralized after cell counting with three different solutions: PBS alone, ES-basal media containing 15% (v/v) FBS (Hyclone), and ES-basal media containing 15% (v/v) FBS (Hyclone) with bFGF, LIF, and SCF. For transplantation, 6-week-old immune-deficient NOD-SCID mice (Charles River, Japan) were maintained under non-specific pathogen-free conditions. The animals were anesthetized with 2% (v/v) isoflurane. Next, 100 µL of the ppESC solution containing 5 × 10^6^ cells combined with same volume of pre-chilled Matrigel without phenol red (Invitrogen) was directly injected into the subcutaneous region of the recipient mice using a 28.5-gauge insulin syringe. The mice were sacrificed after tumours larger than 1 cm^3^ had developed. The tumours were collected, and tumour invasion into other organs including the liver, lungs, and spleen was evaluated. For histological analysis, teratoma tissues were fixed with a 10% (v/v) formaldehyde solution for 2 days and then stained with hematoxylin and eosin (H&E). Expression of markers for the three germ layers was monitored with IF staining.
Statistical analysis
--------------------
The generalized linear model (PROC-GLM) included with SAS software was used to evaluate the effects of each treatment. Significant differences among treatments were identified according to *p* values (*p* \< 0.05).
Results
=======
Effects of FBS on blastocyst development
----------------------------------------
Blastocysts that were cultured in the IVC medium alone were poorly developed. Many had irregular blastomeres and fragments in the perivitelline area (panels A-C in [Fig. 1](#F1){ref-type="fig"}). Conversely, blastocysts maintained in the FBS (+) medium were well developed compared to those grown in the IVC medium alone during the same period (panels D-F in [Fig. 1](#F1){ref-type="fig"}). ICMs were positioned appropriately in each group of blastocysts (panel D in [Fig. 1](#F1){ref-type="fig"}). On the 6th day of IVC, blastocysts in the FBS (+) medium were more mature and had begun to hatch spontaneously from the ZP (panel E in [Fig. 1](#F1){ref-type="fig"}). On the 7th day of IVC, most blastocysts in the FBS (+) medium had matured into fully developed blastocysts and were completely hatched (panel F in [Fig. 1](#F1){ref-type="fig"}).
Effects of FBS treatment on ICM attachment rates
------------------------------------------------
Attachment rates of ICMs isolated from the FBS (+) group were higher than those of the FBS (-) group ([Table 1](#T1){ref-type="table"}, 40% *vs.* 24%). Since we observed a tendency for the FBS (+) group to spontaneously hatch relative to the FBS (-) group (data not shown), we compared the attachment efficiency of spontaneously hatched and artificially hatched blastocysts within the FBS (+) group. ICMs derived from spontaneously hatched blastocysts had a higher attachment rate than those derived from artificially hatched blastocysts ([Table 2](#T2){ref-type="table"}, 42% *vs.* 25%) and generated primitive ppESC colonies ([Table 2](#T2){ref-type="table"}, 39% *vs.* 0%). Hence, we decided to use the spontaneously hatched blastocysts cultured in FBS (+) medium for further experiments.
Effects of cytokines on ICM attachment rates
--------------------------------------------
We evaluated the effect of cytokines on ICM attachment efficiency and propagation of ICM-derived cells. For this, the cells were cultured under four different conditions ([Table 3](#T3){ref-type="table"}). The attachment efficiency of ICMs in bFGF- and LIF-supplemented medium (Medium II) was greater than that of ICMs maintained with bFGF alone (Medium I; 56% *vs.* 11%) or bFGF and SCF (Medium III; 56% *vs.* 34%). The best ICM attachment rate (82%) and greatest formation of primitive ppESC colonies were observed with the bFGF, LIF, and SCF combination (Medium IV; [Table 3](#T3){ref-type="table"}). Therefore, we selected Medium IV as the culture medium for the first ICM implantation and further culturing.
Characterization of ppESC stemness
----------------------------------
Seven ppESC lines were established from spontaneously hatched blastocysts from the FBS (+) group and maintained in Medium IV. Although the trophectoderm cells remained around the ICMs transferred onto MEFs (panel A in [Fig. 2](#F2){ref-type="fig"}), most disappeared between 8 and 12 days after implantation. The ICM-derived colony-forming cells were flat and round (panel B in [Fig. 2](#F2){ref-type="fig"}). After a second sub-passage, the colonies had clarified margins (panel C in [Fig. 2](#F2){ref-type="fig"}). AP activity was detected in all ppESC lines but no reactivity was detected for MEFs around the ppESCs (panel A in [Fig. 3](#F3){ref-type="fig"}). Additionally, all cell lines were positive for stemness markers (OCT3/4 and Nanog) by RT-PCR and IF staining (panels B and C in [Fig. 3](#F3){ref-type="fig"}). Expression of the stem cell surface marker SSEA4 was also detected (panel C in [Fig. 3](#F3){ref-type="fig"}). All of these proteins were highly expressed. Our ppESCs also had high levels of telomerase activity (panel D in [Fig. 3](#F3){ref-type="fig"}) that disappeared after differentiation. The normal porcine chromosome number, 38XY (n = 38), was found in our ppESCs (unpublished data).
Characterization of the ppESC differentiation capacity
------------------------------------------------------
Our ppESCs were able to form EBs (panel A in [Fig. 4](#F4){ref-type="fig"}). mRNA expression of markers for the three germ layers (*i.e.*, NF-H, BMP-4, and GATA6) was highly increased in the EBs. On the other hand, expression of stemness markers (OCT3/4 and Nanog) was dramatically decreased (panel B in [Fig. 4](#F4){ref-type="fig"}). The protein expression of lineage-specific cytoskeleton components (vimentin, desmin, and cytokeratin-17) was also detected in each of the differentiated cells (panel C in [Fig. 4](#F4){ref-type="fig"}).
Among all the NOD-SCID mice subcutaneously injected with ppESCs, only group C (ppESCs in the injection medium with bFGF, SCF, and LIF) produced teratomas ([Table 4](#T4){ref-type="table"}). Typical structures representing the three germ layers, perineurium of the involved nerve, blood vessels, and several tubular epithelial structures formed within the granulation tissue layer (panel A in [Fig. 5](#F5){ref-type="fig"}) were observed in all ppESC-derived teratoma tissues (panel A in [Fig. 5](#F5){ref-type="fig"}). The expression of lineage-specific markers (CK8-18-19, SMA, and MAP-2) was also confirmed (panel B in [Fig. 5](#F5){ref-type="fig"}).
Discussion
==========
ICMs that give rise to definitive fetal structures are generated during the early developmental stage of blastocysts, and ESCs originate from these cell masses \[[@B32]\]. ICM attachment onto feeder cells is the first important step for the production of ESCs from embryos \[[@B23]\]. Therefore, many researchers have explored various culture conditions to derive ICM and obtain the ESCs \[[@B30]\]. However, it is difficult to culture and maintain ICMs *in vitro*.
In previous reports, FBS was shown to exert inhibitory effects on the early cleavage of embryos, but promote the rapid development of morulae into blastocysts, yielding higher quality blastocysts \[[@B6],[@B17]\]. Moreover, the ability of blastocysts to transition into the 4-cell stage and subsequent hatching was dependent on the length of time for which the cells were cultured in FBS-free IVC medium before transfer to FBS-supplemented medium \[[@B6],[@B12]\]. In addition, when fetuses produced in FBS free-medium or medium containing BSA and medium containing FBS from morulae were transplanted into the uterus, successful implantation and further development were achieved for a higher proportion of fetuses produced from blastocysts cultured in FBS-containing medium \[[@B12]\]. Consistent with these previous reports, we also observed the positive effect of FBS on ICM attachment and ESC generation.
Several chemicals and hormones such as insulin are present in FBS \[[@B26]\]. In previous studies of ESC establishment, insulin was found to improve the development and viability of embryos \[[@B13],[@B15]\]. In addition, insulin increases the ICM cell number of blastocysts \[[@B14]\] and persistence of outgrowth of the implanted cells to generate ESCs \[[@B5]\]. Therefore, it was speculated that although we did not check the ICM cell number or hatching rate, insulin in the FBS administered during blastocyst development may have enhanced embryo viability and blastocyst development, resulting in high ppESC attachment and production rates in our study.
Recently, there have been many reports about the effects of cytokines on embryo development and ESC maintenance. FGF is needed for maintenance of human ES cells \[[@B1]\]. SCF is known to have inhibitory effects on apoptosis that are induced by treatment with Fas-L, and enhancing effects on cell recovery during embryo development *in vitro* \[[@B10],[@B11]\]. Consistent with these findings, we observed more viable embryos in SCF-supplemented media (data not shown). In addition, enhancement of cell attachment for blastocysts and bovine embryonic stem-like cells during later passages was observed when culturing with bFGF, SCF, and LIF \[[@B16]\]. Likewise, we observed the same effects on attachment of ICMs and pluripotency maintenance of long-term ppESC cultures up to passage 52.
In our study, we used Matrigel to produce teratomas in mice. Matrigel that consists of extracellular matrix proteins and growth factors has been known to enhance teratoma formation by elevating graft survival and growth rates due to endovascularization to the injection site as well as interaction between the extracellular matrix of the recipient and the injected cells \[[@B27]\]. However, teratoma formation in this study was not significantly affected by the Matrigel. Instead, the addition of bFGF, SCF, and LIF to the Matrigel-based injection medium significantly improved the teratoma formation efficiency. Although further experiments are needed to explain these effects, a more suitable environment that promotes the survival of xenogeneic cells might be achieved by co-injection of LIF, bFGF, and SCF.
In summary, we demonstrated the usefulness of FBS treatment during blastocyst development and ICM attachment. We also observed the effects of LIF, bFGF, and SCF with ES-basal medium on enhancing the attachment of ICMs as well as ppESC generation and long-term culturing. This study is the first to report teratoma formation from ppESCs delivered by injection with the three cytokines. Our data suggest that improved efficiency for ppESC generation using our technique will contribute to the investigation of porcine stem cell biology, and is a safe and reliable method for producing porcine ESCs for clinical applications.
There is no conflict of interest.
{#F1}
{#F2}
![Characterization of ppESC stemness. (A) AP reactivity was observed in ppESC line 1 at passage 18 \[ppESC1 (P18)\]. (B) mRNA expression of OCT3/4 and Nanog was confirmed by RT-PCR \[ppESC1 (P28) and ppESC2 (P33)\]. Distilled water (DW) and MEFs were used as negative controls. (C) Protein expression of OCT3/4, Nanog, and SSEA4 in the ppESCs was detected by immunofluorescence (IF) staining \[ppESC1 (P31)\]. Hoechst was used to stain the nuclei (middle panel). Merged images of Hoechst staining and signals for each stemness marker are shown in the bottom panel. (D) Telomerase activity of the ppESCs was observed. Absorbance greater than 0.2 was considered telomerase-positive. The dotted line indicates the value 0.2. Lysis reagent (LR), synthetic oligonucleotide (SO), heat-treated human embryonic kidney 293 cells (heat 293), and porcine primary keratinocytes (Kera) were used as negative controls. HEK 293 cells (293) were used as a positive control. All experiments were performed in triplicate. ppESC1 and ppESC2: undifferentiated ppESCs \[ppESC1 (P21) and ppESC2 (P25)\]. ppESC1-D and ppESC2-D: differentiated ppESCs \[ppESC1-D (P23) and ppESC2-D (P33)\]. Scale bars = 500 µm (A) and 100 µm (C).](jvs-15-519-g003){#F3}
![Characterization of the ppESC differentiation capacity *in vitro*. (A) Morphology of a representative EB on the 4th day after differentiation induction \[ppESC1 (P23)\]. (B) mRNA expression of three germ layer markers (NF-H, BMP-4, and GATA6) in differentiated ppESCs \[ppESC1 (P25)\] was confirmed by RT-PCR. DW and undifferentiated pPESCs \[ppESC1 (P32)\] were used as negative controls. (C) The expression of lineage-specific markers (vimentin, desmin, and CK17) was detected in differentiated cells \[ppESC1 (P35)\] by IF. Scale bars = 100 µm.](jvs-15-519-g004){#F4}
![Characterization of the ppESC differentiation capacity *in vivo*. (A) Representative structures of three germ layers were observed in H&E-stained ppESC-derived teratomas \[ppESC1 (P17)\]. (B) Expression of lineage-specific markers (MAP2, SMA, and CK8-18-19) was detected by IF \[ppESC1 (P19) and ppESC2 (P23)\]. Scale bars = 100 µm (A) and 50 µm (B).](jvs-15-519-g005){#F5}
######
Effects of FBS on ICM attachment rates
^\*^The blastocysts were obtained by treatment with acid Tyrode\'s solution (TA) on the 5th to 7th day of IVC. ^†^,^‡^Values within the same column with different superscript symbols are significantly different (*p* \< 0.005). All groups included four replicates.
######
Effects of the hatching process on ICM attachment rates
^\*^Formation of primitive ppESCs colonies was determined after the first passage of attached ICMs. ^†^-^‡^,^§^,^∥^Values withinthe same column with different superscript symbols are significantly different (*p* \< 0.005). All groups included four replicates.
######
Effects of cytokine-supplemented medium on ICM attachment rates
^\*^,^†^Values within the same column with different superscript symbols are significantly different (*p* \< 0.05). All groups included two replicates.
######
Teratoma formation efficiency with different injection media
All groups included two replicates except in the case of MEF injection.
[^1]: ^†^The first two authors contributed equally to this work.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
It has been estimated that almost 200 million people in Europe have disabling foot or ankle pain and that this figure will rise with aging societies and the associated increase in prevalence of chronic long term conditions \[[@B1]-[@B5]\]. Foot pain can cause loss of function, discomfort, and a general lowering of the patient\'s quality of life. Custom ankle-foot and foot orthoses are a popularly prescribed conservative treatment intended to alleviate this pain, via a number of purported mechanisms. Currently the design of these devices is largely based around capturing the foot shape using traditional techniques such as plaster casting, and determining abnormal foot function through clinical examination. These approaches may lead to variability in the prescription and restrict design choice and personalised function to simple parameters such as cushioning, support and range of motion control.
Computational modelling of the human body - ranging from the force interactions of joints to the way cells communicate with each other - has advanced significantly in the past few decades and it is now an important and useful tool for researchers and clinicians. These models provide a method of simulating and assessing interventions that are being developed, reducing the time and risk involved with trialling in humans. This approach is particularly appealing for studying foot biomechanics due to the challenging nature of directly investigating the internal loading and movements of the complex structure of bones and soft tissues of the foot that occur during gait.
A small but growing body of research, primarily based around finite element (FE) analysis, has studied the foot using models based on different combinations of gait analysis, pressure distribution measurements, computed tomography (CT) and magnetic resonance (MR) imaging of the foot. This has provided insights into inflammation of the plantar fascia \[[@B6],[@B7]\], pressure assessment of the diabetic foot \[[@B8]\] and therapeutic footwear \[[@B9]\].
Lower limb musculoskeletal biomechanics is an area where extensive modelling work has been successfully carried out, supporting the development and assessment of a range of treatments and interventions \[[@B10]-[@B13]\]. In terms of the foot however, these models have tended to represent it as a single rigid segment, and it is only recently that progress has been made in incorporating some of the intrinsic joints of the foot \[[@B14]\].
With these factors in mind, it is suggested that there is potential for highly detailed biomechanical foot models to be used in the process of designing orthotic interventions for the foot and ankle. These could lead to the development of devices and prescription paradigms which could improve the efficacy of these devices and benefit the patient, as well as reducing long term treatment costs for the healthcare provider.
Models
------
This article describes a protocol that has been developed to generate the biomechanical and anatomical data required to produce two linked foot models: a forward dynamic model that combines a multibody approach with FE analysis; and an inverse dynamic model which describes the musculoskeletal interactions of the system. The forward dynamic model will be developed using the Madymo software platform (TASS, Rijswijk), and the inverse dynamic model in the AnyBody modelling system (AnyBody Technology, Aalborg). Generally, forward dynamic models need joint torques and/or muscle forces as an input to compute kinematics, and for a complex structure like the foot this information can be generated from inverse dynamic models driven by motion capture data. Using the same dataset to construct both models leads to the possibility of a combination of both models in a final application.
### Inverse dynamic model
Standard gait analysis and inverse dynamic models of the lower extremity consider the foot as a single rigid segment. Some models have been developed that describe the foot in more detail, however to the authors\' knowledge no kinematic model has attempted to describe all 26 bones. The proposed kinematic model is scalable and parametric and will integrate into the existing AnyBody whole body musculoskeletal model (Figure [1](#F1){ref-type="fig"}). The model will contain all of the ligaments and muscles of the foot and ankle. By combing the inverse dynamic modelling with an optimisation algorithm the model will provide insight in function of the foot and leg muscles during gait.
{#F1}
### Forward dynamic model
The forward dynamic model is a combined multibody and FE approach. Bones, joints, ligaments and muscles are defined as multibody elements. This multibody model is surrounded by a freeform FE sheet, representing the skin. The soft tissue characteristics are implemented by loading functions that connect the sheet to the multibody elements. This combination leads to a computationally less complex model compared to a full FE model of the foot and ankle. This reduction could be improved by a full multibody representation of the ankle-foot complex, however in this model computation of the plantar pressure would be impossible. In the proposed model this is solved by a FE mesh representing the plantar surface, leading to the possibility of performing a dynamic simulation to compute the deforming plantar surface.
An existing model (Figure [1](#F1){ref-type="fig"}) has previously been developed from a combination of data from a post-mortem human subject and information derived from existing literature \[[@B15]\]. The generation of one complete dataset leads to a consistent model. Beyond this, the generation of datasets from several subjects creates the possibility of scaling the model. This scaling will be extended for patients requiring foot and ankle orthoses.
The overall aim of this work is to develop detailed and accurate biomechanical models of the foot and ankle which can be used to inform the design of foot and ankle orthoses by predicting the biomechanical effects of the device.
Methods/Design
==============
Study Design
------------
This study is a feasibility/pilot study to generate data that will be used to develop the biomechanical foot models described in the previous section. It is a multicentre study with data being collected at the motion capture lab of Glasgow Caledonian University (GCU) and Radiology Department of Glasgow Royal Infirmary, and at the motion capture lab of Maastricht University Medical Centre (MUMC+) and Department of Radiology at MUMC+. The data collection will take a period of six months for each centre. Data collection will last approximately four hours for each subject. The data acquisition will be independent, and after acquisition the data will be pooled and used to develop both models.
Ethical Consideration
---------------------
This study will be conducted in accordance to the Declaration of Helsinki. Ethical approval for this study has been granted by the West of Scotland Research Ethics Committee (application reference 10/S1001/24) and National Health Service Greater Glasgow and Clyde Research and Development Committee (reference GN10RH187) for the UK site. At the Dutch centre, the study was granted approval by the Medical Ethical Committee azM/UM (reference number NL31656.068.10/MEC 08-2-028).
Participants
------------
Two groups of participants will be investigated, healthy individuals and patients with foot and/or ankle problems. Healthy volunteers will be recruited via convenience sampling from staff bodies of the test centres. Participants with pathological foot problems will be informed about the study by their orthopaedic surgeon (MUMC+) or podiatrist (GCU) during the course of attending a routine appointment at a local foot and ankle clinic. Table [1](#T1){ref-type="table"} gives an overview of the study population.
######
Study population
Total Maastricht Glasgow
------------------------------------------- ------- ------------ ---------
Healthy subjects 10 5 5
Patients requiring: 15 7 8
*Pressure releasing orthotics* 7 3 4
*Alignment improving orthotics* 7 4 3
*Ankle foot orthosis for motion control* 1 0 1
### Inclusion criteria
In both groups, healthy feet and group pathological feet, participants will be included if they are physically able to walk at least 20 meters barefoot and unaided. In addition, participants must be in age group 18-50 years and have feet of size 38-44 (EUR). All participants will be fully competent and be able to give informed consent.
In addition to this general inclusion criteria, participants in the patient group must fall into one of the following categories: 1) Patients with metatarsalgia on their right foot, who would be prescribed pressure relieving foot orthoses. Metatarsalgia will be clinically diagnosed as the presence of one or more features of spontaneous pain or tenderness at one or more metatarsophalangeal joints elicited by firm pressure and/or movement of the joint. 2) Patients with flexible flat foot deformities on their right foot, who would be prescribed foot orthoses to improve alignment. For the purposes of this study, flexible flat foot is diagnosed as a correctable relaxed calcaneal stance position greater than six degrees everted with a navicular tuberosity height lower than 35 mm. 3) Hemiplegic stroke patients who would be prescribed an ankle foot orthosis on their right foot to control the motion of their ankle during gait.
### Exclusion criteria
Participants in both groups will be excluded if they have a diagnosable disease with known involvement of the lower limb and foot including, for example, diabetes mellitus, peripheral vascular disease and rheumatoid arthritis. Pregnant and lactating women will not be eligable for participation due to the radiation dose associated with the CT scans that are part of this protocol.
To be eligible for inclusion in the healthy group, participants must not currently be receiving treatment for any foot or ankle conditions or have any significant history of foot or ankle trauma, injury, fracture or dislocation.
Clarification of sample size
----------------------------
The exploratory nature of this study makes it difficult to calculate power requirements for statistical purposes. Therefore, a pragmatic approach has been taken with the number of subjects chosen by the opinion of experts to cover a broad scope of variation of foot problems. A broad range of subjects in terms of foot size, age, BMI, will be included for development of scaling methods and to test the validity of the models.
Study procedure
---------------
Participants will initially be asked to attend to the motion capture laboratory at GCU or MUMC+. Each participant will undergo a clinical foot assessment, 3D surface scanning of the foot and gait analysis. Each participant will then undergo a set of CT scans and a subset of participants, the five healthy subjects tested at GCU, will also undergo MRI scans of the foot at a nearby medical imaging facility.
The full protocol will last approximately four hours. Research teams at both institutions have extensive experience of collecting and processing the functional and imaging measurements required for this study. At GCU, the clinical assessment will be carried out by a clinician with over 20 years\' experience. At MUMC+, the clinical assessment will be carried out by a clinician with over 5 years\' experience.
Clinical foot assessment
------------------------
Each participant will first have an extended clinical foot function assessment during which range of joint motion, muscle strength, posture and impairments such as pain, stiffness and deformity will be recorded. The clinician will also assess the participant\'s ability to carry out the tasks required during the remainder of the protocol, particularly in the case of those with neuromuscular conditions. Each participant will complete the Manchester Foot Pain and Disability Questionnaire \[[@B16]\] and the foot-function index \[[@B17],[@B18]\].
3-D surface geometry
--------------------
Each participant will be asked to stand with their right foot in a 3D foot surface scanner (Easy Foot Scan; OrthoBaltic, Kaunas, Lithuania) and scans will be taken with: minimal weight on the foot (\< 5% body weight); 50% body weight on the foot; and \> 95% body weight on the foot. The participant will be asked before each scan if they are comfortable maintaining the related level of weight bearing on the foot and the scan will only be carried out if they are able to maintain this load. These scans will take approximately one minute each including positioning. Supports will be provided and a researcher will be nearby to reduce the risk of falls.
Gait analysis
-------------
Each participant will undergo a comprehensive assessment of their gait, in both barefoot and shod conditions. During gait analysis, kinematic, kinetic, electromyographic (EMG) and plantar pressure measurements from the participant\'s right foot and leg will be collected simultaneously during the stance phase of gait.
### Kinematic measurements
Kinematic data will be collected using a 12 camera Qualisys system (Glasgow) and an eight camera Vicon system (Maastricht). Residual errors of \< 1 mm are deemed acceptable for both systems.
Bony and tracking landmarks (see Table [2](#T2){ref-type="table"}) will be identified through physical palpation of the relevant areas on the foot and leg by trained researchers. Once identified, these points are indicated on the skin with non-permanent marker. Passive, reflective markers (Qualisys AB, Gothenburg, Sweden) will be attached at these points using double sided tape. The marker model used is an adapted version of that used in the multi-segment foot model described in Hyslop et al (2010) \[[@B19]\]. The model has been extended with additional markers on the thigh, hips, lesser toes and the lateral cuneiform.
######
Kinematics
Number Landmark/Location Label Name Marker Size (mm) Barefoot trials only (B) or shod and barefoot (S)
-------- -------------------------------------- ------------ ------------------- ---------------------------------------------------
1 Right iliac crest RAIC 19 S
2 Left iliac crest LAIC 19 S
3 Right posterior superior iliac spine RPSI 19 S
4 Left posterior superior iliac spine LPSI 19 S
5 Right greater trochanter RGT 12 S
6 Left greater trochanter LGT 12 S
7 Thigh 1^st^ THI1 19 S
8 Thigh 2^nd^ THI2 19 S
9 Lateral knee LKNE 12 S
10 Tibial tuberosity TTUB 7 S
11 Head of fibula HFIB 7 S
12 Shin 1^st^ SHN1 19 S
13 Shin 2^nd^ SHN2 19 S
14 Superior calcaneum SCAL 7 S
15 Inferior calcaneum ICAL 7 S
16 Medial malleolus MMAL 7 S
17 Medial calcaneum MCAL 7 S
18 Tuberosity navicular NAV 7 S
19 Proximal 1^st^met head P1MT 7 B
20 Central 1^st^met C1MT 7 (on 20 mm wand) B
21 Medial 1^st^met head M1MT 7 S
22 Lateral 1^st^met head L1MH 7 B
23 Hallux 1^st^ HLX1 7 B
24 Hallux 2^nd^ HLX2 7 B
25 Hallux 3^rd^ HLX3 7 B
26 Lateral malleolus LMAL 7 S
27 Lateral calcaneum LCAL 7 S
28 Cuboid CUB 7 B
29 Proximal 5^th^met P5MT 7 S
30 Distal 5^th^met D5MT 4 S
31 Intermediate cuneiform ICUN 7 B
32 Lateral cuneiform LCUN 7 B
33 2^nd^met head D2MT 4 B
34 3^rd^met head D3MT 4 B
35 4^th^met head D4MT 4 B
36 2^nd^proximal phalanx D2PP 4 B
37 3^rd^proximal phalanx D3PP 4 B
38 4^th^proximal phalanx D4PP 4 B
39 5^th^proximal phalanx D5PP 4 B
40 2^nd^distal phalanx (on nail) D2DP 4 B
41 3^rd^distal phalanx (on nail) D3DP 4 B
42 4^th^distal phalanx (on nail) D4DP 4 B
43 5^th^distal phalanx (on nail) D5DP 4 B
Overview of the kinematic marker set
For the shod trials, the participants are provided with standardised footwear (Flextop Diabetic Black shoes, Reed Medical, Blackburn, UK). A number of the foot mounted markers used in the barefoot trials will be removed for the shod trials (see Table [2](#T2){ref-type="table"} for details), and holes are cut into the shoes to allow the remaining markers to be visualised by the motion capture system and to move during walking without interference.
### Kinetic measurements
Kinetic measurements will be taken at both centres using Kistler force plates (Kistler Instrument Corp., Amherst, NY) synchronised with and recorded through the QTM software (GCU) or Nexus software (MUMC+) at a frequency of 2400 Hz.
### Electromyographic measurements
All parts of the protocol relating to surface EMG measurement will be carried out in accordance with the guidelines produced by the Surface Electromyography for the Non-Invasive Assessment of Muscles (SENIAM) project \[[@B20]\]. These guidelines cover the location and orientation of electrode placement, skin preparation and signal tests for each muscle.
Trigno wireless EMG systems (Delsys Inc, Boston, MA) will be used to collect the EMG measurements at both centres. The electrode units will be attached to the following muscles: tibialis anterior, gastrocnemius medialis, gastrocnemius lateralis, soleus, peroneus longus, vastus lateralis, rectus femoris, and biceps femoris. Signals from each muscle will be checked in real time using EMGworks software (Delsys Inc, Boston, MA) while performing the exercises described in the SENIAM guidelines.
Reference measurements will be taken for each muscle in the form of maximal voluntary isometric contractions (MVICs). Measurements will be recorded for five seconds in total with the participant being asked to gradually build up the force they apply over the first two seconds, and maintain their maximum effort for the remainder of the contraction. Each contraction is repeated three times in a non-consecutive randomised order with at least one minute recovery time between exercises.
For the MVIC and gait components of the testing, EMG signals from the Trigno sensors will be recorded through the analogue channels of QTM or VICON software at a frequency of 2400 Hz.
### Plantar pressure measurements
For barefoot trials, plantar pressure measurements will be collected using a 0.5 m Footscan^®^plate (RSscan International, Lammerdries, Belgium) recording at 500 Hz. The plate is mounted directly on top of the force plate and secured in place using double sided tape. The effect of this setup on the accuracy of the force plate was assessed using the CalTester^®^quality assurance tool (C-Motion Inc, Germantown, MA) and errors were found to be within acceptable limits. To ensure high levels of accuracy in the pressure measurements, the pressure plate is dynamically calibrated with the vertical force signals from the force plate.
In-shoe plantar pressure measurements will be made using the Pedar^®^system (Novel, GmbH, Munich, Germany) at 50 Hz. In addition, pressure under the sole of the shoe will be recorded during these trials using the Footscan^®^plate.
### Testing
A static trial will be recorded with the participant in a relaxed standing pose in the motion capture area. The participant will then be asked to walk barefoot at a self selected speed along the motion capture area such that their right foot strikes the centre of the pressure plate. Five successful walking trials will be recorded. This will then be repeated for the shod trials.
Imaging
-------
### Computed tomography (CT)
CT scans of the leg and foot will be undertaken in hospital radiology centres. Each participant will undergo four scans under a variety of different conditions. First the knee, shank and foot will be scanned with the participant asked to apply only minimal loading to the plate with the foot in a neutral position (90 degrees flexion). Then three scans imaging the foot and ankle only will be taken in a randomised order: foot loaded to 50% body weight with the foot in the neutral position; foot loaded to 50% body weight with the foot 25° of plantar flexion; 50% body weight with 10° of dorsiflexion.
To allow the participant to apply force on the foot while being scanned, a novel loading rig was developed and fabricated (Figure [2](#F2){ref-type="fig"}). Previously, several investigators have described similar systems (14) but to the authors\' knowledge this is the first device that allows the position of the foot in relation to the lower leg to be easily and quickly manipulated.
{#F2}
The loading rig takes the form of a chair fixed on to a plywood base plate and a plate for the participant to push against with their foot at the other end. The nature of CT means that metallic objects can cause interference in the image, an effect known as scattering. To avoid this, the steel components which allow the loading plate to be repositioned are kept behind the scan plane. The loading plate can be easily moved closer to or further from the chair at fixed 20 mm intervals to suit the participant\'s leg length. A standard bathroom scale with a large LED display is mounted on the loading plate and is used to provide feedback to the participant on the level of loading being applied. The relatively short time frame of the CT scan (5-10 seconds) allows medium level loads to be applied and maintained over its duration. During the scans, a Pedar pressure insole (Novel, GmbH, Munich, Germany) is placed between the foot and the loading plate. In addition, using the pen marks made on the foot to guide placement, radiopaque markers (4 mm diameter Beekley Spots^®^, Oncology Imaging Systems, East Hoathly, UK) are attached at the same points as the motion capture markers during gait analysis.
The following parameters were used to acquire the four scans on an Aquilion 64 slice scanner (Toshiba, Tokyo, Japan) at the Glasgow centre or a Brilliance 64 slices (Phillips, Amsterdam, Netherlands) at Maastricht: 120 kVp, 100 mAs, 1.0 mm collimination, 1.0 mm effective slice thickness, pitch factor 41, rotation time 0.5 seconds, B30S medium smooth reconstruction kernel, 512 × 512 matrix.
### MR
Due to the limitations of soft tissue information that can be inferred from CT imaging, a subset of five participants will also have MR scans taken of their right foot. This will take place on a different day to the rest of the testing, but within six weeks of the initial assessment.
For the MR scans the foot is placed in a suitable imaging coil and foam padding is placed around the foot to prevent movement during the scan. Images will be acquired using a 3T system (Siemens Verio; Erlangen, Germany). Three scans will be taken in total, a T2-weighted scan covering the full foot and ankle, and two T1-weighted scans, one of the rearfoot/midfoot complex and one of the forefoot/midfoot complex.
Scanning parameters for the T2 scan (trueFISP 3D volume) are: repetition time, 9.8 ms; echo time, 4.92 ms; flip angle, 35°; field of view, 290 mm; slice thickness, 0.6 mm (no slice gap); slices per slab, 144; matrix, 256 × 216 (interpolated); phase encoding, anterior to posterior; number of averages, 2.
Scanning parameters for the T1 scan (Space 3D volume) are: repetition time 700 ms; echo time 22 ms; flip angle 105°; field of view, 150 mm; slice thickness 0.8 mm (no slice gap); slices per slab, 94; matrix 320 × 290 (interpolated); phase encoding anterior to posterior; averages, 2.4.
Data Processing
---------------
### Gait analysis data
The kinematic data, the kinetic data and the plantar pressure will be processed by using Nexus (Vicon, Oxford, UK) software (Maastricht) or Qualisys Track Manager (Qualisys, Gothenburg, Sweden) software (Glasgow) and saved in the C3D format.
Kinematic and kinetic measurements will be used as an input for the inverse dynamic model and to validate the forward dynamic model. The dataset of one healthy subject will be used to develop the first model. Afterwards the datasets of the other healthy subjects will be used to refine the initial model, develop kinematic and kinetic rules and a morphing-based scaling facility so the models can be personalised. After the development of the morphing algorithm the combination of this algorithm and the two models will be used to predict the effect of insoles.
Data from the foot assessments in combination with the 3D surface scans will be processed to develop an algorithm for personalisation of the models with a non invasive, low-end method. It is intended to investigate if an algorithm can be developed to drive the musculoskeletal model directly from plantar pressure measurements, allowing the kinematic parameters of the lower extremities to be determined without the need for a full motion capture system.
### Imaging data
CT data will be segmented into the individual bones of the foot using Mimics (Materialise, Leuven, Belgium) image processing software. The CT-data will be used to compute the joint axes of the foot, by correlating the positions of the bones in the various positions. The loaded CT-data in combination with the pressure measurement will be used to gain insight in the soft tissue characteristics of the foot. Insertion and via points will be identified from partial segmentation of the MR data and described as co-ordinates on the bones segmented from the CT data. Insertion points will be defined as the centre of the area of insertion.
Data Analysis
-------------
Initial validation of the inverse dynamic model will be carried out by visual inspection of predicted and measured (via EMG) muscle timings. Formal validation of both models will be carried out by comparing plantar pressure measurements taken during gait analysis to those predicted by the forward dynamic model. Intraclass correlation coefficients (ICC~2,\ k~) will be used to compare peak and average pressures at the hallux, the lesser toes, each of the metatarsal heads, the midfoot region, and the lateral and medial heel. Future studies will investigate the validity of the models for predicting biomechanical changes induced by orthoses.
Discussion
==========
The primary aim of this study is to collect data for the development of two biomechanical foot models. A single dataset that includes a clinical foot assessment, gait analysis and imaging data has, to the authors\' knowledge, not been combined previously for the generation of a biomechanical foot model \[[@B21]\]. The geometry of existing FE models tend to be based on an MRI \[[@B22]\] or CT dataset \[[@B23]\] for one subject, while material properties are obtained from the literature and describe averages of larger groups of subjects. Cheung et al \[[@B24]\] have used an FE model to simulate several phases of the gait cycle, deriving boundary conditions for these static simulations from EMG and ground reaction force measurements.
To the authors\' knowledge, multibody models of the foot describing the level of detail proposed here have not previously been attempted. An inverse dynamic model with three segments has been developed by Saraswat et al. \[[@B14]\], however the anatomical information was derived from literature, making it impossible to produce a personalised model.
Various FE models have been used in combination with insole models \[[@B25]-[@B32]\]. These models have been developed in varying complexity, from 2D FE simulations of the second ray \[[@B26]\] to the inclusion of nonlinear material properties \[[@B27]\]. These studies are mainly parametric studies in which several properties of the insole are simulated in a static simulation of the mid-stance phase. Variations can be made in geometry \[[@B27]\] or material properties \[[@B25]\], or a combination \[[@B28]\]. The computational complexity of highly detailed FE models however, means that only a static simulation can be run.
A driving input of the models that will be developed in this study is the kinematic data. This data is acquired by a set of skin mounted markers. Markers are positioned on bony-landmarks, taking into account the influence of muscles, tendons and ligaments on skin motion. Previously reported bone pin studies show the distinction between the movements of the skin markers and the bones \[[@B33]\]. This distinction has two causes: soft tissue movement \[[@B34]\] and the rigid body violation \[[@B35]\]. The latter point is solved by introducing a 26 segment model. The problem of soft tissue movement is partially addressed by performing loaded CT with radio-opaque markers and having the option to include direction dependent weighting factors in the AnyBody model. A bone pin study involving all bones would be difficult due to the small size of the bones. In addition, the confounding effect of this invasive method on the motion pattern of gait is not known.
Validation of the models is partly performed by comparison of the muscle activity patterns predicted by the inverse dynamic model to those recorded during gait. EMG can be acquired by surface electrodes or by intramuscular measurements. Both methods have advantages and disadvantages. In this study surface EMG is used to minimise the influence of the measurements on normal gait as EMG is not used as an input for the model, only as a validation tool.
During the motion analysis force measurements are performed in 3D by a force plate and in one dimension by the plantar pressure plate. The force plate measures a global force vector of the ground reaction force, averaged in space. A pressure plate measures the individual vertical component of the ground reaction force over the full plantar surface. Ideally these measurements should be combined, yielding a high resolution measurement of three dimensional force vectors. Attempts have been made to develop this type of device, however no suitable and validated system is currently commercially available.
Weight bearing CT scans are performed with approximately 50% body weight. This is lower than would be encountered during normal gait, however the restrictions of the scanner mean that the participant needs to be in a sitting position so that the full foot and ankle can be imaged \[[@B36]\]. Commean et al \[[@B8]\] have demonstrated the reliability of weight-bearing CT in a sitting position.
Despite these limitations, the protocol will produce unique datasets consisting of detailed anatomic and dynamic measurements. These will be used to develop scalable biomechanical foot models to improve understanding of foot function and to attempt to predict the effect of foot and ankle orthotic design so that the design of these devices can be optimised prior to manufacture, potentially removing some of the variability in form and function that is seen with currently prescribed devices. Driving the models using dynamic plantar pressure rather than kinematic measurements could make this approach particularly useful and accessible for clinicians, avoiding the cost and time associated with full motion capture analysis. If validation of the models is successful, the next step will be to run a clinical trial to test if the use of the model for the development of foot and ankle orthoses leads to improved efficacy.
Competing interests
===================
Tørholm is joint founder of AnyBody Technology, a commercial developer of biomechanical modelling software. It is planned that the foot model will be made available through the company\'s repository of biomechanical models. SC is employed by AnyBody Technology.
Authors\' contributions
=======================
MO and Telfer will be responsible for data collection and prepared the initial draft of this manuscript. The original concept for this study was conceived by JW, Tørholm and LWvR as part of the A-FOOTPRINT project. SC has been responsible for the coupling of the modelling with the inverse dynamic model. RM has contributed to the imaging protocol. KM has contributed to the gait analysis protocol. All authors contributed to the development of the protocol and approved the final draft of this manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2474/12/256/prepub>
Acknowledgements and Funding
============================
This work is being funded through the European Commission Framework Seven Program (grant number NMP2-SE-2009-228893) as part of the A-FOOTPRINT project (<http://www.afootprint.eu>). The Easy Foot Scan has been supplied by OrthoBaltic, the Footscan^®^plate by RSscan International, and the Mimics software by Materialise. These companies are partners in the project but did not have any input into the design of this protocol.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s0005}
============
In 1947, Bosworth \[[@bb0005]\] published a report of 5 cases of an unusual ankle fracture-dislocation. In all of the cases, the proximal fibular shaft fragment was locked behind the posterior tibial tubercle. Bosworth fracture\'s diagnosis usually goes unnoticed, even after a complete radiological study \[[@bb0010],[@bb0015]\]. Many published studies have stated the inability to visualize the retrotibial position of the proximal fibular shaft fragment until the surgical act \[[@bb0020]\].
Bosworth injury is known as an uncommon cause of closed reduction failure in ankle fracture-dislocations. Previous studies have described that repeated attempts of close reduction in these patients may not only increase patient\'s discomfort, but also enlarge tissue and cartilage damage, as well as worsen clinical outcomes in the medium-long term \[[@bb0025], [@bb0030], [@bb0035]\].
In this case report, we present the first published case of a Bosworth injury in a pregnant woman. Our aim is to review this infrequent pathology, as well as its injury mechanisms, radiological characteristics and treatment.
Case report {#s0010}
===========
A 34 year-old woman, who was 32 weeks pregnant, was seen at the emergency room for hitting her left ankle after falling down the stairs. As her personal medical history, she suffered from focal epilepsy controlled by the Neurology Department since she was a teenager. During the clinical exam, important deformity with severe swelling and pain in both malleolus was observed. The neurovascular status of the foot was intact.
In the initial radiographs, the lesion was diagnosed as a left trimalleolar fracture-dislocation ([Fig. 1](#f0005){ref-type="fig"}). An attempt to reduce the ankle dislocation was made and a long leg plaster cast was applied afterwards. In the posterior radiological control ([Fig. 1](#f0005){ref-type="fig"}), we observed that the closed reduction had been unsuccessful. Since immediate surgery was an option, we decided not to attempt any more closed reductions, and we got the patient ready for an urgent surgical procedure.Fig. 1A) Initial anterior-posterior (AP) and lateral (L) radiographs. Trimalleolar ankle fracture dislocation. The study has been altered by the patient\'s incapacity to maintain an adequate position. B) Control radiograph after the first closed reduction attempt. As we can observe, there\'s a persistent incongruity and the components of the fracture haven\'t moved from their original position, although classic reduction techniques have been performed.Fig. 1
The surgical procedure and its possible complications, both for the patient and her fetus, were explained to the patient. The patient accepted the risks, signed the informed consent and 2 g of intravenous cefazolin were prescribed as antibiotic prophylaxis.
Under spinal anaesthesia and left lower limb ischemia of 250 mm Hg, the patient was positioned in supine decubitus with both posterior and anterior abdominopelvic X-ray apron. Initially, we attempted a last closed reduction under fluoroscopic control ([Fig. 2](#f0010){ref-type="fig"}), with an unsuccessful result, so we performed an open reduction. In the first place, a lateral approach to the fibula was used, frankly observing the tibial facet of the distal tibiofibular joint ([Fig. 2](#f0010){ref-type="fig"}), as well as the complete breakage of both posterior and anterior syndesmoses and the displacement of the proximal fibular shaft fragment behind the posterior tibial tubercle. To achieve its reduction, a vigorous "lever mechanism" over the proximal fibular fragment was applied to unlock it from its retrotibial position.Fig. 2A) Fluoroscopic images after the second closed reduction attempt in the operating theatre. Unsuccessful reduction. B) Articular surface of the distal tibiofibular joint (intermediate thickness arrow). Proximal fibular shaft entrapped behind the tibia (thick arrow). Talus (thin arrow).Fig. 2
Once the fracture was reduced, an internal fixation with one cortical interfragmentary screw and a seven-hole one-third tubular plate was performed. Since the distal tibiofibular syndesmosis was found unstable, we also used a tricortical syndesmotic screw. Once the fibular fracture was stabilized, the tibial malleolus fracture was spontaneously reduced, not being necessary to perform an open reduction, and it was fixed using two cannulated screws. After checking its stability under the fluoroscope, both the syndesmosis and the anterior capsule were sutured.
After the surgery, the ankle joint was immobilized using a long leg plaster cast. The postoperative period was uneventful and the patient was discharged from the hospital the following day. Ten days after the surgery, the patient came for a clinical review to our medical office, where we removed the plaster cast and checked the good state of the wound and soft tissues, starting flexion-extension non-weight exercises and sending the patient to the Rehabilitation Unit to start physiotherapy. Before coming back to our office for the second time, the patient was in hospital in the Obstetrics and Gynaecology Unit where she underwent an elective caesarean section at 37 weeks. The postoperative period was positive.
Six weeks after the surgery, slight inflammation and pain persisted, mostly during bipedestation. Due to the patient\'s situation (breastfeeding), we decided not to remove the transyndesmal screw, and we allowed partial load of the ankle using walking sticks and Calm-Walker like orthosis. Three months after the surgery, the patient\'s mobility had improved, being able to walk using just one stick, with a pain decrease and the radiograph didn\'t show any differences compared to the previous one ([Fig. 3](#f0015){ref-type="fig"}). Clinically, the patient referred scar dysesthesia, a 10° dorsiflexion limitation and felt mild discomfort around the fibular osteosynthesis. A conservative treatment was decided in the meantime and to carry on with the rehabilitation treatment. Nowadays, eighteen months after the surgery, the patient presents a slight dorsiflexion limitation, has no limp or functional limitation to carry out her daily life, and has been back to work with no further problems.Fig. 3A) X-ray image four weeks after surgery. B) Control radiograph one year after the surgery. We can observe fracture consolidation with good joint congruence.Fig. 3
Discussion {#s0015}
==========
The most frequent cause of irreducibility in ankle fracture-dislocations is the interposition of soft tissues in between fragments. In 1947, Bosworth described a less frequent cause, characterized by the lock of the proximal fibular shaft fragment behind the posterior tibial tubercle. Since the 1950s, more than 60 cases of this pathology have been published, with their own particularities. Recently, Bartonicek et al. \[[@bb0015]\] published a report of six cases in which a patient presented an intact fibula associated to a retrotibial dislocation. Taking into account the specifics of our case, it\'s well known that, during pregnancy, women undergo weight, shape and hormonal changes that are associated to musculoskeletal consequences, including an increase of lumbar lordosis and joint laxity, which could consequently lead to failure and instability of weight-bearing joints (hip, knee and ankle) \[[@bb0040]\]. On the other hand, fluoroscope is needed to perform surgery. It\'s well known that radiation injuries depend on the dose of exposure and the gestation stage in which the exposition occurs \[[@bb0050]\]. Our patient was on the third trimester of pregnancy, so the fetal risk of radiation damage was very low. Nevertheless, a collimator was used to minimize the use of fluoroscopy as well as a double shield to protect the woman.
So far no radiological persistent characteristics have been described that could be helpful for the surgeon to recognise the nature of the injury. Therefore, we think that radiological signs of this lesion aren\'t ease to identify, but there are some clues that could put us on track to diagnose this injury: an increase in the medial joint space, an overlap of the proximal fibular fragment over the distal tibia in the anteroposterior projection and a posterior displacement of the fibula in the lateral projection \[[@bb0055]\]. Khan and Borton introduced a new radiological sign that could be used as an indicator in Bosworth injuries: the "Axilla sign" \[[@bb0060]\]. This sign was described as a cortical density in the axilla of the medial tibial plafond because of the medial permanent rotation of the tibia. To avoid difficulties understanding the configuration of the fracture, many authors support the use of a computerized tomography (CT) \[[@bb0030],[@bb0035]\]. In our case, a series of factors led to urgent surgery: the impossibility to achieve a close reduction, our patient\'s pregnancy, the availability of an operating theatre and a positive preoperative exam.
Bosworth fracture is characteristically irreducible using closed techniques, owing to a fixed dislocation of the proximal fibular fragment posterior to the lateral ridge of the tibia. Hence, an early open reduction is necessary to minimize further complications. During surgery, it is convenient to apply a vigorous "lever mechanism" between tibia and fibula in order to achieve the reduction of the distal tibiofibular joint. Because of the characteristics of this lesion and the instability risk, we decided to use a tricortical transyndesmal screw to protect the breakage of the interosseous membrane. Many authors recommend the removal of the transyndesmal screw 6--8 weeks after the surgery to allow progressive weight-bearing of the operated limb \[[@bb0010],[@bb0015]\]. In our case, surgery to remove the transyndesmal screw was proposed to the patient who, due to her concomitant condition (breastfeeding), decided to postpone it.
Complications such as cutaneous necrosis, superficial infection, compartimental syndrome, avascular necrosis of astragalus, joint stiffness, lesion of the superficial fibular nerve and secondary arthrosis, can appear more frequently in the Bosworth injury than in any other type of ankle fracture-dislocation. The delay for surgical reduction or repetitive close reduction attempts are risk factors for obtaining poor results and a higher risk of complications \[[@bb0045]\].
In conclusion, Bosworth injury is an uncommon condition that should be taken into account in the differential diagnosis of irreducible ankle fracture-dislocations. It is often misdiagnosed in initial radiographs, usually going unnoticed until the surgical act. In order to minimize complications and to obtain a good functional result, an early diagnosis and open reduction is very important, as well as to limit the number of closed reduction attempts.
Declaration of competing interest
=================================
The authors declare no conflict of interest.
| {
"pile_set_name": "PubMed Central"
} |
**(See the major Article by Suarez et al, on pages 781--91.)**
Human cytomegalovirus (HCMV) is a major coinfection in human immunodeficiency virus (HIV)--infected people, in whom, as in other immunocompromised individuals such as transplant recipients, it contributes to morbidity and mortality. HCMV is also the most frequent congenital infection, causing adverse neurodevelopment, including hearing loss, microcephaly, and neonatal morbidity. Postnatal infection generally occurs via milk in breastfeeding populations and is usually asymptomatic. However, it has been linked to morbidity, especially in preterm or underweight infants, and, in recent population studies, to adverse developmental effects, especially in association with HIV exposure in developing countries \[[@CIT0001]\]. The most severe HCMV infections in transplant recipients, whether due to primary infection, reinfection, or reactivation from latency, can result in severe or end-organ diseases such as retinitis, pneumonitis, hepatitis, and enterocolitis \[[@CIT0005]\]. Few studies of HCMV diversity, transmission, and epidemiology have been conducted in relation to developing countries, including those having a high burden of endemic HIV.
HCMV has a double-stranded DNA genome of 236 kbp containing at least 170 protein-coding genes \[[@CIT0006]\]. Diversity among strains is low overall, except in several hypervariable genes that exist as distinct, stable genotypes. These genes encode proteins that are particularly vulnerable to immune selection, including virus entry glycoproteins, other membrane glycoproteins, and secreted proteins. The recombinant nature of HCMV strains was first identified in serological surveys and then in genomic studies, and is a key consideration for vaccine development \[[@CIT0007]\]. However, understanding the pathogenic effects of HCMV diversity is at an early stage \[[@CIT0017]\], and is limited by the fact that most analyses have focused on a few hypervariable genes characterized by polymerase chain reaction (PCR)--based genotyping \[[@CIT0007], [@CIT0012], [@CIT0020]\]. This approach is relatively insensitive to the presence of minor strains in multiple-strain infections, which may have more serious outcomes.
High-throughput sequencing studies at the whole-genome level have started to facilitate a broader view of HCMV diversity, but most have involved isolating the virus in cell culture, which is prone to strain loss or mutation, or have depended on direct sequencing of PCR amplicons generated from clinical samples \[[@CIT0011], [@CIT0014], [@CIT0016], [@CIT0021]\]. Recent studies have avoided these limitations by using target enrichment to enable direct sequencing of strains present in clinical samples, most of which originated from patients in developed countries with congenital or transplantation-associated infections \[[@CIT0011], [@CIT0022]\]. Here, we use this and new methods to examine HCMV strain diversity in a developing country by analyzing breast milk from women in Zambia, who constitute an HIV-endemic population in sub-Saharan Africa, where we have previously demonstrated the negative developmental effects of early infection of infants with HCMV, particularly alongside HIV exposure \[[@CIT0001], [@CIT0003]\].
METHODS {#s1}
=======
Patients and Samples {#s2}
--------------------
Anonymized breast milk samples were collected with informed consent as a substudy of the Breast Feeding and Postpartum Health study conducted at the University Teaching Hospital, Lusaka, Zambia, as approved by the ethical committees of the University Teaching Hospital and the London School of Hygiene and Tropical Medicine. This substudy included 28 HCMV-positive breast milk samples donated from one or both breasts by 15 HIV-infected and 7 HIV-negative mothers at 4 and/or 16 weeks postpartum ([Table 1](#T1){ref-type="table"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 3--6\]) \[[@CIT0003]\].
######
Characteristics of Donors, Samples, and Datasets
Donor^a^ HIV Status Breast Sample Weeks Postpartum HCMV Load, ge/mL^b^ Dataset Strains^c^ Detected
---------- ------------ --------------- ------------------ --------------------- ---------------- ---------------------
158 Negative Left 16 818 244 158L16 2
166 Negative Left 16 282 252 166L16 1
193 Negative Right 16 215 217 **193R16** ^d^ **1**
232 Negative Right 4 470 150 232R4 2
239 Negative Right 4 4 752 875 239R4 1
263 Negative Left 4 5 285 775 263L4 1
280 Negative Right 4 7 505 536 280R4 1
141 Positive Right 16 319 888 **141R16** ^e^ **2**
154 Positive Left 16 2 195 856 154L16 2
173 Positive Right 16 349 532 **173R16** ^d^ **5**
**174** Positive Left 16 371 027 **174L16** ^d^ **3**
**174** Positive Right 16 642 516 **174R16** ^d^ **3**
181 Positive Left 4 1 972 365 181L4 2
**243** Positive Left 16 643 895 **243L16** ^e^ **2**
**243** Positive Right 4 65 511 020 **243R4** ^d^ **1**
**243** Positive Right 16 795 092 **243R16** ^d^ **1**
248 Positive Right 4 441 679 **248R4** ^d^ **1**
258 Positive Right 4 610 613 **258R4** ^d^ **2**
**259** Positive Left 16 2 366 193 **259L16** ^d^ **3**
**259** Positive Right 16 5 053 047 **259R16** ^d^ **3**
264 Positive Left 16 388 519 **264L16** ^d^ **2**
277 Positive Right 16 250 377 **277R16** ^d^ **2**
**278** Positive Left 16 3 751 776 **278L16** ^d^ **2**
**278** Positive Right 4 294 246 272 **278R4** ^d^ **2**
**278** Positive Right 16 4 370 800 **278R16** ^d^ **2**
281 Positive Right 4 20 291 530 **281R4** ^d^ **2**
283 Positive Right 16 274 391 **283R16** ^d^ **1**
288 Positive Right 4 31 574 022 **288R4** ^d^ **3**
Abbreviations: HCMV, human cytomegalovirus; HIV, human immunodeficiency virus; ge, genomic equivalent.
^a^Donor IDs in bold and underlined are sequential or from paired tissue samples.
^b^Median loads are higher in HIV-positive compared to negative and also in week 4 compared to week 16 as shown previously \[[@CIT0003]\].
^c^Number of strains detected are from [Table 3](#T3){ref-type="table"}, only those meeting quality thresholds noted are in bold, with the original data from the [Supplementary Tables 1](#sup1){ref-type="supplementary-material"} and [3](#sup3){ref-type="supplementary-material"}.
^d^Met all quality thresholds.
^e^Met all quality thresholds except that unique fragment coverage depth was 10--20 rather than ≥20 reads/nt.
DNA Extraction and Viral Load Quantification {#s3}
--------------------------------------------
DNA was extracted from 200 µL breast milk using a QIAamp DNA mini kit (Qiagen), and viral DNA load measured using an HCMV gB TaqMan assay on an Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems), as described ([Table 1](#T1){ref-type="table"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 7\]) \[[@CIT0003]\].
High-Throughput DNA Sequencing {#s4}
------------------------------
The SureSelect version 1.7 target enrichment system (Agilent) was used to prepare sequencing libraries ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 8--10\]) \[[@CIT0022]\]. These were sequenced using a MiSeq (Illumina) with version 3 chemistry generating original datasets of paired-end reads of 300 nucleotides (nt) ([Table 1](#T1){ref-type="table"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 11--12\]).
Phylogenetic Analysis {#s5}
---------------------
UL73 and UL74 genotypes \[[@CIT0007], [@CIT0012]\] were investigated in 243 different HCMV strains with complete genome sequences available \[[@CIT0024]\]. MEGA 6.06 \[[@CIT0025]\] was used to generate muscle-derived amino acid sequence alignments and phylogenetic trees based on the Jones--Taylor--Thornton model and discrete gamma distribution with 5 categories.
Strain Characterization Using Sequence Motifs {#s6}
---------------------------------------------
Original datasets were quality-checked and trimmed using Trim Galore (<http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/;> length = 21, quality = 10 and stringency = 3) ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 13\]). Bowtie2 \[[@CIT0026]\] was used to remove reads mapping to the Genome Reference Consortium Human Reference 38 sequence, quality-checked and trimmed to create purged datasets ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 14\]). Dataset quality parameters were set on thresholds described in the Results ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 19--23\]) \[[@CIT0024]\].
The number of genotypes was analyzed by counting reads containing conserved, genotype-specific sequence motifs or their reverse complements. One short motif (14 nt) for each UL73 genotype and 3 short motifs (12--13 nt) for each UL74 genotype were identified by initially examining nucleotide sequence alignments and polymorphism plots derived from the 163 HCMV complete genome sequences in GenBank Release 211 (15 December 2015). Motif conservation was confirmed in the 243 genome set as described \[[@CIT0024]\] plus 383 UL73 and 72 UL74 single-gene sequences available in GenBank, which originated from various tissues, including milk (the UL73/74 single gene set only), and various locations worldwide, including Zambia (single gene set only) \[[@CIT0003], [@CIT0007], [@CIT0012]\]. The sequences of the short motifs are listed in [Table 2](#T2){ref-type="table"} with their frequency of occurrence.
######
Short Motif Sequences in UL73 and UL74
Gene Position^a^ Genotype Motif Sequence^b^ Sequences, No.^c^ Occurrences, No.^c^ Frequency, %^c^
------ ------------- ---------- ------------------- ------------------- --------------------- -----------------
UL73 5′ G1 GCGTATCAACTACC 121 121 100
G2 GTGTGTCGACGAGT 53 53 100
G3A GCGTGTCAACAAGC 104 104 100
G3B GTGTATCAACGGTA 47 47 100
G4A GCACCTTAACAACC 114 113 99
G4B ACACCTCAACGACC 55 55 100
G4C GCACCTCAACAACC 39 38 97
G4D ACGCCTCAACAACC 93 92 99
UL74 5′ G1A AAACGACWATTT 47 43 91
G1B AAAAGGATATCT 60 60 100
G1C AAAGGGAACCTT 19 19 100
G2A AACCTATTCCTT 27 27 100
G2B AGAGCGACATAT 38 38 100
G3 CGAGCCAGGATT 66 64 97
G4 AAACAGGTGATT 19 19 100
G5 TGTCTACATCAT 38 38 100
UL74 C G1A CCTTGTGGTACTG 47 47 100
G1B TCTTGCGGTACGG 60 60 100
G1C TCTTGTGGTACAG 19 19 100
G2A TCGTGTGGCGCAG 27 27 100
G2B CCTTGCGGTACAG 38 38 100
G3 TCTTGTGGCACTG 66 66 100
G4 TCCTGTGGYACGA 19 19 100
G5 CCTTGYGGCACAG 38 38 100
UL74 3′ G1A TATTACTACCGCC 47 47 100
G1B TGTTACTACCACC 60 60 100
G1C GGTTACCACCAGC 19 19 100
G2A TGTTACCACCACC 27 27 100
G2B TGTTACAACCACC 38 38 100
G3 TGCTACCACCACT 66 66 100
G4 TCCTATTGTCCCA 19 19 100
G5 TGCTACCGCTGCT 38 38 100
^a^5′, toward the 5′ end of the protein-coding region; C, in the central part of the protein-coding region; 3′, towards the 3′ end of the protein-coding region; in reference strain Merlin, the UL73 5′ motif is located at 104--117 nucleotides (nt) in UL73 (408 nt; G4D), and the UL74 5′, C and 3′ motifs are located at 206--217, 443--454, and 906--918 nt, respectively, in UL74 (1419 nt; G5).
^b^UL73 and UL74 are transcribed rightward and leftward, respectively, in the human cytomegalovirus genome; the sequences are presented 5′-3′ in relation to the direction of transcription; international union of pure and applied chemistry nucleotide codes are used.
^c^The total number of sequences in the 243 genome set plus single-gene sequences, followed by the number and percentage of these sequences possessing the motif; one UL74 intergenic recombinant (BE/23/2010) was excluded. This provides a measure of motif sensitivity.
In addition to counting short motifs ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 25--56\]), long motifs (20--24 nt) at one per genotype were counted in UL73 and UL74 and a further 10 hypervariable genes (RL5A, RL6, RL12, RL13, UL1, UL9, UL11, UL120, UL146, and UL139) ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 58--166\]) \[[@CIT0024]\]. Long motifs identifying common gene-disrupting mutations in 3 genes (RL5A, UL111A, and US9) \[[@CIT0024]\] were also counted ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 167--174\]). Strain numbers were estimated from long motif counts using thresholds described in the Results ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 17\]).
Variant Analysis {#s7}
----------------
Replacement of one strain by another as the major population (genotype switch) in compartmental or longitudinal samples from the same individual, were investigated by variant analysis \[[@CIT0021], [@CIT0027]\]. The original datasets were quality checked using FASTQC (<http://www.bioinformatics.babraham.ac.uk/projects/fastqc/>), trimmed to not less than 100 nt using Trimmomatic \[[@CIT0028]\], optimized using VelvetOptimiser parameters (<http://www.vicbioinformatics.com/software.velvetoptimiser.shtml>), and assembled de novo using Velvet \[[@CIT0029]\], producing contigs that were ordered by reference genome mapping using ABACAS \[[@CIT0030]\]. The resulting contigs were verified by reference mapping using BWA \[[@CIT0031]\] and SAMtools/BCFtools \[[@CIT0032]\]. GATK \[[@CIT0033]\] was used for indexing, mapping and variant calling, defining variant nucleotides as follows: prevalence \<50%, overall read depth ≥50, average nucleotide quality ≥30, variant frequency ≥1% for read depths \>1000 and \>10% for read depths 50--1000, and minimum SNP depth ≥10. Artemis \[[@CIT0034]\] was used for visualization.
Data Deposition {#s8}
---------------
The human purged datasets were deposited in the European Nucleotide Archive under project number PRJEB31143 ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 15\]). Complete genome sequences were assembled as described \[[@CIT0022]\] and deposited in GenBank ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 16\]) under accessions [MK290742](MK290742)--[MK290744](MK290744) and [MK422176](MK422176).
RESULTS {#s9}
=======
Dataset Assessment {#s10}
------------------
In a recent study, we highlighted the importance of monitoring dataset quality produced directly from clinical material by target enrichment and high-throughput sequencing \[[@CIT0024]\]. We implemented this here by assembling datasets against the reference strain Merlin genome (GenBank accession [AY446894](AY446894)), noting numbers of matching HCMV reads ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[line 19\]), and deriving 2 parameters: (1) percentages of matched HCMV reads in the dataset and (2) percentages of the reference genome represented ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[lines 20--21\]). Also, since sequencing methodology is highly PCR based, the number of HCMV fragments producing the data was monitored by additional parameters: (3) coverage depth of the reference genome by all HCMV reads, and (4) coverage depth of the reference genome by reads generated from unique HCMV DNA fragments ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[lines 22--23\]).
Quality threshold values were set at (1) ≥50%, (2) ≥95%, (3) ≥1000 of the total fragment reads/nt, and (4) ≥20 unique fragment reads/nt. Eighteen datasets generated from 13 women met all 4 criteria, and 2 datasets (141R16 and 243L16) met criteria (1) to (3) but exhibited lower values (10--20) for criterion (4). These 20 datasets (from one HIV-negative woman and 19 from 13 HIV-infected women) ([Table 1](#T1){ref-type="table"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 11\]) are analyzed further.
Genotypic Structure of UL73/UL74 {#s11}
--------------------------------
Our previous study involving Sanger sequencing of single HCMV genes in breast milk samples obtained at multiple time points postpartum pointed to the presence of multiple strains \[[@CIT0003]\]. We extended this here by using sequence differences between the genotypes of hypervariable genes across the genome to characterize the strains represented in the datasets. We focused first on UL73 and UL74, as our earlier work had shown that these adjacent genes are markedly hypervariable, are almost always genotypically linked, grouping into 8 genotypes, also identified in milk samples \[[@CIT0003], [@CIT0007], [@CIT0012]\]. The nucleotide sequences were extracted from the set of 243 genome sequences for which complete genome sequences were available \[[@CIT0024]\] and analyzed phylogenetically ([Figure 1](#F1){ref-type="fig"}). This confirmed the existence of 8 genotypes for each gene ([Table 2](#T2){ref-type="table"}), their strong linkage (only 7 recombinants were noted), and high levels of intergenotypic diversity and low levels of intragenotypic diversity as observed initially in small datasets \[[@CIT0007], [@CIT0012]\]. In the UL73 phylogeny, a single G4B strain (HAN; GenBank accession number [KJ426589](KJ426589)) fell outside the genotypes due to 3 nucleotide differences that are characteristic of G4A strains and probably represent homoplasies. In the UL74 phylogeny, a single strain (BE/23/2010; GenBank accession [KP745697](KP745697)) fell outside the genotypes potentially from intragenic recombination between G1C and G1A. The distances between genotypes and the branching patterns in the 2 phylogenies also supported our previous inference that an ancestral recombination event had given rise to the linkage between UL73 G4C and UL74 G1C \[[@CIT0012]\].
{ref-type="table"}), and the frequencies of the deduced linkages in the samples (milk; 26 in total; [Table 3](#T3){ref-type="table"}). The frequency of each genotype in the milk set was not significantly different (above *P* = .05) from that in the 243 plus 383 single gene set, as determined by random subsampling analysis (10 000 samplings of 26 genotypes from the set of 626). \*Although no examples of this linkage were present in the datasets at levels in excess of the thresholds, at least one patient (258) was infected at subthreshold levels by a relevant strain ([Supplementary Table 1](#sup1){ref-type="supplementary-material"}).](jiz209f0001){#F1}
######
Genotypes and Haplotypes Assigned to Datasets
Donor Dataset Strains^a^ Genotypes^b^
------- ------------- ------------ -------------- ------------ ----------------- ------------ --------- --------------- --------- --------------- -------------- ---------- ------------- --------------
193 193R16^c,d^ 1 1 3 8 8 8 4 1 3A 1B 2B 12 3
141 141R16 2 1, 6 2, 3 2, 4B 2 2, 4 2, 3 5, 6 2, 3A 1B, 2B 4B 2, 12 3, 8
173 173R16 5 1, 2, 6 2, 3, 4 1B, 3, 4B, 6, 8 1, 6, 8 1, 6, 8 2, 4, 6, 7, 9 1, 3, 5 1, 3A, 4A, 4B 1A, 1B, 3, 4 1A, 4B 3, 7, 9, 12 1A, 1B, 3, 7
174 174L16 3 2 2, 4 1B, 6 1, 6, 10 1, 4, 6 4, 6 1, 4 1, 2, 4A 2B, 3, 4 2B, 3A 7, 9 5, 7
174 174R16 3 1, 2 1, 2, 3, 4 1B, 6 1, 6, 10 1, 6 4, 6, 7 1, 4 2, 4A 1B, 2B, 3 2B, 3A 7, 9 5, 7
243 243L16^c^ 2 1 6 2, (10) (1), 2 2 3 6 4B 4 2A 8, 9 7
243 243R4^c,d^ 1 1 6 2 2 2 3 (1), 6 4B 4 2A 8 7
243 243R16^c^ 1 1 6 2 2 2 3 6 4B 4 2A 8 7
248 248R4^c,d^ 1 1 1 4A 4A 4 1 1 3B 2A 4B \[5\] 4, (7)
258 258R4^c^ 2 6 3 3 3 3 3 6 3A, (4A) 1B, (3) (2A), 2B (2), 9 5
259 259L16 3 1, 2 2, 3, 4 1A, 6, 8 1, 6, 8 1, 6, 8 1, 3, 6 1, 4, 6 1, 2, 4B 1A, 2B, 4 4B 1, 9, 12 2, 8
259 259R16 3 1, 2 2, 3, 4 1A, 6, 8 1, 6, 8 1, 6, 8 1, 3, 6 1, 4, 6 1, 2, 4B 1A, 2B, 4 4B 1, 9, 12 2, 8
264 264L16 2 1 1, 2 8, 10 8, 10 10 8 4, 7 3A, 4B 4 1A, 3A 10 3
277 277R16 2 1 3 4A, 6 4A, 6 4, 6 6, 9 1, 4 1, 4A 1A, 3 3A, 4A 1, 9 3, 4
278 278L16^c^ 2 (2), 6 3, (4) (1A), 9 (1), \[9\] (1), 9 (1), 9 (1), 6 3A, (4D) 1B, (5) (3A), 4B 8, (9) 2, (2)
278 278R4^c^ 2 (2), 6 3, (4) (1A), 9 (1), \[9\] (1), 9 (1), 9 (1), 6 3A, (4D) 1B, (5) (3A), 4B 8, (9) 2, (2)
278 278R16^c^ 2 (2), 6 3, (4) (1A), 9 (1), \[9\] (1), 9 (1), 9 (1), 6 3A, (4D) 1B, (5) (3A), 4B 8, (9) 2, (2)
281 281R4^c^ 2 (1), 6 3 1A, (6) 1 1, (10) 4, (7) 1 4D (1B), 5 4B 11 5, (7)
283 283R16^c,d^ 1 \[2\] 4 4B \[4B\] 4 2 5 3A, (3B) 1B 3A 1 5
288 288R4 3 1, 2 3, 4 6, 7 6, 8 5, 6, 8 4, 6, 9 1 1, 4B, 4D 1A, 4, 5 2B, 4B 1, 3 4, 5
^a^Determined using long motifs for 12 genes ([Supplementary Table 1](#sup1){ref-type="supplementary-material"}).
^b^Genotype (G) prefix omitted; round brackets indicate an assigned minority genotype; multiple genotypes with none in round parentheses indicate that majority and minority genotypes could not be distinguished; square brackets indicate a single mismatch in the motif.
^c^Datasets from which haplotypes were assigned.
^d^Datasets from which complete genome sequences were derived.
Genotyping Using Sequence Motifs {#s12}
--------------------------------
Having established a comprehensive view of UL73 and UL74 hypervariation, we developed short motifs capable of identifying individual genotypes. These consisted of a single motif near the 5′ end of each UL73 genotype and 3 separate motifs near the 5′ and 3′ ends and in the central region of each UL74 genotype, and successfully genotyped the majority of sequences used in the phylogenetic analyses ([Table 2](#T2){ref-type="table"}). We then extended the analysis to a further 10 hypervariable genes, using a single, long, nonredundant motif for each genotype to improve discrimination.
The original datasets were trimmed (to create trimmed datasets) or purged of human reads and trimmed (to create purged datasets). The relative frequencies of individual genotypes were then estimated by counting motifs in each dataset with threshold requirements ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[lines 25--56 and 58--166\], respectively). Purging human reads had little effect, except when short motifs were used with datasets containing a significant proportion of residual nonviral reads. The UL74 5′ motif offered the least accurate genotypic discernment in such samples, perhaps from its minimal length (12 nt). The number of strains in each sample was scored from the purged datasets using the long motifs with threshold requirements ([Table 3](#T3){ref-type="table"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[row 17\]). A genotype was considered to be present when represented by \>25 reads and \>5% of the total number of reads detected for all genotypes of that gene, and the number of strains was scored as being the greatest number of genotypes detected using long motifs for at least 2 genes. Thus, strains present at \<5% were unlikely to score. There was a high degree of congruence between the results obtained using short and long motifs with datasets meeting the quality thresholds ([Supplementary Table 2](#sup2){ref-type="supplementary-material"}).
Strain Complexity in HIV-Infected Women {#s13}
---------------------------------------
The majority of HIV-infected women (11/13) were infected by multiple HCMV strains ([Table 3](#T3){ref-type="table"} and [Supplementary Table 1](#sup1){ref-type="supplementary-material"}). The mode was at least 2 strains, and one woman was infected by 5 strains. In the dataset meeting quality thresholds, the only HIV-negative woman was infected by a single strain. This was also indicated in the datasets from the 6 other HIV-negative women, but these were below quality thresholds, partly from lower viral loads, and not compared further. Even among this small cohort, 89 of the 109 possible genotypes for the 12 hypervariable genes were detected. It was possible to assign with confidence fully linked genotypes (haplotypes) to 8 strains represented in 11 datasets from 7 donors, on the basis of complete genome sequences (4 datasets) or the presence of a single strain or major and minor strains (when the former was highly predominant) in multiple-strain infections ([Table 3](#T3){ref-type="table"}). Consideration of all the other datasets from multiple-strain infections where both major and further minor strains could be identified, allowed haplotypes to be assigned to a further 12 strains, but with less confidence, 20 total ([Supplementary Table 3](#sup3){ref-type="supplementary-material"}).
Genotypic linkage was detected only in 2 loci where recombination has been shown to occur rarely, namely, those containing the 2 respective sets of adjacent, hypervariable genes UL73/UL74 \[[@CIT0012], [@CIT0017], [@CIT0035]\] and RL12/RL13/UL1 \[[@CIT0011], [@CIT0016]\]. The overall frequencies of UL73/UL74 genotypes in the milk samples were not significantly different from those in the 243 genome set plus the 383 single-gene sequences ([Tables 1](#T1){ref-type="table"} and [3](#T3){ref-type="table"}). Comparisons to only the 243 genome set, which does not include milk or African samples, showed some evidence for increased proportions of UL73/UL74 G4B-G4 and RL12/RL13/UL1 G2-G2-G2 in milk (*P* = .001 and *P* = .02, respectively), but case-controlled cohorts are required to confirm.
The use of 3 short motifs in UL74 facilitated an examination of intragenic recombination, and confirmed that strain BE/23/2010 is a recombinant with a G1C motif near the 5′ end and G1A motifs in the central region and near the 3′ end. In addition, compartmental stability was revealed by the use of both short and long motifs, in the form of genotypic conservation in samples from both breasts of 4 HIV-infected women ([Figure 2](#F2){ref-type="fig"}). Small differences may be accounted for by minor strains present at levels nearing the detection threshold. Longitudinal stability was observed in 2 donors (243 and 278) with samples taken at weeks 4 and 16 postpartum ([Table 3](#T3){ref-type="table"}); small differences in one (243) were probably due to threshold effects. This stability also showed in variant analysis, which demonstrated the absence of genotype switches in all donors.
{ref-type="table"}). The inner and outer rings show the results obtained using short and long motifs, respectively. Short motif 3′ was used for UL74 ([Table 2](#T2){ref-type="table"}). The color key for genotypes is shown at the foot. Reads that did not meet the inclusion criteria for genotyping are shown as "other."](jiz209f0002){#F2}
Finally, additional long sequence motifs were used to investigate whether any strains contained gene-disrupting mutations detected previously in the 243 genome set, and resulting in pseudogenes \[[@CIT0024]\]. Such mutations are more common in certain genes, most frequently in UL9, RL5A, UL1, RL6, US9, and UL111A \[[@CIT0014], [@CIT0016], [@CIT0024]\]. The use of motifs representing 3 mutations in RL5A (present in 37 members of the 243 genome set), 2 in US9 (35 members) and one in UL111A (5 members), demonstrated the presence of the RL5A and US9 mutations, but not that in UL111A, encoding viral interleukin 10 ([Supplementary Table 1](#sup1){ref-type="supplementary-material"} \[rows 167--174\]).
DISCUSSION {#s14}
==========
Analysis of HCMV genomes directly from clinical samples is necessary for characterizing infectious natural populations while avoiding the mutational artefacts arising from laboratory adaptation to cell culture. Target enrichment has proven successful in this regard \[[@CIT0011], [@CIT0022], [@CIT0024]\], but accurate genome analysis can be confounded by multiple strains, particularly in immunosuppressed groups in whom additional complexity may accumulate by reinfection or reactivation \[[@CIT0017], [@CIT0021], [@CIT0024]\]. We have shown previously that HIV-infected women in sub-Saharan Africa have higher HCMV loads in breast milk than HIV-negative women, and that this is associated with adverse infant development \[[@CIT0001], [@CIT0003]\]. However, genomic studies of HCMV in milk samples or, indeed, samples from Africa, are scarce. We examined milk because of its importance in HCMV transmission, with the aim of understanding strain diversity and the burden of infection in HIV-infected (immunosuppressed) mothers, which may affect their infants. The sequence datasets were generated from 28 samples donated by 22 women, and 20 datasets from 14 women meeting quality thresholds were analyzed.
The analysis focused on counting reads containing motifs specific to the genotypes of hypervariable genes. Short motifs were developed initially for sensitive characterization of UL73 and UL74, which encode glycoproteins N and O (gN and gO), respectively, and then long motifs were used for further resolution of these 2 genes and 10 others. Since, as shown further here, UL73 and UL74 are linked and behave as a single genotype, haplotypes could not be determined using solely the short motifs ([Supplementary Table 2](#sup2){ref-type="supplementary-material"}) \[[@CIT0007], [@CIT0012]\]. However, mapping 3 short motifs to each UL74 genotype was uniquely useful for detecting intragenic recombination. The use of long motifs in a larger number of genes allowed increased resolution and also haplotype determination. These were less compromised by residual human reads in the datasets, but more susceptible to mismatches in target genomes ([Table 3](#T3){ref-type="table"}).
Genotypic and haplotypic complexity in this small cohort was remarkable. Most (82%) of the genotypes possible in the 12 hypervariable genes were detected, and 85% of the HIV-infected donors were infected by multiple strains. The level of multiple strain infection exceeded that in previous cohort analyses, including congenitally infected and transplantation patients from developed countries \[[@CIT0022], [@CIT0024]\]. Each of the 20 fully characterized haplotypes identified was unique in this cohort and in the set of 243 strains, in which most strains (223) are also unique \[[@CIT0024]\]. These observations testify to the huge number of HCMV haplotypes that may exist, possibly exceeding that related to immune diversity, as was recognized long before the high-throughput sequencing era \[[@CIT0007], [@CIT0012], [@CIT0013], [@CIT0015]\]. No evidence emerged for the existence of novel African genotypes, consistent with the view that HCMV genotypes are distributed throughout the world, although their relative prevalence may vary \[[@CIT0007], [@CIT0008], [@CIT0012]\].
Strain composition in individual women was essentially stable, both compartmentally (in milk samples from both breasts) and longitudinally (at 4 and 16 weeks postpartum). This indicates that the strains detected were present in the donor prior to viral reactivation, which peaks at 4 weeks in breast tissue during lactation \[[@CIT0003]\]. Leukocyte infiltrates have been characterized during this period \[[@CIT0036]\], and may be the source of reactivated virus. It may also be that the strains in blood differed from those in milk, but this was not investigated. A saliva-based study conducted in Uganda by PCR and antibody assays indicated that HCMV secretion was induced in seropositive mothers after exposure to their HCMV-excreting children \[[@CIT0037]\]. However, even though multiple-strain infections were common in the cohort and opportunities for fresh infection existed at home or in the hospital because all the mothers were HCMV-infected and had young children at home, there was limited evidence for reinfection or reactivation with new strains during the 4- to 16-week period postpartum. These observations differ from those made in developed countries in transplantation patients. A proportion of transplant-associated infections involve multiple strains, and these exhibit substantial longitudinal dynamism \[[@CIT0022], [@CIT0024]\] and are also associated with increased viral loads and the pathological outcomes of HCMV disease \[[@CIT0018], [@CIT0020]\]. The contrasting observation, that most congenital or postnatal infections involve single strains \[[@CIT0024], [@CIT0038]\], suggests that only certain strains cross the placenta or are transmitted by breast milk, urine, or saliva, perhaps due to the competence of a few virions to establish infection \[[@CIT0038], [@CIT0039]\]. This also implies that the HIV-infected women were exposed to a high burden of HCMV superinfection.
Whole-genome analyses and earlier PCR-based studies showed a high degree of linkage within the UL73/UL74 \[[@CIT0007], [@CIT0012], [@CIT0024], [@CIT0035]\] and RL12/RL13/UL1 loci \[[@CIT0011], [@CIT0024]\]. This is consistent with the involvement of homologous recombination during HCMV evolution, and may also reflect the functional constraints imposed on proteins that interact with each other or have interdependent functions. UL73/gN and UL74/gO are part of the viral entry complex and have roles in viral exocytosis, cellular tropism, and modulation of antibody neutralization, and the RL12, RL13, and UL1 proteins are known or suspected to be involved in aspects of immune evasion probably mediated by an immunoglobulin-like binding domain shared by these proteins and other members of the RL11 family \[[@CIT0009], [@CIT0013], [@CIT0040]\]. In addition, RL13 may influence the effect of UL74 on the growth of HCMV \[[@CIT0044]\]. It is possible that different genotypes of hypervariable genes, and different combinations of genotypes, provide variable growth properties leading to higher viral loads and specific pathologies. For example, UL74 genotypes differentially affect viral growth properties in vitro \[[@CIT0045]\], and genotypes of UL146, which is the most hypervariable gene in HCMV and encodes a vCXCL1 chemokine, affect neutrophil chemotaxis efficiency \[[@CIT0046]\]. Similarly, human genetic variation is higher in Africa than other regions and may affect susceptibility as a result of HCMV genotype-specific interactions, for example with immunoglobulin variants \[[@CIT0047]\].
Although information on genotypes and mutants could be extracted from the datasets regardless of strain complexity, complete genome assembly was possible for only 4 datasets because of a high frequency of confounding multiple infections. To our knowledge, these are the first complete HCMV genome sequences to be determined from people living in Africa. Moreover, one of these originated from an HIV-negative woman (193) and thus represents the first from an immunocompetent adult lacking HCMV-associated pathology. Future research is likely to focus on understanding the differences in HCMV transmission in immunosuppressed and immunocompetent settings to define the interplay between viral strain and host immunotype diversity in controlling disease.
Supplementary Data {#s15}
==================
Supplementary materials are available at *The Journal of Infectious Diseases* online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
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***Acknowledgments.*** We thank the participants and clinical staff, as managed by Dr L. Kasonka at the University Teaching Hospital, Lusaka, for facilitating the Breastfeeding and Postpartum Health Study, as directed by Professor S. Filteau, London School of Hygiene and Tropical Medicine, and for enabling the follow-up analyses. We also thank Dr J. Hughes, Medical Research Council--University of Glasgow Centre for Virus Research, for advice on bioinformatic analysis, and Drs T. Clark and J. Phelan, London School of Hygiene and Tropical Medicine, for facilitating genomics UNIX cluster access and Perl support.
***Financial support.*** This work was supported by the Commonwealth Scholarship Commission; a Bloomsbury Studentship Award; the Medical Research Council (grant number MC_UU_12014/3); and the Wellcome Trust (grant number 204870/Z/16/Z).
***Potential conflicts of interest.*** All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
Published as a bioRxiv preprint on 23 December 2018 and revised on 19 February 2019 ([https://doi.org/10.1101/493742](10.1101/493742)).
[^1]: N. M. S. and K. G. M. contributed equally to this work.
[^2]: Present affiliation: Public Health England, Porton Down, Wiltshire, United Kingdom.
[^3]: Present affiliation: Virokine Therapeutics Ltd., London Bioscience Innovation Ctr, Royal Veterinary College, University of London, 2 Royal College St, London NW1 0NH, UK.
| {
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Opioids are small endogenously produced peptide molecules that are widely known for their analgesic and psychoactive properties ([@bib3]; [@bib17]; [@bib12]). It has been shown that opioids can promote the growth of tumour cells ([@bib10]). On the other hand, it has been controversially reported that opioids induce the apoptosis of immunocytes, cancer cells and neuroblastoma cells ([@bib1]). Thus, it is becoming increasingly recognised that opioids have a role in tumour growth ([@bib13]).
Three major types of opioid receptors, *μ*, *δ* and *κ*, have been well characterised. *κ*-Opioid receptors (KORs) are widely expressed throughout the central nervous system ([@bib2]; [@bib4]). It has been reported that KOR is also expressed in the human adenocarcinoma breast cancer cell line MCF7 and small cell lung carcinoma ([@bib8]). Furthermore, KOR agonist has been shown to inhibit the growth of H157 cell, which is a non-small cell lung cancer (NSCLC) cell ([@bib11]). However, little is known about the mechanism that underlies the inhibitory effect of KOR stimulation on the growth of NSCLC cells.
Epidermal growth factor receptor (EGFR) is a major target of molecular anti-NSCLC therapy ([@bib16]). Non-small cell lung cancer patients with L858R or exon 19 deletion mutations in EGFR show good responses to the tyrosine kinase inhibitor gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are eventually resistant to treatment with gefitinib. In this study, we examined whether the selective KOR agonist U50,488H could inhibit the growth of gefitinib-sensitive and EGFR mutant (delE746-A750, L858R) NSCLC cells (HCC827) and gefitinib-resistant and EGFR mutant (T790M) NSCLC cells (H1975), and investigated the signalling mechanism of the KOR-mediated inhibitory effect on tumour cell growth.
Materials and methods
=====================
Cell culture
------------
The human NSCLC cell lines HCC827 and NCI-H1975 (H1975; both from American Type Culture Collection Co., MD, USA) were cultured in HEPES-modified RPMI 1640 medium (Sigma-Aldrich Co., St Louis, MO, USA) with 10% fetal bovine serum (FBS; Invitrogen Life Technologies Co., Carlsbad, CA, USA) and 1% penicillin-streptomycin (Invitrogen Life Technologies Co.). Normal human lung fibroblasts (NHLF; Lonza Inc., Allendale, NJ, USA) were cultured in fibroblast basal medium with insulin, rhFGF-B, GA-1000 and FBS (all from Takara Bio Inc., Tokyo, Japan). All cells were maintained under a humidified atmosphere of 5% CO~2~ at 37°C.
Reagents
--------
The reagents used in the present study were gefitinib (Toronto Research Chemicals Inc., Canada), (±) trans 3,4-dichloro-*N*-methyl-*N*-(2-(1-pyrrolidinyl) cyclohexyl)-benzeneacetamide (U50,488H) methanesulfonate (Sigma Chemical Co.), nor-binaltorphimine dihydrochloride (nor-BNI; Tocris Cookson Ltd., St Louis, MO, USA), and 6-bromoindirubin-3′-oxime (BIO; WAKO Pure Chemical Industries Ltd., Osaka, Japan).
Cell viability assay
--------------------
Cell viability was determined by a cell proliferation assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellow tetrazole (MTT). A 20-*μ*l of MTT solution (5 mg ml^−1^) was added to each well of the culture medium. After incubation for an additional 2 h, the medium was removed and 100 *μ*l of DMSO was added to resolve the formazan crystals. Optical density was measured using a microplate reader with an absorption wavelength of 600 nm. In each experiment, three replicates were prepared for each sample. The proportion of living cells was determined based on the difference in absorbance between the samples and controls.
Immunohistochemistry
--------------------
The procedure for immunohistochemistry is described in the [Supplementary Methods](#sup1){ref-type="supplementary-material"}.
RNA preparation and semiquantitative analysis by reverse transcription (RT)-PCR
-------------------------------------------------------------------------------
The RNA preparation and RT-PCR method are described in the [Supplementary Methods](#sup1){ref-type="supplementary-material"}.
Western blotting
----------------
Sample preparation and loading for western blotting are described in the [Supplementary Methods](#sup1){ref-type="supplementary-material"}. For immunoblot detection, membranes were blocked in Tris-buffered saline (TBS) containing 1% non-fat milk (Bio-Rad Laboratories, Hercules, CA, USA) containing 0.1% Tween 20 (Sigma-Aldrich Co.) for 1 h at room temperature with agitation. The membrane was incubated with primary antibody diluted in TBS (1 : 1000 phosphorylated-EGFR (Cell Signaling Technology Inc., Boston, MA, USA), 1 : 500 p-Akt (Cell Signaling Technology Inc.), 1 : 1000 p-GSK3*β* (Cell Signaling Technology Inc.), 1 : 2000 p-STAT3 (Cell Signaling Technology Inc.), 1 : 750 GSK3*β* (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 1 : 5000 Akt (Cell Signaling Technology Inc.) and 1 : 3500 Stat3 (Cell Signaling Technology Inc.) containing 1% non-fat dried milk with 0.1% Tween 20 overnight at 4°C. The membrane was washed in TBS containing 0.05% Tween 20, and then incubated for 2 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates Inc., Birmingham, AL, USA) diluted 1 : 10 000 in TBS containing 1% non-fat dried milk containing 0.1% Tween 20. The antigen--antibody peroxidase complex was finally detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA) and visualised by exposure to Amersham Hyperfilm (Amersham Life Sciences, Arlington Heights, IL, USA).
Results
=======
Localisation of KORs in NSCLC cells
-----------------------------------
KORs were found in gefitinib-sensitive HCC827 cells, gefitinib-resistant H1975 cells and NHLF cells, as detected by RT-PCR ([Figure 1A](#fig1){ref-type="fig"}) and immunoreactivity towards KOR antibody ([Figure 1B](#fig1){ref-type="fig"}). The expression of KOR mRNA was significantly increased in HCC827 cells (*P*\<0.01 *vs* NHLF) and H1975 cells (*P*\<0.001 *vs* NHLF) compared with NHLF ([Figure 1](#fig1){ref-type="fig"}).
Effect of KOR agonist on the growth of the EGFR exon 19 mutant NSCLC cell line HCC827
-------------------------------------------------------------------------------------
Addition of the KOR agonist U50,488H to HCC827 cells for 2 days produced a concentration-dependent decrease in tumour cell growth ([Figure 2A](#fig2){ref-type="fig"}, *P*\<0.001 *vs* non-treated group). This effect was abolished by co-treatment with the selective KOR antagonist nor-BNI ([Figure 2B](#fig2){ref-type="fig"}, ^\*\*\*^*P*\<0.001 *vs* non-treated group, ^\#\#\#^*P*\<0.001 *vs* U50,488H-treated group). In contrast, treatment of NHLF cells with U50,488H did not affect their growth ([Figure 2C](#fig2){ref-type="fig"}). In experiments that compared the inhibition of cell growth in cells treated with gefitinib and cells treated with a combination of gefitinib and U50,488H, the growth-inhibitory effects in HCC827 cells were further enhanced in a dose-dependent manner ([Figure 2D](#fig2){ref-type="fig"}, *P*\<0.001 *vs* gefitinib-treated cells).
Changes in the growth of gefitinib-resistant H1975 cells by treatment with KOR agonist
--------------------------------------------------------------------------------------
Treatment of gefitinib-resistant H1975 cells with U50,488H for 2 days produced a concentration-dependent and dramatic decrease in tumour cell growth ([Figure 3A](#fig3){ref-type="fig"}, *P*\<0.001 *vs* non-treated group). This effect was blocked by co-treatment with nor-BNI ([Figure 3B](#fig3){ref-type="fig"}, ^\*\*\*^*P*\<0.001 *vs* non-treated group, ^\#\#\#^*P*\<0.001 *vs* U50,488H-treated group).
Effect of KOR agonist on the levels of phosphorylated Akt, GSK3*β* and Stat3 in H1975 cells
-------------------------------------------------------------------------------------------
There were no changes in the levels of either p-Akt or p-Stat3 in H1975 cells by treatment with U50,488H for 2 days ([Figures 3C and E](#fig3){ref-type="fig"}). However, the addition of U50,488H to H1975 cells produced a significant and concentration-dependent decrease in p-GSK3*β* ([Figure 3D](#fig3){ref-type="fig"}, *P*\<0.001 *vs* non-treated group). Furthermore, treatment with a specific GSK-3*β* inhibitor BIO produced a concentration-dependent and significant decrease in tumour cell growth ([Figure 3F](#fig3){ref-type="fig"}, *P*\<0.001 *vs* non-treated group).
Discussion
==========
In the present study, we investigated the role of KOR in NSCLC cells using gefitinib-sensitive HCC827 and gefitinib-resistant H1975 cells. We found that KORs were highly expressed in both cell lines. Under these conditions, addition of the selective KOR agonist U50,488H to either HCC827 or H1975 cells produced a concentration-dependent decrease in tumour cell growth. Although some of the doses of U50,488H were relatively high, these effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. These results support the idea that U50,488H can pharmacologically act on KORs to decrease tumour growth. Additionally, the inhibition of tumour growth by gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. These findings suggest that the stimulation of KOR may provide unique opportunities for the prevention and treatment of NSCLC.
GSK3*β* is a multifunctional serine/threonine kinase that phosphorylates and thereby regulates the functions of many metabolic, signaling, and structural proteins and transcriptional factors ([@bib6]). EGF can inactivate GSK3*β*, leading to the degradation of c-Myc and *β*-catenin, which are overexpressed in tumour cells. Furthermore, the tumour suppressor p53 can be inactivated because of inactive GSK3*β*. It has been reported that the progressive inactivation of GSK3*β*, which is related to the increase in phosphorylation of GSK3*β*, is critical for the progression of lung cancer ([@bib14]). In this study, treatment of H1975 cells with U50,488H produced a significant decrease in the phosphorylation of GSK3*β*. It has been recognised that activated protein kinase A (PKA) leads to phosphorylation of GSK3*β* ([@bib5]), whereas activated JNK increases GSK3*β* activity ([@bib7]). It should be noted that the stimulation of KOR suppresses cAMP production through Gi proteins, which leads to the inactivation of PKA ([@bib15]). Furthermore, the stimulation of KOR invokes the JNK cascade ([@bib9]). Although the exact mechanism of KOR-mediated GSK3*β* activation remains unclear at this time, we propose that the stimulation of KOR may activate GSK3*β* through inhibition of the cAMP/PKA pathway and/or activation of the JNK pathway in NSCLC, resulting in the prevention of cancer.
In conclusion, the present results suggest that stimulation of KOR reduces the growth of NSCLC cells through the activation of GSK3*β*. Furthermore, KOR agonist might be a valuable candidate for preventing gefitinib-resistant NSCLC.
[Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies the paper on British Journal of Cancer website (http://www.nature.com/bjc)
This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License.
Supplementary Material {#sup1}
======================
######
Click here for additional data file.
{#fig1}
{#fig2}
{#fig3}
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Foreword 2014 01 {#S0001}
================
Abstracts and proceedings from VI Scandinavian COPD Research Symposium {#S0001-S20001}
----------------------------------------------------------------------
### [Magnus Sköld]{.ul} ^1^, Johny Kongerud^2^, Thomas Ringbaek^3^ and Terttu Harju^4^ <magnus.skold@ki.se> ^1^Department of Medicine, Karolinska Institutet, Stockholm, Sweden; ^2^Department of Respiratory Medicine, Rikshospitalet, Oslo, Norway; ^3^Department of Medicine, Hvidovre Hospital, Copenhagen, Denmark; ^4^Department of Pulmonary Medicine, University of Oulu, Oulu, Finland
###
On 21--22 November 2014, the sixth Scandinavian COPD Research Symposium was held at Holmenkollen Park Hotel Rica, Oslo. Like the previous five meetings (1--5), the purpose was to let young scientists from Denmark, Finland, Norway, and Sweden come together and present their current COPD-related research. The meeting should also facilitate collaboration and stimulate further research in the field of COPD. Eleven young scientists from our countries presented their latest data and three state-of-the-art lecturers covered two sessions: 'Inflammation and structural changes in COPD' and 'COPD not caused by cigarette smoke'. To better facilitate interactions in a smaller format, three 'break-out sessions' was also arranged. The meeting was generously supported by grants from Boehringer Ingelheim, which also made publication of this supplement to *European Clinical Respiratory Journal* possible.
**References**
1\. Sköld M, Bakke P, Kongerud J, Ringbaek T. Scandinavian COPD research symposium. Respir Med. 2005; 99: 918--25.
2\. Sköld M, Bakke P, Ringbaek T. Abstracts and proceedings from II Scandinavian COPD research symposium. Respir Med COPD Update. 2007; 3: 119--27.
3\. Sköld M, Kongerud J, Ringbaek T, Pietinahlo A. Proceedings and Abstracts from III Scandinavian COPD Research Symposium. Respir Med. 2009; 103(Suppl 1): S1--7.
4\. Sköld M, Kongerud J, Ringbaek T, Myllärniemi M. Abstracts and Proceedings from IV Scandinavian COPD Research Symposium. Clin Respir J. 2011; 5(Suppl 1): 1--10.
5\. Sköld M, Kongerud J, Ringbaek T, Myllarniemi M. Abstracts and Proceedings from the V Scandinavian COPD Research Symposium. Respir Med. 2013; 107: S1--7.
ABSTRACTS STATE OF THE ART 02 {#S0002}
=============================
Inflammation in COPD {#S0002-S20001}
--------------------
### [Dirkje S. Postma]{.ul} <d.s.postma@med.umcg.nl> Department of Pulmonary Medicine and Tuberculosis, University Medical Center Groningen, Groningen, The Netherlands
###
COPD reflects airway obstruction due to an inflammatory and remodelling process in the airways and lungs that becomes a symptomatic disease at adult age. It is caused by cigarette smoking or other environmental factors, and recently even pesticides were reported of importance. Inflammatory cells play a role in COPD development and progression and it generally accepted that neutrophils and CD8+ lymphocytes are higher in sputum and airway wall biopsies of the large airways in smokers with than without COPD. Moreover, increased numbers of macrophages populate the airways. Neutrophils are particularly found in the mucous glands and their factors likely contribute to increased mucus hypersecretion. During exacerbations increased numbers of eosinophils have been reported. There exists even a subset of COPD patients with blood eosinophilia and this has been suggested to predict a good response to inhaled steroid treatment to prevent exacerbations. In the peripheral airways and lung tissue, neutrophil, lymphocyte, and mast cell numbers are increased, next to a B cell response in the large and small airways; plasmacytoid dendritic cell numbers in the pulmonary lymphoid follicles are increased. Recently, the epithelial cells have become a target of interest given their pro-inflammatory and inflammatory function and possible role in COPD as a barrier against inhaled toxic substances. Besides local effects on the airway wall, inflammation also increases stretching and ultimately the loss of alveolar septal attachments to the small airways with emphysema as a consequence. Inflammation can also be defined by the production of cytokines, proteases, and so on. The cytokines currently thought of importance include (TNF)-α, INF-γ, interleukin (IL)-1β, IL-6, IL-17, IL-18, IL-32, TSLP, and growth factors such as TGF-β and VEGF. Given the complex nature and intricate pathways leading to the different forms of COPD gene expression profiling of lung tissues and specific cell types and other techniques to define pathways of inflammation and repair in COPD are of utmost importance. Only by integratively combining approaches of clinical sub-phenotyping, genetic, epigenetic, and proteomic approaches, we will make a next step in understanding the inflammatory mechanisms of tissue breakdown and repair associated with COPD.
03 {#S0003}
==
Environmental exposures and COPD {#S0003-S20001}
--------------------------------
### [Paul K. Henneberger]{.ul} <pkh0@cdc.gov> National Institute for Occupational Safety and Health Centers for Disease Control and Prevention, Morgantown, WV, USA
###
Chronic obstructive pulmonary disease (COPD) is responsible for substantial morbidity and mortality worldwide. Cigarette smoking is the leading cause, but its dominance in industrialised countries has waned as smoking rates have declined. Occupational exposures continue to be important contributors to the burden of COPD. Moreover, the identification of harmful occupational exposures offers the possibility that reduction or elimination of such exposures could prevent disease. This presentation will review the major occupational causes of COPD as revealed primarily by epidemiologic studies. The summary will emphasise findings from Scandinavian research, including population-based studies and investigations conducted in specific occupations and industries. Methods for the characterisation of occupational exposures in these studies include self-reports and job exposure matrices, as well as workplace measurements of airborne dusts and gases. Different approaches have also been used to determine COPD status, such as spirometry, self-reported symptoms and disease, and cause-specific mortality. Scandinavian researchers have published multiple papers about COPD among construction, cement, smelter, and tunnel workers, but they have also identified many other occupations and industries with an elevated prevalence or incidence of obstructive disease. A topic addressed in several studies is the effect of occupational exposure by smoking status, with some results suggesting that non-smokers have a greater relative risk for occupational COPD than their co-workers who smoke. Interventions to reduce exposures and prevent COPD will be discussed.
04 {#S0004}
==
Early life origin of COPD {#S0004-S20001}
-------------------------
### [Andrew Bush]{.ul} <A.Bush@rbht.nhs.uk> Imperial College, National Heart and Lung Institute, Royal Brompton & Harefield NHS Foundation Trust, London, UK
###
The key questions are 1) what is COPD; 2) what is early life?; and 3) are they connected? *What is COPD?* Modern definitions rely heavily on an FEV1/FVC ratio of \<70%, ignoring the developmental absurdities of a fixed ratio and the fact that many diseases cause airflow obstruction \[Thorax 2012;67:88--9\]. This presentation will refer to the early life origin of premature airflow obstruction. *What is early life?* Any meaningful definition must think before early childhood, to antenatal events, and even transgenerational influences on childhood development. *What is the connection?* Normal lung growth requires the child to be born with normal airway function; for lung growth in childhood to go at a normal pace; and for there to be no premature lung aging. Many COPD patients in fact have a normal rate of lung aging, underscoring the importance of early life events causing premature airflow obstruction \[*NEJM* 2011; 365: 1184--92; *Am J Respir Crit Care Med* 2011; 184: 1015--51\]. Transgenerational, antenatal, early postnatal and late childhood and adult factors impacting normal lung growth will be reviewed. In particular, five childhood factors have been shown to influence COPD risk at least as much as heavy smoking in adulthood \[*Thorax* 2010; 65: 14--20\], namely maternal, paternal and childhood asthma, maternal smoking and childhood respiratory infections. The Melbourne cohort have recently reported that nearly 50% of children with severe asthma have COPD, independent of smoking, and with no accelerated decline in lung function \[Tai A, Thorax, epub\]. Chronic lung disease of prematurity will also be a cause of premature airway obstruction, but the airway pathology is very different from asthma; so is the 'COPD' resulting from these two diseases the same, and is it the same as that in term, non-atopic smokers? *Conclusions*: If premature airflow obstruction is to be prevented, efforts must be focussed antenatally and in the pre-school years. We must not assume that all chronic airflow obstruction is the same disease.
ABSTRACTS OF OTHER LECTURES 05 {#S0005}
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Potential biomarkers of asthma-COPD overlap syndrome {#S0005-S20001}
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### [Jing Gao^1^]{.ul}, Tarja Laitinen^2^, Hiroshi Iwamoto^3^, Harri Alenius^4^, Witold Mazur^1^ and Ville Pulkkinen^1^ <jing.gao@helsinki.fi> ^1^Department of Medicine, Pulmonary Division, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; ^2^Department of Pulmonary Diseases, Turku University Central Hospital, Turku, Finland; ^3^Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; ^4^Unit of Systems Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland
###
*Background*: The underlying inflammatory mechanisms of asthma-COPD overlap syndrome (ACOS) are poorly characterised. There is a clear unmet need to find novel biomarkers for clinical assessment and management of ACOS. We have investigated two panels of potential sputum and/or plasma biomarkers for ACOS phenotype: COPD-related biomarkers and asthma-related biomarkers. The four COPD-related biomarker are surfactant protein A (SP-A), soluble receptor for advanced glycation end-products (sRAGE), myeloperoxidase (MPO), and neutrophil gelatinase-associated lipocalin (NGAL). The four asthma-related biomarkers are periostin and interleukin 13 (IL-13), chitinase-like protein (YKL-40), and interleukin 6 (IL-6). *Aim*: To clarify the similarity and differences between asthma, COPD, and ACOS; and to identify reliable, specific, and sensitive biomarkers for ACOS. *Method*: Induced sputum and/or plasma levels of biomarkers were measured by ELISA/Luminex assays in 132 study subjects into five groups: non-smokers (*n*=24), smokers (*n*=23), asthma (*n*=32), COPD (*n*=39), and ACOS (*n*=14). *Results*: In patients with ACOS, plasma sRAGE levels were reduced whereas, the other measured biomarkers were elevated when compared with non-smokers. However, sputum NGAL, YKL-40, and IL-6 were significantly elevated in ACOS and could differentiate ACOS from asthma and COPD, and further independently with reduced lung function in the multivariate stepwise analysis. *Conclusion*: Elevated sputum levels of NGAL, YKL-40, and IL-6 are characteristic for ACOS and are related to enhanced neutrophilic and/or eosinophilic airway inflammation, remodelling, and airway obstruction. Monitoring of sputum biomarkers could be useful in clinical practice for identifying ACOS.
**References**
1\. Iwamoto H\*, Gao J\*, Koskela J, Kinnula V, Kobayashi H, Laitinen T, et al. Differences in plasma and sputum biomarkers between COPD and COPD-asthma overlap. Eur Respir J. 2014; 43: 421--9. \*Co-first authors.
2\. Iwamoto H, Gao J, Pulkkinen V, Toljamo T, Nieminen P, Mazur W. Soluble receptor for advanced glycation end-products and progression of airway disease. BMC Pulm Med. 2014; 14: 68.
06 {#S0006}
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Adaptive immune responses in COPD largely depend on current smoking status and not airway obstruction {#S0006-S20001}
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### [H. Forsslund]{.ul}, M. Yang, M. Mikko, J. Grunewald, J. Wahlström, Å. M. Wheelock and C. M. Sköld <helena.forsslund@gmail.com> Respiratory Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
###
T cells are important cells in smoking-induced inflammation and COPD. We have previously shown that the distribution of T-cell subtypes in BAL and blood is more related to current smoking status than airway obstruction, at least in GOLD stage I--II (1). More recently, gender differences have been gaining increased attention (2). Many factors, such as symptoms and mortality rate, differ between men and women and for unknown reasons, women are more susceptible to develop the disease. We studied the distribution on T-cell subsets along with chemokines and its receptors, which are important for the recruitment of T cells to the lung. Thirty-seven never-smokers, 38 smokers with normal lung function, and 32 patients with COPD, GOLD stage I--II (23 smokers and 9 ex-smokers) underwent bronchoalveolar lavage (BAL). Using multicolour flow cytometry, BAL and blood T cells were analysed for the expression of CD4 and CD8 and the chemokine receptors CXCR3, CCR4, and CCR5. The levels of chemokines, as well as a number of cytokines and growth factors, were analysed using Luminex bead-based technology. Smokers with or without COPD had higher percentages of CD8 T cells in BAL than did never-smokers and ex-smokers with COPD (*p*\<0.01 for both). Conversely, the percentages of CD4 T cells were lower in both smoking groups than in the non-smokers (*p*\<0.001 for both). Furthermore, VEGF, IL-12, and IL-13 were lower in both groups of smokers compared to never-smokers and COPD ex-smokers. In addition, female smokers with normal lung function had higher levels of inflammatory mediators, including the chemokine CXCL10 and the cytokine IL-13 compared to males, indicating a gender difference in the inflammatory response to cigarette smoke. Smoking has a larger impact on the adaptive immune response than airway obstruction *per se* and there seems to be gender difference mediators important for cell recruitment. Ongoing multivariate analysis will help us combine our findings with clinical data.
**References**
1\. Forsslund H, Mikko M, Karimi R, Grunewald J, Wheelock ÅM, Wahlström J, et al. Distribution of T cell subsets in bronchoalveolar lavage fluid of patients with mild to moderate COPD is dependent on current smoking status and not airway obstruction. Chest. 2014; 145: 711--22.
2\. Kohler M, Sandberg AS, Kjellqvist S, Thomas A, Karimi R, Nyrén S, et al. Gender differences in the bronchoalveolar lavage cell proteome of patients with COPD. J Allergy Clin Immunol. 2013; 131: 743--51.
07 {#S0007}
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COPD and vitamin D status {#S0007-S20001}
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### [M. Moberg]{.ul}, T. Ringbaek, J. Vestbo, L. Ferrucci, S. Ladelund, P. Lange, G. Martinez, N. B. Roberts, E. P. A. Rutten, M. A. Spruit, J. E. A. Williams and E. F. Wouters <miamoberg@hotmail.com> University of Copenhagen, Hvidovre University Hospital, Copenhagen, Denmark
###
Vitamin D deficiency is common in patients with COPD. Vitamin D deficiency has been associated with increased risk of inflammation, infection, and mortality. However, it is unknown whether vitamin D deficiency is specifically associated with COPD disease mechanism or whether it is a general marker of poor health. We wanted to investigate the prognostic value of vitamin D in patients with stable COPD and in patients hospitalised due to acute exacerbation of COPD, to examine the relation between vitamin D and COPD phenotypes and to examine the relation between vitamin D deficiency and airflow limitation in a population of older subjects. Studies I and II: a cohort of patients from the pulmonary rehabilitation program at Hvidovre Hospital. Study I: 674 patients were included. Study II: 423 patients were included. Study III: a subgroup of 91 patients from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints study. Study IV: 89 patients were hospitalised due to acute exacerbation of COPD at Hvidovre Hospital. Study V: 897 subjects from the Baltimore Longitudinal Study of Aging. Vitamin D predicted neither hospitalisation nor mortality. We did not find any association between COPD phenotypes and vitamin D status. Airflow limitation was not an independent determinant of vitamin D deficiency. Our results do not support a role for vitamin D as a predictor in COPD as other clinical characteristics seem to be more important. Vitamin D deficiency is possibly representing poor health rather than being specific to COPD disease mechanism.
08 {#S0008}
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Selective inhibition by simvastatin of IRF3 phosphorylation and TSLP production in dsRNA-challenged bronchial epithelial cells from COPD donors {#S0008-S20001}
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### [A. Brandelius]{.ul} ^1^, I. Mahmutovic Persson^1^, J. Calvén^1^, L. Bjermer ^2^, C. G. Persson^2^, M. Andersson^2^ and L. Uller^1^ <angelica.brandelius@med.lu.se> ^1^Department of Experimental Medical Science, Lund University, Sweden; ^2^Department of Respiratory medicine, Lund University Hospital, Sweden
###
Possibly reflecting anti-inflammatory properties, statin treatment may ameliorate COPD exacerbations (1, 2). Viral infections are common triggers of exacerbations in COPD and cause Th2-type inflammation (3). We have shown that thymic stromal lymphopoietin (TSLP), a cytokine linking innate and adaptive immunity and switching on Th2-type inflammation, is overproduced in virally- and dsRNA-stimulated cells from patients with GOLD stage IV COPD (4). In this study, we test the hypothesis that simvastatin inhibits dsRNA-induced TSLP in human bronchial epithelial cells. Primary human bronchial epithelial cells (HBECs), obtained by fibre optic bronchoscopy from COPD (GOLD stage II) (*n*=7) and healthy smokers (*n*=8) individual donors, were grown until confluent in 12-well plates. The cells were stimulated with the synthetic viral surrogate molecule dsRNA (10 µg/ml) for 3 and 24 hours to induce cytokine gene expression (analysed by RT-qPCR) and protein release and production (analysed by ELISA). Simvastatin (0.2--5 µg), alternatively dexamethasone, was added prior to dsRNA. dsRNA induced TSLP, TNFα, CXCL8, and IFNβ. TSLP was overproduced in dsRNA-exposed COPD cells compared with control. Simvastatin, but not dexamethasone, inhibited dsRNA-induced TSLP. Unexpectedly, simvastatin did not affect dsRNA-induced NFκ-B activation nor did it reduce the production of TNFα and CXCL8. Instead, simvastatin inhibited dsRNA-induced IRF3 phosphorylation and generation of IFNβ. The pharmacology of simvastatin may unravel paths of inhibition of TSLP production in COPD epithelium.
**References**
1\. Blamoun AI, Batty GN, DeBari VA, Rashid AO, Sheikh M, Khan MA. Statins may reduce episodes of exacerbation and the requirement for intubation in patients with COPD: evidence from a retrospective cohort study. Int J Clin Pract. 2008; 62: 1373--8.
2\. Bartziokas K, Papaioannou AI, Minas M, Kostikas K, Banya W, Daniil ZD, et al. Statins and outcome after hospitalization for COPD exacerbation: a prospective study. Pulm Pharmacol Ther. 2011; 24:625--31.
3\. Papi A, Luppi F, Franco F, Fabbri LM. Pathophysiology of exacerbations of chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2006; 3: 245--51.
4\. Calvén J, Yudina Y, Hallgren O, Westergren-Thorsson G, Davies DE, Brandelius A, et al. Viral stimuli trigger exaggerated thymic stromal lymphopoietin expression by chronic obstructive pulmonary disease epithelium: role of endosomal TLR3 and cytosolic RIG-I-like helicases. J Innate Immun. 2012; 4: 86--99.
09 {#S0009}
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The role of inflammasomes in pulmonary hypertension associated with COPD {#S0009-S20001}
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### [F. T. Cero]{.ul} ^1,2,3^, V. Hillestad^2,3^, I. Sjaastad^2,3^, A. Yndestad^3,4,5^, P. Aukrust^4,5^, E. M. Løberg^6^, G. Christensen^2,3^, K. O. Larsen^1^ and O. H. Skjønsberg^1^ <cero.fadila@medisin.uio.no> ^1^Department of Pulmonary Medicine, Oslo University Hospital, Ullevål, Oslo, Norway; ^2^Institute for Experimental Medical Research, Oslo University Hospital, Ullevål, Oslo, Norway; ^3^Center for Heart Failure Research, University of Oslo, Oslo, Norway; ^4^Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; ^5^K.G. Jebsen Inflammation Research Centre, University of Oslo, Oslo, Norway; ^6^Department of Pathology, Oslo University Hospital, Ullevål, Oslo, Norway
###
*Background*: Inflammasomes are molecular platforms which are part of the innate immunity and the best characterised is the NLRP3 inflammasome consisting of the receptor (NLRP3), the adaptor protein (ASC) and caspase-1, the protease which activate IL-18 and IL-1β. Increased levels of IL-18 and IL-1β are found in the lungs of COPD patients. Uncorrected chronic hypoxemia in COPD patients is associated with the development of pulmonary hypertension. *Objective*: To study role of NLRP3 inflammasome in chronic hypoxia-induced pulmonary hypertension. *Methods*: Active caspase-1, IL-18, and IL-1β were measured by western blot analysis of lung homogenates in WT, NLRP3^−/−^ and ASC^−/−^ mice exposed to 1 months of hypoxia in a tightly sealed chamber containing 10% oxygen. Right ventricular systolic pressure (RVSP) was measured with a microtipped transducer catheter after 3 months of hypoxia exposure. *Results*: Chronic hypoxia induced increased protein levels of caspase-1, IL-18 and IL-1β in WT, and NLRP3^−/\ −^ mice. However, hypoxia-induced activation of the inflammasome was absent in ASC^−/\ −^ mice shown by no increase of active caspase-1, IL-18, or IL-1β. RVSP of ASC^−/\ −^ exposed to hypoxia was significantly lower than WT hypoxia (40.8±1.5 mmHg versus 55.8±2.4 mmHg, *p*\<0.001), demonstrating a lesser degree of pulmonary hypertension. RVSP of NLRP3^−/\ −^ exposed to hypoxia was not significantly altered compared to WT hypoxia (52.4±2.3 mmHg versus 55.8±2.4 mmHg). *Conclusions*: Altogether, our results show that ASC^−/\ −^ mice are protected against hypoxia-induced pulmonary hypertension, suggesting inflammasome activation to be involved in the pathogenesis of the disease.
10 {#S0010}
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Emphysema progression is visually detectable in continuous but not in former smokers in a five-year time frame {#S0010-S20001}
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### Inter-observer analysis and time-trend of visual assessments of CT scans from The Danish Lung Cancer Screening Trial
### [Laura H. Thomsen]{.ul} ^1^, Mathilde M. W. Wille^1^, Asger Dirksen^1^, Jens Petersen^2^, Jesper Holst Pedersen^3^ and Saher B. Shaker^1^ <laurahohwu@gmail.com> ^1^Department of Respiratory Medicine, Gentofte Hospital, Copenhagen University, København, Denmark; ^2^Department of Computer Science, Copenhagen University, København, Denmark; ^3^Department of Thoracic Surgery, Rigshospitalet, Copenhagen University Hospital, København, Denmark
###
To evaluate interobserver agreement and time-trend in chest CT assessment of emphysema, airways, and interstitial abnormalities in a lung cancer screening cohort. Visual assessments of baseline and fifth-year scan of 1990 participants were performed independently by two observers. Results were standardised by means of an electronic score sheet; kappa and time-trend analyses were performed. We found that interobserver agreement was substantial in early emphysema diagnosis; highly significant (*p*\<0.001) time-trends in both emphysema presence and grading were found (higher prevalence and grade of emphysema in late scans). Significant progression in emphysema was seen in continuous smokers, but not in former smokers. Agreement on centrilobular emphysema subtype was substantial, agreement on paraseptal subtype moderate. Agreements on panlobular and mixed subtypes were only fair. Agreement was fair regarding airway analysis. Interstitial abnormalities were infrequent in the cohort and agreement on these fair to moderate. Highly significant time-trend was found regarding interstitial abnormalities, which were more frequent in late scans. In conclusion, visual scoring of chest CT scan characterise presence, pattern, and progression of early emphysema. Continuous smokers progress, whereas former smokers do not. Early airways disease probably requires supplementary automated computer analysis. Further studies are needed on reliability in visual assessment of interstitial lung disease.
**References**
1\. Barr RG, Berkowitz EA, Bigazzi F, Bode F, Bon J, Bowler RP, et al. A combined pulmonary-radiology workshop for visual evaluation of COPD: study design, chest CT findings and concordance with quantitative evaluation. COPD. 2012; 2: 151--9.
2\. Gietema HA, Müller NL, Fauerbach PV, Sharma S, Edwards LD, Camp PG, et al. Quantifying the extent of emphysema: factors associated with radiologists' estimations and quantitative indices of emphysema severity using the ECLIPSE cohort. Acad Radiol. 2011; 18: 661--71.
3\. Sverzellati N, Devaraj A, Desai SR, Quigley M, Wells AU, Hansell DM. Method for minimizing observer variation for the quantification of high-resolution computed tomographic signs of lung disease. J Comput Assist Tomogr. 2011; 5: 596--601.
4\. Mets OM, Smit EJ, Mohamed Hoesein FA, Gietema HA, Bokkers RP, Attrach M, et al. Visual versus automated evaluation of chest computed tomography for the presence of chronic obstructive pulmonary disease. PLoS One. 2012; 7: e42227.
5\. Hansell DM, Bankier AA, MacMahon H, McLoud TC, Müller NL, Remy J. Fleischner Society: glossary of terms for thoracic imaging. Radiology. 2008; 3: 697--722.
11 {#S0011}
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Combined effect of smoking and occupational exposure to dusts, gases or fumes on the incidence of COPD {#S0011-S20001}
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### [Annette Kainu]{.ul} ^1^, Paula Pallasaho^2^, Anssi Sovijärvi^3,4^, Ari Lindqvist^5^ and Päivi Piirilä^3,4^ <annette.kainu@helsinki.fi> ^1^HUCH Heart and Lung Center, Peijas Hospital, Helsinki, Finland; ^2^Team for Control of Hypersensitivity Diseases, Finnish Institute of Occupational Health, Helsinki, Finland; ^3^Department of Clinical Physiology and Nuclear Medicine, HUS Medical Imaging Center, Helsinki University Central Hospital, Helsinki, Finland; ^4^University of Helsinki, Helsinki, Finland; ^5^Research Unit of Pulmonary Diseases, Helsinki University Central Hospital, Helsinki, Finland
###
To assess risk factors related to the development of chronic obstructive pulmonary disease (COPD) including smoking and occupational exposure (OE) to dusts, gases, or fumes, we performed a longitudinal 11-year follow-up postal survey. The original study population was a random population sample of 8,000 inhabitants of Helsinki aged 20--69 years in 1996. Participants of the first postal questionnaire were invited to this follow-up survey in 2007 with 4,302 (78%) answers obtained. Cumulative incidence of COPD in 11 years was 3.43% corresponding to an incidence rate of 3.17/1,000/year after exclusion of those with self-reported physician-diagnosed COPD and ever COPD in 1996. Smoking and age, but not gender, were associated with incident COPD. Reported family history of COPD increased the cumulative incidence to 8.55% versus 3.04% among those without a family history (*p*\<0.001). In multivariate analysis, significant independent risk factors for incident COPD were: current smoking in 1996 (OR 4.40 \[95% CI 2.89--6.71\]), age over 50 (OR 3.42 \[95% CI 2.22--5.26\]), family history of COPD (OR 2.08 \[95% CI 1.27--3.43\]), ever asthma (OR 2.28 \[1.35--3.86\]), and self-reported OE (OR 2.14 \[1.50--3.05\]). Occupational exposure to dusts, gases, or fumes, assessed both based on self-reported exposure and a job exposure matrix using reported professions, was an independent risk factor for incident COPD. Smoking and OE together yielded an additive effect on incidence of COPD.
**Reference**
1\. Pallasaho P, Kainu A, Sovijärvi A, Lindqvist A, Piirilä PL. Combined effect of smoking and occupational exposure to dusts, gases or fumes on the incidence of COPD. COPD. 2014; 11: 88--95. doi: <http://dx.doi.org/10.3109/15412555>
12 {#S0012}
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Occupational exposure and CT in patients with and without COPD {#S0012-S20001}
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### [Øistein Svanes]{.ul} ^1,2,3^, Trude Duelien Skorge^1^, Ane Johannessen^4^, Thomas Blix Grydeland^2^, Amund Gulsvik^3^ and Per Bakke^3^ <oistein.svanes@k2.uib.no> ^1^Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway; ^2^Department of Thoracic Medicine, Haukeland University Hospital, Bergen, Norway; ^3^Department of Clinical Science, University of Bergen, Bergen, Norway; ^4^Centre for Clinical Research, Haukeland University Hospital, Bergen, Norway
###
*Background*: There is limited knowledge on the effect of occupational exposure on quantitative CT (qCT) measurements of emphysema. *Aims*: To examine the effect of occupational exposure assessed by a job exposure matrix (JEM) on CT measurements of emphysema. *Methods*: In the Norwegian GenKOLS study 2003--2005, 951 ever-smokers (49% with COPD) aged 40--85 years performed spirometry and CT examination; of these 941 completed a full occupational history. In this study, a JEM was used to assess occupational exposure in longest held job, and CT measured emphysema (% low-attenuation areas, %LAA) was the main outcome. Multiple linear regression was used for the multivariate analyses, adjusting for sex, age, and smoking (pack years and previous/current smoker). *Results*: Multiple linear regression showed significant associations between occupational exposure to biological and dust gas/fumes in longest held job, and %LAA. In COPD subjects, the unadjusted regression coefficients for %LAA in subjects with exposure to biological dust as compared to those with no exposure were 0.67 (95% CI: 0.43--0.91). The corresponding figures for gas/fumes exposure were 0.30 (0.037--0.57). After adjustments, the associations still remained statistically significant for COPD subjects exposed to biological dust and gas/fumes; 0.29 (0.031--0.55) and 0.29 (0.037--0.54). In subjects without COPD, no relationship between occupational exposure and level of emphysema was found. *Conclusions*: COPD subjects with occupational exposure to biological dust and gas/fumes in their longest held job were found to have higher levels of CT measured emphysema compared to COPD subjects with no exposure.
13 {#S0013}
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Preterm birth and lung function development {#S0013-S20001}
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### [Per Thunqvist]{.ul} <per.thunqvist@sodersjukhuset.se> Department of Clinical Science and Education, Karolinska Institutet, Stockholm, Sweden; Department of Pediatrics, Sachs' Children\'s Hospital, Stockholm, Sweden
###
Due to a tremendous development of neonatal care, the survival rate of infants born extremely preterm (\<28 weeks of gestation) has more than doubled in the last decades. The majority of these infants are diagnosed with and classified for severity of bronchopulmonary dysplasia (BPD) (1). In a longitudinal cohort study, we found no association between BPD classification and level of reduced lung function using infant lung function tests at 6 and 18 months of age. However, we report on a clear association between decreased lung function and respiratory symptoms (2). At school age, we found lower lung function for all levels of BPD severity for children with BPD compared to preterm children without BDP; however, only a minority of all children reported respiratory symptoms (3). Hence, early classification of BPD severity provides limited information on forthcoming lung function. We also compared lung function in individuals born moderate-to-late preterm (gestation week 32--36) to those born at term at eight and 16 years of age. The moderate-to-late preterm group did not report increased respiratory symptoms but presented with lower lung function at both ages without catch-up growth between the two ages (4). A recently published longitudinal follow-up studies from childhood to adult life has demonstrated that reduced airway function measured during childhood is still present in early adulthood (5). Besides not reaching the same level of lung function peak, it is still unclear if former preterm individuals also are at risk of more rapid decline. Future research should aim to study the mechanism for altered lung function after preterm birth.
**References**
1\. Fellman V, Hellstrom-Westas L, Norman M, Westgren M, Kallen K, Lagercrantz H, et al. One-year survival of extremely preterm infants after active perinatal care in Sweden. JAMA. 2009; 301: 2225--33.
2\. Thunqvist P, Gustafsson P, Norman M, Wickman M, Hallberg J. Lung function at 6 and 18 months after preterm birth in relation to severity of bronchopulmonary dysplasia. Pediatr Pulmonol. 2015; 50: 978--86.
3\. Brostrom EB, Thunqvist P, Adenfelt G, Borling E, Katz-Salamon M. Obstructive lung disease in children with mild to severe bpd. Respir Med 2010; 104: 362--70.
4\. Thunqvist P, Gustafsson PM, Schultz ES, Bellander T, Berggren-Broström E, Norman M, et al. Lung function at age 8 and 16 years after moderate-to-late preterm birth. Submitted.
5\. Vollsaeter M, Roksund OD, Eide GE, Markestad T, Halvorsen T. Lung function after preterm birth: development from mid-childhood to adulthood. Thorax 2013; 68: 767--76.
14 {#S0014}
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COPD in never-smokers {#S0014-S20001}
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### [Yunus Çolak]{.ul} ^1,2^ and Peter Lange^1,2^ <yunuscol@gmail.com> ^1^The Copenhagen General Population Study, Herlev Hospital, Copenhagen University Hospital, Copenhagen, Denmark; ^2^Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
###
Tobacco smoking remains to be the single most important risk factor for chronic obstructive pulmonary disease (COPD) (1). However, a substantial proportion of individuals with COPD in western countries are never-smokers, indicating that other risk factors may exist (2). A recent publication based on data from 69,000 participants in the Copenhagen General Population Study (CGPS) reports that never-smokers comprise 22% of individuals with COPD (3). Although, never-smokers with COPD had less severe disease with a better prognosis compared to ever-smokers with COPD, risk of lung-related hospital admissions were substantial. Compared to never-smokers without COPD, hazard ratios (HRs) for hospital admission with COPD were 8.6 (95% confidence interval: 5.3--14) in never-smokers with COPD, 30 (22--41) in former-smokers with COPD, and 43 (32--59) in current-smokers with COPD. HRs for hospital admission with pneumonia were 1.9 (1.4--2.6), 2.8 (2.3--3.4), and 3.4 (2.9--4.2), respectively. However, compared to never-smokers without COPD, never-smokers with COPD did not differ with regard to risk of lung cancer, cardiovascular comorbidities, and all-cause mortality. Surprisingly, never-smokers with COPD had a lower exposure to passive smoking and to occupational exposure compared to ever-smokers with COPD. These observations suggest that never-smokers with COPD constitute a different phenotype than ever-smokers with COPD. In an ongoing PhD project, we plan to investigate never-smokers with COPD in more details with special focus on the presence of undiagnosed asthma in this subgroup. In addition, we will present data on the impact of tobacco smoking on clinical characteristics and prognosis of individuals with asthma in the CGPS.
**References**
1\. Vestbo J, Hurd SS, Agusti AG, Jones PW, Vogelmeier C, Anzueto A, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2013; 187: 347--65.
2\. Salvi SS, Barnes PJ. Chronic obstructive pulmonary disease in non-smokers. Lancet. 2009; 374: 733--743.
3\. Thomsen M, Nordestgaard BG, Vestbo J, Lange P. Characteristics and outcomes of chronic obstructive pulmonary disease in never smokers in Denmark: a prospective population study. Lancet Respir Med. 2013; 1: 543--50.
15 {#S0015}
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Epidemiological aspects of COPD among never-smokers {#S0015-S20001}
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### [Stig Hagstad]{.ul} <stig.hagstad@gu.se> Krefting Research Centre, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
###
The main risk factor for developing COPD is active tobacco smoke. However, COPD also occurs among subjects who are never-smokers. Globally, use of biomass fuel for heating and cooking as well as tuberculosis has been associated with COPD among never-smokers although the risk profile in developed countries is not yet fully established. My doctoral thesis concerns the epidemiology of COPD among never-smokers and is based on data from the Obstructive Lung Disease in Northern Sweden (OLIN) studies. The first study examined prevalence, risk factors, and symptomatology of COPD in never-smokers. We found that one in five subjects with COPD had never smoked themselves. The overall prevalence of COPD among never-smokers was 6.9%, and for GOLD stage ≥II 3.5%, compared with 14.0 and 7.3% respectively among all subjects. Respiratory symptoms (i.e. sputum production, recurrent wheeze, and dyspnoea) were highly prevalent among never-smokers with GOLD stage ≥II. Risk factor analysis found COPD among never-smokers to be significantly associated with age ≥66 years (OR 5.56, 95% CI 2.56--12.21) and a previous physician diagnosis of asthma (OR 2.96, 1.44--6.08). The subsequent study examined the role of passive smoke as a risk factor for COPD in 2,118 lifelong never-smokers. Passive smoking in various settings was associated with COPD among never-smokers in a dose-dependent manner. Exposure to passive smoke both ever at home and at both current and previous workplace was, after belonging to the oldest age group, the strongest risk factor for COPD among never-smokers (OR 3.80, 1.29--11.2).
| {
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Introduction
============
Cerebral ischemic insults represent an immense burden for national health care systems. The total European annual health care cost of traumatic brain injury (TBI) is over 33 billion € ([@B8]). The incidence of cranio-cerebral traumas in industrialized countries is 200--300 per 100,000 humans per year ([@B6]). It was estimated that every year 2.5 million people sustain a TBI leading to 53,000 TBI-related deaths in the United States ([@B32]).
Traumatic brain injury mediated damages of the brain proceed in two phases. Initial direct mechanical impact such as brain contusion causes parenchymal damage, which becomes evident as tissue destruction, axonal shearing, and hemorrhage associated with increased cerebrovascular permeability and impaired cerebral blood flow ([@B84]). Within hours to days the primary brain damage induces a set of secondary processes on the cellular, metabolic, and molecular level such as oxidative stress, inflammation, and apoptosis. These events could cause further cerebrovascular disruption and neuronal loss leading, eventually, to disability or even death ([@B92]; [@B16]; [@B30]; [@B52]; [@B83]). Cerebral edema formation is one of the most serious and difficult to control secondary effects of TBI and one of the main factors for the high mortality and morbidity after TBI. The causes of edema in TBI patients are complex, but it is well appreciated that leakage of the blood-brain barrier (BBB) induced by cerebral ischemia or hypoxia is a significant factor in the development of vasogenic edema ([@B5]; [@B18]; [@B36]).
Several therapeutic strategies for TBI have been developed in the last decades. However, numerous animal studies and clinical trials were unsuccessful, and no effective therapy for TBI patients has emerged ([@B37]; [@B56]; [@B12]; [@B100]; [@B54], [@B55]; [@B97]; [@B83]). Therefore, the therapeutic focus is still set on activities to reduce brain edema and intracranial pressure ([@B84]).
In the development of novel therapeutic approaches it could be useful to evaluate treatment strategies for other diseases with similar events during the disease. In the case of TBI and cerebral edema, the comparison with stroke seems to be meaningful. Although the primary cause of TBI and stroke are substantially different, the secondary mechanisms of brain infarction are overlapping. In particular, excitotoxicity, inflammation, free radical damage, and BBB disruption are hallmarks of both TBI and stroke ([@B77]). In case of stroke current treatments are only based on thrombolysis by, e.g., tissue plasminogen activator (t-PA) in the acute phase of stroke. Another approach to decrease the number of cerebral ischemic insults is to minimize risk factors. The most important risk for stroke is high blood pressure. In addition, high concentrations of lipids in blood have been identified as another main risk factor ([@B49]; [@B4]). In recent years, it was hypothesized that low triglycerides, high HDL levels or lower LDL/HDL ratios were more protective than low concentrations of total cholesterol or LDL-bound cholesterol ([@B48]; [@B25]). Clinical studies showed that lipid-lowering compounds were able to reduce the number of fatal stroke, but not the incidence of stroke ([@B102]). Therefore, it is believed that lipid-lowering substances such as peroxisome proliferator-activated receptor (PPAR) agonists are able to attenuate stroke damage. Some studies reported that the combination of statins with PPAR agonists show additional beneficial effects on adverse stroke outcome, especially in cases of high risk groups with, e.g., atherogenic dyslipidemia ([@B58]; [@B91]; [@B15]).
Peroxisome proliferator-activated receptors are divided into the three subtypes α, β/δ, and γ, whereby especially PPARα activation leads to a decrease of lipids. PPARs belong to the group of orphan nuclear receptors which regulate DNA transcription after dimerization with retinoic acid-X-receptors (RXR) ([@B33]). Several studies revealed that PPAR agonists were not only beneficial via their lipid-lowering effects. They additionally exerted pleiotropic mechanisms modulating inflammatory, immune and oxidative pathways ([@B82]; [@B24]; [@B28]; [@B93]; [@B21],[@B22]). Since PPARs are expressed in neurons as well as in glial and endothelial cells, PPAR agonists were not only neuroprotective, they also reduced microglial activation or BBB permeability ([@B72]; [@B34]). This could be of immense relevance, because clinical studies focusing only on neuroprotection were not successful in the last decades indicating that future therapeutic strategies should be extended toward additional target cells ([@B90]).
In this regard the term neurovascular unit describes the fact that the functionality of the BBB is highly regulated by its microenvironment, and that also brain capillary endothelial cells could regulate the functionality of CNS cells as well. That's why microglial cells as well as brain endothelial cells got into the focus of recent research about novel targets. Indeed, the breakdown of the BBB contributes to vasogenic edema formation during ischemic insults and the stabilization of the BBB could reduce edema formation and brain tissue damage ([@B51]; [@B2]; [@B68]; [@B1]). Brain capillary endothelial cells are distinctly different compared to peripheral endothelial cells. Their intercellular gaps are sealed by tight junctions, the pinocytotic activity is remarkably decreased and they do not form fenestrae ([@B45]). In addition, they possess a significant array of active transporter proteins and exhibit an increased metabolic activity. All these features contribute to the barrier function which is generally comprised by its physical (paracellular), transporter and metabolic components ([@B66]).
*In vivo* as well as *in vitro* studies confirmed that preventive treatment, administration during the acute phase as well as therapeutic application of PPARα agonists such as fenofibrate could decrease brain damage after cerebral ischemia ([@B72], [@B71]; [@B34]). It was shown that treatment during the acute phase was necessary to prevent BBB breakdown and that this was dependent on the presence of PPARα, because fibrate medications were not effective in PPARα knock-out mice or in models based on brain endothelial cells derived from PPARα knock-out mice ([@B42]; [@B60]; [@B38]). With regard to TBI, therapy with fenofibrate in a TBI-rat model revealed also beneficial effects on the adverse outcomes ([@B10]; [@B17]). However, despite these efforts the underlying BBB protecting mechanisms are still not clarified.
In this regard, the choice of an appropriate *in vitro* model to simulate ischemia induced BBB breakdown is of pivotal importance. A huge array of *in vitro* models for TBI exists comprising of different scratch, strain/stretch or organotypic hippocampal slice models ([@B59]; [@B84]). Most models are simplified and are not capable to include or handle all relevant parts during a brain injury. To investigate the ischemic component, cells in these models are treated by hypoxia or oxygen/glucose deprivation (OGD). This could be applied alone or in combination with mechanical stress models ([@B84]). Recently, we have analyzed the effects of stretch, OGD and the combination of stretch with OGD on brain endothelial cells as a TBI *in vitro* model of the BBB. Results revealed that the effects of OGD were significantly more prominent than the influence of stretch. Moreover, only OGD led to a distinct breakdown of the paracellular barrier of brain endothelial cell layers ([@B75]). In addition, measurement of the transendothelial electrical resistance (TEER) as important tightness parameter was only applicable in the Transwell model, but not in the stretch or in the combined stretch/OGD-set-up. Based on these facts it was decided to investigate the effects of PPARα agonist WY-14643 in our recently validated *in vitro* BBB OGD-model ([@B65]).
Our data describe for the first time underlying molecular mechanisms of PPARα agonist WY-14643 for the stabilization of the BBB under ischemic conditions. In correspondence to the literature, we show two types of effects. On the one hand we found inhibition of functional barrier breakdown determined by TEER and fluorescein permeability, alteration of tight junction protein localization and changes of enzyme activities (MAP kinases, proteasomal enzymes). These effects were not inhibited by PPARα antagonist GW6471. On the contrary, we found that WY-14643 upregulated the expression of plasminogen activator inhibitor-1 (PAI-1) in brain endothelial cells for the first time. This was blocked by PPARα antagonist GW6471 and led to decreased t-PA activity. Mouse experiments confirmed beneficial effects of WY-14643 in a TBI model. Data supported that WY-14643 acted via pathways which either were or were not inhibited by PPARα antagonist GW6471. This opens the discussion about probably involved cellular processes which require the presence of PPARα, but not its direct transcriptional activity.
Materials and Methods {#s1}
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Material
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Collagen IV from human placenta (C5533), PBS (D8537), Triton-X 100 (T8787), DMEM (D5796), DAPI (D8417), β-mercapto-ethanol (M6250), fluorescein sodium (F6377), *N*-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (S6510), Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin (B4636), Z-Leu-Leu-Glu-7-amido-4-methylcoumarin (C0483), MG132 (C2211, Z-Leu-Leu-Leu-al), Lactacystin (L6785), WY-14643 (C7081), GW6471 (G5045), rhPAI-1 (A8111), albumin from bovine serum for immunofluorescence microscopy (fraction V, A9647) and for western blotting (A7906) were purchased from Sigma--Aldrich. DMEM without glucose (11966-025, Gibco^®^) was obtained from Life technologies (USA). FCS Gold EU approved was bought from PAA Laboratories (A15151, Lot A15111-2018, Linz, Austria) and was heat-inactivated in a water-bath at 56°C for 30 min. Penicillin/streptomycin (100X, 10,000 Units/mL, 10,000 μg/mL, A2213) and 0.05% Tyrpsin/0.02% EDTA-solution (L2143) were from BioChrom AG (Berlin, Germany). 6-well and 24-well plates and 24-well Transwell^®^ inserts (0.4 μm pore size, PET) were obtained from Becton and Dickinson (REF353046, REF353035, REF353226, USA). Gelatine was from SERVA (22151, Heidelberg, Germany), nuclease-free water was purchased from Ambion (AM9937, USA). All other substances were of analytical grade.
Cell Culture
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Mouse brain endothelial cell line cerebEND was cultured in DMEM medium supplemented with 10% FCS and 1% penicillin/streptomycin in 0.5% gelatine coated cell culture tissue flasks, rat glioma cell line C6 (ATCC) was grown with the same culture medium also in 0.5% gelatine coated tissue flasks ([@B79]). Cells were maintained in an incubator at 37°C, 95% humidity and a 5% CO2/95% air atmosphere and subcultivated 1:3 or 1:20, respectively, once a week ([@B63]). Transwell experiments were accomplished as recently reported ([@B65]). In brief, cerebENDs were cultivated on collagen-IV coated 24-well inserts, on day 9 inserts with cerebENDs were placed over C6 cell containing well-plates to form the co-culture set-up. OGD experiments were conducted on day 13. After washing steps cells were subjected to serum-free DMEM with or without glucose supplemented with the substances. All solutions had the same end-concentrations of solvents. TEER was measured using chopstick electrodes from Millipore after 30 min of equilibration at RT as previously published ([@B62], [@B65]). After 4 h OGD treatment (1% O~2~) TEER was determined followed by a transport study with 10 μM fluorescein. TEER \[Ohm × cm^2^\] and permeability coefficients Pe including blank insert values were calculated according the clearance principle as previously reported ([@B67]; [@B69]). Due to comparison reasons, TEER values of normoxic mono-cultured cerebEND controls and permeability coefficients of OGD-treated co-cultures (OC6) were set to 100%.
Quantitative Polymerase Chain Reaction (qPCR)
---------------------------------------------
For quantitative polymerase chain reaction (qPCR) experiments cerebEND and C6 cells were cultivated on 6-well plates. NC6 is the abbreviation for normoxia-treated cerebEND cells with the supernatant of normoxic treated C6 cells, OC6 means OGD-treated cerebEND cells with the supernatant of OGD-treated C6 cells. In these cases C6 cells were incubated for 4 h normoxia or OGD to obtain their supernatants which were directly applied on PBS washed cerebEND cell layers to guarentee fresh soluble factors of C6 cells for further 4 h incubation. RNA isolation, cDNA transcription and qPCR were accomplished as recently published ([@B65]). Used FAM-labeled probes from Taqman^®^ (Applied Biosystems) were listed in Supplementary Table [S1](#SM1){ref-type="supplementary-material"}. In case of PPARα and LRP1, cDNA was preamplified using Taqman PreAmp Master mix (2x) prior qPCR analysis according to [@B63], Supplementary Part). Each sample was analyzed as triplicate. Relative mRNA abundances to β-actin were calculated by the ddCt method using following formula: 2^(Ct\ of\ β-actin--Ct\ of\ gene\ of\ interest)^, where Ct is the threshold cycle value.
Western Blotting
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Western blotting was conducted as recently published ([@B65]). In brief, cells were scraped after the treatments in RIPA buffer on ice. In case of membran protein enrichment, proteins were extracted with 1% Triton-X 100 followed by lysis of the residual proteins in RIPA buffer. All lysis buffers were supplemented with protease inhibitor cocktail and PhosphoSTOP. Protein concentrations were measured by a detergent-compatible Pierce BCA assay (Fisher Scientific). Twenty microgram protein of total lysates or 10 μg of Triton-X 100 and RIPA fractions per lane were loaded onto 7.5, 10, or 12% SDS--PAGE gels. After gel electrophoresis proteins were immunoblotted onto polyvinylidene difluoride membranes. Incubations with primary and secondary antibodies (Supplementary Table [S2](#SM1){ref-type="supplementary-material"}) were carried out as previously described ([@B64]). ECL-solutions were used for the visualization of the bands using a FluorChem FC2 Multiimager II (Alpha Innotech, Hessisch Oldendorf, Germany). Density values of single protein bands were calculated with the software Alpha View and were related to the corresponding β-actin bands.
Proteasomal Activity
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Proteasomal activities were measured according to [@B41] with slight modifications. Stock solutions of proteasomal enzyme substrates *N*-succinyl-Leu- Leu-Val-Tyr-7-amido-4-methylcoumarin (5mM, chymotrypsin-like), Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin (5mM, trypsin-like), Z-Leu- Leu-Glu-7-amido-4-methylcoumarin \[5mM, peptidylglutamyl-peptide hydrolyzing activity (PGPH)\] as well as proteasomal inhibitors lactacystin (5mM) and MG132 (50mM) were prepared in DMSO. cerebEND and C6 cells were seeded on gelatin coated 6-wells, cultivated for 6 days and subjected to normoxia or OGD treatments as recently published ([@B65]). After the treatments cells were washed with ice-cold PBS twice and lysed with 300 μL proteasome activity buffer \[PAB; 10 mM Tris-HCl (pH = 7.8), 1 mM EDTA, 0.5 mM dithiothreitol, 0.5% Triton X-100, 5 mM MgCl~2~\] per 6-well on ice with moderate shaking (52 rpm) for 30 min. While supernatants were stored on ice, protein concentrations were determined by BCA protein assay kit from Pierce (Cat. No. 23227) using 25 μL sample per 96-well. Then, volume for 50 μg protein per sample was supplemented with according volumes of WY-14643 and GW6471 stock solutions in DMSO and filled up with PAB to 196 μL per well to reach a total DMSO concentration of 0.4% in all samples. Four microliter enzyme substrate stock solution per well were added on ice, and the reaction started by putting the black 96-well plate in a prewarmed microplate reader (GeniosPro, Tecan, Austria) at 37°C. Fluorescence increase was recorded at 360 nm/460 nm every 10 min for 2 h. Control blank values were substracted from cell lysate values and slopes between 10 and 120 min were calculated after linear regression analysis. Finally, slopes of control lysates set to 100%. Each sample was measured as duplicate from three independent conducted experiments.
MMP and t-PA Activity
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Enzymatic activity of matrix metalloproteinases (MMP) or of t-PA of concentrated medium supernatants were measured by using 520 MMP FRET substrate SB-14 (Anaspec, USA) or component A of Sensolyte AMC t-PA activity assay kit (72160, Anaspec, USA) as recently described ([@B65]). Increasing fluorescence was recorded over 120 min at 37°C in a microplate reader (GeniosPro, Tecan, Austria). Data between 10 and 120 min were used to calculate the slope by linear regression analysis. Slopes of media supernatants of OC6-treated cerebEND cells were set to 100%.
Animals
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All animal procedures were conducted in compliance with the institutional guidelines of the Johannes Gutenberg-University, Mainz, Germany and approved by the State Agency for Consumer and Health Protection (approval number: G12-1-041), and performed in accordance with the German Animal Welfare Act. The group sizes of the study were calculated prior to approval with the analysis of variance sample size. 16.2 ± 7.7 weeks old 48 male C57Bl6/N mice (Charles River Laboratories, Sulzfeld, Germany; 26.4 ± 8.3 g) and 26 weeks old 20 PA-1 deficient mice (B6.129S2-*Serpine1^tm1Mlg^*/J) with 26--32 g weight, purchased from Jackson Laboratories (Bar Harbor, ME, US), were included in the study. The compliance with the ARRIVE guidelines is confirmed. Animals were held in scantainer ventilated cabinets with exercise wheels and plastic houses at the animal facility of the Department of Experimental Surgery. The animals were exposed to a 12-h dark/light interval and ingested H~2~O and feed *ad libitum.*
Traumatic Brain Injury
----------------------
Animals were anesthetized with 5 mg/kg midazolam (Siegfried Hameln, Hameln, Germany), 0.05 mg/kg fentanyl (Merck, Darmstadt, Germany), and 0.5 mg/kg medetomidine (Zoetis, Kirkland, QC, Canada) intraperitoneally. Rectal temperature was maintained at 37°C with a thermostatically regulated, feedback-controlled heating pad (Hugo Sachs, March-Hugstetten, Germany). The TBI was induced as described before ([@B87]). The cranium was fixed in a stereotactical frame, a craniotomy was generated between lambdoid, sagittal and coronal sutures with a saline cooled high speed drill. The trauma was induced with an electromagnetic cortical impact device (Impact OneTM Stereotaxic Impactor, Richmond, IL, USA) diameter of the impactor tip: 3 mm; impact velocity: 6 m/sec; impact duration: 200 msec, and displacement: 1.5 mm. The craniotomy was closed with the initially removed bone flap using conventional tissue glue (Histoacryl, Braun-Melsungen, Germany). The skin was carefully closed with four single button sutures, anaesthesia antagonized (0.5 mg/kg Flumazenil, Siegfried Hameln, Hameln, Germany, and 2.5 mg/kg Atipamezole hydrochloride, Pfizer, Freiburg, Germany, i.p.) and the animals were transferred back into their cages. The animals were placed for 1.5 h in a neonatal incubator (IC8000, Draeger, Luebeck, Germany) with controlled air temperature (35°C) and ambient humidity (35%) to maintain constant body temperature and avoid hypothermia. The experimenter performing the CCI surgery was blinded to the treatment. A separate experimenter, also blinded to treatment, performed tissue preparations.
Drug Preparation
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WY-14643 (Tocris Bioscience, Bristol, UK) was dissolved in DMSO, warmed in 37°C water-bath and diluted in 0.9% NaCl solution to 10% DMSO end-concentration. GW6471 (Tocris Bioscience, Bristol, UK) was also dissolved in 10% DMSO with 0.9% NaCl solution. As vehicle control 10% DMSO in 0.9% NaCl solution was administered i.p.. WY-14643 or vehicle was administered 30 min before CCI. In experiments with both substances GW6471 or vehicle was administered 60 min before CCI and WY-14643 or vehicle 30 min before CCI. The experimentator was blinded to treatment.
Euthanasia and Tissue Preparation
---------------------------------
The animals were reanaesthetized as described and killed by cervical dislocation. For the quantification of brain water content and further investigations the cerebellum was separated and the hemispheres were cut along the interhemispheric plane slightly modified as described before ([@B88]). Both hemispheres were separated again in the middle of the contusion area and weighed to assess their wet weight. One half of each hemisphere was dried in a vacuum-centrifuge (Univapo 100 H, UniEquip, Planegg, Germany) for 48 h at 39°C to determine the dry weight. On the basis of the gravimetrical differences, brain water content was obtained by the following calculation: Hemispheric water content (%): (WW - DW)/WW × 100, where WW is the wet weight (g) and DW is the dry weight (g) of the brain hemispheres ([@B86]). The other half of the hemispheres were frozen in liquid nitrogen and stored at -80°C for tissue analysis.
Statistical Analyses
--------------------
Statistical tests were performed using SigmaPlot 12.5 including SigmaStat tools applying One-Way ANOVA multiple comparison versus control with Holm--Sidak method. Data sets showing either no normal distribution or equality of variances were analyzed using Kruskal--Wallis One-Way ANOVA Rank Test followed by an all pairwise multiple comparison procedure with Dunn's Method. Results are given as mean ± standard deviation (SD), or if indicated, as standard error of mean (SEM). The level of statistical significance was set at *p* \< 0.05, indicated with asterisk (^∗^).
Results
=======
WY-14643 Inhibits Barrier Breakdown during OGD
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At the beginning of the experiments TEER values were about 27.6 ± 2.1 Ω × cm^2^ (raw data differences of ∼80 Ωs between empty inserts and inserts with cells). After 4 h of OGD treatment TEER was decreased to 56 ± 4% (mean ± SEM, *n* = 36, *p* \< 0.05), which was abolished by addition of PPARα agonist WY-14643 to 63 ± 7% (30 μM, *n* = 6, n.s.), 90 ± 10% (100 μM, *n* = 6, *p* \< 0.05), and 83 ± 7% (300 μM, *n* = 15, *p* \< 0.05) in a concentration dependent manner (**Figure [1A](#F1){ref-type="fig"}**). In concordance to this, permeability of paracellular marker fluorescein was increased by OGD treatment from 66.6 ± 5.8% (=2.03 ± 0.17 × 10^-5^ cm/s) to 100.0 ± 3.2% (mean ± SEM, *n* = 27, *p* \< 0.05). Application of WY-14643 during OGD reduced fluorescein permeability to 84.6 ± 2.6% (30 μM, *n* = 6, n.s.), 76.7 ± 11.5% (100 μM, *n* = 6, n.s.), and 78.9 ± 3.6% (300 μM, *n* = 15, *p* \< 0.05) (**Figure [1B](#F1){ref-type="fig"}**). Notably, addition of PPARα antagonist GW6471 did not reverse the BBB stabilizing effects of WY-14643. Therefore, it was verified whether WY-14643 activated PPARα in our model. Addition of 300 μM WY-14643 induced translocation of PPARα to the nucleus (Supplementary Figure [S1](#SM1){ref-type="supplementary-material"}). Moreover, mRNA expression of PPARα was decreased by OC6 and was further reduced by 300 μM WY-14643. The effect of WY-14643 on PPARα expression was blocked by 10 μM GW6471, whereas mRNA of PPARγ was upregulated by OC6 and not affected by addition of WY-14643 (Supplementary Table [S3](#SM1){ref-type="supplementary-material"}). These data indicated that WY-14643 induced PPARα activation in our model. In summary, WY-14643 reduced OGD-induced barrier damage significantly, but probably not by mechanisms dependent on the transcriptive activity of PPARα.
{#F1}
Effects of WY-14643 on Tight Junction Proteins after OGD
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Barrier functionality is linked to the expression and localization of tight junction proteins ([@B96]; [@B44]). In order to investigate effects of WY-14643 on tight junction proteins, mRNA levels were measured by qPCR, and protein expression and distribution was determined by western blotting. As recently shown, 4 h of OC6-treatment decreased mRNA expression of claudin-5, ZO-1, and occludin significantly ([@B65]), but addition of 300 μM WY-14643 did not change mRNA expression of any of the investigated tight junction proteins (claudin-3, claudin-5, claudin-12, occludin, and ZO-1) after 4 h of OC6-treatment (data not shown).
Western blotting analysis revealed a slight, but not significant upregulation of ZO-1 protein by OC6-treatment in comparison to the normoxic control (0.77 vs. 1.00-fold, **Figure [2A](#F2){ref-type="fig"}**). Addition of 300 μM WY-14643 during OC6 significantly increased total ZO-1 protein to 1.12 ± 0.05-fold, and the addition of GW6471 did not reverse this effect. In order to test for a change in cellular localization after the treatments, cells were lysed first in triton-X 100 and residual cell components were dissolved in RIPA buffer. The fraction soluble in triton-X 100 was enriched with plasma membrane proteins, whereas the fraction insoluble in triton-X 100, so-called RIPA fraction, contained mainly cytoskeleton/cytosolic proteins allowing to investigate the movement of proteins between these two fractions. For example, a significant enrichment of plasma membrane marker Na^+^/K^+^-ATPase was shown in the triton-X 100 fraction, whereas cytoskeleton protein β-actin was mainly found in the RIPA-fraction (see Supplementary Figure [S2](#SM1){ref-type="supplementary-material"}).
![**Influence of WY-14643 and PPARα antagonist GW6471 on effects of OC6 treatment on the protein expression of tight junction proteins ZO-1, occludin and claudin-5 of cerebEND cells.** OC6 treatment of cerebENDs accords to 4 h OGD treatment with medium supernatants derived from 4 h OGD-treated C6 cells, whereas NC6 treatment means cerebEND cells incubated for 4 h under normoxic conditions with medium supernatants of 4 h normoxic treated C6 cells. Results for ZO-1 **(A)**, occludin **(B)** and claudin-5 **(C)** were displayed. Densitometric analyzed protein expression of total cell lysates were compared to RIPA and triton-X 100 fractions. Moreover, protein distributions \[%\] between RIPA and triton-X 100 fractions were presented. Values were related to β-actin and according representative western blot images are depicted. Dotted lines indicated cuts of images of the same blot due to presentation reasons of selected bands. Statistical significance was labeled with ^∗^ vs. NC6 and ^\#^ vs. OC6 (*p* \< 0.05). Data are presented as means ± SEM (*n* = 3--8).](fnmol-10-00149-g002){#F2}
In case of ZO-1, the distribution between the triton-X 100 fraction and the RIPA-fraction was changed. OC6, OC6+WY-14643, and OC6+WY14-643+GW6471 stepwise reduced the amount of ZO-1 in the triton-X 100 fraction. Together with the expression data, it was shown that the amount of ZO-1 in the RIPA-fraction was significantly increased by WY-14643 during OC6 by 22% to 1.22 ± 0.06-fold, whereas the triton-X fraction remained unaffected. Addition of 10 μM GW6471 to WY-14643 during OC6 treatment did not change the effects of WY-14643 again. In case of occludin (65 kDa), its expression decreased in the RIPA-fraction from 1.49+0.11-fold (NC6) to 1.00-fold (OC6) due to the OC6-treatment. This change in expression was again prevented by addition of 300 μM WY-14643 (1.44 ± 0.14-fold). Interestingly, the effect of WY-14643 on occludin expression was reversed by PPARα antagonist GW6471 to 1.10 ± 0.20 indicating a PPARα dependency. Moreover, OC6 treatment resulted in a movement of occludin into the triton-X 100 fraction from 21.8 ± 1.5% (NC6) to 31.5 ± 1.9% (OC6), which was blocked by WY-14643 (22.8 ± 1.5%), but not reversed by GW6471 (23.8 ± 3.6%) (mean ± SEM, *n* = 3--6, **Figure [2B](#F2){ref-type="fig"}**). Based on these results, it could be assumed that occludin expression was PPARα dependent, but the protein distribution was PPARα independent. Protein expression of claudin-5 was not regulated either by OC6 nor by the addition of WY-14643 during OC6 treatment in comparison to the NC6 control (**Figure [2C](#F2){ref-type="fig"}**). In summary, changes of tight junction proteins by WY-14643 were only found on the protein level. The redistribution of tight junction proteins might play an important role for the observed barrier breakdown.
Effects of WY-14643 on the Phosphorylation of MAP Kinases after OGD
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Tight junction protein localization is dependent on the level of intracellular calcium and is associated with their phosphorylation status ([@B31]; [@B57]; [@B81]). These parameters are linked to the activation of kinases which could be deduced from the protein expression ratio of phosphorylated kinase per total kinase. Therefore, effects of 300 μM WY-14643 on the phosphorylation ratio of MAP kinases -- known for their role during cerebral ischemia ([@B29]; [@B99]) -- during OGD were investigated. The amount of phosphorylation of all four investigated kinases ERK1/2, Akt, p38, and SAPK/JNK in cerebEND cells were upregulated by 4 h of OC6 treatment (ERK1/2: from 0.28 to 1.00-fold, Akt: from 0.82 to 1.00-fold, p38: from 0.55 to 1.00-fold, and SAPK/JNK: from 0.55 to 1.00-fold), which was only for Akt not statistically significant (**Figure [3](#F3){ref-type="fig"}**). Addition of WY-14643 during OC6 treatment significantly reduced phosphorylation of ERK1/2 from 1.00 to 0.75-fold, but increased phosphorylation of p38 and SAPK/JNK to 2.33- and 1.42-fold, respectively. Addition of 10 μM GW6471 did not reverse the effects of WY-14643 on ERK1/2 or p38, it increased the phosphorylation of SAPK/JNK only in the RIPA-fraction and reduced the pAkt/Akt ratio for all fractions to 0.54-, 0.52-, and 0.55-fold. Interestingly, administration of 300 μM WY-14643 led to an increased amount of pSAPK/JNK only in the Triton-X 100 (membrane) fraction. In summary, addition of PPARα agonist WY-14643 modulated the phosphorylation grade of ERK1/2, p38 and SAPK/JNK that was only reversed in the RIPA-fraction of SAPK/JNK by PPARα antagonist GW6471.
![**Influence of WY-14643 and PPARα antagonist GW6471 on effects of OC6 treatment on phosphorylation ratios of MAP kinases ERK1/2, Akt, p38, and SAPK/JNK of cerebEND cells.** OC6 treatment of cerebENDs accords to 4 h OGD treatment with medium supernatants derived from 4 h OGD-treated C6 cells, whereas NC6 treatment means cerebEND cells incubated for 4 h under normoxic conditions with medium supernatants of 4 h normoxic treated C6 cells. Results for ERK1/2 **(A)**, Akt **(B)**, p38 **(C)**, and SAPK/JNK **(D)** were displayed. Densitometric analyzed protein expression of total cell lysates were compared to RIPA and triton-X 100 fractions. Moreover, protein distributions \[%\] between cytoskeleton (RIPA) and membrane enriched fractions (triton-X 100) were presented. Values were related to β-actin and according representative western blot images are depicted. Dotted lines indicated cuts of images of the same blot due to presentation reasons of selected bands. Statistical significance was labeled with ^∗^ vs. NC6, ^\#^ vs. OC6 and ^§^ vs. OC6+WY (*p* \< 0.05). Data are presented as means ± SEM (*n* = 3--6).](fnmol-10-00149-g003){#F3}
WY-14643 Is able to Inhibit Activity of Proteasomal Enzymes
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Recent work showed that blockade of the proteasome could contribute to the stabilization of the BBB during TBI as well as stroke ([@B46]; [@B85]). Therefore, effects of WY-14643 on proteasomal activity was investigated. Interestingly, no significant changes were found in proteasomal activity when 300 μM WY-14643 were added during 4 h of OC6 treatment followed by enzyme isolation and activity measurement. Thus, in order to test whether WY-14643 inhibition of proteasomal enzymes could be reversible and was abolished by the enzyme isolation procedure, WY-14643 was added after isolation of proteasomal enzymes of cerebEND cells. This procedure led to a significant blockade of chymotrypsin-like (300 μM WY-14643: 64.6 ± 0.59%) and trypsin-like activity (300 μM WY-14643: 81.2 ± 1.8 %) by WY-14643 in a concentration dependent manner (**Figure [4](#F4){ref-type="fig"}**), whereas PGPH activity was not altered. Addition of 10 μM of PPARα antagonist GW6471 did not reverse effects by WY-14643, it even further blocked the activity of chymotrypsin-like (300 μM WY-14643+10 μM GW6471: 57.9 ± 1.7%) and trypsin-like (300 μM WY-14643+10 μM GW6471: 72.3 ± 1.1%) proteasomal activity significantly. In summary, WY-14643 could inhibit proteasomal activity that was not reversed by PPARα antagonist GW6471.
{#F4}
WY-14643 Reduces Activity of Tissue-Plasminogen Activator (t-PA) after OGD
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It is rather well established that MMPs and t-PA contribute to BBB breakdown during cerebral ischemia ([@B73]; [@B3]). Therefore, firstly effects of WY-14643 on mRNA expression of MMP2, MMP3, and MMP9, and of their endogenous inhibitors TIMP1 and TIMP3 were investigated. Three hundred micrometer WY-14643 decreased expression of MMP3 to 0.75-fold and TIMP1 to 0.68-fold after 4 h OC6 treatment. Addition of 10 μM GW6471 did not reverse effects of WY-14643, it further significantly decreased TIMP1 expression to 0.48-fold and increased TIMP3 expression from 1.11-fold (300 μM WY-14643 during OC6) to 1.37-fold. In concordance with the concomitant decrease of MMP3 and TIMP1 WY-14643 did not reduce total MMP-activity after 4 h of OC6 treatment. This indicated a minor role of MMP-activity for the beneficial effects of WY-14643 to prevent BBB breakdown *in vitro* (Supplementary Table [S3](#SM1){ref-type="supplementary-material"}). Then, expression of t-PA and its endogenous inhibitor PAI-1 was analyzed. mRNA expression of t-PA was significantly upregulated from 0.22 ± 0.04 (NC6) to 1.00-fold by OC6 treatment, however, WY-14643 addition showed no effect on t-PA mRNA expression (**Figure [5](#F5){ref-type="fig"}**). Western blotting confirmed mRNA expression results. OC6 treatment significantly upregulated protein expression of t-PA on the protein level in cerebENDs from 0.74 ± 0.09 to 1.00-fold, whereas addition of WY-14643 had no effects on total protein expression. Interestingly, application of 300 μM WY-14643 resulted in a significant redistribution of t-PA toward the RIPA (cytosol/cytoskeleton) fraction which was still consistent after addition of GW6471. In case of PAI-1, 4 h of OC6 treatment did not regulate mRNA expression, but application of 300 μM WY-14643 significantly upregulated PAI-1 from 1.00-fold (OC6) to 2.15 ± 0.44-fold. This upregulation was abolished by addition of 10 μM GW6471 indicating a PPARα dependent effect. Western blotting confirmed a slight PAI-1 upregulation by WY-14643 from 1.00 to 1.17 ± 0.06-fold (n.s.), which was significantly reduced by GW6471 to 0.74 ± 0.07-fold. Interestingly, OC6 treatment revealed a substance independent redistribution of PAI-1 toward the triton-X 100 (membrane) fraction. Moreover, OC6 led to a significant protein reduction of PAI-1 in cerebEND cells from 1.84 ± 0.12-fold (NC6) to 1.00-fold (OC6) indicating a significant release into the growth medium. Therefore, t-PA activity of cell culture medium supernatants was measured after the treatments. According to expression results of t-PA and PAI-1, OC6 treatment increased t-PA activity in cell supernatants from 74.7 ± 5.9% (NC6) to 100 ± 3.9% (OC6) which was reduced by WY-14643 to 62.1 ± 8.7%. Data suggested a major role of upregulated PAI-1 for WY-14643 mediated barrier stabilization. Addition of 1 μg/mL rhPAI-1 decreased t-PA activity in supernatants to 6.9 ± 1.6%. Moreover, 1 μg/mL rhPAI-1 blocked barrier breakdown significantly. In detail, decrease of TEER by OC6 treatment was attenuated from 55.8 ± 3.6% to 77.7 ± 9.9%, and fluorescein permeability was reduced from 100.0 ± 8.8% (OC6) to 53.3 ± 3.8% by addition of 1 μg/mL rhPAI-1 in comparison to 68.8 ± 5.7% of the normoxia control NC6. In summary, data suggested that upregulation of PAI-1 and subsequent deceased t-PA activity could be one major mechanism of the blockade of BBB breakdown by PPARα agonist WY-14643.
![**Influence of WY-14643 on t-PA and PAI-1 expression and activity in the blood-brain barrier (BBB) *in vitro* ischemia model.** OC6 treatment of cerebENDs accords to 4 h OGD treatment with medium supernatants derived from 4 h OGD-treated C6 cells, whereas NC6 treatment means cerebEND cells incubated for 4 h under normoxic conditions with medium supernatants of 4 h normoxic treated C6 cells. Results on mRNA as well as protein expression of t-PA **(A)** and PAI-1 **(B)** of cerebEND cells after OC6 treatment were displayed. Effects of WY-14643 on t-PA activity and of rhPAI-1 on barrier functionality were shown in **(C)**. Densitometric analyzed protein expression of total cell lysates were compared to RIPA and triton-X 100 fractions. Protein distributions \[%\] between cytoskeleton (RIPA) and membrane enriched fractions (triton-X 100) were presented. Values were related to β-actin and according representative western blot images are depicted. Dotted lines indicated cuts of images of the same blot due to presentation reasons of selected bands. Statistical significance was labeled with ^∗^ vs. NC6, ^\#^ vs. OC6 and ^§^ vs. OC6+WY (*p* \< 0.05). Data are presented as means ± SEM (mRNA: *n* = 4--8, protein expression: *n* = 3--6, t-PA activity: *n* = 8--10, and barrier functionality: *n* = 12--36).](fnmol-10-00149-g005){#F5}
WY-14643 Reduces Brain Edema Formation after Traumatic Brain Injury
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In order to test the *in vivo* relevance of the found BBB stabilizing properties of WY-14643 during OGD, effects of addition of WY-14643 were investigated in a controlled cortical impact mouse model of TBI. This model comprises a significant cerebral ischemic component. Brain edema formation was measured as a major indicator of TBI after 24 h of the impact. The injury significantly increased brain water content in the vehicle control group about 4.61 ± 0.54% (mean ± SEM, *n* = 12, from 78.74 ± 0.62% in the contralateral hemisphere to 83.35 ± 0.26% in the ipsilateral hemisphere, **Figure [6A](#F6){ref-type="fig"}**). Administration of 60 mg/kg body weight of WY-14643 reduced the increased water amount significantly to 3.06 ± 0.25% (contralateral: 78.34 ± 0.34%, ipsilateral: 81.40 ± 0.45%, mean ± SEM, *n* = 12). Similar to BBB functionality *in vitro* data, combined treatment of 60 mg/kg body weight WY-14643 with 30 mg/kg body weight GW6471 did not reverse sole effects of WY-14643 in a significant manner. In this group, difference of water content between ipsilateral (82.36 ± 0.37%, mean ± SEM, *n* = 6) and contralateral hemisphere (79.04 ± 0.16%, mean ± SEM, *n* = 6) after TBI was 3.32 ± 0.36%. In order to verify the *in vivo* role of upregulated PAI-1 by WY-14643 found in the BBB *in vitro* model, effects of WY-14643 on edema formation in the same mouse TBI model using PAI-1 knockout (PAI-1^-/-^) mice were investigated. Addition of 60 mg/kg body weight WY-14643 decreased the increase of brain water content from 5.27 ± 0.59% (mean ± SEM, *n* = 7, ipsilateral: 80.08 ± 0.44%, contralateral: 74.81 ± 0.93%) to 4.52 ± 0.95% (mean ± SEM, *n* = 6, ipsilateral: 79.08 ± 0.92%, contralateral: 74.56 ± 1.26%, **Figure [6B](#F6){ref-type="fig"}**) in a non-significant manner and addition of GW6471 showed also no effect. In summary, these data revealed that WY-14643 was able to decrease edema formation in a TBI model which was not only PPARα dependent. Moreover, animal experiments with PAI-1^-/-^ mice supported the hypothesis about participation of PAI-1 in WY-14643 mediated effects. These *in vivo* data were in concordance with *in vitro* BBB results suggesting contribution of PPARα independent mechanisms as well as PPARα dependent PAI-1 regulation to multifaceted effects of WY-14643 during cerebral ischemia.
![**Influence of WY-14643 on brain water content \[%\] 24 h after controlled cortical impact in a mouse traumatic brain injury model.** 60 mg/kg body weight of WY-14643 reduced the increase of brain water content in the ipsilateral brain hemisphere of wild type C57Bl/6 mice significantly. Addition of PPARα antagonist GW6471 did not reverse the beneficial effects of WY-14643 **(A)**. Administration of 60 mg/kg body weight WY-14643 did not decrease brain water content of PAI-1 knockout mice (PAI-1^-/-^) after TBI. **(B)**. Statistical significance was labeled with ^∗^ (*p* \< 0.05). Data are presented as means ± SEM (wild type: *n* = 6--12; PAI-1^-/-^: *n* = 6--7).](fnmol-10-00149-g006){#F6}
Discussion
==========
In seventy percent of brain trauma cases the neurovascular unit is compromised and the extent of neurovascular permeability correlates with that of cortical dysfunction ([@B89]). Moreover, the disruption of the neurovascular unit is linked to several long-term consequences of TBI such as Alzheimer's disease and post-traumatic epilepsy ([@B78]; [@B76]). Cerebral ischemia or hypoxia during TBI induces leakage of the BBB which subsequently contributes to the development of cerebral edema. The formation of cerebral edema is one of the main factors for the high mortality and morbidity after TBI. Therefore, the preservation of the functionality of the neurovascular unit after TBI seems to be a promising strategy to significantly improve the clinical outcome.
In this context, PPARs have been considered as new pharmacological targets in cerebral ischemic insults ([@B11]). Treatments with either fenofibrate, WY-14643 or glitazones reduced adverse outcomes ([@B28]; [@B34]). For example, administration of fenofibrate after TBI led to improved neurological scores, reduced cerebral edema, lesion volume and ICAM-1 expression in a rat model. In the same animal model fenofibrate acted also in an antiinflammatory and antioxidative manner ([@B10]; [@B17]). Similar effects have been found in stroke models. PPARα agonists enhanced vascular function and were neuroprotective by modulating inflammatory, immune and oxidative pathways. They decreased cerebral infarct volume, microglial activation, neutrophil infiltration and improved synaptogenesis, neurogenesis, neurorepair, motor function (short-term effect), cognitive post-stroke consequences and angiogenesis. Antioxidative and antiinflammatory properties of PPARα agonists were shown by increased superoxid-dismutase and decreased NFkb and AP-1 activity. Effects of PPARα agonists on the brain endothelium confirmed that the BBB significantly contributes to the disease progression. For example, PPARα agonists improved acetylcholine induced vascular relaxation by nitric oxide and enhanced cerebral blood flow in the ischemic penumbra, but also decreased superoxide generation and inhibited the upregulation of adhesion molecules ICAM-1 and VCAM-1 ([@B82]; [@B24]; [@B28]; [@B93]; [@B21],[@B22]; [@B26]; [@B11]; [@B72], [@B71]; [@B38]). Moreover, experiments with Apo-E deficient mice proved that fenofibrate could also act in a lipid/glucose metabolism independent manner, since the infarct size was still reduced by the PPARα agonist after these mice developed hyperglycemia ([@B26]). In addition, PPARα agonists induced protective effects against ischemia/reperfusion damage in peripheral organs including heart, kidney, and intestine ([@B80]; [@B95]; [@B98]; [@B23]).
However, although previous studies showed that the brain endothelium and its permeability was affected by PPARα treatments during cerebral ischemic insults, detailed information about underlying mechanisms are still missing. [@B60] reported that fenofibrate was able to block BBB breakdown in an *in vitro* OGD model in a PPARα dependent manner, because brain endothelial cells isolated from PPARα knockout mice were not susceptible for that treatment. They further showed that fenofibrate acted directly on brain endothelial cells, but they have not found any PPARα target to explain the observed effects ([@B60]). Therefore, the aim of the present study was to find ischemia relevant mechanisms modulated by PPARα agonists that could cause the protection of the BBB. For our studies, it was decided to use PPARα agonist WY-14643. In a previous study WY-14643 decreased the infarct volume in a MCAO model of permanent focal cerebral ischemia which was not found in PPARα knockout mice ([@B42]). Moreover, WY-14643 reduced tissue damage after ischemia/reperfusion in liver, heart, and gut, but also increased angiogenesis in zebrafish ([@B95]; [@B23]; [@B70]; [@B13]; [@B61]; [@B74]). In the present study the comprehensively validated BBB *in vitro* OGD model based on a co-culture of mouse brain endothelial cerebENDs cells and rat glioma C6 cells was used to investigate the effects of WY-14643 ([@B65]).
Similarly to the results of [@B60] the addition of a PPARα agonist -- in our case WY-14643 -- inhibited BBB barrier breakdown *in vitro* by OGD. This was shown by TEER and fluorescein permeability data. Surprisingly, PPARα antagonist GW6471 was not able to reverse WY-14643 mediated protection of barrier tightness. Paracellular tightness is linked to the expression and localization of tight junction proteins ([@B96]; [@B44]). Interestingly, WY-14643 did not prevent the decrease of mRNA levels of tight junction proteins indicating no direct transcriptional PPARα dependent effects. On the contrary, western blotting results showed different effects on expression and protein distribution of tight junction proteins. Especially, effects on occludin were very interesting. Regulations of the RIPA-fraction by WY-14643 and GW6471 suggested a PPARα dependency, whereas the effects on protein distribution were according to TEER data indicating a PPARα independent occludin distribution. Corresponding to our data, [@B14] recently found changes in TEER that was related to altered tight junction protein localization. At the same time they did not detect a significant regulation of tight junction protein expression using a human BBB model based on endothelial cells differentiated from human induced pluripotent stem cells.
The localization status of tight junction proteins is dependent on their phosphorylated sites. Therefore, we investigated as next step the influence of WY-14643 on MAP kinases. Previous studies showed that kinases such as ERK1/2, Akt, p38, and SAPK/JNK could be involved during BBB breakdown. According to literature, phosphorylation of all four analyzed kinases increased after OC6 treatment in our model ([@B47]; [@B43]; [@B20]; [@B29]; [@B50]; [@B103]; [@B19]; [@B101]; [@B94]; [@B99]). Interestingly, WY-14643 decreased the phosphorylation of ERK1/2, but increased phosphorylation of p38 and SAPK/JNK indicating a complex regulatory network which should be decoded in future studies. Recent work revealed that inhibition of proteasomal activity could contribute to decelerated degradation of proteins relevant for tight junction protein expression ([@B46]; [@B85]). Therefore, proteasomal inhibitory properties of WY-14643 were tested and indeed it was found for the first time that WY-14643 was capable of blocking proteasomal activity. However, the addition of proteasome inhibitors itself were not able to prevent BBB breakdown or tissue damage. It was suggested that the less degraded glucocorticoid receptor had to be still activated in order to induce expression of occludin in the murine BBB under ischemic conditions ([@B46]). If and how this pathway is modulated by WY-14643 and plays a role in our model could be elucidated in further studies. In general, the relevance of the modulation of MAP kinases and proteasomal enzymes by PPARα and PPARγ for BBB stabilization were confirmed in previous studies concerning HIV mediated BBB breakdown ([@B41], [@B39], [@B40]).
One major finding of this study was that we were able to show for the first time that WY-14643 increased PAI-1 expression of brain endothelial cells after OC6 treatment and that this was associated with a decrease of t-PA activity. On the one hand t-PA is used as a drug to lyse thrombi during stroke treatment, on the other hand it is known that t-PA contributes to BBB breakdown via MMP activation or cleavage of membrane receptors of the brain endothelium ([@B73]; [@B3]). Recent publications underlined the possible importance of this finding. Fenofibrate therapy was successful in an acute phase stroke model with a thrombolysis-induced hemorrhage, where thrombolysis was induced by t-PA -- treatment. Here, fenofibrate reduced the risk of hemorrhage after thrombolysis and decreased infarct volume, the stroke-induced vascular endothelium dysfunction, microglial activation and neutrophil infiltrations ([@B34]). In this case, it could be speculated that fenofibrate increased PAI-1 expression leading to less t-PA mediated damage.
An interesting question is whether the amount of PAI-1 derived from brain endothelial cells would be enough to inhibit its breakdown during an ischemic insult. In this regard, it was published that WY-14643 upregulated PAI-1 in cells of other tissues such as cardiomyocyte-like cells or hepatocytes ([@B7]; [@B27]). Moreover, it was reported that stroke-induced upregulation of acute phase proteins in the liver was inhibited by fenofibrate leading to decreased leukocytosis in the brain ([@B53]). Another study showed that fenofibrate treatment of salt-loaded spontaneously hypertensive stroke-prone rats reduced brain damage. In these animals an initially renal failure is linked to a cerebral ischemic insult. During the process they develop systemic inflammation followed by hypertension and proteinuria leading to end-organ injury ([@B35]). Altogether these data support the hypothesis that organ--organ interactions might play an important role for the beneficial effects of PPARα agonists to decrease adverse outcomes after cerebral ischemic insults. In this context, we also determined possible effects of WY-14643 on astrocyte mimicking glioma C6 cells being part of our co-culture model. Although it was published that fenofibrate acted mainly directly on brain endothelial cells to block BBB breakdown ([@B60]), we found several targets such as t-PA, PAI-1, Angpt1, PDGFb, GDNF, ApoE, and TNFα in C6 cells which were regulated at the mRNA level by WY-14643 after OGD treatment (see Supplementary Table [S4](#SM1){ref-type="supplementary-material"}).
An important observation in our study was that several WY-14643 effects were not reversed by PPARα antagonist GW6471 indicating that for these effects no transcriptional activity of PPARα was necessary. Moreover, it could be speculated that WY-14643 at the effective concentrations (100--300 μM) activated also other targets than PPARα, since distinctly lower EC~50~ values (5.38 μM) were reported for WY-14643-mediated PPARα activation ([@B9]). To evaluate our *in vitro* data in an *in vivo* model, mice were subjected to CCI-TBI and treated with WY-14643 and GW6471 before. WY-14643 reduced edema formation significantly, which was also not inhibited by addition of PPARα antagonist GW6471. Western blotting of total brain samples revealed that similar targets (PAI-1, ZO-1, and ERK1/2) were affected by WY-14643 treatment *in vivo* as already found *in vitro* (data not shown). Moreover, experiments with PAI-1 knockout mice proved the relevance of PAI-1 for WY-14643 induced decrease of edema formation. It has to be mentioned that the CCI-TBI model also comprises tissue damage and inflammatory components which can influence edema formation, and that edema formation is only partly linked to BBB breakdown. However, we have chosen to analyze edema formation in order to evaluate whether the data obtained with a BBB *in vitro* OGD model could be predictive for a general parameter of CCI-TBI mediated damage.
In summary, we present several novel findings at the molecular level about how a PPARα agonist could prevent BBB damage in cerebral ischemic insults. Future studies might elucidate the underlying pathways to understand the processes in more detail and the data relevance for humans.
Author Contributions
====================
WN contributed to the study via its initial planning, experimental planning, collection of data, data analysis/interpretation and via the writing of the manuscript. TK contributed to the study via experimental planning, collection of data, data analysis/interpretation and via the writing of the manuscript. CG contributed to the study via experimental planning, collection of data and data analysis/interpretation. ST and CF contributed to the study via data analysis/interpretation and revision of the manuscript. AN contributed via the collection and analysis of data.
Conflict of Interest Statement
==============================
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
**Funding.** The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007--2013) under Grant agreement No. HEALTH-F2-2009-241778 to CF.
We would like to thank Prof. Dr. Dr. h.c. Norbert Roewer for his general support as a Chair of the Department of Anaesthesia and Critical Care, University Hospital Würzburg.
Supplementary Material
======================
The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fnmol.2017.00149/full#supplementary-material>
######
Click here for additional data file.
[^1]: Edited by: *Nicola Maggio, The Chaim Sheba Medical Center, Israel*
[^2]: Reviewed by: *Maria A. Deli, Biological Research Centre (HAS), Hungary; Samaneh Maysami, University of Manchester, United Kingdom*
| {
"pile_set_name": "PubMed Central"
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Introduction {#s1}
============
Every day \~100 billion cubic meters of bottom seawater are transported down into the permeable upper oceanic crust. Within this gigantic aquifer system oxic seawater circulates and reacts with reduced igneous rocks before eventually recharging back into the oceans 10^3^--10^4^ years later (Wheat et al., [@B58]; Orcutt et al., [@B43]). Consequently, the chemical composition of fluids and rocks are strongly altered, with wide-reaching ramifications throughout the marine system (Fisher and Becker, [@B18]; Bach and Edwards, [@B3]; Bach et al., [@B4]). Strong evidence exist for an abundant microbial community residing within this subsurface crustal basaltic aquifer (Giovannoni et al., [@B23]; Torsvik et al., [@B56]; Fisk et al., [@B20]; Lysnes et al., [@B37]; Orcutt et al., [@B41]; Nigro et al., [@B40]; Lever et al., [@B35]) where microbial activity is believed to influence basalt alteration and mineral dissolution rates (Thorseth et al., [@B55]; Fisk et al., [@B19]; Furnes et al., [@B22]; Storrie-Lombardi and Fisk, [@B53]; Kruber et al., [@B31]).
Endolithic microorganisms in subsurface basalt were first reported two decades ago from a drilling expedition to the Costa Rica Rift zone (Ocean Drilling Program Leg, ODP Leg 148). The presence and activity of microorganisms were inferred via detection of biosignatures including: (i) microscopic tubular structures in which DNA could be detected by staining (Thorseth et al., [@B55]; Giovannoni et al., [@B23]), (ii) targeting and localization of intact and active cells via *in situ* fluorescent hybridization (FISH) (Torsvik et al., [@B56]), and (iii) site-specific nitrogen and carbon enrichment in the altered tubular structures (Giovannoni et al., [@B23]; Torsvik et al., [@B56]). These results were later supported by drilling in the Australian Antarctic Discordance (ODP Leg 187) where, in addition to corroborating textural, geochemical, and molecular observations (Furnes et al., [@B21],[@B22]; Thorseth et al., [@B54]), microbial DNA (16S rRNA genes) from subsurface samples was for the first time successfully amplified and sequenced (Lysnes et al., [@B37]). Despite limited sequencing depth, this analysis revealed a unique microbial population dominated by the bacterial phyla *Gammaproteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi*, and *Firmicutes*, and different from those in the above sediment and seawater.
Besides the pioneering work outlined above only a few additional microbial studies have directly investigated native subsurface igneous rocks (Fisk et al., [@B20]; Mason et al., [@B38]; Lever et al., [@B35]; Orcutt et al., [@B44]). Thus, this habitat is heavily under-studied, a fact that can be largely attributed to the immense technical and economic challenges involved in the sampling of deeply buried oceanic crust. Consequently, most of our knowledge about the crustal biosphere originates from samples exposed at the seafloor (Orcutt et al., [@B41]; Edwards et al., [@B10]; Orcutt and Edwards, [@B42]). Seafloor-derived samples, however, are not representative of the subseafloor crustal environment and constitute only a small fraction of the 10^9^ km^3^ of the upper oceanic crust that has been suggested to be habitable (Heberling et al., [@B25]). In an effort to address these concerns, *in situ* subseafloor observatories, installed primarily at Juan de Fuca Ridge (JdFR), have expanded our understanding of subseafloor water--rock--microbe interactions in a more representative setting (Orcutt et al., [@B41]). Despite such technological advancement, a number of basic questions cannot easily be inferred from subseafloor observatories, including cell abundances and community structure in native material.
The first dedicated microbial investigation of a low-temperature young ridge flank system was undertaken by the International Ocean Drilling Program (IODP) expedition 336 to North Pond in the North Atlantic gyre (Expedition 336 Scientists, [@B17]). The basement in North Pond is covered by a sediment layer (up to 300 m) and is characterized by vigorous crustal fluid circulation driven mainly by advection (Edwards et al., [@B11]). The fast fluid circulation results in relatively low fluid temperatures (10--15°C) and seawater-like fluid chemistry, such as high dissolved oxygen concentration at discharge zones \[55--191 μM\] (Orcutt et al., [@B45]). Further, a recent study revealed an active and distinct bacterial community in the crustal fluids underneath North Pond (Meyer et al., [@B39]).
In the present study, we analyse the abundance and structure of microbial communities in deeply buried basaltic rocks in a total of 33 different samples retrieved from the basement underneath North Pond and compare these to the communities in the above sediments. We analyse the samples by means of 16S rRNA gene amplicon libraries and quantitative PCR (qPCR). Our results are among the first to quantify microbial abundances in native subsurface basalt, thus guiding biomass constrains for this globally significant system. Furthermore, our data elucidate the taxonomic identity of native microbial inhabitants suggesting a community capable of facilitating a diverse range of redox reactions. Lastly, we provide evidence that the dispersal scenarios of the sedimentary and crustal microbial inhabitants are intertwined and potentially closely linked.
Materials and methods {#s2}
=====================
Sample location, collection, and description
--------------------------------------------
We investigated a total of 33 subsurface samples (27 from igneous crust and 6 from a sedimentary breccia) collected from North Pond on the west flank of the Mid-Atlantic Ridge. Samples originate from Holes 1382A (22°45.353′N, 46°04.891′W) and 1383C (22°48.1241′N, 46°03.1662′W; Figure [1](#F1){ref-type="fig"}), both were retrieved using rotary core barrel (RCB) coring. Florescent microspheres were added to the drilling fluid in order to assess potential contamination as described in details elsewhere (Expedition 336 Scientists, [@B16]). In order to remove potential contamination introduced during drilling operation, all samples were washed three times in sterile seawater on-board before being sub-sampled into smaller pieces using a chisel and hammer under sterile conditions, as described in details elsewhere (Expedition 336 Scientists, [@B15]). The presence of fluorescent microspheres in the wash solution was investigated by microscopy after the last wash. Subsamples (\~2 cm^3^) were placed in sterile Whirlpak bags and immediately frozen at −80°C before further processing at the home institute.
![**Bathymetric map with the location of the investigated sites**. Red circles indicate the geographic location of the investigated sites. Insert: global map indicating the position of North Pond in the Mid-Atlantic Ocean (Source: National Centers for environmental Information - NCEI). Modified from Bach ([@B2]).](fmicb-07-00820-g0001){#F1}
Samples follow a depth gradient ranging from \~110 to 200 meter below seafloor (mbsf) in Hole 1382A (16 samples) and 70 to 300 mbsf in Hole 1383C (17 samples). The igneous crust at the two sites likely originates from different volcanic centers fed by a mantle source of variable composition (Expedition 336 Scientists, [@B15]). A short overview of sample depths and lithological characteristics can be found in Table [1](#T1){ref-type="table"}. For a comprehensive description the reader is recommended to consult the IODP proceedings volume 336 (<http://publications.iodp.org/proceedings/336/336title.htm>). Following is a brief characterization of each site.
######
**General sample description including depth, lithological unit, onboard sample description, and in which group in the hierarchical cluster analysis the microbial community is located**.
**Sample** **Depth (mbsf)** **Unit** **Specific sample description** **Cluster**
------------ ------------------ ---------- ---------------------------------------------------------------------------------- -------------
**1382A**
2R_1C 110 I Massive, minor red-yellow-brown alteration 4
3R_2B 115 I Massive, yellow-white-brown alteration in vein 4
3R_3A 117 I Massive, patchy orange-brown alteration 4
3R_4B 117 I Massive, mostly brown oxidized halo 4
4R_1B 123 II Aphyric cryptocrystalline basalt, gray-brown alteration 4
5R_1B 133 II Massive, aphyric, red alteration 4
6R_1A 142 II Aphyric, cryptocrystalline, less vesicular, patchy alteration 4
7R_2B 153 III Aphyric, glassy margin, red, and orange-brown alteration 4
8R_1A 161 IV Ultramafic, pyroxene, evidence of low-temperature alteration 1
8R_1B 161 IV Porphyritic basalt 4
8R_2F 162 IV Orange-brown sediment with small (\< 1 mm) basalt clasts 2
8R_3G 163 IV Gray-brown + orange-brown sediment with small (\< 1 mm) basalt clasts 1
8R_4D 163 IV Sedimentary breccia with basalt clasts, rusty colored, extensive carbon 2
9R_1C 172 IV Sediment near serpentinized breccia 3
10R_3D 183 V Medium-grained basalt, massive, porphyritic, pervasive alteration 4
12R_1A 199 VI Porphyritic basalt, minor alteration 4
**1383C**
2R_2E 72 I Aphyric basalt, highly altered, vein with red alteration, light brown alteration 5
3R_1B 77 I Aphyric basalt, slight alteration, tan alteration deposits 4
4R_1B 87 I Light tan micrite breccia with altered glass clasts 4
5R_1B_I 97 I Aphyric basalt, moderately altered, vein with red alteration 5
5R_1B_II 97 I Aphyric basalt, moderately altered 5
6R_1A 105 I Aphyric basalt, slight alteration, altered chilled margin, ochre alteration 6
10R_1A 144 II Light tan micrite with large clasts of altered glass 5
10R_1D 145 II Phyric basalt, moderately altered, multiple veins, slight red alteration 5
11R_1C 154 II Phyric basalt, extensive alteration, orange, and olive alteration 5
19R_1B 212 III Aphyric basalt, highly oxidized, light brown alteration, relatively brittle 5
19R_1A 212 III Two small pieces, mostly glass, rust alteration 5
20R_1A 219 III All basalt glass, rust alteration 5
24R_1B 257 III Aphyric basalt, oxidized, dark orange-brown alteration, fractured 5
24R_1A 256 III Aphyric basalt glass with rust alteration, vesicles 5
27R_1A 285 III Aphyric massive basalt, alteration 4
29R_1A 300 III Aphyric basalt, highly oxidized, thin carbonate veins 6
30R_1A 304 III Aphyric basalt, oxidized, some fractures, dark orange alteration 5
*Cluster number corresponds to numbers in Figure [4](#F4){ref-type="fig"}. The sample description presented here is modified from IODP 336 site summaries (Expedition 336 Scientists, [@B13],[@B14])*.
In Hole 1382A basement was located 90 mbsf, however the interval from 93 to 99 mbsf are inferred to be sedimentary (Expedition 336 Scientists, [@B17]). A total of 32 meter of upper crustal material was recovered between 110 and 210 mbsf (recovery 32%). From this material we analyzed 16 samples covering six of the eight lithological units encountered in this Hole (I, II, IV, V, VI, and VII). Unit V consist of sedimentary breccia; likely as a result of a rock slide deposit, whereas all other units are represented by basalt, either as varying volcanic pillow basalt or massive flows with geochemical and petrographic distinct characteristics. Rock alteration can be assigned to low temperature processes.
In Hole 1383C the sediment/basement interface was found at 38.3 mbsf and 50.3 meter of hard rock was recovered from the interval between 69.5 and 331.5 mbsf (recovery 19%). The 17 samples investigated from this Hole are glassy to fine-grained basalts with variable content of phenocrysts, which divides them into three major lithological units (I, II, and III).
DNA extraction
--------------
Small pieces of sample material was pulverized in a flame sterilized steel mortar and \~0.5 g (0.49--0.91 g) from each sample was subjected to genomic DNA extraction using the FastPrep soil DNA isolation kit (MP Biomedicals) following the manufacturer\'s instruction with two modifications. First, we used a special bead coating, similar to the G2 DNA/RNA enhancer (Amplicon A/S, Odense, Denmark, available from June 2016) that increases yield, by reducing DNA binding to the beads (Baælum and Jacobsen, [@B1]; Bælum et al., [@B5]; Hjelmso et al., [@B27]). Next, 200 μg of sterile filtered polyadenylic acid (PolyA; Sigma) was added to each lysis mixture prior to bead beating, to avoid DNA binding to the sample matrix (Hugenholtz et al., [@B29]). Bead beating was performed using the MP-Biomedical FastPrep®-24 for 45 s (speed setting 6). DNA was finally eluted into 75 μl PCR-grade double-distilled water (ddH~2~O), and preserved at −80°C until further analysis. In order to assess potential contamination introduced from the extraction kit, two blank extractions were included using the same batch of chemical reagents as for the samples.
Quantitative PCR
----------------
Bacterial and Archaeal 16S rRNA genes were quantified individually using quantitative real time PCR applying the StepOne Real Time PCR system (Applied Biosystems). All samples and standards were run in triplicates using SYBR Green Hot Start master mixture (Qiagen) and with the standards, primers, and thermal conditions described in details elsewhere (Jørgensen et al., [@B30]). In short, a dilution series (10--10^6^ target copies) containing *Escherichia coli* PCR amplified full-length 16S rRNA genes and a linearized archaeal fosmid (54d9) was used as bacterial and archaeal standards, respectively. Bacterial SSU rRNA genes were targeted with the primers bac341F (5′-CCTACGGGWGGCWGCA) and 518R (5′-ATTACCGCGGCTGCTGG). For archaeal SSU rRNA gene amplification the primers Un515F (5′-CAGCMGCCGCGGTAA) and Arc908R (5′-CCCGCCAATTCCTTTAAGTT) were used. All *R*^2^ were \>0.95 and the amplification efficiency between 90 and 104%.
Ion torrent SSU rRNA amplicon library preparation and sequencing
----------------------------------------------------------------
All DNA extracts were PCR amplified in duplicates with the SSU rRNA gene specific primers 519f (5′-CAGCMGCCGCGGTAA) and 805r (5′-GACTACHVGGGTATCTAATCC) in order to generate an amplicon library for subsequent sequencing using the Ion Torrent PGM Personal Genome Machine (PGM) platform technology (Life Technologies). We used a two-step amplification approach as described by Berry et al. ([@B6]), to minimize bias introduced by the long adaptor sequence. The first-round PCR was carried out in duplicate for each sample, to minimize PCR drifting, and each reaction (20 μl) contained 10 μl 2x HotStarTaq® master mixture (Qiagen), 0.2 μl of each primer (100 μM stock), 2 μl template and ddH~2~O. The PCR program was initiated with a hot start activation step for 15 min at 95°C followed by an optimized number of PCR cycles (36--37) of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. The duplicate PCR products were pooled and purified using QIAquick PCR purification kit (Qiagen). In the second-round PCR attaching the Multiplex Identifiers (MIDs), seven cycles were run, where each reaction (25 μl) contained 12.5 μl 2x HotStarTaq® master mixture (Qiagen), 0.2 μl 806r-B-Key (100 μM stock), and 2 μl 519f MID primer (10 μM stock), with 5 μl of purified PCR products from first-round amplification as the template, according to the Ion Torrent protocol. The PCR amplicons were purified using AMPure XP bead Purification Kit (Agencourt), following manufactures protocol, before all samples were pooled in equimolar concentrations (26 pmol). We note, that due to the PCR and the subsequent equimolar pooling, the number of reads do not reflect the original concentration of DNA, which based on gel band intensity after PCR was much higher in the samples than in the blank extractions. Raw reads generated in this study were deposited at the NCBI Sequence Read Archive under the project number SRP070121.
OTU filtering, clustering, and taxonomic assignment
---------------------------------------------------
Sequence reads obtained from the Ion Torrent sequencing were cropped at 220 bp and quality filtered with a 0.5 quality cut-off, chimera checked, and Operational Taxonomic Units (OTUs) clustered (97% similarity) using UPARSE/USEARCH (Edgar, [@B8]). The resulting OTUs were taxonomically assigned using the CREST software, with a lowest common ancestor algorithm implementing the SilvaMod reference database (Lanzen et al., [@B32]). A fasta sequence file of the represented OTUs can be found in the Supplementary Material (Data sheet [S2](#SM5){ref-type="supplementary-material"}).
Contamination assessment of sequence pool
-----------------------------------------
Contamination issues have previously hampered progress in deep marine research. In an attempt to avoid such obstacles this study applies several measures to prevent and assess the degree of potential contamination, as outlined in the sample handling and collection section above. Further, as a broad reaching contamination control for drilling protocols we extracted and sequenced DNA from the drill mud and a recovered microsphere bag (exposed to bottom seawater, drill fluid, and mud). The purpose of this control is to address potential inadvertent contamination of samples introduced during standard IODP drilling protocols. The results allow estimating the ratio of the inferred natural community that is likely to arise from contamination. Any OTU present in the control and in the native sample material was removed from the dataset. We note that true overlap between communities in the control and native samples may exist, which would lead to culling of legitimate sequences. Additionally, in order to assess potential contamination in the 16S rRNA gene amplicon preparation procedure two blank extractions (no sample material) were subjected to the same amplification protocol as the samples. Amplified DNA from these blanks may represent contamination originating from DNA extraction kit and/or PCR mix reagents (Champlot et al., [@B7]; Lusk, [@B36]; Salter et al., [@B47]). Therefore, any OTU found in both the extraction blanks and the native sample material was removed prior to any further downstream analysis, with the exception of OTUs that was found to be more than 50 times as abundant in the basalts than in the above-described controls. These were retained in the dataset, due to the plausible scenario of cross-contamination from controls to samples. This approach is similar to that described by Lee et al. ([@B33]) with modified increased stringency addressing the assumed lower biomass in our sample set. However, in order to evaluate the impact of this stringent filtering, ordination, and clustering were likewise performed on the full dataset prior to the above described filtering.
Ordination and hierarchical clustering analysis
-----------------------------------------------
The relative abundances of individual OTUs in each sample were clustered based on unconstrained Bray-Curtis, Jaccard, and Dice dissimilarity index using the software PAST version 3.08 (Hammer et al., [@B24]), before and after removal of potential contaminant reads. The basalt-hosted microbial communities were compared to those in the overlying sediments (Hole 1383E), using non-metric dimensional scaling (NMDS) applying Bray-Curtis dissimilarity. The data from the sediment samples was generated following identical protocols (e.g., the same primers, extraction kit, PCR mix), sequencing platform and downstream analysis, thereby enabling a valid comparison. The concentrations of major and trace elements from rocks provided by the IODP data report (Expedition 336 Scientists, [@B13],[@B14]) originating from the same core sections, but separated from the samples used for microbiology (between 20 and 70 cm distance), were used in cluster analysis using Bray-Curtis and Jaccard.
Results {#s3}
=======
16S rRNA gene abundance
-----------------------
The total abundance of 16S rRNA gene copy numbers (Archaea plus Bacteria) estimated by qPCR varies between 0.3 and 8.3 × 10^4^ copies per gram in Hole 1382A and 0.6--3.9 × 10^4^ copies per gram in 1383C (Table [2](#T2){ref-type="table"}), with the majority of all samples (80%) falling within the range of 1.9--5.8 × 10^4^ copies. Three samples from the sedimentary breccia between 162.8 and 163.8 mbsf in Hole 1382A (8R-2F, 8R-3G, and 8R-4D) had notably lower numbers (0.3--0.6 × 10^4^ copies per gram) than the remaining samples from this site. Based on our quantification the 16S rRNA genes are predominantly of bacterial origin, comprising between 92.6--100% and 91--100% in Hole 1382A and 1383C, respectively (Table [2](#T2){ref-type="table"}). Assuming an average copy number per genome of 4.2 for Bacteria and 1 for Archaea (Stoddard et al., [@B52]), these copy numbers suggests cell abundances ranging \~0.1--2 × 10^4^ per gram of sample material (average 0.71 × 10^4^).
######
**General molecular characteristic of the samples investigated**.
**Sample ID** **Depth (mbsf)** **Unit** **16S rRNA copies (× 10^4^)** **% Bacteria** **Reads after filtering** **OTU \#** **Shared with sediment**
--------------- ------------------ ---------- ------------------------------- ---------------- --------------------------- ------------ -------------------------- ----- ----
**U1382A**
2R_1C 110 I 5.7 98 100 8,598 371 198 72
3R_2B 115 I 8.3 100 100 12,966 174 114 81
3R_3A 117 I 2.7 100 99 16,574 96 66 80
3R_4B 117 I 2.4 100 100 11,349 224 133 81
4R_1B 123 II 4.8 96 97 15,330 215 128 78
5R_1B 133 II 3.9 92 99 19,517 279 168 83
6R_1A 142 II 2.1 100 100 20,437 175 101 69
7R_2B 153 IV 3.8 100 100 10,968 193 127 85
8R_1A 161 V 1.8 100 100 23,251 42 23 80
8R_1B 161 V 4.1 95 100 12,495 261 142 80
8R_2F 162 V 0.3 100 100 19,858 16 10 99
8R_3G 163 V 0.5 100 100 19,145 25 18 22
8R_4D 163 V 0.6 100 86 25,028 27 20 31
9R_1C 172 V 4.2 92 99 16,873 314 185 82
10R_3D 183 VI 5.8 97 99 18,080 283 148 79
12R_1A 199 VII 3.0 92 98 19,506 273 153 75
**U1383C**
2R_2E 72 I 2.8 99 100 19,163 319 165 63
3R_1B 77 I 3.1 98 100 18,240 141 75 75
4R_1B 87 I 1.9 98 97 19,144 124 72 72
5R_1BI 97 I 3.1 91 96 15,164 177 124 77
5R_1BII 97 I 2.6 99 99 19,553 330 168 75
6R_1A 105 I 2.2 100 100 23,627 243 146 71
10R_1A 144 II 2.1 90 100 25,841 158 99 86
10R_1D 145 II 2.9 98 100 17,447 326 161 83
11R_1C 154 II 1.7 99 100 24,686 204 116 87
19R_1B 212 III 3.0 98 100 19,107 264 130 84
19R_1A 212 III 3.3 99 100 18,946 126 81 88
20R_1A 219 III 2.9 100 100 3916 148 90 81
24R_1B 257 III 1.9 100 100 12,775 162 99 86
24R_1A 256 III 3.2 100 100 20,900 213 121 86
27R_1A 285 III 3.9 99 100 9,811 162 113 87
29R_1A 300 III 0.6 100 98 11,218 69 48 82
30R_1A 304 III 3.3 100 100 22,903 229 117 88
*Including gene copy numbers of total prokaryotic 16S rRNA gene copies per gram of sample material. Percent of the total community related to the domain Bacteria reported both from the qPCR and the amplicon library. The total number of reads after filtering. Number of OTUs in each sample (97% similarity). The number of OTUs and the % of total read that is shared with the samples in the above sediment from North Pond (1383E)*.
Sequence reads, filtering, and OTU clustering
---------------------------------------------
The total number of sequence reads per sample after filtering and potential contaminants removal (OTUs present in the four controls) varied between 8598 and 25,841 with an average of 17,649 (Table [2](#T2){ref-type="table"}). A total of 1,804 OTUs (\>97% sequence similarity) were found across all samples, of which 1,643 OTUs (91% of total OTUs) were assigned to the bacterial domain, 44 OTUs (2.5% of total OTUs) to the archaeal domain, while 60 OTUs (3.4% of total OTUs) were Eukaryotic. The remaining 57 OTUs were classified as "no hits" which means that the sequence is \< 80% similar to any in the database (Data sheet [S1](#SM1){ref-type="supplementary-material"}). The number of OTUs in the individual samples varied between 16 and 371 (Table [2](#T2){ref-type="table"}). Blank extractions contained a combined total of 221 OTUs of which 142 were removed according to the criteria outlined in the Materials and Methods Section. An additional 50 OTUs were removed from the original data, as they were present in either the drill mud and/or on the recovered microsphere bag (12 and 46 OTUs, respectively).
Microbial community composition
-------------------------------
As the microbial communities are spread across 41 different phyla, 73 classes, 155 orders, and 218 families, it is far beyond the scope of this work to address the community composition in all samples in detail. Therefore, only the results of the most abundant groups are listed here. However, a full list of OTUs present and their taxonomic assignments can be found in the Supplementary Material (Data sheet [S1](#SM1){ref-type="supplementary-material"}). In general the communities are relatively homogenous on higher taxonomic level across all samples, however in the sedimentary breccia the diversity of microorganisms and number of OTUs are extraordinarily low, especially in the middle section of that lithological unit (Figure [2](#F2){ref-type="fig"} and Table [2](#T2){ref-type="table"}), in many ways causing these samples to deviate substantially from the rest.
{#F2}
All samples are dominated by *Proteobacteria* (35--99% of the total communities) of which the class of *Gammaproteobacteria* is the most abundant, followed by *Alphaproteobacteria, Deltaproteobacteria*, and *Betaproteobacteria*, respectively (Figure [2A](#F2){ref-type="fig"} and Data sheet [S1](#SM1){ref-type="supplementary-material"}). Only very low abundances, if any, were assigned to the class of *Epsilonproteobacteria* and *Zetaproteobacteria*. Analysing the different classes of *Proteobacteria* with higher taxonomic resolution show that many (average 60%) of the *Gammaproteobacteria* could not be assigned below class level (Figure [2B](#F2){ref-type="fig"}). The majority of these were represented by OTU5, which showed high similarity (100%) to *Pseudomonas* when performing NCBI blast search. The limited taxonomic resolution of OTU5 in our analysis is likely due to the high stringency used by the lowest common ancestor algorithm applied to assign taxonomy. Most of the reminding *Gammaproteobacteria* in Hole 1382A belonged to the two orders *Oceanospirillales*, mainly affiliated with the SAR86 clade and *Pseudmonadales*, (largely divided between the families *Moraxellaceae* and *Pseudomonadaceae*). In contrast, the most abundant Gammaproteobacterial order from Hole 1383C is *Alteromonadales*, whereof most can only be assigned to family level (*Alteromonadaceae*) and to a lesser extent *Marinobacter* (\< 1% of total community). Although, abundance-variation between samples is present within the *Alphaproteobacteria*, as for all taxonomic groups, *Rhizobiales, Rhodobaceriales*, and *Rhodospirilialles* were in general the most abundant orders (Figure [2C](#F2){ref-type="fig"}). At both sites, *Burkholderiales* was by far the most prominent member of the *Betaproteobacteria* at the order level, with the genus *Variovorax* accounting for approximately half of this group and the family *Oxalobacteraceae* representing the other half (Figure [2D](#F2){ref-type="fig"} and Data sheet [S1](#SM1){ref-type="supplementary-material"}). The overall abundance of *Deltaproteobacteria* was relatively low and in Hole 1382A it was dominated primarily by *Myxococcales* and the *SAR324* clade whereas *Bdellovibrionales* and *Desulfobacterales* were the most abundant orders in Hole 1383C (Figure [2E](#F2){ref-type="fig"}). In addition to the mentioned members of the *Proteobacteria* the following phyla were found in relatively high abundances; *Actinobacteria, Firmicutes, Bacteroidetes*, and *Acidobacteria* along with a number of less abundant groups, but still representing more than 1% of all reads in one or more samples, such as *Planctomycetes* and *Chloroflexi* (see Figure [2A](#F2){ref-type="fig"}).
The archaeal community constitute only a minor fraction of the entire community (max. 5%, avg. 0.6% of all reads) and is represented by five phyla; *Ancient Archaeal Group* (AAG), *Crenarchaeota, Euryarchaeota, Thaumarchaeota*, and the newly proposed *Lokiarchaeota* (Spang et al., [@B51]; Figure [2A](#F2){ref-type="fig"} and Data sheet [S1](#SM1){ref-type="supplementary-material"}). Of these, Thaumarchaeal *Marine Group I* is by far the most abundant.
A comparison between the rank abundance of the 576 OTUs shared between the basalt and the overlying sediment was performed using the average abundances of the OTUs across all basalt and across all sediment samples, excluding the sedimentary breccia from Hole 1384A (**Figure 5**). The shared OTUs represented between 63 and 86% (average 80%) of all reads in the basalt and 44--71% (average 58%) in the sediments, but show a markedly different rank abundance distribution (Table [2](#T2){ref-type="table"} and **Figure 5**).
Ordination and clustering analysis
----------------------------------
The variation in the microbial community structure (relative abundance of OTUs) found in the basaltic samples beneath North Pond was compared to the composition in a number of sedimentary horizons in Hole 1383E directly overlying the basaltic crust by means of NMDS. The sedimentary community was investigated at 17 different depths, spanning from the top of the sediment to a few meters above the sediment basement interface and clearly shows a separation from those observed in the underlying upper crust (Figure [3](#F3){ref-type="fig"}). The microbial composition in the sedimentary breccia (lithological unit V) in Hole 1382A are markedly different from the rest of the samples from this sites and also from the basaltic rocks from Hole 1383C, causing these samples to cluster alone, with the exception of sample 8R_1B (the only basalt sample within the breccia unit). An NMDS analysis was also carried out on the full dataset, before removal of any of the OTUs found in the four controls, as described in the Materials and Methods Section. This result shows the same pattern, as with the "cleaned" dataset presented in Figure [3](#F3){ref-type="fig"}, and verifies that all control samples are markedly different from any of the indigenous samples (Figure [S1](#SM2){ref-type="supplementary-material"}).
{#F3}
In order to investigate any link between community composition and lithology a hierarchical clustering analysis based on the relative abundance of OTUs was executed. The results show several minor (group 1, 2, 3, 6) and two major (group 4 and 5) clusters separated by high bootstrap value (Figure [4](#F4){ref-type="fig"}). The major clusters largely distinguish the two sites from one another. However, three samples from Hole 1383C cluster within 1382A. The bootstrap values are generally low and clustering according to lithology or depth cannot be inferred. The clustering using the full dataset, before removal of potential contaminant reads in general shows the same clustering pattern, however a higher degree of mixing between the two sites is observed (Figure [S2](#SM3){ref-type="supplementary-material"}). The results from clustering based on Jaccard and Dice dissimilarity indexes, showed no clear difference in clustering pattern as compared to Bray-Curtis (data not shown). Using the geochemical data (major and trace elements) from the same core sections as those used for microbiology, in a hierarchical clustering, shows only very small variation in composition. No clear clustering pattern between sites or lithology could be observed and most branching points were unsupported (Figure [S3](#SM4){ref-type="supplementary-material"}).
{ref-type="table"}.](fmicb-07-00820-g0004){#F4}
Contamination control
---------------------
The presence of fluorescent microspheres in the sterile seawater used to wash the basalt rock surface was investigated on board the ship. After three washing rounds, microspheres were detected in 36% of the samples (Figure [4](#F4){ref-type="fig"}) and more frequently observed in Hole 1383C than 1382A.
Drill mud and the microsphere bag yielded a total of \~47,500 high-quality reads comprising a number of different bacterial taxa (Data sheet [S1](#SM1){ref-type="supplementary-material"}). After removal of extraction blanks, 12 OTUs were obtained from the drill mud and 46 from the microsphere bag, most of these were affiliated with *Streptococcus*, a group that are often associated with human pathogens. A larger number of OTUs were obtained from the microsphere plastic bag, many of which were affiliated with different SAR clades. However, also here a number of reads were associated with bacterial groups often associated with humans (e.g., *Streptococcus* and *Dermabacter*; Data sheet [S1](#SM1){ref-type="supplementary-material"}). Standard drilling protocols inevitably introduce contaminants to drilling components and we propose this as an explanation for the detection of human associated taxa.
Extraction blanks were represented by \~16,000 reads whereof the vast majority could be assigned to the following three taxonomic groups: *Ralstonia, Enterobacteriaceae*, and *Methylobacterium*. On average 38% of all reads were removed during the cleaning procedure (total of 158 OTUs) highlighting the importance of performing operation controls and analysing blank extractions, especially when working with low biomass sample material (Data sheet [S1](#SM1){ref-type="supplementary-material"}).
Discussion {#s4}
==========
Microbial abundances in subsurface basaltic crust
-------------------------------------------------
Based on our qPCR results of prokaryotic 16S rRNA gene abundances, we estimate that the samples contain \~10^4^ cells per gram of rock sample, with *Bacteria* outnumbering *Archaea* in all samples (Table [2](#T2){ref-type="table"}). However, as with all cell estimates based on a primer-based approach it is prone to bias and the numbers should be evaluated with this in mind. It is difficult to compare our estimates with earlier reports since, to the best of our knowledge, only one previous study has been conducted in which direct cell abundances were estimated from native cold subsurface basalt (Fisk et al., [@B20]). Based on amino acid concentration they suggest \~10^5^ cells per gram sample. A recent study estimated cell numbers in the basaltic fluids from both investigated sites, to be between 1.4 and 2.2 × 10^4^ per ml fluid, based on direct cell counts (Meyer et al., [@B39]). Considering the average basaltic porosity of 4% and assuming a density of 3, implies that our quantification is not merely representing cells in the fluid, but that the majority must be attached to the rock surfaces.
Due to the restricted number of sites in our study we have restrained ourselves from the tantalizing prospect of extrapolating the cell abundance to global biomass. However, we note that a previous estimate based on thermodynamic and bioenergetic models was suggested to translate into \~10^7^--10^9^ cells per gram rock (Santelli et al., [@B49]). In other words, 3--5 orders of magnitudes off our estimations. In support of a lower cell abundance is the relatively low oxygen consumption (\< 1 nmol O~2~ cm^−3^ rock d^−1^) estimated beneath North Pond (Orcutt et al., [@B45]). Another interesting observation related to the cell numbers is the relative consistency across all samples, which suggest that the cells are limited by a common vital anabolic or catabolic resource.
Microbial community composition in subsurface basaltic crust
------------------------------------------------------------
Based on the 16S rRNA amplicon libraries the microbial communities in the subsurface basalt in North Pond are all dominated by Bacteria, in general leaving the archaeal domain represented by \< 1%. Although, our qPCR estimations vary slightly from this, both analyses confirm the bacterial dominance (Table [2](#T2){ref-type="table"}). By far the most abundant phylum was *Proteobacteria*, with *Gamma-, Beta*-, and *Alphaproteobacteria* constituting the majority within this phylum. In addition *Actinobacteria, Firmicutes, Bacteroidetes*, and *Acidobacteria* were all represented by relatively high abundances. To the best of our knowledge, only one previous published study has successfully amplified DNA from native subsurface material and determined the community composition (Lysnes et al., [@B37]). By means of DGGE that study reported sequences related to *Gammaproteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi*, and *Firmicutes*, of which *Gammaproteobacteria* was the most abundant. A more recent study compiled taxonomic data from a number of studies regarding surface-exposed basalts and identified a set of commonly found abundant microbial groups, including *Gamma-, Alpha*-, and *Deltaproteobacteria*, as well as *Actinobacteria, Bacteroidetes, Acidobacteria, Planctomycetes, Gemmatimonadetes*, and *Nitrospirae* (Lee et al., [@B33]). Finally, the microbial community composition in the fluids underneath North Pond has revealed a similar dominance of *Proteobacteria*, also with *Gammaproteobacteria* being most abundant, then followed by *Alpha*-, *Epsilon*-, *Beta*-, and *Deltaproteobacteria* (Meyer et al., [@B39]). Further, relative high abundances of *Actinobacteria, Bacteroidetes, planctomycetes, Gemmatimonadetes*, and *Chloroflexi* were observed. Based on this it is tempting to suggest that a basalt-hosted taxonomic core group exists, including surface, subsurface fluids, and hard rocks. However, it is important to stress that the similarity of microbial groups in all cases is based on high taxonomic rank and future in-depth phylogenetic comparison, founded on full-length 16S rRNA sequences, are needed to resolve this issue.
The origin of the subsurface oceanic crustal community is an open question and different scenarios have been debated, one of them being that dispersion may occur via the above sediment column (Huber et al., [@B28]; Schrenk et al., [@B50]). When we compared the relative community composition in the basaltic rocks with that in overlying sediment striking differences were observed (Figure [3](#F3){ref-type="fig"}). However, this is by no means indicative of community isolation between the crust and sediment. On the contrary, a high degree of overlap was revealed from the OTUs distribution pattern. Of the 1,802 OTUs found across all 33 crustal samples, 576 OTUs were shared with the sedimentary habitat above, comprising as much as 63--86% (average 80%) of all reads in the basaltic samples. However, the relative distributions of the shared OTUs in the two environments are markedly different, which might reflect environment-specific taxon recruitment based on availability of electron donors and acceptors (Figure [5](#F5){ref-type="fig"}). Based on these results the two components of the deep biosphere do indeed share many of their microbial inhabitants. Although, the dynamics and mechanisms of taxon dispersal between the crustal and sedimentary subseafloor regimes is beyond the scope of this study, we note that a recent study support the dispersal of sedimentary bacteria via the ocean (Walsh et al., [@B57]).
{#F5}
Regarding the heterogeneity of crust-associated microbial communities beneath North Pond, we note that although the communities from the two different sites to a large degree cluster together, (Figure [3](#F3){ref-type="fig"}), there are also clear differences (Figure [4](#F4){ref-type="fig"}). Two major clusters, supported by high bootstrap values (\> 90) were found, largely separating the two investigated sites from one another. Notable differences are the presence of a few highly abundant OTUs affiliating with an uncultured *Alteromonas* lineage (*Gammaproteobacteria*) in Hole 1383C and a much higher occurrence of *Rhodobacterales* (*Betaproteobacteria*) in Hole 1383C than in Hole 1382A. The significance of this is not known, but it is possible that the physico-chemical nature, and associated redox coupling, of advective crustal fluids, drive community differentiation.
Potential metabolic traits and dominant groups
----------------------------------------------
The vast majority of lineages reported here do not group within taxonomic clades with known metabolism, and therefore their potential role in the ecosystem is unresolved. However, a number of less abundant groups with relatively constrained metabolic potential are present, thereby allowing us to assign their function with some degree of certainty. For example, as putative sulfate reducers in other environments a number of different genera were observed, including, *Desulfotomaculum, Desulfurispora, Desulfosporosinus, Desulfobaca, Desulfobulbus, Desulforomonas, Desulfovibrio*, and *Desulfobacula*, suggesting that at least the potential for active sulfate reduction is present. The classical sulfur oxidizers (mainly within *Epsilonproteobacteria*), on the other hand, were only sporadically observed. This observation is worth mentioning in the context of the relatively high abundances of sulfur oxidizers found in the fluids by Meyer et al. ([@B39]) and in the overlying sediments, suggesting different functions between the free-living and the surface-attached communities.
Iron and hydrogen (beside sulfur) has been proposed to be important electron donors in this type of habitat (Bach and Edwards, [@B3]; Edwards et al., [@B9]). However, known metal reducers such as, *Marinobacter, Shewanella, Geobacter*, and *Ferruginibacter* made up only a minor fraction of the entire community (*Marinobacter* up to 0.3%). Hydrogen utilization is another widespread trait that is difficult to pinpoint based on taxonomy alone, hence we could only assign this to *Hydrogenophilus, Hydrogenophaga*, and *Paracoccus*, all represented in the dataset, but in low abundances.
Finally, we observe a number of groups with the ability to transform nitrogen compounds. This includes members of the putative ammonium-oxidizing archaeal Marine Group I (\< 5% of total community) and nitrifiers (*Nitrospira*), which was detected in discrete samples up to 0.5%. Despite, the relative low abundances, their presence suggest an active nitrogen cycle, which is in congruence with the low concentration of organic carbon measured in this environment (Orcutt et al., [@B44]; Sakata et al., [@B46]).
Contamination control
---------------------
A great concern related to investigations of subsurface crustal material (and the deeply buried biosphere in general) has been the challenge of overcoming issues related to contamination constrains (Lever et al., [@B34]; Santelli et al., [@B48]). In order to delineate potential sources of contamination (acquired during drilling or subsequent sample processing) we analyzed two sample processing controls (extraction blanks) and two drilling operation controls (drill mud and a recovered empty microsphere bag). Designation of potential contaminants facilitates the tracking of their source in addition to their downstream removal from the bioinformatic pipeline as described before. As more reads were removed from the native samples due to their overlap with extraction blanks rather than with the drill mud and microsphere bag, we conclude that more contamination was introduced during DNA extraction and amplification than from the actual drilling procedure. However, we acknowledge that contamination could have originated from drilling or downstream procedures not accounted for by our contamination controls.
Despite, the observed contamination, several lines of evidence suggest that the final community structure is not corrupted: (i) the bifurcation of community structure of the two crustal sites, (ii) drastic differences between the composition and structure of sedimentary and crustal samples, despite equally low biomass, (iii) congruence between our results and taxonomic identity of enriched organisms from independent studies at this site, including *Pseudomonas, Burkholderia, Bacillus, Salinibacterium, Sphingomonas, Moraxella*, and *Methylobacterium* (Hirayama et al., [@B26]), (iv) agreement with core-taxa hitherto identified in basalt hosted environments (Lysnes et al., [@B37]; Lee et al., [@B33]) and in the fluids under North Pond (Meyer et al., [@B39]).
In sum, the data shows that contamination in low abundance habitats is a concern, and we encourage that both extraction and operation controls are performed. If such measures are taken the influence of contamination in data analysis seems manageable.
We also note that the presence of microspheres, a general measure of contamination deployed during IODP drilling operations, does not seem to be reflected in the magnitude of contamination in the 16S rRNA gene libraries (measured as number of reads removed in the filtering due to drill mud contamination).
Conclusions {#s5}
===========
Our study gives some of the first insights into the microbial inventory of the subsurface oceanic crust in a young cool ridge flank system and show a community dominated by *Proteobacteria* (*Gammaproteobacteria, Alphaproteobacteria*, and *Betaproteobacteria*), followed by *Actinobacteria, Firmicutes, Bacteroidetes*, and *Acidobacteria*. In general, the same phyla are present in high abundances on seafloor-exposed basalts and in the crustal fluids, suggesting the possibility for a common basalt-hosted microbial biome. However, more data is needed to establish if a core group also exist at deeper taxonomic levels. Within the crustal communities we find microbial representatives that are likely to be involved in iron, sulfur, hydrogen, and nitrogen cycling but all in relative low abundances. However, the activity levels might be considerably higher than what the relative abundance implies.
Our estimated cell abundances are on average 0.7 × 10^4^ cells per gram of igneous rock, several orders of magnitude lower than what has been found on seafloor-exposed basalt (e.g., Einen et al., [@B12]; Santelli et al., [@B49]). The abundance is relatively consistent across all samples and we therefor speculate that the available energy is equal across the different samples and/or cell numbers are limited by a common nutrient factor.
Further, the variations in community structure between the samples do, to a large extent, separate the two investigated sites (1382A and 1383C) into two major clusters with no apparent link to differences in lithology. Comparing the basalt-hosted community structure to the overlying sediment using the relative distribution of individual OTUs clearly separates the two environments. However, \~1/3 of all OTUs in the basalt, representing an average of 80% of all sequences, were also present in the overlying sediment, indicating coupling between the two compartments of the deep biosphere. This observation is interesting with respect to the origin of the microorganisms inhabiting the crustal aquifer, however, deciphering the direction of cell dispersal, the source and the nature and underlying mechanisms of such links needs further investigation.
Author contributions {#s6}
====================
SJ designed the research; SJ and RZ performed the research; SJ and RZ analyzed the data; and SJ and RZ wrote the manuscript.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This article is dedicated to the late Katrina Edwards. We thank the entire scientific party, especially the basaltic sampling crew and all crewmembers onboard Joides Resolution during IODP expedition 336, for their help and expertise. Co-chief scientists Katrina Edwards and Wolfgang Bach were instrumental in realizing this great undertaking, and are gratefully acknowledged. We thank Håkon Dahle for helping to develop the bioinformatic pipeline. The comments and suggestions from Gustavo Ramirez on the final version of this article are much appreciated. This study used samples and data provided by the IODP and was funded by the Norwegian Research Council through the Centre for Geobiology, University of Bergen.
Supplementary material {#s7}
======================
The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fmicb.2016.00820>
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[^1]: Edited by: Beth Orcutt, Bigelow Laboratory for Ocean Sciences, USA
[^2]: Reviewed by: William Brazelton, East Carolina University, USA; Hazel Barton, University of Akron, USA
[^3]: This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology
| {
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Background {#Sec1}
==========
Construction of technological pipelines is one of the main fields of polymeric material application in the world \[[@CR1]\]. Among polymers used for pipe production, polyethylene (PE) is one of the most commonly used \[[@CR2]\]; this material has a perfect correlation between prices, mechanical properties, and weldability and, hence, has considerable advantage comparing with other polymers.
Pipes produced of various types of high-density polyethylene (HDPE; so-called "pipe" polyethylene) are used for pipeline construction \[[@CR3]\]. Pipes for the first technological pipelines have been produced from the raw material marked as PE-63. Later on, the next brands, namely PE-80 and PE-100, have been developed and widely applied \[[@CR4],[@CR5]\]. Currently, all these three types of polyethylene are used in the pipe industry \[[@CR6]\].
Welding is the main method of PE pipes joining for pipeline construction. As for today, the following welding methods are sufficiently developed from technological point of view and are commonly used in practice: hot tool butt welding, hot tool socket welding, and resistance welding \[[@CR7],[@CR8]\]. The last two methods require some special coupling details, such as socket and resistance fittings. Butt welding is the most simple and multipurpose method and can be used for pipes of all diameters (except the thin-wall pipes).
Performance characteristics of polyethylene pipelines are considerably dependent on the welded joint quality. As a rule, the declared pipeline lifetime is at least 50 years, and all factors that could promote pipe or weld destruction are permanently investigated and can be eliminated \[[@CR9]\]. In cases when destruction has occurred, it is important to have an efficient and reliable repair technology \[[@CR10]\]. Since pipes are produced of various types of polyethylene, it is required to develop welding technology providing reliable welds of dissimilar PE types.
All abovementioned welding methods have their own technological peculiarities and typical defects of welded joints \[[@CR11]\]. Numerous scientific studies aim to improve hot tool butt welding method. Empiric methods are used by researchers in order to optimize main welding parameters for various technological conditions \[[@CR12],[@CR13]\] as well as to investigate peculiarities of various-sized pipe welding \[[@CR14]\]. Mechanical and thermal properties of the pipe material also strongly affect the hot tool butt welding process \[[@CR15],[@CR16]\]. This should be taken into account when dissimilar types of polyethylene are welded with each other. PE-63, PE-80, and PE-100 have different technological characteristics, like, for example, shrinkage degree at cooling \[[@CR17]\] and different melt flow indexes, so special welding technology and equipment should be developed for the cases when dissimilar PE types have to be welded together.
In spite of the numerous developed technologies and wide pipes' welding practical application, the detailed research of polyolefin welding nature is still not completed; mechanism of welds' formation is not explored sufficiently. Investigations of morphology, as a rule, enable to study the PE pipe macrostructure, fusion lines, and heat-affected zone geometry \[[@CR18],[@CR19]\]. In some works, the PE macromolecular structure affecting on material weldability has been investigated \[[@CR20]\] as well as the internal deformations in PE welded joints \[[@CR21]\], but general mechanism of welded joint formation and macromolecular structures \[[@CR22],[@CR23]\] in the weld are still studied insufficiently.
Hereby, there is still no complete understanding of PE and other polyolefin welded joint formation and structural peculiarities. Welding process of more complicated chemical system than polyethylene is even less explored. In this work, the results of complex investigations (by means of differential scanning calorimetry, thermogravimetric and thermomechanical analyses, as well as wide-angle X-ray scattering) of dissimilar technical PE type weld structure and their properties are presented. Basing on analysis of the results obtained, some new hypothesis concerning nature and mechanism of welds' formation and polymer structuring in such welds are proposed.
Methods {#Sec2}
=======
Materials and processing {#Sec3}
------------------------
The following samples have been used for welding experiments, for structure analysis, and for investigations of mechanical and thermal properties: polyethylene pipes produced from two types of high-density polyethylene (HDPE) with different minimum required strengths (MRS)---PE-80 (MW~bimodal~ 300000 g/mol, density 0.953 g/cm^3^, MRS = 8 MPa), and PE-100 (MW~bimodal~ 300000 g/mol and density 0.960 g/cm^3^, MRS = 10 MPa).
The welding experiments have been carried out with 63 mm diameter and 6 mm wall thickness of PE-80 and PE-100 pipes using traditional hot plate butt welding under the following conditions: 200°C welding temperature, 0.2 MPa welding pressure, and 60 s upset time. Change over time was 3 s. The cooling time under pressure was 6 min. SAT-1 hot plate butt welding device produced by Experimental Welding Equipment Factory of E.O.Paton Electric Welding Institute of the NAS of Ukraine has been used for welding. Photo of PE-80 and PE-100 pipes' weld is presented in Figure [1](#Fig1){ref-type="fig"}.Figure 1**The welded joint of PE pipes.** Dissimilar pipe weld (PE80 and PE-100, 63 mm in diameter).
Equipment and measurements {#Sec4}
--------------------------
PE structure of PE (types PE-80 and PE-100) as well as of PE-80/PE-100 welds has been studied by means of wide-angle X-ray scattering (WAXS) using X-ray diffractometer DRON-4.07 (Burevestnik, Saint Petersburg, Russia) with X-ray optical scheme according to the Debye-Scherer method, using CuK~α~ emission (*λ* = 0.154 nm), monochromated by Ni-filter. X-ray tube BSV27Cu working at *U* = 30 kV and *I* = 30 mA has been used like a source of characteristic X-ray radiation. X-ray measurements have been carried out by step-by-step scanning with scattering angles (2*θ*) from 2.6° to 40°, with an exposure time of 5 s at temperature *T* = 20 ± 2°С.
Thermal properties of the initial specimens and of the welds have been explored by means of differential scanning calorimetry (DSC) on DSC Q2000 device from TA Instruments (New Castle, DE, USA), in the inert atmosphere (higher-purity nitrogen, GOST 9293--74) under temperatures from 40 to 200°C with linear heating rate 20°C/min. Specimen weight was between of 6 and 10 mg each. Temperature measurement precision was ±0.01°С, heat flow precision ±0.01 J/g.
Thermal stability and thermal-oxidative breakdown (TGA) of the initial specimens and of the weld have been studied with TGA Q50 device by TA Instruments (New Castle, DE, USA), in the dried air atmosphere under temperatures from 30 to 700°C with a linear heating rate of 20°C/min. Specimens' weight was approximately 6--12 mg each. Temperature measurement precision was ±0.01°С, and weight loss precision was ±0.0001 mg.
Thermomechanical behavior and deformational characteristics (TMA) of the initial specimens and of the weld have been investigated with TMA Q400 EM device by TA Instruments (New Castle, DE, USA), in the dried air atmosphere, with a linear heating rate of 10°C/min under temperatures from 30 to 250°C. Measurements have been carried out in thermal expansion mode with the use of quartz indenter of 2.8 ± 0.01 mm in diameter. The applied indenter pressure to the specimen was permanent and equal to 10^−1^ MPa. Temperature measurement precision was ±0.01°С, and deformation control precision was ±0.01 μm. All the devices from TA Instruments have been certified according to the international standard ISO 9001:2000.
Mechanical properties (strength and elongation at break) of initial and welded specimens have been evaluated by means of tensile axial test (according to DBN B.2.5-41 standard) with a 50 mm/min tension rate at room temperature with FP-10 tension machine (Germany). Welding quality was also estimated basing on visual geometrical parameters. All investigations were repeated three times with different specimens for each time to enhance accuracy of the measurements.
Results and discussion {#Sec5}
======================
Results of thermogravimetric investigations for PE-100, PE-80, and their weld are presented in Figure [2](#Fig2){ref-type="fig"}a. It is evidently that under temperatures 280-500°C, a curve of PE-80/PE-100 weld is located between curves of pure PE-80 and PE-100, which correspond to the thermal-oxidative breakdown process. Such behavior of curves is logical and is not a subject to any discussion. But in the starting area of thermal-oxidative breakdown process (up to 280°C), there is a certain increased stability of PE-80/PE-100 weld comparing to the pure polyethylenes. As one can see in the insert in Figure [2](#Fig2){ref-type="fig"}a, PE-80/PE-100 weld has lower weight loss in the beginning of the breakdown and increased (up to 10°C) temperature of the breakdown start comparing both with PE-80 and PE-100. Such curve's pattern indicates that some structures with higher thermal stability are formed in the weld.Figure 2**Resulting plots of the TGA and TMA studies.** Thermogravimetric **(a)** and thermomechanical **(b)** results for pure PE-80, PE-100, and their PE-80/PE-100 weld.
A similar behavior of materials is observed in the thermomechanical test (Figure [2](#Fig2){ref-type="fig"}b). Relative strain curve of PE-80/PE-100 weld during the melting at temperatures higher than *T* = 140°C is located between respective curves for pure PE-80 and PE-100. Wherein under temperatures 25-130°C, PE-80/PE-100 weld has the maximum values of thermal expansion (insert in Figure [2](#Fig2){ref-type="fig"}b) comparing to the pure PE-80 and PE-100. It can be explained by the existence of the internal stresses in "frozen" areas of the amorphous part of polymer, which appear during welding process. Relaxation and unfreezing of these areas at heating lead to enhancing of molecular mobility and increasing of the material volume.
Basing on the abovementioned data, we can assume that under welding of dissimilar types of polyethylene, like PE-80 and PE-100, areas with higher thermal stability (evidently, in crystalline phase) and areas with internal stresses (in amorphous phase) are formed in the welding zone. In order to verify this idea, all specimens (both pure polyethylene types and their welds) have been studied by means of differential scanning calorimetry (Figure [3](#Fig3){ref-type="fig"}a) and wide-angle X-ray spectroscopy (Figure [3](#Fig3){ref-type="fig"}b).Figure 3**Resulting plots of the DSC and WAXS studies.** DSC **(a)** and WAXS **(b)** spectra of pure PE-80, PE-100, and of their PE-80/PE-100 weld.
For all three specimens on DSC curves, two minimums corresponding to the melting processes of crystalline structures within PE can be observed, wherein these two melting temperatures at all specimens indicate their polycrystallinity. The first minimum *T*~*m*1~ indicates the melting point for the easier-melting fraction with a melting temperature of 117-125°C. The second minimum *T*~*m*2~ corresponds to the melting of better-ordered (better-packed) crystallites with higher thermal stability with melting temperature between 133°C and 138°C. Melting temperatures corresponding to both types of crystallites for all specimens are presented in Table [1](#Tab1){ref-type="table"}.Table 1**Thermal characteristics (melting temperatures and enthalpies) of both types of polyethylenes and of their weld obtained from DSC studiesSpecimenMelting temperature** ***Т*** ~***m*1**~ **, °СMelting temperature** ***Т*** ~***m*2**~ **, °СMelting enthalpy, J/g**PE-80117.10133.13114.90PE-100124.34136.21134.90PE-80/PE-100 weld118.90138.06144.60
Increased melting temperature *T*~*m*2~ of the weld comparing to respective *T*~*m*2~ of both pure polyethylene types is an important confirmation of assumption that the weld contains areas with higher thermal stability and, respectively, with crystallites with higher order (packing).
Similar trend is also found for integral melting enthalpies, defined from the melting areas on DSC curves that enabled us to calculate degree of crystallinity using the classical equation \[[@CR24]\] (see Table [1](#Tab1){ref-type="table"}). For PE-80/PE-100 weld integral melting enthalpy is the biggest among three polymers that evidently indicates higher thermal stability of crystalline phase of the PE-80/PE-100 weld comparing both with pure PE-80 and PE-100, and, in turn, this can be explained by formation of better-packed crystallites in the welded joint. Degrees of crystallinity presented in Table [2](#Tab2){ref-type="table"} are calculated basing on the integral melting enthalpies for each specimen using classical equation \[[@CR24]\]. One can see that the degree of crystallinity of the weld is the highest among three specimens and, respectively, higher than of pure matrixes of both PE types.Table 2**Structural and mechanical characteristics of polyethylenes and of their weldSpecimenDegree of crystallinity (DSC) %Degree of crystallinity (WAXS) %Crystallites size** ***L*** **1 (2** ***θ*** **= 21.2°) nmCrystallites size** ***L*** **2 (2** ***θ*** **= 23.6°) nmTensile strength MPaRelative tensile strength %**PE-8042567.27.219.6100PE-10051577.27.223.1100PE-80/PE-100 weld53667.28.0Destroyed on the basic material\>100
Other arguments, which confirm our assumption, are WAXS results (Figure [3](#Fig3){ref-type="fig"}b). PE-80, PE-100, and PE-80/PE-100 weld's spectra analysis show that they have amorphous-crystalline structure (presented by diffraction maximums at scattering angles 2*θ*max = 21.2°, 23.6°, 29.7°, and 36.7° against the background of virtual amorphous halo).
Relative crystallinity degree (*X*cr) was determinated by Matthews's method \[[@CR25]\]:$$\documentclass[12pt]{minimal}
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\begin{document}$$ {X}_{\mathrm{cr}}={Q}_{\mathrm{cr}}{\left({Q}_{\mathrm{cr}}+{Q}_{\mathrm{am}}\right)}^{-1}\cdot 100 $$\end{document}$$
where *Q*cr is the area of diffraction maximums describing crystalline structure of polymer and *Q*~cr~ + *Q*~am~ is the total area of diffraction pattern within the scattering angles, where amorphous-crystalline structure of polymer is appearing. This determination has shown that crystallinity degree for both PE-80 and PE-100 is almost equal (approximately 56% for PE-80 and 57% for PE-100) and is quite different to such degree of the PE-80/PE-100 weld (66%), and these data correlate with results of DSC investigations. Differences in crystallinity degrees calculated from DSC and WAXS studies, as reported in \[[@CR26]\], are quite typical and can be explained by unequal research conditions and macromolecule state at room (WAXS) and elevated (DSC) temperatures.
In turn, effective crystallite size (*L*1 and *L*2) evaluation, made by Scherer's method \[[@CR27]\], is presented as follows:$$\documentclass[12pt]{minimal}
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\begin{document}$$ L=K\lambda {\left(\beta \cos {\theta}_{\max}\right)}^{-1} $$\end{document}$$
where *K* is a constant related to the crystallite's shape (if shape is not determined, *К* = 0.9), and *β*, which is the angular half-width (width of half-height) of diffraction maximum, has shown that average values of *L*1 ≈ 7.2 nm for PE-80, PE-100, and PE-80/PE-100 weld and average values of *L*2 ≈ 7.2 nm for PE-80 and PE-100, while for PE-80/PE-100 weld, *L*2 ≈ 8.0 nm (diffraction maximums at 2*θ*max = 21.2° and 23.6° have been used for the calculation).
In order to evaluate the difference between the experimental X-ray diffraction pattern of PE-80/PE-100 weld and diffraction patterns of PE-80 and PE-100 mechanical blends (under conditions of zero interaction between them), further calculations of such blends' X-ray diffraction pattern have been done assuming that both components (both types of PE) are making an additive contribution into the diffraction picture:$$\documentclass[12pt]{minimal}
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\begin{document}$$ {I}_{\mathrm{add}}={w}_1{I}_1+{w}_2{I}_2 $$\end{document}$$
where *I*~1~ and *I*~2~ are intensities of wide-angle X-ray scattering of PE-80 and PE-100; *w*~1~ and *w*~2~ are mass content of the components in the system (*w*~1~ + *w*~2~ = 1). Comparing experimental and calculated X-ray diffraction patterns, one can see in Figure [3](#Fig3){ref-type="fig"} that non-additive change in the experimental diffraction curve comparing with the theoretical one takes place; it is an important result since it confirms that interaction between PE-80 and PE-100 macromolecules occurs in the PE-80/PE-100 weld. Analyzing PE-80/PE-100 weld experimental diffraction curve, it is obvious that intensity of the first diffraction maximum (2*θ*max = 21.2°) is decreasing and intensity of the second diffraction maximum is considerably increasing (2*θ*max = 23.6°) comparing to the respective diffraction maximums on both pure PE spectrums. Apparently, this factor indicates that the restructuring of PE-80 and PE-100 crystalline phases occurs when these two materials are welded and that better-packed crystallites (comparing to the pure materials) are formed in PE-80/PE-100 weld. By this fact, the increased strength of dissimilar polymers joint identified earlier by specialists and confirmed experimentally before the start of current investigations can be explained (see Table [2](#Tab2){ref-type="table"}). The values of crystallite size (*L*1 and *L*2) for each specimen calculated basing on separate diffraction maximums are also presented in Table [2](#Tab2){ref-type="table"}. Thus, the increased crystallite size characterizes for PE-80/PE-100 weld.
Conclusions {#Sec6}
===========
Results of complex thermal and structural investigations for two technical PE types (PE-80 and PE-100) and of their weld have been represented. The welded joint was produced by means of traditional hot tool butt welding. It is revealed that during the welding process, restructuring of crystalline phases occurs and crystalline areas with higher mechanical and thermal properties appear due to the increase of quantity of crystallites and to their bigger size and better ordering (packing).
DSC
: differential scanning calorimetry
HDPE
: high-density polyethylene
PE-80
: high-density polyethylene with MW 80000 g/mol and density 0.953 g/cm^3^
PE-100
: high-density polyethylene with MW 100000 g/mol and density 0.960 g/cm^3^
PE
: polyethylene
TGA
: thermogravimetric analysis
TMA
: thermomechanical analysis
WAXS
: wide-angle X-ray scattering
**Competing interests**
The authors declare that they have no competing interests.
**Authors' contributions**
AG performed butt fusion welding of PE pipes. NK provided valuable discussions and wrote the manuscript. VK assisted in tensile testing and interpretation of its results. VD fulfilled the WAXS investigations and analysis of the WAXS data. AS and MI provided valuable discussions and contributed in the results' analysis comparing to the structural peculiarities of the samples. AV provided visual imaging and helped with the results analysis. All authors read and approved the final manuscript.
**Authors' information**
AG is the leading engineer at the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine. NK is PhD in Technical Sciences, senior staff scientist at the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine. VK is junior researcher at the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine. VD is PhD in Polymer Physics, staff researcher at the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine and at the Institute of Macromolecular Chemistry of the NAS of Ukraine. AS is PhD in Technical Sciences, staff researcher at the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine. AV is leading engineer at the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine. MI is Doctor in Physics, Head of Department, Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine, and senior staff scientist at the Institute of Macromolecular Chemistry of the NAS of Ukraine.
Submitted results were obtained during the fulfillment of the budget supported project in the Plastics Welding Department of the E.O.Paton Electric Welding Institute of the NAS of Ukraine.
| {
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Endemic goiter is a major health problem in India. Widespread national salt iodization programmes implemented by the Government of India have not shown a dramatic decline in the prevalence of endemic goiter[@ref1]. The reason for the high prevalence of goiter is not fully understood. A genetic predisposition of the Indian population for development of autoimmune goiter has been suggested[@ref2]. Secondly, iodine itself could induce autoimmunity in patients. Iodine induced thyroiditis is well established in animal studies[@ref3]. It has also been seen that iodine supplementation in iodine deficient areas increases the prevalence of lymphocytic infiltration of thyroid by three-fold; with 40 per cent increase in prevalence of antithyroid antibodies in serum over 0.5 to 5 years[@ref4].
This study was carried out at a tertiary care hospital located in the western coastal area known for endemicity of goiter, where sea food is the staple diet of the population. Moreover, there is a widespread use of iodized salt in the population. In spite of this, increasing numbers of goiters diagnosed to have Hashimoto\'s thyroiditis (HT) have been observed. We undertook this study to understand the clinical presentation, biochemical status and ultrasonographic picture of HT in this area. We also did a cytological analysis on a few patients to analyse the typical cytomorphologic features of HT and associated the clinical features, biochemical thyroid status and cytological parameters with each other to understand the pathogenesis of the disease.
Material & Methods {#sec1-1}
==================
This study was conducted in the department of Otorhinolaryngology, Head and Neck surgery, Kasturba Medical College, Mangalore, Karnataka, India, on 144 patients with HT. This was a cross-sectional study conducted on patients with cytologic diagnosis of Hashimoto\'s thyroiditis/lymphocytic thyroiditis (LT) during January 2001 to July 2011. Retrospective analysis of the medical records of patients was carried out from 2001 to 2009 and prospective study of patients was done from July 2009 to July 2011. Patients with cytology proven HT/LT with or without associated pathology were included in the study. Patients with cytology of multinodular or colloid goiter were excluded. The study protocol was approved by the ethics committee of the hospital. Informed written consent was obtained from patients of prospective study group.
Information on clinical presentation with emphasis on age, sex, presenting complaints, duration of complaint, clinical signs of hyperthyroidism or hypothyroidism, presence of goiter and tenderness was recorded. Investigations noted were thyroid function tests namely T3 (triiodothyronine) (normal values 0.6-2.02 IU/ml), T4 (thyroxine) (normal values 5.13-14.06 IU/ml), TSH (thyroid stimulating hormone) (normal values 0.27-5.5 IU/ml), FreeT4 (normal values 0.93-1.71 IU/ml). Antithyroid antibody levels namely anti thyroid peroxidase (anti TPO) (normal values \<35 IU/ml) and antithyroglobulin (anti Tg) antibody levels (normal values \<35 IU/ml) were documented. The thyroid function tests and antithyroid antibody levels were estimated in our hospital lab by ECLIA (Electro chemilumniscence immune assay) method. (Automated Roche Cobas e411 Analyser, Roche Diagnostics, USA).
Ultrasonographic reports were also evaluated. Volume of gland, echo texture, echogenicity and vascularity were recorded in all possible cases. Volume was calculated using the formula: length x width x thickness x 0.52 (correction factor) for each lobe. A volume of 18.6 ± 4.5 ml was considered to be normal[@ref5]. Treatment modalities offered to patients were also evaluated. A detailed cytological study was done in selected 52 cases from fine needle aspiration (FNA) smears from the last two years (2009-2011). The main parameters studied were amount of lymphocytes, lymphocytic infiltration and destruction of follicular cells and Hurthle cell change.
Statistical analysis was done using SPSS 17.0 (Chicago: SPSS Inc). Chi-square test and ANOVA were used and *P*\<0.05 was considered as significant. Biochemical values of thyroid function, ultrasound characteristics of HT, cytological parameters and antithyroid antibody levels were correlated with one another using the Karl Pearson\'s correlation coefficient.
Results {#sec1-2}
=======
A total of 144 patients with cytology proven HT/LT were included in the study. Of these, 46 patients were included retrospectively, while the prospective group had 98 patients. These were 129 females and 15 males ([Table I](#T1){ref-type="table"}), which indicated a significantly higher percentage of females having HT as compared to males (*P*\<0.001). The age of the patients ranged from 9 to 75 yr. Mean age was 34.18 ± 12.71 yr. Maximum number of cases were noted in the 3^rd^ decade (20-30 yr age group, n=46) followed by the 4^th^ decade (31-40 yr age group, n=40). Patients in the third decade were significantly more affected than those in other age groups (*P*\<0.001).
######
Clinical findings in Hashimoto\'s thyroiditis in our study population (n=144)
Ninety seven patients had documentation about the duration of their symptoms; 87 (89.6%) patients presented to hospital within 5 years of symptom manifestation ([Table I](#T1){ref-type="table"}). The difference was significant when compared with patients presenting at a later date (*P*\<0.001).
Goiter was present in 100 cases, 14 cases were documented as absent goiter and no comment was documented in 30 cases. A significant number of patients had a goiter at presentation (*P*\<0.001). Of these 100 cases, goiter was diffuse in 68 cases, multinodular in 28 cases and solitary nodule in four cases. Clinically majority (n=100) of patients had no features of hypothyroidism or hyperthyroidism, only 12 cases were identified to have hypothyroid features and 22 had hyperthyroid features ([Table I](#T1){ref-type="table"}). This showed that a significant number of patients were clinically euthyroid (*P*\<0.001). Vague symptoms like generalized fatigue and malaise were reported by some patients. Pain/tenderness of thyroid gland was present in seven cases among the total 144 cases.
Based on TSH levels, there were 64 cases of hypothyroidism, 46 cases of euthyroidism and 30 of hyperthyroidism. Information was incomplete in rest of the cases ([Table II](#T2){ref-type="table"}). There was a significant difference in the thyroid function status of the patients (*P*\<0.01). The hypothyroid patients were classified into mild, moderate and severe based on the TSH levels (mild 5.5 - 45.5, moderate 45.5 - 85.5, severe \>85.5 IU/ml). Significant number of patients had mild hypothyroidism (*P*\<0.01). T4 levels were normal in 32 cases (subclinical hypothyroidism) and low in 24 cases (overt hypothyroidism) ([Table II](#T2){ref-type="table"}). Volume, echo texture, echogenicity and vascularity of the gland were also recorded ([Table II](#T2){ref-type="table"}).
######
Biochemical and ultrasonographic investigations in Hashimoto\'s thyroiditis patients
AntiTg antibody levels were available in 33 cases, 31 cases had high value. Anti TPO antibody levels were available in 52 cases, of whom 48 had high value indicating that a significant number of patients had high antithyroid antibody levels (*P*\<0.001).
Of the 81 patients treated thyroxine supplementation was given to 63 patients, antithyroid drugs (neomercazole/carbimazole) to 19 and symptomatic treatment given to six patients (propranolol, alprazolam, paracetamol, diclofenac); 46 patients received no treatment and surgery was done in 17 cases. Among patients with cases with cytologic diagnosis of HT, nine were proven to be HT by histopathology also while two were shown to be multinodular goiter and colloid goiter. Among patients diagnosed as papillary carcinoma with LT, two cases underwent total thyroidectomy with neck dissection and one was treated with completion thyroidectomy after hemithyroidectomy. One patient of papillary carcinoma with LT was proven to be Hurthle cell adenoma with colloid adenomatous goiter with LT after histopathologic examination. Another patient of papillary carcinoma with LT also had a lymph node with adult T cell lymphoma.
The various aspects studied in cytology were cellularity of smear, presence of follicular cells, Hurthle cells, degree of lymphocytes, presence of lymphocytic infiltration and destruction of follicular cells, presence of giant cells, colloid and lymphoepithelial (L:E) ratio ([Table III](#T3){ref-type="table"}). Cellularity of smears was assessed as good, adequate, low and poor. Lymphocytes seen in the smears were graded as sparse, mild, moderate, moderately dense or abundant ([Fig. 1](#F1){ref-type="fig"}). Hurthle cells were absent in some smears, seen as occasional cells or small groups, or seen in many large groups ([Fig. 2](#F2){ref-type="fig"}). Lymphocytic infiltration and destruction were graded as present or absent ([Fig. 3](#F3){ref-type="fig"}). Centroblasts were absent, occasional or many. Colloid was absent in most with only two slides showing small blobs. Lymphoepithelial ratio ranged from 1:1 to 30:1.
######
Cytomorphological features in Hashimoto\'s thyroiditis
{#F1}
{#F2}
{#F3}
The duration of symptoms was found to have a significant association with presence of goiter (*P*\<0.05). There was no significant association between thyroid status and echogenicity of thyroid gland, antithyroid antibody, and intensity of lymphocytes, Hurthle cells, lymphocytic infiltration and destruction of follicular cells.
Discussion {#sec1-3}
==========
Most of our patients with HT belonged to the 21-40 yr age group. In a study from Puducherry, maximum patients with HT were in their 3^rd^ decade[@ref6]. In another study from Chandigarh, majority of patients were in 3^rd^ and 4^th^ decades[@ref7]. Unlike the Indian studies, studies from the West reported Hashimoto\'s thyroiditis being diagnosed between the 4^th^ and 6^th^ decades of life[@ref8]. Thyroid diseases are always reported to be higher in the female population. The reported female to male ratio in Hashimoto\'s thyroiditis ranges from 15:1 to 3:1[@ref6][@ref7]. Our gender ratio falls in this range. Most of our patients (61%) presented with symptoms of one year duration. The association of goiter with duration of symptoms before presentation was significant. But the biochemical thyroid status when associated with the same variable was found to have no significance. This shows that in our population, goiter but not symptoms of altered thyroid hormone status is a reason for seeking medical care.
The high occurrence (61%) of diffuse goiter in our study was similar to previous studies from India. In the study from Chandigarh on cytology proven Hashimoto\'s thyroiditis[@ref7], 93 per cent of patients had goiter, of whom 90 per cent were diffuse and 3 per cent were nodular. Nodules are seen in early stages of disease when clinical and hormonal changes are not yet established[@ref7]. A study from Malaysia has reported that Indians have increased prevalence of diffuse goiter while Chinese have more of nodular presentation[@ref2].
Most of our patients were hypothyroid (subclinical and overt), and of these only 38 per cent showed overt hypothyroidism. These results were similar to previous studies from India[@ref6][@ref7] and abroad[@ref2]. In our study, 21 per cent patients had hyperthyroidism, not seen in other studies. It is interesting to note that 66 per cent of the hyperthyroid patients presented in less than one year duration for evaluation.
We found 93 per cent of our patients were anti TPO positive and 92 per cent of them were anti Tg positive. There is a well-recognized discrepancy between antibody levels and cytology in Hashimoto\'s thyroiditis in children and young adults[@ref9]. This could be because that in early stage of disease, antibody production is confined to intrathyroidal lymphocytes[@ref10]. On the other hand, patients with significant titres may not have cytology proven Hashimoto\'s thyroiditis. This can be explained by the fact that focal LT which is an early lesion may be missed by cytology[@ref9].
Ultrasonography reports were studied in a limited number of cases. In our study, the volume of gland was enlarged in 41 per cent and normal in another 41 per cent of patients. Studies have shown that thyroid volume changes as the disease progresses from euthyroid to overtly hypothyroid with smaller volumes found in overt hypothyroidism[@ref11]. Our study showed 64 per cent cases with normal thyroid echogenicity and 36 per cent cases with diffuse hypoechogenicity. Low echogenicity is due to altered thyroid structure because of disruption of follicles and diffuse lymphocytic infiltration[@ref12]. Studies from India[@ref13] and abroad[@ref12] have shown 80-85 per cent of cases having normal echogenicity.
In fine needle aspiration cytology of thyroid, L:E ratio (lymphoid:epithelial ratio) in Hashimoto\'s thyroiditis ranges from 2:1 to 10:1[@ref6]. We found moderate to abundant lymphocytic infiltrate in 69.3 per cent cases and lymphocytic infiltration of follicular cells in 57.7 per cent cases. An increase in number of epithelial cells raises suspicion of epithelial proliferative process associated with HT like malignancy[@ref6]. Plasma cells were seen in 75 per cent of our cases. Plasma cells are useful in diagnosing early Hashimoto\'s thyroiditis where lymphocytic infiltration of follicles is insignificant[@ref6]. A wide range of Hurthle cell change in Hashimoto\'s thyroiditis (48-98%) has been reported[@ref6]. As seen in our study, absent or scanty colloid is a usual feature in Hashimoto\'s thyroiditis, but paradoxical presence of colloid[@ref14][@ref15] is not unusual due to a combination of iodine supplementation and autoimmunity[@ref6].
Our study showed no association between the thyroid status and antibody positivity, sonographic hypoechogenicity or cytologic parameters. Though many previous studies resemble ours[@ref6][@ref7][@ref16], a few studies have shown positive correlation of biochemical parameters with reduction in echogenicity in sonography[@ref12][@ref13], lymphocytic infiltration[@ref13][@ref14] and antibody status[@ref13]. A study with long term follow up has reported presence of raised TSH along with positive TPO antibodies which raises the risk of developing overt hypothyroidism[@ref17].
Various neoplastic and non-neoplastic lesions are shown to be associated with HT on FNA like colloid goiter, cellular adenoma, follicular neoplasm, Hurthle cell neoplasm, papillary carcinoma, non-Hodgkins lymphoma (NHL) and follicular carcinoma[@ref6]. Therefore four cases of malignancies associated with HT on cytology in our study group, three of whom were papillary carcinoma and one was medullary carcinoma.
The most commonly encountered neoplasms in association with HT are papillary thyroid carcinoma (PTC) and primary thyroid lymphoma. Several studies have attempted to define relationship between HT and PTC. In a population of patients with thyroid cancers, 1.9 times higher rate of HT were seen in patients with PTC, when compared with other cancers. On the other hand, there is a 2-3 times increased risk of development of PTC in presence of HT[@ref18]. The presence of a dominant nodule, decreased response to suppressive therapy or clinical signs of metastasis should raise suspicion of PTC in patients with HT. FNA is useful in diagnosis of patients with HT associated lesions with a sensitivity of \>90 per cent[@ref18]. Many studies have been done on the prognosis of LT in PTC, majority of these reporting protective role of autoimmunity in terms of less aggressive disease at presentation, low recurrence[@ref19] and improved survival[@ref18].
Our study had several limitations. The lack of complete data for all variables in all patients has reduced the sample size in the analysis of the various parameters. Antithyroid antibody levels were available only in a few patients, cost constraints being the main limiting factor. T3, T4, TSH and antibody levels at the time of presentation to hospital were taken.
In conclusion, Hashimoto\'s thyroiditis was diagnosed in goiter in our patients who came from the iodine sufficient western coastal belt. The presence of goiter and hypothyroidism points towards the necessity of further evaluation of disease. Ultrasound compliments diagnosis with its characteristic features but cytology is confirmatory. Association of HT with malignancy requires a close follow up of these patients.
The study was partially supported by the TMA Pai Research grant (Manipal University), Manipal, India.
| {
"pile_set_name": "PubMed Central"
} |
###### Significance
The mechanism of action potential generation of nerves has been explained based on the conductance change of Na^+^ (and K^+^) channel in the membrane with time. However, the substantial source of the change isn't necessarily obvious. It is estimated that the change in the state of molecular particles at its gate would cause the conductance change, but it could be also explained from a view that the Na^+^ channel for action potential generation is represented by the series circuit of resistance (R), inductance (L), and capacitance (C). The current pattern of the RLC circuit as a model of the Na^+^ channel simulates well the conductance change in the voltage clump examination of nerves.
The action potential event of nerves is discussed supposing that the plasma membrane has micro pores selectively permeable to ion species, which are called (ion) channels represented by the registers. It is well known that depolarization of the inside of nerve above a certain voltage, so called the threshold, activates Na^+^ channels, leading to the generation of an action potential. When a nerve fiber is stimulated by a battery or other electric devices using paired electrodes placed on the axon, at switch-on (onset) an action potential is generated at the place corresponding to the negative pole (cathode) of the battery. On the other hand, at the switch-off (break), it is generated at the place corresponding to the positive pole (anode). The former is called a cathode circuit-closed excitation (CE), and the latter, an anode break excitation (AE) \[[@b1-14_147],[@b2-14_147]\]. The action potential generation in CE is understandable as the depolarization occurs (or outward current flows) at the electrode, but AE is a strange phenomenon as the action potential is produced even though the membrane potential does not reach its threshold voltage level. The AE phenomenon is interpreted commonly as follows. During a rectangular pulse stimulation, the inside of the axon at the positive electrode is under hyperpolarization which decreases the degree of inactivation of Na^+^ channels, and activation of Na^+^ channels occurs when the hyperpolarization returns to the resting level by the break of the stimulus \[[@b3-14_147],[@b4-14_147]\].
A phenomenon similar to the AE is observed when a hyperpolarizing rectangular pulse voltage is applied directly to the inside of nerve, in which the action potential is generated at the return of the stimulus to the resting potential \[[@b5-14_147],[@b6-14_147]\]. Here, the question arises "Doesn't the nerve membrane possess inductance factor?" If a nerve membrane possesses an inductance factor, another discussion about the AE and hyperpolarizing break stimulation becomes possible.
An essential property of an inductor is generation of a terminal voltage (*V~L~*) to prevent the "change" of current. It is proportional to its rate, *V~L~*=−*L*·*di/dt*: *L* is inductance measured in henrys (H) if the voltage is in volts (V) and the current change is in amperes per second (A/sec). The inductor produces a transient terminal voltage at the onset of a voltage pulse, and that of opposite polar voltage at its break as the current changes abruptly at both edges of the pulse. In a serial circuit of a resister (R) and an inductor (L) ([Fig. 1A](#f1-14_147){ref-type="fig"}), the voltage of the inductor in relation to time (*t*) in the rectangular pulse of *V~E~* volts is expressed as +*V~E~*·*e*^−^*^Rt/L^* for the onset and as −*V~E~*·*e*^−^*^Rt/L^*, for the break ([Fig. 1B](#f1-14_147){ref-type="fig"}). In the pulse stimulation of nerve with paired electrodes, the inside positive and negative transient voltages are generated at the place for the cathode electrode to the onset and to the break, respectively, if the membrane has an inductance factor. We understand in the stimulation that an action potential is generated at the place for the cathode electrode only in the onset. The opposite potential events occur at the place for the anode electrode, and the action potential should be originated at the break of the stimulus ([Fig. 1C](#f1-14_147){ref-type="fig"}). The same could be said for the hyperpolarizing break stimulation of the cell inside.
There is another noticeable phenomenon for action potential generation. A slow-rising stimulating current applied across the membrane fails to trigger the action potential nevertheless it depolarizes the membrane to its usual threshold membrane potential, which is called accommodation \[[@b3-14_147],[@b7-14_147],[@b8-14_147]\]. This phenomenon is interpreted as follows. During the slow approach to the threshold membrane voltage, inactivation of the Na^+^ channel and activation of the K^+^ channel advance before the voltage reached to the threshold \[[@b3-14_147]\]. Based on the idea that the membrane possesses an inductance factor responsible for action potential generation, the following interpretation is also possible: As the potential produced on the inductor is proportional to the rate of change of the current, the slow increment of the current cannot produce enough voltage for triggering the action potential generation. In the circuit shown in [Figure 1A](#f1-14_147){ref-type="fig"}, the resister also originates the potential that is linear to the current, so it is rational to think that the potential produced on the inductance plays a main role in triggering the action potential generation, which must become obvious especially in the pulse stimulation.
Phenomena such as the AE, hyperpolarizing break stimulation, and accommodation demonstrate that so called the threshold for action potential generation doesn't mean an absolute depolarizing level of the membrane potential, but certain amount of positive change from its preceding (or holding) potential. This feature of the threshold seems to be well explained by supposing that the membrane of nerve has an inductance factor, potential produced on which leads to action potential generation. It is understandable that the plasma membrane has both factors of resistance and capacitance, but there is no discernible structure resembling a coil anywhere in the plasma membrane. It is, however, known that electric components such as resistors, capacitors, and connecting wire possess their own intrinsic inductance. Circuits where these electric elements are not discriminated obviously are called "distributed constant circuits", and every material is considered to have more or less all factors of resistance, capacitance, and inductance. It also seems to be conceivable that the plasma membrane of nerves possesses an inductance factor. In this study, the possibility that the Na^+^ channel would contain inductance factor involving to the conductance (or current) change in action potential generation was examined by way of computer simulation.
Methods
=======
As an inductor is merely a wire to a DC signal and shorts sooner or later the potential difference such as a resting potential, it is difficult to think that the Na^+^ channel would be represented by only an inductance (plus resistance) element. In this study, the examination was performed supposing that the Na^+^ channel would be represented by a circuit composed serially of resistance (R \[Ω\]), inductance (L \[Henry\]), and capacitance (C \[Farad\]). The electric property of the circuit was studied using a personal computer system. The circuit current and voltages produced in the inductor and the capacitor of the serial RLC circuit in supplying variable magnitudes of rectangular pulse signal were analyzed by solving a basic formation obtained by applying the Kirchhoff's law on the circuit. The patterns of the current and voltages with time were drawn with the software, LabVIEW-ver.8.5 (National Instruments Corporation). The values of R, L, and C were varied properly referring to the results reported mainly by Hodgkin & Huxley \[[@b3-14_147]\].
Results and Discussion
======================
Hodgkin & Huxley's hypothesis
-----------------------------
In the investigation of the mechanism of action potential generation, a technique, called the voltage clamp, has been frequently used, where ionic currents across the membrane in fixing cell inside with a step voltage of various levels are measured. It was first introduced by Cole (1949) \[[@b9-14_147]\] and Marmont (1949) \[[@b10-14_147]\] and was subsequently developed by Hodgkin & Huxley (1952) \[[@b3-14_147]\] and Hodgkin, Huxley & Katz (1952) \[[@b11-14_147]\] using the squid giant axon. The method revealed that phasic inward current of Na^+^ ions occurs immediately after the effective depolarizing clamp, and outward K^+^ current follows with a short delay, which increases gradually towards the steady level. Graphs of [Supplementary Figure S1B](#s1-14_147){ref-type="supplementary-material"}, C presented first by Hodgkin \[[@b12-14_147]\] and redrawn by Kuffler *et al.* \[[@b13-14_147]\] illustrate these current manners which were also ensured pharmacologically by Hill \[[@b14-14_147]\]. As the potential across the membrane is fixed in the test, the change of the Na^+^ and K^+^ currents could be understood as the changes of conductance for the corresponding ionic current. (It is conventional, in electric studies of neurons, to use the term "conductance" instead of resistance as its reverse, so we also use "conductance" henceforth.)
Supposing that ions flow through the exclusive channels for each ion, Hodgkin and Huxley described the membrane potential (*V~m~*) and time (*t*) dependences of conductances for Na^+^ channel (*g~Na~*) and K^+^ channel (*g~K~*) using the following equations, respectively \[[@b3-14_147]\].
g
N
a
(
V
m
,
t
)
=
G
N
a
·
m
(
V
m
,
t
)
3
·
h
(
V
m
,
t
)
,
g
K
(
V
m
,
t
)
=
G
K
·
n
(
V
m
,
t
)
4
,
where *G~Na~* and *G~K~* represent the maximum Na^+^ and maximum K^+^ conductance, respectively; *m* and *n*, activation parameters for Na^+^ and K^+^ conductance, respectively, which vary from 0 to 1 as a function of *Vm* and *t*; and *h*, an inactivation parameter for Na^+^ conductance that also varies from 0 to 1 as a function of *Vm* and *t*. The time dependence of *m^3^h* and *n^4^* to step voltages match well to the curves estimated from Na^+^ current and K^+^ current in voltage clamp tests, respectively, when *m*, *n*, and *h* are given by *m*=*1*--*exp*(−*t/τ~m~*), *n*=*1*--*exp*(−*t/τ~n~*), and *h*=*exp*(−*t/τ~h~*): *τ~m~*, *τ~n~*, and *τ~h~* are time constants for their exponential curves which are decided from transfer rates in two states, for instance, activation and inactivation, which can be calculated on the base of voltage clamp data \[[@b3-14_147],[@b15-14_147]\]. [Supplementary Figure S1D, E](#s1-14_147){ref-type="supplementary-material"} are samples of the conductance changes reported by Hodgkin and Huxley, showing that experimental estimates for Na^+^ and K^+^ conductance changes match well to their theoretical curves. As for the powers of *m*, *h*, and *n*, they explained as follows: the gates of ion channels are considered to be controlled by the shift of charged particles, where the gate of Na^+^ channel would be composed of the three activation particles, *m^3^*, and one inactivation particle, *h*, and the gate of K^+^ channel, by the four activation particles, *n^4^*.
Serial RLC circuit model as Na^+^ channel for action potential generation
-------------------------------------------------------------------------
Apart from the Hodgkin & Huxley hypothesis, let us, here, examine the electric property of a circuit composed serially of resistance (R), inductance (L), and capacitance (C) as an electric model of Na^+^ channel per unit area. [Figure 2A](#f2-14_147){ref-type="fig"} shows this RLC circuit model, where a battery formed by its equilibrium potential is omitted as it is not essential in examining the circuit property. (A notice is that the capacitor element in this RLC circuit model is different from the capacitor composed by a lipid bilayer of the membrane, which doesn't involve directly in action potential generation.) In supplying step voltage (*V~e~*) to this RLC circuit, the following formation is formed for time (*t*) from the Kirchhoff's law supposing that the capacitor is initially charged with *V~0~* volts and circuit current (*i*) flows clockwise.
V
e
=
i
·
R
\+
L
·
d
i
/
d
t
\+
(
V
0
\+
1
/
C
·
∫
i
d
t
)
Differentiating this by *t* and rearranging the terms, we get,
L
·
d
2
i
/
d
t
2
\+
R
·
d
i
/
d
t
\+
1
/
C
·
i
=
0
This is known as a second order linear differential equation for describing oscillatory movements with a resistance factor \[[@b16-14_147]\]. Its solution is established, so we shall take, in this article, only its result.
The solution separates two cases, *R^2^/4L^2^≥1/LC* and *R^2^/4L^2^*\<*1/LC*. In the former case, the circuit current doesn't exhibit the oscillation and converges to zero with simple time course, whereas in the latter case, the circuit current oscillates, amplitude of which decreases exponentially with time \[[@b17-14_147]\]. In this study, we shall restrict our examination to the former case, because oscillation isn't observed in the Na^+^ current in voltage clamp test. The established solution of the former case is,
i
=
A
1
·
e
(
\-
α
\+
β
)
t
\+
A
2
·
e
(
\-
α
\-
β
)
t
(
A
1
,
A
2
:
integration constants
,
α
=
R
/
2
L
,
β
=
R
2
/
4
L
2
\-
1
/
L
C
)
*A~1~* and *A~2~* can be obtained from the circuit conditions, *i*=*0* in *t*=*0*, and *V~C~* (capacitor voltage) = *Ve* in *t*=*∞*. The final form of *i* becomes the following.
i
=
V
e
\-
V
0
2
R
2
4
\-
L
C
{
e
(
\-
α
\+
β
)
t
\-
e
(
\-
α
\-
β
)
t
}
=
V
e
\-
V
0
R
2
4
\-
L
C
e
\-
α
t
s
i
n
h
β
t
We get also voltages produced in the inductor (*V~L~*) and the capacitor (*V~c~*). As the resolution processes are complicated, only the results are presented.
V
L
=
L
·
d
i
/
d
t
=
L
V
e
\-
V
0
R
2
4
\-
L
C
e
\-
α
t
(
\-
α
s
i
n
h
β
t
\+
β
c
o
s
h
β
t
)
V
c
=
V
0
\+
1
/
C
·
∫
i
d
t
=
V
0
\+
V
e
\-
V
0
C
R
2
\-
4
L
C
{
1
α
\-
β
(
1
\-
e
(
\-
α
\+
β
)
t
)
\-
1
α
\+
β
(
1
\-
e
(
\-
α
\-
β
)
t
)
}
Although there are many combinations of R, L, and C values, time courses of the circuit current, inductor potential, and capacitor potential are similar for whatever values we chose if they satisfy the condition, *R^2^/4L^2^*≥*1/LC*. As a sample, we shall briefly examine them in a case of R=20 \[Ω\]), L=1500 \[μH\]), and C=50 \[μF\]); the resistance of 20 Ω is adopted referring to the peak current in 76\~100 mV reported in voltage clamp test ([Supplementary Fig. S1D](#s1-14_147){ref-type="supplementary-material"}), and the values of inductance and capacitance are selected to fit approximately to the manner of Na^+^ current ([Supplementary Fig. S1C](#s1-14_147){ref-type="supplementary-material"}). [Figure 2B, C, and D](#f2-14_147){ref-type="fig"} show the graphs respectively of the circuit current (*i*), inductor potential (*V~L~*), and capacitor potential (*V~C~*) in supplying step voltages of −15, 0, 15, 30, 45, and 60 mV, in no initial charges of the capacitor (*V~0~*=0). (Although the initial charge has be set to 0 mV, it is also possible to set it to a resting potential, for instance, −65 mV, where 65 mV will substitutes for *V~0~* and the value will be added to each of the supplying voltages.)
A possibility that nerve membrane has an inductance factor, inside positive potential produced mainly on which leads to the action potential generation has mentioned above. Potential response of the inductor in this RLC circuit is similar to that in serial RL circuit ([Fig. 1B](#f1-14_147){ref-type="fig"}), but biphasic ([Fig. 2C](#f2-14_147){ref-type="fig"}). Deflection of the early transient potential is larger than the following opposite polar potential which approaches gradually to the base, 0 mV. (The return to 0 mV is independent on initial charges of the capacitor as the inductor cannot hold the potential to DC signal.) The polar becomes negative-positive when the supplying voltage is negative ([Fig. 2C](#f2-14_147){ref-type="fig"}, −15 mV). Negative supplying voltage produces anticlockwise current which means, in nerves, an outward current, and the negative potential of the early phase, depolarization. Supposing that only the positive potential produced on the membrane inductor would link to generate action potential, the early negative potential produced on the inductor of the RLC circuit must be responsible for the action potential generation.
Then, we shall examine the circuit current of the RLC circuit. In clamp test, membrane potential (V~m~) of nerve is fixed, in regular, positive (depolarization) to activate the Na^+^ channel with various values. Na^+^ ions are thought to move according to the formation, *I~Na~*=*g~Na~*(*V~m~*--*E~Na~*) where *E~Na~* and *g~Na~* are respectively Na^+^ equilibrium potential and Na^+^ conductance \[[@b3-14_147]\]. Na^+^ current is known to flow inward in early phase when *V~m~* is lower than *E~N~*~a~, and it changes, in regular, to the outward current around at the condition, *V~m~*=*E~Na~*. Supplying voltage of *Ve* and positive currents in the RLC circuit correspond respectively to *V~m~*--*E~Na~* and to the inward current in clamp test. In supplying the positive voltages, circuit current exhibits an early positive peak decreasing gradually to zero (15, 30, 45, and 60 mV in [Fig. 2B](#f2-14_147){ref-type="fig"}). As *Ve* is constant during the supply, the current pattern for each supplying voltage expresses simultaneously the change of conductance in time. Although it depends on combination of R, L, and C values, current patterns of the RLC circuit to step voltages simulate well the manner of Na^+^ conductance changes estimated from voltage clamp test ([Supplementary Fig. S1D](#s1-14_147){ref-type="supplementary-material"}). The change of Na^+^ conductance was reported to depend on the membrane potential; its peak becomes faster with the magnitude of depolarizing clamps, and the peak value saturates at high depolarizations. Simulating these features is possible in RLC circuit by changing R, L, and (or) C values with *Ve* magnitudes using any function. [Figure 3](#f3-14_147){ref-type="fig"} shows samples of the circuit currents in changing simply values of the inductance (A) and capacitance (B) in 45 mV step voltages, where we can observe shift of the peak and change of the time course.
About K^+^ channel activity
---------------------------
From the voltage clamp test, Na^+^ inward current is followed by K^+^ outward current that increases gradually towards the constant level ([Supplementary Fig. S1B](#s1-14_147){ref-type="supplementary-material"}). The capacitor in the RLC circuit is charged gradually toward the supplying voltages ([Fig. 2D](#f2-14_147){ref-type="fig"}). We notice that its time course is similar to the K^+^ current in voltage clamp test. Then an idea arises that the voltage produced in the capacitor of the RLC circuit model would link to the activation of K^+^ channel through any intermediating mechanism. In short, a Na^+^ channel for action potential generation can be represented by a serial RLC circuit, where the inductance element would be responsible for controlling the Na^+^ channel activity and the capacitance element would link remotely to the K^+^ channel activity.
Additional considerations in RLC circuit model
----------------------------------------------
Up to here the argument mainly has focused on the electric responses of RLC circuit as Na^+^ channel for action potential generation to step signals. The current property of serial RLC circuit to alternating signals is also known. Its essence is a change of circuit impedance with signal frequencies. It is established knowledge that a serial RLC circuit exhibits the lowest impedance at a certain frequency, in which the circuit current becomes maximum. The frequency for the maximum current is called the resonance frequency (*f~0~*), value of which is calculated from the equation, $f_{0} = 1/(2\pi\sqrt{LC})$. The action potential once originated propagates along the nerve fiber, mechanism of which is explained as following: Currents formed around the action potential, so called the local currents, stimulate neighboring portion by flowing across the membrane from the inside to outside, generating a new action potential if the current makes the inside depolarize enough \[[@b18-14_147],[@b19-14_147]\]. From the view of the resonance, an additional idea that the preceding action potential stimulates neighboring Na^+^ channels with the resonation current might be possible. It doesn't contradict the local current theory as the neighboring Na^+^ channel simply stimulated stronger by the resonance current. Incidentally, the resonance frequency of the circuit in L=1500 μH and C=50 μF is 581Hz which seems not to be an inadequate value, considering that the electric event of almost action potentials completes within a few msec. Examining the optimum frequency for eliciting the action potential will add an evidence on this RLC circuit model.
In this article, electric responses of serial RLC circuits have been studied restricted to the condition exhibiting no oscillations. As mentioned, the circuit current oscillates with decreasing amplitude in condition, *R^2^/4L^2^*\<*1/LC*. There are many kinds of neuron in the nervous system, most of which originate, in a regular physiological state, a train of impulses in response to effective synaptic (or receptor) potentials. It may be a consideration that the RLC circuit as the Na^+^ channel at a place initiating the action potential, for instance at the axon hillock or the initial segment of the axon \[[@b20-14_147],[@b21-14_147]\], would satisfy the oscillating condition, and the impulse trains of nerves would be any reflection of the oscillation of the circuit. It is a consideration that the action potential could be elicited successively till the positive potential on the corresponding inductor in the oscillation becomes lower than the threshold level, where the frequency of the impulse train is decided by the L and C values. This seems to be another interesting subject for the RLC circuit model as a representation of the Na^+^ channel.
Conclusion
==========
The action potential event could be also explained from the view point that the Na^+^ channel is composed of resistance, inductance, and capacitance elements, where the inductance element would function in controlling the Na^+^ channel activity, and the capacitance element, the K^+^ channel activity remotely.
Supplementary Information
=========================
I would like to offer thanks to Dr. Yuji Masuda, professor of Oral Medicine at Matsumoto Dental University, for helpful discussions on the manuscript, and Dr. Takashi Mochizuki, chief of researchers at MST Private Institute, for advices regarding circuit analyses. I also thanks to David Carlson, professor of English at Matsumoto Dental University, for his kind support in the writing of this article.
**Conflict of interest**
The author declares no potential conflicts of interest.
**Author Contributions**
The author wrote all part of the manuscript.
{#f1-14_147}
{#f2-14_147}
{#f3-14_147}
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Phylogenomic datasets comprising hundreds to thousands of genome segments produced through high throughput sequencing technology have shown promise to resolve difficult phylogenetic problems (e.g., [@ref-15]; [@ref-14]; [@ref-18]; [@ref-21]; [@ref-32]). At the same time, novel and refined inference tools including implementations of the multispecies coalescent model to address incomplete lineage sorting (ILS) through Gene Trees-to-Species Tree (GT-ST) methods ([@ref-29]) continue to extend the power and complexity of phylogenetic research. Despite these advances in genomic-scale dataset production and phylogenetic inference, difficult areas of the tree of life remain unresolved ([@ref-8]; [@ref-44]; [@ref-45]). Relationships among early-branching euteleost lineages remain nebulous (e.g., [@ref-4]; [@ref-7]; [@ref-33]; [@ref-42]) and stand out as one of the most contentious regions of the fish tree of life. Although this question has been studied from morphological and molecular perspectives consensus has yet to emerge.
The name Euteleostei was first applied to a diverse group of fishes that includes all teleosts outside of the superorders Elopomorpha, Osteoglossomorpha and Clupeomorpha by phyletic analysis ([@ref-19]; [@ref-20]). [@ref-47] excluded esocoids from the Euteleostei based on cladistic analyses of morphological characters, while [@ref-27] included esocoids but excluded ostariophysans. Subsequent phylogenetic studies of mitochondrial (e.g., [@ref-38]; [@ref-31]) and nuclear DNA (e.g., [@ref-4]; [@ref-42]) supported a monophyletic Euteleostei including esocoids but excluding Ostariophysi and the Alepocephaliformes (previously classified in Argentiniformes nested in the Euteleostei).
Recent phylogenetic studies based on molecular evidence consistently support the monophyly of five major euteleost lineages ([@ref-4]; [@ref-7]; [@ref-33]; [@ref-42]): (1) a clade formed by Esociformes and Salmoniformes; (2) the Stomiatii *sensu* [@ref-4] consisting of Osmeriformes (excluding Galaxiiformes) and Stomiiformes; (3) the Argentiniformes (excluding the Alepocephaliformes); (4) the Galaxiiformes (excluding *Lepidogalaxias*); and (5) the Neoteleostei. In addition, these studies agree on placing the monotypic *Lepidogalaxias* as the sister group of all other euteleosts. Aside from the placement of *Lepidogalaxias,* there is little congruence among different studies regarding relationships among the five lineages (e.g., [@ref-4]; [@ref-7]; [@ref-33]; [@ref-42]). The early branching patterns of euteleosts are still in need of further study and represent a difficult problem for traditional morphological and molecular phylogenetics.
Here we apply the "new and general theory of molecular systematics" ([@ref-10]) to examine early-branching euteleost relationships using multi-locus datasets generated by targeted enrichment of conserved nuclear DNA sequences. Concatenated and GT-ST phylogenetic inference frameworks are used to assess the stability and strength of evidence for alternative arrangements in this poorly resolved section of the fish tree of life.
Material and Methods
====================
Taxon and character sampling
----------------------------
We targeted species representing five of the six major euteleost lineages as well as several non-euteleost outgroups ([Table S1](#supp-1){ref-type="supplementary-material"}). We prepared genomic DNA libraries with 500--600 bp inserts by shearing total genomic DNA extracts to size using a sonicator (Diagenode, Inc) and ligating a set of custom-indexed Illumina Tru-Seq compatible adapters ([@ref-13]) to the sheared DNA using reagents from a library preparation kit (KapaBiosystems, Inc.). Adapter-ligated DNA was amplified with 16--18 cycles of PCR. To obtain sequences from homologous loci across the taxonomic sample, we performed targeted enrichment of ultraconserved element (UCEs) loci shared among acanthopterygians following protocols outlined in [@ref-15]. We modified the capture protocol by pooling eight, indexed sequencing libraries at equimolar ratios prior to enrichment and performing 12--16 cycles of PCR-recovery after enrichment. Following the enrichment procedure, we quantified enriched, amplified libraries using a commercial qPCR quantification kit (KapaBiosystems, Inc.), and we prepared an equimolar pool of pooled libraries for sequencing on an Illumina HiSeq 2500 instrument using 100 base pair, paired-end sequencing chemistry in rapid run mode (UCLA Neuroscience Genomics Core). To extend our taxon sampling, we included previously published UCE data ([@ref-15]) in our analyses ([Table S1](#supp-1){ref-type="supplementary-material"}).
Raw sequence data processing
----------------------------
Demultiplexed reads were edited for length, overall quality and adapter contamination using Trimmomatic v. 0.32 ([@ref-5]). We assembled a subset of cleaned reads across various kmers with Velvet v. 1.2.10 ([@ref-59]) to establish a range of suitable kmers for assembly. We then assembled sequences for each species using two different approaches. For non-salmonids, we assembled reads using VelvetOptimiser v. 2.2.5 across the optimal range of kmers we identified (57 to 83). For salmonids, assemblies from Velvet were produced for each value between 57 and 83. However, as the optimization performed by VelvetOptimiser is designed for haploid or diploid organisms, an alternative selection criterion of the maximum number of single copy UCE loci was chosen to accommodate the effect of ancestral polyploidy in salmonid genomes ([@ref-3]). A single dataset assembly was retained downstream analyses from each alternative approach to data assembly. We identified homologous UCE loci and prepared sequences for alignment with the PHYLUCE pipeline ([@ref-12]). During orthology assessment, the PHYLUCE package screens for and removes from analysis reciprocally duplicate enriched loci, which may represent paralogs.
Alignment and phylogenetic analysis
-----------------------------------
Following orthology assessment, the taxon set consisted of thirty-four Operational Taxonomic Units (OTUs) representing outgroups and basal euteleost lineages. We ensured this taxon set included loci sequenced in at least 31 of the 34 OTUs. We aligned data from all loci in with MAFFT v. 7.130b ([@ref-28]) through the PHYLUCE pipeline ([@ref-12]).
We analyzed the 34-OTU dataset under the Maximum-Likelihood (ML) framework as implemented in RAxML v. 8.1.24 ([@ref-55]). Each UCE locus was modeled as a partition evolving under the general time reversible (GTR) model of sequence evolution with gamma distributed rate variation (Γ). We set ML pseudoreplicate searches to automatically stop when stable bootstrap indices were detected (autoMRE). A joint gene trees and species tree estimation was conducted in a Bayesian framework with \*BEAST ([@ref-22]) as implemented in BEAST v. 2.1.3 ([@ref-9]). We analyzed data using a constant coalescent model under a Hasegawa-Kishino-Yano (HKY) model of sequence evolution with a four-category gamma distributed rate variation (Γ) and empirical base frequencies to each locus. Convergence and sufficient effective sample sizes (ESSs, \>200) of all parameters were reached by combining three chains of 800 million generations with 40% burn-in. Two additional analyses were conducted. To verify that partitioning in the ML analysis by gene does not influence early-branching euteleost relationships and support values, objective partitioning was investigated. To verify that the use of the coalescent model in \*BEAST resulted in an alternative arrangement of early-branching euteleost lineages, not the choice of nucleotide evolution model, a concatenated Bayesian analysis with BEAST 2 with the same nucleotide evolution model for each UCE locus as \*BEAST (HKY + Γ with empirical base frequencies) was undertaken ([Document S1](#supp-2){ref-type="supplementary-material"}).
Topology tests and occurrence of particular arrangements in the Bayesian tree posterior sample
----------------------------------------------------------------------------------------------
To determine the significance of UCE evidence corroborating or refuting alternative phylogenetic arrangements, we tested the following topologies resulting from concatenated and GT-ST analysis against each other: (1) the best-scoring ML topology; (2) the consensus species-tree topology from \*BEAST; and (3) a Protacanthopterygii *sensu* [@ref-4] as the sister lineage to the Stomiatii. A best scoring ML tree (1 from above) and constrained trees (2 and 3 from above) were generated with RAxML v. 8.2.3 partitioned by UCE using a GTR + Γ model of nucleotide evolution. We tested the trees against each other by generating per site likelihoods with RAxML and analyzing the output with CONSEL v. 0.20 ([@ref-53]). CONSEL implements several hypothesis tests allowing a more rigorous comparison between alternative hypotheses than solely comparing likelihood values.
As the \*BEAST posterior tree presented as the consensus species-tree topology represents the combination of many different species trees, we searched the combined post burn-in posterior tree sample from the separate \*BEAST chains (180,003 trees) for alternative phylogenetic hypotheses to determine if the \*BEAST algorithm considered these alternatives. The \*BEAST posterior tree sample was searched for the best scoring ML topology and a monophyletic Protacanthopterygii *sensu* [@ref-4] with Python scripts ([@ref-41]).
Summary coalescent analyses
---------------------------
To further examine the potential impacts of small regions in concatenated alignments on key relationships differing between RAxML concatenated analyses and the \*BEAST species tree, we examined our data through summary coalescent analyses. These methods function on independently estimated gene trees, therefore decreasing the influence of small regions in concatenated analyses ([@ref-52]) and accounting for ILS. Furthermore, \*BEAST and BEAST assume a molecular clock that may contribute to discrepancies between these analyses and RAxML analyses. For each of the UCE loci, we generated a gene tree with nucleotide evolution modeled with the GTR nucleotide evolution model and gamma distributed rate variation (Γ) with RAxML version 8.0.19. A set of rooted gene trees were also generated by specifying *Polypterus senegalus* as the outgroup. Four summary coalescent analysis frameworks were applied to the gene trees. The Accurate Species TRee ALgorithm (ASTRAL) and Neighbor Joining species tree (NJ*st*) tree methods accept unrooted trees with missing taxa. The unrooted gene trees were analyzed program ASTRAL version 4.10.12 ([@ref-39]; [@ref-40]) and with NJ*st* ([@ref-35]) via the Species TRee Analysis Web server (STRAW) ([@ref-51]). The rooted trees were analyzed with both STAR ([@ref-37]) and MP-EST ([@ref-36]) through STRAW.
Results
=======
Characteristics of UCE dataset
------------------------------
Following orthology assessment and filtering for loci not present in 31 of 34 OTUs, the dataset is composed of a total of 53 UCE loci, 17,957 characters, 9,576 distinct alignment patterns and 22.11% gaps or missing data. We present details of the number of UCE loci recovered for each taxon, the average length of UCE matching contigs, average coverage of contigs matching UCEs and number of duplicate loci removed in [Table S1](#supp-1){ref-type="supplementary-material"}. The assemblies and alignment are available within the [Data S1](#supp-3){ref-type="supplementary-material"}.
Early-branching euteleost relationships
---------------------------------------
Concatenated ML analysis supports a monophyletic Euteleostei, excluding Ostariophysi and Alepocephaliformes (Bootstrap Support \[bs\] = 100%). [Figure 1](#fig-1){ref-type="fig"} shows the inferred branching pattern among main euteleost groups from the 34-OTU dataset. Relationships among main euteleost lineages in the concatenated ML topology are (*Lepidogalaxias*, ((Esociformes, Salmoniformes), (Argentiniformes, (Stomiatii, Neoteleostei)))) with all nodes among those lineages receiving strong support (bs = 100%).
{ref-type="fig"}) are indicated in blue.](peerj-05-3548-g001){#fig-1}
GT-ST analysis of the dataset in \*BEAST indicates a monophyletic Euteleostei with high support, posterior probability (*pp*) = 1.00 ([Fig. 2](#fig-2){ref-type="fig"}). A topology of (*Lepidogalaxias,* ((Esociformes, Salmoniformes), ((Argentiniformes, Stomiatii), Neoteleostei))))) is generated in this analysis. Support values for the placement of main euteleost lineages are high throughout the consensus tree. The placement of *Lepidogalaxias* and the Esociformes + Salmoniformes receive very high support (*pp* = 1.00). Argentiniformes + Stomiatii as the sister lineage of the neoteolosts received strong support (*pp* = 0.99). A sister relationship between the Argentiniformes and Stomiatii was also well supported ( *pp* = 0.96). The GT-ST and ML inferred phylogenies differ on the relationships among argentiniforms, stomiatians and neoteleosts.
{ref-type="fig"}) are indicated in blue.](peerj-05-3548-g002){#fig-2}
Through the additional concatenated analyses presented in the [Document S1](#supp-2){ref-type="supplementary-material"}, conflicts between ML and GT-ST results presented in [Figs. 1](#fig-1){ref-type="fig"} and [2](#fig-2){ref-type="fig"} are shown to be the product of the distinct analytical frameworks and do not result from how data were modeled. The additional concatenated analyses in [Document S1](#supp-2){ref-type="supplementary-material"} show identical branching patterns for main early-branching euteleost lineages to the concatenated ML analysis presented in [Fig. 1](#fig-1){ref-type="fig"} with high support values. Retaining the same model but changing the partitioning strategy with RAxML demonstrates that the inferred phylogeny from the ML analysis presented in [Fig. 1](#fig-1){ref-type="fig"} is not sensitive to partitioning ([Fig. S1](#supp-3){ref-type="supplementary-material"}). Not implementing a \*BEAST model, while retaining the same nucleotide evolution and partitioning scheme for a concatenated analysis with BEAST 2 also produces a phylogeny ([Fig. S2](#supp-3){ref-type="supplementary-material"}) with the branching of main early-branching euteleost lineages matching that of the concatenated ML analysis presented in [Fig. 1](#fig-1){ref-type="fig"}, not the \*BEAST GT-ST analysis presented in [Fig. 2](#fig-2){ref-type="fig"}. Consequently, the topological differences between phylogenies shown in [Fig. S2](#supp-3){ref-type="supplementary-material"} and [Fig. 2](#fig-2){ref-type="fig"} may be attributed to whether a concatenated or coalescent approach is implemented. There are three key locations in the inferred trees that differ: (1) within the Otomorpha, the placement of *Bajacalifornia megalops*, (2) the arrangement of early-branching euteleost lineages, and (3) the arrangement of the three percomorph lineages.
Topology tests and occurrence of particular arrangements in the Bayesian tree posterior sample
----------------------------------------------------------------------------------------------
Testing with CONSEL indicates the best-scoring ML tree, with a topology of (*Lepidogalaxias,* ((Esociformes, Salmoniformes), (Argentiniformes, (Stomiatii, Neoteleostei)))), is significantly better than the topology generated by GT-ST analysis with both the approximately unbiased test (*p* = 1 × 10^−5^) and the weighted Shimodaira-Hasegawa test (*p* = 1 × 10^−3^). A monophyletic assemblage of protacanthopterygian taxa *sensu* [@ref-4] sister to the Stomiatii is significantly worse than the best-scoring ML tree with both the approximately unbiased test (*p* = 8 × 10^−6^) and the weighted Shimodaira-Hasegawa test (*p* = 1 × 10^−4^). The posterior set of 180,003 trees generated by \*BEAST did not include a single occurrence of either the ML best tree topology or a monophyletic Protacanthopterygii *sensu* [@ref-4].
Summary coalescent analyses
---------------------------
Fifty-three unrooted gene trees were analyzed by ASTRAL and NJ*st*, the requirement of rooting by *Polypterus senegalus* reduced the number of available gene trees that are rooted to forty-four for STAR and MP-EST. The summary coalescent results vary with regards to the three key differences identified previously as presented in [Figs. 1](#fig-1){ref-type="fig"} and [2](#fig-2){ref-type="fig"} ([Table 1](#table-1){ref-type="table"}). ASTRAL ([Fig. 3](#fig-3){ref-type="fig"}) and NJ*st* resolves the otomorphs as found by RAxML concatenation analyses, the early-branching euteleost relationships as in RAxML and BEAST concatenation, while the percomorph taxa are found in an arrangement from \*BEAST and BEAST. ASTRAL posterior probability values for the placement of the Argentiniformes and Stomiatii + Neoteleosteii are low (0.58 and 0.56 respectively). STAR resolves the otomorphs and percomorphs as BEAST and \*BEAST analyses, but early-branching euteleosts match the concatenated RAxML results. MP-EST matches the concatenated RAxML results for otomorphs and BEAST / \*BEAST for the percomorphs while producing a unique topology of early-branching euteleost lineages.
10.7717/peerj.3548/table-1
###### Comparison of results from all phylogenetic analyses presented in this study of topological differences within the Otomorpha, the early-branching euteleost lineages and the Percomorpha. Concatenation or Gene Trees-to-Species Tree (GT-ST) framework and analysis program are indicated in the Method column.
If a tree is presented, it is indicated in the Figure column. The three key topological differences determined between concatenation in RAxML presented in [Fig. 1](#fig-1){ref-type="fig"} and \*BEAST presented in [Fig. 2](#fig-2){ref-type="fig"} are indicated and color-coded to analysis method. Results consistent with the RAxML results are blue and those consistent with the \*BEAST analysis are orange. A unique topology is not color-coded for early-branching euteleosts present only in the results from MP-EST.
Method Figure Otomorpha Early-Branching Euteleosts Percomorpha
--------------------- ------------------------------------------------------- ----------------------------------------------- -------------------------------------------------------------------------------------------------- ------------------------------------------
Concatenation RAxML [Fig. 1](#fig-1){ref-type="fig"} (Bajacalifornia, (Anchoa, (Astyanax, Danio))) (Lepidogalaxias, (Esociformes + Salmoniformes, (Argentiniformes, (Stomiatii, Neoteleosteii)))) (Taenianotus, (Acanthurus, Antennarius))
GT-ST \*BEAST [Fig. 2](#fig-2){ref-type="fig"} ((Anchoa, Bajacalifornia), (Astyanax, Danio)) (Lepidogalaxias, (Esociformes + Salmoniformes, ((Argentiniformes, Stomiatii), (Neoteleosteii)))) (Antennarius, (Acanthurus, Taenianotus))
Concatenation RAxML [Fig. S1](#supp-3){ref-type="supplementary-material"} (Bajacalifornia, (Anchoa, (Astyanax, Danio))) (Lepidogalaxias, (Esociformes + Salmoniformes, (Argentiniformes, (Stomiatii, Neoteleosteii)))) (Taenianotus, (Acanthurus, Antennarius))
Concatenation BEAST [Fig. S2](#supp-3){ref-type="supplementary-material"} ((Anchoa, Bajacalifornia), (Astyanax, Danio)) (Lepidogalaxias, (Esociformes + Salmoniformes, (Argentiniformes, (Stomiatii, Neoteleosteii)))) (Antennarius, (Acanthurus, Taenianotus))
GT-ST ASTRAL [Fig. 3](#fig-3){ref-type="fig"} (Bajacalifornia, (Anchoa, (Astyanax, Danio))) (Lepidogalaxias, (Esociformes + Salmoniformes, (Argentiniformes, (Stomiatii, Neoteleosteii)))) (Antennarius, (Acanthurus, Taenianotus))
GT-ST NJ*st* (Bajacalifornia, (Anchoa, (Astyanax, Danio))) (Lepidogalaxias, (Esociformes + Salmoniformes, (Argentiniformes, (Stomiatii, Neoteleosteii)))) (Antennarius, (Acanthurus, Taenianotus))
GT-ST STAR ((Anchoa, Bajacalifornia), (Astyanax, Danio)) (Lepidogalaxias, (Esociformes + Salmoniformes, (Argentiniformes, (Stomiatii, Neoteleosteii)))) (Antennarius, (Acanthurus, Taenianotus))
GT-ST MP-EST (Bajacalifornia, (Anchoa, (Astyanax, Danio))) (Lepidogalaxias, (Argentiniformes, (Esociformes + Salmoniformes, (Stomiatii, Neoteleostei)))) (Antennarius, (Acanthurus, Taenianotus))
{ref-type="fig"} and [2](#fig-2){ref-type="fig"} are indicated in blue.](peerj-05-3548-g003){#fig-3}
Discussion
==========
Hypotheses of early-branching euteleost relationships
-----------------------------------------------------
Our phylogenomic analysis provides strong support for relationships of early diverging euteleosts that consist of *Lepidogalaxias* and esociforms + salmoniforms as successive sister lineages to a clade containing argentiniforms, stomiatiids and neoteleosts. Despite the most intensive character sampling of this group to date, our analyses do not resolve two conflicting hypotheses for relationships among the Argentiniformes, Stomiatii and Neoteleostei. The concatenated ML derived topology resolves argentiniforms and stomatiids as successive sister lineages to the neoteleosts as do most summary coalescent methods, while the \*BEAST GT-ST analysis recovers an argentiniform + stomiatiids clade as the sister group to neoteleosts.
Combined, our analyses yield strong support for the Esociformes + Salmoniformes clade, which has found robust and consistent support in molecular phylogenetic studies ([@ref-38]), reviewed by [@ref-7], despite weak or conflicting evidence from morphology ([@ref-27]; [@ref-58]). We also recover the Stomiatii (Osmeriformes + Stomiiformes) with high support values in both analyses in this study. On the other hand, we do not find a close relationship between the clade of Esociformes + Salmoniformes and any other major group of early-branching euteleosts such as Argentiniformes ([@ref-42]). Instead, as shown in mitogenomic phylogenies ([@ref-7]; [@ref-25]) or analyses of combined mitochondrial and nuclear data ([@ref-6]), we find Esociformes and Salmoniformes as sister to all other euteleosts in the study, with the exclusion of *Lepidogalaxias.*
Support for hypotheses of early-branching euteleost lineages
------------------------------------------------------------
Unlike other molecular (and morphological) studies of the euteleost phylogeny (e.g., [@ref-4]; [@ref-33]; [@ref-42]), our conflicting topologies are strongly supported by both bootstrap values and Bayesian posterior probabilities.
Earlier studies typically yield low or moderate support for relationships along this section of the teleost phylogeny backbone. For example, the placement of the Argentiniformes and Salmoniformes + Esociformes sister to the remaining three major euteleost lineages (Stomiati, Galaxiiformes, and neoteleosts) receives a bootstrap support value between 70--89% in [@ref-42]. Other nodes supporting the branching order of the five major euteleost lineages are supported by 90--99% bootstrap support values. The bootstrap support for a sister Protacanthopterygii *sensu* [@ref-4] and Stomiatii is 36%, and the monophyly of Stomiatii receives a bootstrap support of 73% in the hypothesis presented by [@ref-4]. In a mitochondrial genome based study, a sister relationship of Argentiniformes to the Salmoniformes + Esociformes receives a bootstrap support of 74% ([@ref-33]). In the same study, the Argentiniformes, Salmoniformes and Esociformes are the sister lineage of the Stomiatii, supported by an 81% bootstrap support value ([@ref-33]).
While we find uncharacteristically high support for branching relationships among all of the four major euteleost lineages represented in this study in a concatenated ML framework, gauging the significance of high bootstrap values in analyses of large data matrices is problematic. Bootstrap values may be high even with conflict or systematic error ([@ref-16]; [@ref-23]; [@ref-24]). Concatenated ML phylogenomic analysis has previously been demonstrated with 1,070 genes in yeasts to produce 100% bootstrap support for all internodes, despite incorrect branching likely present ([@ref-50]). The \*BEAST GT-ST analysis also produces high support values; however, posterior probability values themselves are both conditioned on the model of evolution and are not guaranteed to have good frequentist statistical behavior ([@ref-2]; [@ref-1]) and may be misleading under certain conditions ([@ref-56]; [@ref-50]). Another potential issue in phylogenetics is that a few loci may substantially influence results in concatenated analyses ([@ref-52]). To mitigate the influence of small regions of large effect in a concatenated analysis, we evaluated individual gene trees with four summary coalescent analyses. Three of the four summary coalescent analyses were in agreement with the RAxML concatenated analyses and the concatenated BEAST analysis hypothesis of early-branching euteleost lineages. The MP-EST analysis produced a unique result regarding early-branching euteleost lineages among all analyses. While the otomorph taxa are not consistently found to have a certain topology in the summary coalescent methods, three of the four analyses match the concatenated RAxML results. The summary coalescent methods indicate that the early-branching euteleost topology of \*BEAST may be a result of the underlying assumption of a molecular clock in this analysis or other issue. The consistent results for percomorph relationships across GT-ST methods suggests that a particular locus or subset of loci and/or incomplete lineage sorting is affecting the concatenated analysis in RAxML.
Hypothesis testing and alternative topologies in the Bayesian posterior tree sample
-----------------------------------------------------------------------------------
In a hypothesis testing framework, the optimal topology from the \*BEAST GT-ST framework is a significantly worse fit compared to the concatenated ML best tree. Conversely the concatenated ML best tree topology is absent from the 180,003 posterior trees produced in the \*BEAST GT-ST analysis. Combined, these demonstrate that strong conflicting signal underlies these topological differences. Recent studies have alternatively suggested that concatenation may perform better than GT-ST when individual loci are not long enough to resolve phylogenies ([@ref-17]), that concatenation and GT-ST methods should behave similarly under a range of conditions ([@ref-57]), and that phylogenomic scale data sets may exacerbate problems of model misspecification ([@ref-34]). For additional discussion around these issues see also [@ref-11] and [@ref-54]. At present, the relationships of the argentiniforms and stomatiids to neoteleosts remain unclear and may depend strongly on the inclusion of the Galaxiidae. The placement of galaxiids has been unstable ([@ref-4]; [@ref-6]; [@ref-7]; [@ref-26]; [@ref-33]; [@ref-38]; [@ref-42]), although independent studies (e.g., [@ref-7]; [@ref-42]) suggest that galaxiids may be the sister lineage of the Neoteleostei.
Lack of evidence for the monophyly of protacanthopterygians
-----------------------------------------------------------
The Protacanthopterygii is an historically important taxon of early-branching euteleosts with its definition and content repeatedly modified (e.g., [@ref-20]; [@ref-27]; [@ref-30]; [@ref-46]; [@ref-48]; [@ref-49]). Protacanthopterygian monophyly as defined by morphology (e.g., [@ref-27]) was questioned by molecular phylogenetics ([@ref-26]). More recently, the Protacanthopterygii was redefined by [@ref-4] with molecular phylogenetics (bs of 37%) containing the Argentiniformes, Galaxiiformes, Esociformes and Salmoniformes. Although we were unable to obtain representatives of Galaxiiformes, our analyses demonstrate that the Argentiniformes are not most closely related to the Esociformes + Salmoniformes. A topology test using available taxa in this dataset further rejected the Protacanthopterygii *sensu* [@ref-4].
Conclusions
===========
The two approaches (concatenation and GT-ST) implemented in this study indicate large areas of congruence in topology resolving several relationships within early-branching euteleost relationships. However, the disagreements highlight some of the potential caveats in resolving all relationships of the early-branching euteleosts. We report the first study using a joint GT-ST method to examine the question of early-branching euteleost relationships. A joint estimation of species tree and gene trees can be preferred over summary methods ([@ref-17]) and produced a slightly different hypothesis of relationships when compared to concatenated analyses. A test of topology rejects the species-tree topology over the best scoring concatenated ML topology. Likewise, posterior support for the \*BEAST Bayesian species tree hypothesis is high for early-branching euteleost nodes, indicating very few occurrences of alternative topologies in the tree search. For major euteleost lineages, relationships among Argentiniformes, Neoteleostei and Stomiatii differed in the results of concatenated ML and Bayesian joint GT-ST analyses. This is in line with previous research on early-branching euteleost relationships. The lack of agreement between studies of early-branching euteleost relationships may be caused by short internode distances deep in the evolutionary past, leading to the formation and preservation of few informative characters linking these old lineages. A related but less likely possibility is that short internodes associated with very rapid diversification created conditions conducive to pervasive ILS at the base of the euteleost radiation resulting in conflicting histories across euteleost genomes and incongruent results between studies of early-branching euteleost relationships.
We evaluated identical datasets under concatenated and GT-ST frameworks and found three areas of incongruence: (1) argentiniform sister lineage, (2) the placement of the alepocephaliform lineage *Bajacalifornia*, and (3) the arrangement of the three neoteleost lineages *Antennarius*, *Acanthurus* and *Taenianotus*. The percomorph taxa (*Antennarius*, *Acanthurus* and *Taenianotus*) belong in a set of fish lineages whose relationship have been particularly difficult to elucidate ([@ref-43]). The incongruent inferences we observed between approaches may be differential effects of ILS on coalescent versus non-coalescent phylogenetic approaches.
In terms of main early-branching euteleost lineages, only the placement of Argentiniformes between concatenated and GT-ST hypotheses varied. The placement of the argentiniform fishes is unresolved by this study and the branching of the Neotelostei, Stomiatii and Argentiniformes may be considered a soft polytomy. We find that phylogenomics and the application of the coalescent model in phylogenetics strengthen support for the earliest splits in the euteleostean radiation. However, key aspects of early euteleost phylogeny remain unresolved and leave open the question of whether extant genomes from these lineages retain historical signal that can be retrieved above the noise accumulated over hundreds of millions of years of independent evolution.
Supplemental Information
========================
10.7717/peerj.3548/supp-1
###### Sampling scheme with classification of taxa included in this study following [@ref-4]
######
Click here for additional data file.
10.7717/peerj.3548/supp-2
###### Methods and results of additional phylogenetic analyses
######
Click here for additional data file.
10.7717/peerj.3548/supp-3
###### Assemblies of sequence data generated for this study, the alignment examined for this study, and PartitionFinder configuration and partition files
######
Click here for additional data file.
We would like to acknowledge the following institutions and individuals that contributed to sampling in this project: Burke Museum of Natural History and Culture, Kansas University Biodiversity Institute and Natural History Museum, Academy of Natural Sciences of Philadelphia, Robert Marcotte (Univeristy of Alaska Fairbanks), Molly Hallock (Washington Department of Fish and Wildlife, retired), Motohiro Kikuchi (Chitose Salmon Park) and Peter Unmack (University of Canberra). Sébastien Lavoué (National Taiwan University), Thaddaeus Buser (Oregon State University) and Kerry Reid (University of California Santa Cruz) provided very helpful comments on drafts of the manuscript. We would like to thank Kevin McCracken (University of Miami) for his support in promoting Next-Generation Sequencing and Bioinformatics at the University of Alaska Fairbanks.
Additional Information and Declarations
=======================================
The authors declare there are no competing interests.
[Matthew A. Campbell](#author-1){ref-type="contrib"} conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
[Michael E. Alfaro](#author-2){ref-type="contrib"} and [J. Andrés López](#author-4){ref-type="contrib"} conceived and designed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
[Max Belasco](#author-3){ref-type="contrib"} conceived and designed the experiments, performed the experiments, reviewed drafts of the paper.
The following information was supplied regarding data availability:
Assembled sequences are included in the [Data S1](#supp-3){ref-type="supplementary-material"}. Data available from the Dryad Digital Repository: [10.5061/dryad.7p511](http://dx.doi.org/10.5061/dryad.7p511).
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#S1}
============
Gliomas and Glioblastoma Multiforme (GM) {#S1-1}
----------------------------------------
Gliomas (glial tumors) are the most common malignant brain tumors and account for about 60% of all primary central nervous system (CNS) cancers ([@B1]). Around 23,880 new cases of primary CNS tumors are expected to be diagnosed in the United States in 2018 ([@B2]). Although rare---accounting for approximately 1.4% of all cancers ([@B3])---they generally have a poor prognosis that leads to a disproportionately high morbidity (patients often exhibit compromised basic and critical functions such as movement and speech) and high mortality (CNS tumors are 10th leading cause of death in the USA) ([@B1]). The 5-year survival rate for primary malignant brain and CNS tumors is the sixth lowest among all types of cancers after pancreatic, liver and intrahepatic bile duct, lung, esophageal, and stomach, making gliomas some of the most devastating types of cancers ([@B2]). Gliomas originate from astrocytes, oligodendrocytes, and ependymal cells and are consequently classified as astrocytomas, oligodendrogliomas, or ependymomas, respectively ([@B4]). According to the World Health Organization (WHO) criteria, gliomas are histologically graded into four grades (grade I--IV). Tumor grading correlates well with tumor morphology, biology, and prognosis.
Glioblastoma multiforme, a fatal grade IV glioma, is the most common glial tumor (accounting for 50--60% of all gliomas) and has the worst prognosis with a median survival of 12--15 months and a 5-year survival rate of less than 5% in adults and 16% in children ([@B5]--[@B8]). The current standard-of-care includes surgical reduction of the tumor mass following craniotomy and then radiation and chemotherapy with temozolomide ([@B9]). GBM is morphologically characterized by increased cellularity, marked nuclear atypia, abundant mitotic activity of neoplastic cells followed by the neoangiogenesis, and/or tumor necrosis.
Recent advances in molecular profiling of brain tumors has led to better disease stratification by allowing a more clear distinction between the low-grade and high-grade gliomas (GBM) ([@B10]). As a result, the 2016 WHO classification of glial tumors has integrated the classical tumor morphology with genomic alterations derived from molecular profiling studies ([@B11], [@B12]). Most gliomas harbor molecular alterations disrupting key signaling pathways involved in regulation of cell growth (e.g., receptor tyrosine kinases, MAPK/ERK PIK3CA/AKT/PTEN signaling pathways), cell cycle/DNA repair/apoptosis (e.g., retinoblastoma/E2F/p53), metabolism \[e.g., isocitrate dehydrogenase (IDH1)\], chromatin, and telomere length ([@B13]). Among the most relevant genetic alterations affecting GBM are mutations of the *IDH* gene that may be linked to survival ([@B14], [@B15]). The enzyme catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate and reduces NAD(P)+ to NAD(P)H ([@B16]). IDH has two isoforms (IDH1 and IDH2) of which mutations in *IDH1* are the most common (Figure [1](#F1){ref-type="fig"}). *IDH* gene mutations are present in only 5% primary and approximately 80% of secondary GBMs ([@B14]).
{#F1}
Epidermal growth factor receptor (EGFR) is commonly overexpressed in GBM, most frequently due to *EGFR* gene amplification and/or the *EGFR variant III* deletion mutation (EGFRvIII). *EGFR* gene amplification is observed in approximately 50% of GBMs, whereas *EGFRvIII* (Figure [1](#F1){ref-type="fig"}), a constitutively active truncated form of the EGFR protein that lacks the extracellular domain, occurs in 20--30% of cases ([@B11], [@B17]--[@B19]). Indeed, targeted inhibition of EGFR or the tumor-specific EGFRvIII holds therapeutic promise and several clinical trials with specific tyrosine kinases as well as monoclonal antibodies are ongoing ([@B20], [@B21]).
O^6^-methylguanine-DNA methyltransferase (MGMT) is an enzyme that is involved in DNA repair. MGMT promoter methylation is commonly detected in GBMs (\~35--50%), particularly among the secondary GBMs ([@B10], [@B12]). Epigenetic silencing of the MGMT DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive alkylating agents including temozolomide (Figure [1](#F1){ref-type="fig"}) ([@B22]--[@B24]).
Telomerase reverse transcriptase (TERT) is an enzyme that is responsible for adding nucleotides to telomeres. Telomerases are usually inactive in the adult normal cells, but can be reactivated (e.g., by mutations) in various cancers to promote oncogenesis ([@B11]). TERT gene mutations in GBMs are activating (usually in the TERT promoter region) ([@B11]). TERT gene mutations are particularly common in primary GBMs (Figure [1](#F1){ref-type="fig"}) ([@B11], [@B14]). Several therapeutic strategies for the inhibition of telomerases have been attempted ([@B25]).
In addition, GBMs are frequently affected by the various copy number aberrations (CNA). These involve gains at chromosomes 7 (EGFR/MET/CDK6), 12 (CDK4 and MDM2), and 4 (PDGFRA), while deletions are commonly observed at chromosomes 9 (CDKN2A/B) and 10 (PTEN) ([@B13]). A subset of GBMs may also have genetic alterations of *PIK3CA, PIK3R1, NF1*, and *RB1* genes ([@B13], [@B19]).
Along with improved understanding of the role of cells in the tumor microenvironment (e.g., reactive astrocytes, activated macrophage, and glioma stem cells), micro RNAs, and long non-coding RNAs in glioma progression ([@B26]--[@B28]), the above genomic and molecular changes are thought to be of growing importance in the diagnosis, development, classification, and therapy of gliomas. However, what actually triggers these molecular changes and oncogenesis in brain tumors remains poorly understood.
Possible Viral Etiology of Gliomas and Scope of Review {#S1-2}
------------------------------------------------------
Although little is known about the etiology of GBM or other gliomas, increased risk has been observed following exposure to ionizing radiation ([@B8]) or chemical agents or through genetic predisposition (e.g., germline *TP53, NF1*, and *NF2* mutations) in a small proportion of the patients with GBM (e.g., Li--Fraumeni syndrome, neurofibromatosis type 1 and type 2) ([@B8]). More recently, increasing emphasis has been placed on a viral etiology of gliomas as they might serve as oncomodulators ([@B29], [@B30]). Oncomodula-tion refers to the ability of viral proteins and non-coding RNAs to promote oncogenic processes without direct oncotransformation, but through disturbances in various intracellular signaling pathways ([@B30]).
Viruses may contribute toward oncogenesis and tumor development in humans by inducing immunosuppression, modifying host cells through inducing oncoproteins, or altering the expression of host cell proteins at viral integration sites ([@B29], [@B31]). Pagano and colleagues have recently reviewed the most common cancer-causing viruses ([@B31]). Viruses such as human papillomavirus (HPV) and human cytomegalovirus (CMV)---also known as human herpes virus-5 are strongly linked to the etiology and progression of cervical and colorectal cancers, respectively ([@B32], [@B33]). Several viruses have been linked to the etiology of brain tumors including CMV and other herpes viruses, such human herpes virus 6 (HHV-6 or roseolovirus), John Cunningham Virus (JCV; a polyomavirus); adenoviruses and Simian virus 40 (SV40), and others ([@B30], [@B34]). However, in the case of brain tumors, there is contradictory and/or controversial evidence linking many of these viruses, especially CMV---a ubiquitous herpes virus ([@B32], [@B35]). Because of its affinity for glial cells and its ability to reduce apoptosis, increase cell invasion, activate telomerase, and enhance angiogenesis in tumor cells ([@B36], [@B37]), several studies have investigated the role of CMV in glioma etiology. The first-ever study by Cobbs et al. in 2002 reported that CMV gene products and nucleic acids were present in all 27 glioma samples investigated, without being detected in other brain tissue ([@B38]). Despite confirmatory reports from other research groups ([@B39], [@B40]), recent conflicting reports showing no association of CMV in brain tissues ([@B35], [@B41]) have cast doubt on the role of CMV in brain tumors.
While the majority of the literature concerning viruses in glioblastoma thus far had focused on CMV, more recently attention has shifted to another potential oncovirus, EBV, and its role in the etiology of gliomas (Figure [1](#F1){ref-type="fig"}). In this review article, we will focus on providing a comprehensive review of the literature pertaining to EBV in gliomas and discuss the possibilities of this virus being causative, simply associative, or even an experimental artifact has been suggested by some recent highly sensitive "*state of the art*" next-generation sequencing-based virome detection assays.
EBV and Tumorigenesis {#S1-3}
---------------------
Epstein--Barr virus, named after Michael Anthony Epstein and Yvonne Barr is also known as HHV-4, and was the first recognized human oncovirus ([@B42]). It belongs to the group of gamma-herpes viruses and is present in more than 90% of the human adult population who largely remain asymptomatic ([@B43]) with the main mode of transmission being *via* salivary and genital fluids ([@B44]). EBV, along with other herpes virus family members, is responsible for infections widely spread in the general population. Exposure mostly occurs in childhood or young adulthood followed by lifelong persistence of the virus. Thus, EBV has two distinct life cycles in humans: an acute lytic cycle, during which the production of new virions occurs; and a latent form, in which the EBV remains "hidden" in the host. Although, EBV typically remains in memory B-cells, in a latent phase, it may also be detected in epithelial cells (oropharynx) as well as in certain subsets of T and NK cells ([@B44]).
Epstein--Barr virus is a DNA virus whose genome is approximately 172 kb in length ([@B44]). Binding of its surface protein gp350 with CD21 receptor \[also known as complement receptor 2 (CR2)\] followed by viral glycoprotein gp42 interaction with cellular MHC class II molecules represents the major cellular fusion and entry mechanism into B-cells, whereas entry into epithelial cells is facilitated by viral protein BMRF-2 binding to cellular β1 integrins ([@B44], [@B45]). Subsequent to primary infection and replication within the lytic cycle, most of EBV genes are turned off as the virus switches to the latent phase ([@B29]).
During latency, EBV genome circular DNA resides in the cell nucleus as an episome and is copied by cellular DNA polymerase. In latency, only a portion of EBV's genes including the six EBV nuclear oncoproteins (EBNA1, -2, -3A, -3B, -3C, and -LP) and the three latent membrane proteins (LMP1, -2A, and -2B), as well as several non-coding RNAs (EBERs and miRNAs) ([@B46]--[@B49]) are expressed in one of three patterns, known as latency programs (namely latency I, latency II, and latency III). Each latency program, therefore, leads to the production of a limited, distinct set of viral proteins, and viral RNAs. As mentioned EBV can latently persist within B cells and epithelial cells, but different latency programs are possible in the two types of cell ([@B50], [@B51]). In cases of EBV-associated cancers, there is differential expression of viral latency genes. However, emerging evidence suggests that of these, LMP1 is a major EBV-oncoprotein, as it provokes a multitude of effects enhancing cell growth, protecting cells from apoptosis, promoting cell motility and angiogenesis, and it is frequently expressed in EBV-linked human oral carcinomas ([@B52]--[@B54]).
Severe infections with EBV can cause infectious mononucleosis (glandular fever), and its latent state can revert (i.e., reactivate virus) to yield multiple lymphoid and epithelial malignancies, including B-cell lymphomas (Burkitt's lymphoma, Hodgkin's lymphoma (HL), post-transplant lymphoproliferative disorder), various T-cell/NK lymphoproliferative disorders, undifferentiated nasopharyngeal, and gastric carcinomas ([@B55]--[@B57]). Recent investigations including three from the Middle East, suggest that EBV is also present in around 40% of human breast malignancy where its occurrence is linked with more aggressive pheno-types ([@B58]--[@B64]).
Epstein--Barr virus can induce several molecular signaling changes in tumors such as those described in HL and undifferentiated nasopharyngeal carcinoma. Approximately 50% of HLs are associated with EBV infection, particularly its lymphocyte-depleted and mixed-cellularity variants. Reed--Sternberg (RS) giant cells represent characteristic B lymphocyte transformed neoplastic cells in HL, which are infected by EBV. Activation and survival of these cells are largely dependent on NF-ĸB upregulation through the intimate interaction of CD40 receptor and LMP1 oncoprotein of EBV ([@B65]). In addition, several signaling pathways may also be upregulated by this interaction, including MAPK/ERK, PIK3CA/AKT, JAK/STAT, and Notch pathways ([@B44]). EBNA-1 is another important EBV product that is required for the replication and maintenance of EBV genome in cancer cells ([@B44]). Thus, in case of undifferentiated nasopharyngeal carcinoma, LMP1, LMP2, and EBNA1 products of EBV are actively involved in promotion of cell growth and anti-apoptotic effects in neoplastic cells ([@B66]), while LMPA2A is responsible for preventing the differentiation of the epithelial cells ([@B46]). All these EBV products are also involved in other processes (e.g., immune evasion, metastasis) that contribute a highly aggressive phenotype and poor clinical outcome of undifferentiated nasopharyngeal carcinomas ([@B66]). Of note, EBV presence has been well documented in several other cancers, including breast, prostate, oral, and salivary gland carcinomas ([@B67]--[@B70]).
EBV and Gliomas {#S2}
===============
Although the role of EBV in B-cell lymphomas and nasopharyngeal carcinomas is well-defined, its role in gliomas is only recently being explored. EBV, whose main latent reservoir is thought to be B-cells in the bone marrow, is also known to be present in the brain. Although rare, EBV infections can be found in the CNS especially in immunocompromised patients as exemplified by a case of EBV-induced encephalitis ([@B71]). Further, EBV is causally associated with a number of other CNS disorders \[infectious mononucleosis, acute encephalitis, acute cerebellar ataxia, demyelinating disease, myelitis or meningitis, and some CNS neuropathies ([@B72])\]. The major cellular receptor for EBV, compliment receptor 2 (CR2) appears to be present on astrocytes ([@B73]) facilitates entry to infect astrocyte cell lines ([@B74]), and leads to increased proliferation. Importantly, primary CNS lymphomas (e.g., diffuse large B-cell lymphomas and lymphoid granulomatosis) are frequently EBV-positive ([@B75]). Thus, the fact that EBV is also thought to be the causative agent for primary CNS lymphomas in immune-deficient patients has led to its investigations in other brain tumors including gliomas.
Literature Survey of EBV in Gliomas {#S2-1}
-----------------------------------
In this section, we provide a detailed review of the key studies on EBV in gliomas (see Table [1](#T1){ref-type="table"}). We searched the PubMed/MEDLINE databases using the following key words, such as glioma(s), glioblastoma multiforme, brain tumors/cancers, EBV, and neurotropic viruses. Our literature search was not time limited.
######
Selected examples of studies investigating Epstein--Barr virus (EBV) in gliomas.
Reference Glioma type Sample size/tissue sampled Methodology Main findings
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Strojnik et al. ([@B34]) High-grade 45 adult patients, tumor biopsy *ebna* RT-polymerase chain reaction (PCR) 3/45 (6.7%) positive
Wrensch et al. ([@B76]) High-grade 57 adult patients, serum analysis Enzyme-linked immunosorbent assay (ELISA) for IgG in sera 86% positive
Poltermann et al. ([@B77]) High-grade 35 patients, serum analysis ELISA for IgG in sera 90% positive
Zavala-Vega et al. ([@B78]) High-grade 21 patients, tissue biopsy Detected latent membrane proteins (LMP-1) by immunohistochemistry and EBER expression by *in situ* hybridization, RT-PCR 6/21 (28.6%) positive
Fonseca et al. ([@B79]) Low-grade and high-grade 75 patients, tissue biopsy EBV using PCR with confirmation using direct sequencing 6/11 (55%) low-grade positive3/22 (13.6%) high-grade positive
Cheng-Te Major Lin et al. ([@B41]) High-grade 19 patients, formalin-fixed glioma tissue EBV *lmp1* DNA with multiplex droplet digital PCR 4/19 (21%) positive
Neves et al. ([@B80]) Pilocytic astrocytoma 35 children, tissue biopsy RT-PCR, LMP1 by immunohistochemistry 9/35 (26%) positive by PCR, but none by immunohistochemistry
Cimino et al. ([@B81]) High-grade 21 patients, tissue biopsy Next-generation sequencing/PCR/*in situ* hybridization 5/21 (24%) positive, but none by *in situ* hybridization
Strong et al. ([@B35]) High-grade 170 patients, tissue biopsy Next-generation sequencing/RT-PCR None positive
Cosset et al. ([@B82]) High-grade 20 patients, tissue biopsy/serum analysis PCR None positive
Khoury et al. ([@B83]) Low- and high-grade 215 patients/tissue biopsy RNA-Seq database analyses None positive
Hashida et al. ([@B84]) High-grade 39 patients/tissue biopsy PCR analyses of *LMP1* gene None positive
Several, but not all, of the studies conducted across different geographical locations, such as North America, South America, Europe, and Japan, have shown a positive association of EBV in patients with gliomas (Table [1](#T1){ref-type="table"}). Recently, Stojnik et al. ([@B34]) studied the presence of EBV, along with HSV-2, HHV-6, and one human enterovirus (hEV) in high-grade gliomas in 45 adult patients (12 with grade III and 33 with grade IV) at the University Clinical Centre in Maribor, Slovenia. Glioma tissue samples were obtained either from tumor biopsies (19/45) or following surgical tumor reduction (26/45) from patients with a median age of 60 years (ranging from 22 to 86 years). Tissue was either used within 24 h for assaying of viral genes by rt-PCR (in the case of EBV, a 166 bp fragment of the *ebna* gene was amplified). Serum analyses of C-reactive protein (CRP) was measured for all patients (24 whom were females) and 30/45 patient samples were also analyzed for specific antibodies for each of the viruses by enzyme immunoassays and complement fixation. PCR studies of gliomas revealed only 3/45 patients were positive for EBV *ebna* gene: a 66-year-old male with GBM located in the left temporal and parietal lobes; a 68-year-old female with GBM located in the right temporal and parietal lobes; and a 77-year-old male with GBM located in the right temporal, parietal, and occipital lobes. Common features were that all samples were attained following craniotomy and surgical tumor reduction. Importantly, all three EBV+ patients had grade IV gliomas (GBM) and no virus was detected in any of the 12 grade III gliomas, implying this virus is preferentially associated with most aggressive CNS tumors. However, none of the patients were found to be seropositive for EBV antibodies ([@B34]). This was in contrast to an earlier report by Wrensch et al. ([@B76]), who used serological IgG antibody binding using ELISA assays to demonstrate that about 90% of their GBM patients, from the USA, San Francisco Bay Area Adult Glioma Study from 1991 to 1995, were seropositive for EBV ([@B76]). Another study conducted by Poltermann et al. ([@B77]) showed the presence of IgG antibodies to EBV in serum of 89% (64/72) of patients with glial tumors (*n* = 35), meningiomas (*n* = 31), and acoustic schwanommas (*n* = 6) though this was not considered significantly different to antibody levels in the general population ([@B77]).
Strojnik et al. ([@B34]) also found HHV-6 in 2/45, HSV2 in 1/45, and hEV in 1/45 glioma tissue samples tested. All positive tests were in grade IV gliomas but of varying origin. However, viral copy numbers for all viruses, including EBV, detected in glioma tissue samples were generally very low (mostly below 2 copies per 5 µL DNA with only the 66-year-old male with EBV having a copy number of 27 copies per 5 µL DNA). Again, none of the patients' positive for HHV-6 or HSV2 in glioma tissues developed antibodies in serum samples though five positive results for HSV2 antibodies were noted even in the absence of virus in the tumor samples. Furthermore, the presence of adenoviruses, HSV-1, CMV, and VZV was not confirmed in any of the 45 tissue samples studied ([@B34]).
Another recent study by Zavala-Vega et al. ([@B78]) reported on presence of EBV, along with CMV and HSV1/2 in Mexican patients with GBM. They performed a retrospective study using brain tissue from 21 adults aged on average 52 years (range 23--83 years). To indicate EBV infection, they detected LMP-1 by immunohistochemistry and EBER expression by *in situ* hybridization in 6/21 (28.6%) of patients. Mixed infections of EBV and HSV-1/2 were noted in 4/21 patients (19%), whereas EBV and CMV in 5/21 (23.8%) patient samples. A particular limitation of this study was that IgG and IgM antibody levels could not be determined in patients with viral infections as this was a retrospective study based on paraffin-embedded tissue samples only. However, the value of measuring antibody titers may not correlate with disease as antibodies produced in the case of the related CMV during early stages of infections have a protective effect, thereby preventing viral reactivation and subsequent development of glioblastoma ([@B85]).
A study by Fonesca et al. ([@B79]) aimed to screen 75 primary glioma biopsy specimens from a cancer centre in Rio de Janeiro, Brazil, for the presence of EBV using PCR with confirmation using direct sequencing. To detect EBV in tumor samples, a 288 bp fragment of EBV bam region was amplified and later sequenced to confirm viral DNA in GeneBank data sets. Using this strategy in fresh frozen tissue samples, 11/75 gliomas (14.7%) were positive for EBV with the majority being low-grade gliomas (6/11), followed by 2/11 for grade III, oligoastrocytoma (1/11), ependymoma (1/11), and only 1/11 being grade IV (GBM). These results are in contrast to the study from Slovenia where only high-grade gliomas were positive for EBV ([@B34]). In addition, Fonesca et al. ([@B79]) also found EBV in one oligoastrocytoma and one ependymoma, but none at all in other CNS tumors including two non-HL---a tumor type in which EBV association has been reported previously ([@B75]). The amplified EBV gene sequences obtained from gliomas were well matched with published EBV genome sequences with an identicalness rate of 95.5% implying that EBV virus was indeed present in these samples.
Cheng-Te Major Lin et al. ([@B41]) used multiplex droplet digital PCR (ddPCR)---a highly precise diagnostic tool that enables the absolute quantification of target DNA in a high throughput setting---to show positivity of EBV *lmp1* DNA in 4/19 (21.1%) of formalin-fixed paraffin-embedded (FFPE) GBM samples and not in any controls. Samples were obtained from the George Washington University Hospital and the National Institutes of Health, USA. Interestingly, two GBM tumor specimens were positive for both HHV-6B and EBV indicating that the possibility of multiple viral infections being associated with GBMs.
Pilocytic astrocytoma of the cerebellum is one of the most common pediatric brain tumors. In FFPE tumor samples analyzed by two different PCR methodologies and immunohistochemistry, EBV was detected by PCR in about 30% of these tumors (9/35) from patients with an average age of 15.5 years; however, none of the samples were positive for EBV by immunohistochemistry (anti-LMP1 antibody) ([@B80]). Most of the astrocytoma (33/35) was of low-grade malignancy. This study suggested that EBV was the most frequent herpes virus found in pilocytic astrocytoma though at levels apparently too low to be considered responsible for tumor induction ([@B80]).
Because polymerase chain reaction (PCR) analyses and viral-specific immunohistochemical assays are biased in that only selected or targeted genes or proteins of viruses are investigated in tumors, more state-of-the art methods with high sensitivity that may avoid these bias are being used to detect infectious agents in tumors. A less biased approach would be to fully sequence brain tumors and search for any EBV virome nucleic acid sequences present. One such methodology that allows this rather unbiased approach is next-generation sequencing (NGS)---a non-Sanger-based high-throughput DNA sequencing technology. There are a number of different NGS platforms, a detailed discussion of which is beyond the scope of this article, but the reader is referred to some recent review ([@B86]--[@B88]). Nonetheless, in all NGS platforms sequencing of millions of small fragments of DNA in parallel is followed by bioinformatics analyses to piece together these fragments and mapping the individual reads to the reference genome. NGS can be used to sequence entire genomes or constrained to specific genes or regions of interest. Recently, NGS studies have been used to study the presence of EBV sequences in gliomas ([@B71], [@B81]).
A NGS study by Cimino et al. ([@B81]) examined viral sequences in 21 high-grade gliomas (mostly glioblastomas) at the University of Washington, St Louis, MO, USA. Unmapped sequencing reads, obtained from FFPE samples, identified EBV in 5/21 (24%) of high-grade gliomas (all GBM). They also found one case of Roseolovirus, but no CMV in any of their glioma tissues. However, further examination of the four of EBV-sequence-positive tumors for virus by *in situ*-hybridization failed to detect EBV-encoded RNA implying that EBV in malignant high-grade gliomas might be transcriptionally inactive and more characteristic of a dormant state that could also be present in the general population ([@B81]). However, since the authors examined only one non-coding EBER RNA, the possibility that other EBV RNAs may be produced still remains unexplored.
Contrary to the findings of Cimino et al. ([@B81]), a more recent and very comprehensive NGS study by Strong et al. ([@B35]) suggested that no major viruses were associated with high-grade gliomas. These authors undertook a large-scale virome assessment in publically available The Cancer Genome Atlas (TCGA) sequencing data sets for 157 primary glioblastomas (GBM) and 13 recurrent GBM as well as whole genome sequencing (WGS) data sets for 51 primary GBM, and 10 recurrent GBM. Finally, they also analyzed fresh frozen tissue from three primary GBM samples (one from a patient at the Louisiana Cancer Research Consortium and two samples from the commercial supplier BioServe, USA). In this comprehensive and detailed study, the authors aimed to address many of the major experimental concerns in detecting viruses in tumor tissues ([@B35]). For instance, to account for heterogeneity within GBM tumor mass that might give rise to differential transcriptome profiles ([@B89]), they used data sets from 92 MRI-localized biopsies from either the core or margins of multiple GBM patients; and to account for the possibility that viruses may lay hidden within cancer stem cells, they also analyzed RNA-seq data sets from a cohort of short-term glioma stem cell cultures freshly isolated from nine patients with primary GBM. Despite these precautionary measures, as well as running NGS experiments at low viral read thresholds (that could have been associated with increased risk of low-level contamination), no major virus associations could be identified. However, in their attempt to account for the possibility that viruses infecting brain tissue become transcriptionally dormant and thus avoid detection in RNA-seq data sets, they also looked at WGS data sets for virome assessment. Analyses of the virus at the DNA level did show low level presence of EBV DNA in samples (at viral reads below 40) from 9 primary GBM and 6 matched blood samples as well as 3 recurrent GBM each from the TCGA and WGS data sets with only one having a moderate EBV viral read of 1,454. However, the presence of EBV DNA in the case of the three recurrent GBM from WGS data could not be validated by the corresponding RNA-seq data. As true, EBV association would normally lead to much higher viral reads (\>10 for RNA-seq and \>1,000s for DNA-seq) and given the presence of EBV in blood and tumor specimens was roughly equivalent, the authors concluded that the detected EBV likely originated from infiltrating EBV-infected B-cells and/or from possible library or sequencing sample cross-contamination.
Similarly, they also dismissed low-level viral reads of several other viruses in gliomas, as likely artifacts or non-pathological incidental infections. For example, they noted that all of the sporadic low-level CMV reads were found to map to the immediate early promoter intimating that they likely originated from laboratory expression vector contamination. In addition, human herpes virus 6 and 7 aligned viral reads were likely false-positives due to their homology with human telomeric-like repeats ([@B35]). These data argue against associations between most known viruses and GBM or meningiomas, but interestingly, the authors highlighted that the most robust virus findings were the detection of HPV and hepatitis B in the occasional low-grade gliomas. Thus, although these findings cast doubt on EBV association in gliomas they rather, open the door for the further in-depth studies on the possible association of HPV and hepatitis B, two viruses that have received little attention in CNS tumors including gliomas.
Several other studies have reported on the complete absence of EBV in gliomas. Cosset et al. ([@B82]) studied 20 GBM biopsies including the corresponding patient serum, where available, by standard clinical diagnostic methods (semi-qPCR) for the presence of the following common neurotropic viruses: CMV, EBV, HSV, HHV6, MeV, PeV, JC virus, EV, and VZV. Although some biopsies were associated with a type I IFN-response, none of the above-mentioned viruses were detected in any sample of GBM or of three other low-grade gliomas, one oligodendroglioma, two meningiomas, one ependymoma, and one oligoastrocytoma ([@B82]). Similarly, Khoury et al. ([@B83]) reported no EBV or any other virus after screening of TCGA malignant tumors including low- and high-grade gliomas on which RNA-Seq data were available. They showed no evidence of transcribed viral elements in any of the low-grade gliomas and glioblastoma multiforme. Further, a study by Hashida et al. ([@B84]) in Japanese subjects with GBMs failed to detect EBV in tumors using real-time PCR analyses of *LMP1* gene. However, these authors did show the presence of high risk HPV16 and HPV18 in 21% (8/39) of the GBMs studied---results that are consistent with the findings of Vidone et al. ([@B90]) in Italian glioma patients and reaffirmed in the NGS study of Strong et al. ([@B35]) discussed above (see also Table [1](#T1){ref-type="table"}).
Perspectives and Concluding Remarks {#S3}
===================================
It is clear from the studies examining EBV in gliomas conducted thus far that, as is the case with other viruses like CMV, there are discordant results on viral association in these malignancies. Reasons for these discordant findings may lie within population/geographic differences, individual genetic variability, inherent heterogeneity of gliomas, variations in samples including anatomical location from which tumor specimen was removed, differences in the actual viral genes probed, as well as sensitivity and precision of the methodologies used. In addition, differences in processing or preparation of samples (such as section thickness, fixation conditions, and antibody dilution) and difficulties with paraffin-embedded tumor samples may have caused the observed discrepancies ([@B32], [@B91]). Are these variables really the explanation? Probably, in part but surely, an ideally robust association of EBV in gliomas would have resulted in sufficiently high viral levels to the extent that the effects of many of the above variables would be minimal or at least mitigated to some extent. However, a few studies have shown no virus and most have shown only low levels of the EBV either in the glioma tissue or as antibodies in serum including the recent elegant and comprehensive study by Strong et al. ([@B35]) that aimed to account for many of the concerns mentioned above.
Serological studies measuring EBV antibodies in glioma patients were also discordant. Given the fact that 90% of the population is carriers of EBV in its latent state, why are we not, therefore, detecting a similar proportion of seropositive tumor patients as the general population in all studies? For example, in one study, the risk of glioma patients being seropositive for EBV was less than the control population implying that the tumor may actually modulate EBV infections. The presence of lower levels of EBV in tumor than in control samples could also be explained if the virus was lost during tumor progression. Such a "hit and run" model has been proposed in HPVs ([@B28], [@B92]). There is also evidence indicating the presence of EBV antibodies early on may actually be protective in tumors ([@B93]). In any case, the high seroprevalence of EBV in controls makes it difficult to make a firm association based on the serum antibody data presented for EBV in gliomas.
Thus, can we really exclude a clinical role of EBV in gliomas based on these findings or could the relatively low levels of EBV, as generally reported in gliomas, still lead to gliomagenesis and/or oncomodulation? A recent report by Shumilov et al. ([@B94]) suggests that EBV might exert some of its oncogenic effects, such as inducing centrosome amplification and chromosomal instability, without having to establish a chronic infection, thereby conferring a risk for development of tumors that do not necessarily carry the viral genome ([@B94]).
Lytic replication, the process by which viral progeny is produced, is a strong risk factor for EBV-associated tumors ([@B31]). This process activates cellular cancer-associated changes such as chromosomal instability, but lytic replication also leads to cell death rendering the link between replicating cells and oncogenesis not so obvious. Shumilov et al. ([@B94]) presented the data that removed this conceptual difficulty by showing that the EBV virions themselves conferred the risk induced by lytic replication to non-replicating cells, i.e., the effects of EBV virions extended to EBV-negative cells. Thus, their paradigm-changing study implies that EBV could be a risk factor for the development of gliomas without being present in the tumor. If others confirm these findings, then this would fundamentally change our view of the role played by EBV in tumors and offer a more rational explanation for the near absence of EBV in gliomas reported in several of the studies reviewed herein.
Since direct viral association studies have generally been discordant, another approach to establish viral association with tumors has been to study the role of antiviral therapies on disease ([@B91]). A recent report has suggested that glioma patients at the Karolinska University Hospital receiving 6 months of antiviral therapy as an add-on to standard radiation and temozolamide therapy exhibited marked increases in survival rates ([@B95]), though the study design and mathematics used have been questioned ([@B23], [@B95]). Some other studies, but not all, have also shown improved outcomes in cancer patients on antiviral therapy ([@B32], [@B96]). However, while the rates of many AIDS-associated malignancies have been declining with the use of highly active anti-retroviral therapy, the rates of EBV-positive Burkitt's and HL in this population have not declined ([@B97], [@B98]). These data may imply that the oncogenic effects of EBV---at least in B-cell lymphomas---are not affected by antiretroviral drugs. It should, however, be noted that while viral therapy may improve clinical outcome in some cases, it does not necessarily imply a viral cause as survival benefit might be explained by secondary or "off-target" effects of the therapy alone unrelated to viral infection.
In contrast, there is also evidence to suggest that prior exposure to stress and/or immunodeficient status induced by therapies may actually predispose patients to EBV-induced oncogenesis. For example, Zakaria et al. ([@B99]) described a patient who within 2 months of undergoing radio-chemotherapy for glioblastoma developed an EBV-positive primary diffuse large B-cell CNS lymphoma ([@B99]). These findings suggest that probably the immunosuppression and/or stress induced by the treatments for GBM, or even co-morbidities, can lead to EBV reactivation.
A corollary of this is the idea that stress resulting from co-infections may also be important in viral reactivation and oncogenesis. Although the low levels of EBV infections reported in gliomas, by themselves may not be sufficient, they likely require additional stress-causing risk factors, such as the co-presence of other oncoviruses, to influence oncogenesis or oncomodulation. Thus, latent EBV viruses may be reactivated when cells experience co-infection with, for example, CMV or HSV1/2, as has been reported in some glioma studies ([@B78]).
In this regard, a preventative vaccine against EBV and/or co-infecting agent may be useful. Vaccines against specific viruses may, therefore, offer a more targeted approach for association studies and clinical therapy \[for review see Cohen ([@B100])\]. For example, an EBV vaccine has been tested (in a phase I clinical trial) on Chinese nasopharyngeal carcinoma patients to determine the safe and immunogenic dose ([@B101]). In that study, it was concluded that the vaccine is both safe and immunogenic, thus paving the way for further clinical testing of the EBV vaccine that may be of clinical benefit in EBV-positive tumors including glioma patients. The first prophylactic EBV vaccine based on virus-like particles (VLPs) that mimic the structure of the EBV virus, but lack its genome has also been reported to be effective in preclinical models ([@B102]) and may represent a safer alternative.
Finally, could it be that by looking for EBV and other herpes viruses like CMV, in gliomas we might have been focusing on the wrong viruses? Recent NGS sequencing data seems to suggest that most of the viruses especially CMV are completely absent from gliomas and many of the positive associations reported are likely artifactual as they may be rationally explained otherwise \[e.g., high homology of detected viral sequences to host as in the case of chromosomal telomere repeats ([@B35])\]. The reported low level presence of EBV does not completely rule it out from being associated with oncogenesis or oncomodulation in gliomas \[indeed, it may not even need to be present to exert its effects as suggested by the study of Shumilov et al. ([@B94])\], but recent reports suggest that HPV infection might be more robustly associated with some gliomas. Thus, additionally more detailed and comprehensive studies are needed to fully implicate EBV and/or other viruses such as HPV in having a direct association in gliomagenesis and oncomodulation. Understanding the role of EBV and other oncoviruses in the etiology of gliomas, that generally have a poor prognosis, would likely open up new avenues for the treatment and management of these, often fatal, CNS tumors.
Author Contributions {#S4}
====================
SA and A-EM conceived the review. SA and SV searched the literature. SA, SV, FC, and A-EM critically appraised the literature, wrote and approved final version of the manuscript.
Conflict of Interest Statement {#S5}
==============================
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
[^1]: Edited by: Ali A. Sultan, Weill Cornell Medicine-Qatar, Qatar
[^2]: Reviewed by: Lital Keinan-Boker, University of Haifa, Israel; Alireza Sadjadi, Tehran University of Medical Sciences, Iran
[^3]: Specialty section: This article was submitted to Cancer Epidemiology and Prevention, a section of the journal Frontiers in Oncology
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"pile_set_name": "PubMed Central"
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{#sp1 .188}
{#sp2 .189}
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INTRODUCTION
============
Analyses of a growing number of high resolution ribosome structures coupled with kinetic studies are revealing that the ribosome is highly dynamic, capable of assuming \>40 different conformational states through the translation elongation cycle \[reviewed in ([@B1])\]. Thus, a critical question is how the ribosome coordinates all of these states to ensure the directionality and fidelity of protein synthesis. The general answer lies in allostery: the formation and breaking of specific intermolecular contacts in response to different ligand binding states serves as a series of switches to ensure that the ribosome is optimally configured to proceed to the next functional state. The current challenge is to identify and functionally map the specific allosteric switching components.
Numerous researchers have employed elegant biochemical, biophysical, structural, genetic, and computational approaches to this problem, a few of which are highlighted here. For example, biochemical approaches have been employed to define the kinetic parameters governing every step of the elongation cycle, revealing that selection of the appropriate aminoacyl--tRNA (aa-tRNA) depends on a two step kinetic process \[reviewed in ([@B2])\]. Biophysical methods have revealed that conformational switching by rRNA and ribosomal protein L1 ensures exit of deacylated tRNA from the ribosome ([@B1],[@B3]), while real time single molecule fluorescence and force measurements are revealing dynamic motions of the ribosome and tRNAs, and directly probing the forces stabilizing ribosomal complexes \[reviewed in ([@B4])\]. Biochemical, computational and high resolution structural methods have been employed to map changes in rRNA structures during the transit of tRNAs through the ribosome and during the peptidyltransfer reaction ([@B5; @B6; @B7; @B8; @B9; @B10]). Pertinent to this study, we have previously used a combination of molecular genetics, biochemical and biophysical approaches to identify the contributions of specific ribosomal proteins and rRNA bases involved in coordinating the stepwise process of accommodation of aa-tRNA into the ribosomal A-site, activation of the peptidyltransferase center, and recruitment of the trans-acting translocase (EF-G in bacteria, eEF2 in eukaryotes) using yeast ribosomes as a model ([@B11; @B12; @B13; @B14; @B15; @B16]).
Ribosomal protein L3 in particular appears to have an important function in this process. [Figure 1](#F1){ref-type="fig"}A shows L3 within the context of the large ribosomal subunit (LSU), including the following structural and functional elements; the peptidyltransferase center (PTC), the aa-tRNA accommodation corridor which lies in between Helix 89 and the complex Helix 90--92 structure, Helix 95 (the Sarcin/Ricin Loop or SRL), and the GTPase-associated center (GAC). The latter two structures interact with the eEF1A--aa-tRNA--GTP ternary complex and eEF2 (the eukaryotic translation elongation factor homologs of bacterial EF--Tu-aa-tRNA--GTP ternary complex and EF-G, respectively). The 3D rendering in [Figure 1](#F1){ref-type="fig"}B shows that L3 contains a globular domain that interfaces with the solvent side of the LSU, and two structures, the N-terminal extension and a central loop that extend deep into the central core of the LSU. The central loop can be subdivided into two domains, the 'tryptophan finger' (W-finger) positioned at the tip of the central extension, and a cluster of basic amino acids that protrudes from the center of the internal loop like a thumb roughly perpendicular to the W-finger that we call the L3 'basic thumb'. [Figure 1](#F1){ref-type="fig"}C shows that this basic thumb is nestled in the core of a cagelike structure formed by elements of three different 25S rRNA helices: H61, H73 and H90. We previously proposed a 'rocker switch' model describing how structural rearrangements of the N-terminal extension and the W-finger of L3 function to coordinate the stepwise processes of translation elongation ([@B17]). Missing from the prior analyses was an explanation of how the movements of these extensions of L3 are communicated to functional centers of the ribosome. In this current study, analysis of the basic thumb of L3 illuminates this question. Mutagenesis of the indicated amino acids to alanines followed by genetic, biochemical and structural analyses suggests that the basic thumb acts as a molecular clamp to play a role in allosterically communicating the ribosome\'s tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, thus facilitating coordination of their functions through the elongation cycle. Figure 1.The L3 basic thumb. (**A**) Crown view of the yeast 60S subunit from ([@B36]). L3 is indicated in green. The aa-tRNA accommodation corridor is framed by Helix 89, and the complex structure formed by Helices 90--92. Elongation factors bind to the GTPase Associated Center (GAC) and the Sarcin/Ricin Loop (SRL) at the tip of Helix 95. The peptidyltransferase center (PTC) is in the center of the large subunit. (**B**) The 3D view of isolated L3, heat map colored from the N-terminus (blue) to the C-terminus (red). The N-terminal extension and the basic thumb and tryptophan (W) finger of the central extension are indicated. (**C**) The 3D view of interactions between amino acid residues of the L3 basic thumb investigated in this study. It is surrounded by a cagelike structure formed by 25S rRNA Helices 61--64, H73 and H90. Amino acids mutated in this study are labeled. Figure 2.Effects of L3 basic thumb mutants on cell growth, virus propagation and programmed --1 ribosomal frameshifting. (**A**) Ten-fold dilution spot assays (10^4^ → 10^0^ CFU) of cell growth at 30°C. (**B**) Killer assay. Cells were replica plated onto a lawn of diploid Killer^−^ indicator cells and grown at 20°C for 3 days. Zone of growth inhibition indicates presence of the Killer virus. (**C**) Dual luciferase assays were used to measure percent programmed --1 ribosomal frameshifting from an L-A virus derived --1 PRF signal (20;21). Error bars denote standard errors.
MATERIALS AND METHODS
=====================
Strains, plasmids, genetic manipulation and media
-------------------------------------------------
*Escherichia coli* DH5α was used to amplify plasmid DNA. Transformation of *E. coli* and yeast, and preparation of yeast growth media (YPAD, synthetic drop out medium, and 4.7 MB plates for testing the Killer phenotype) were as reported earlier ([@B18]). Restriction enzymes were obtained from MBI Fermentas (Vilnius, Lithuania). The QuikChange XL II site-directed specific mutagenesis kit was obtained from Stratagene (La Jolla, CA, USA). Macrogen Inc. (Piscataway, NJ, USA) performed DNA sequence analysis. Oligonucleotide primers were purchased from IDT (Coralville, IA, USA). The yeast strains used in this study were all derived from the *rpl3*-gene disruption (*rpl3Δ*) strain JD1090 (*MATα ura3-52 lys2-801 trp1δ leu2^−^ his3 RPL3::HIS3* p*RPL3-URA3-CEN6* \[L-A HN M~1~\]) ([@B19]). Mutants of *rpl3* were generated using the wild-type *RPL3* gene in pJD225 ([@B19]), synthetic oligonucleotides, and the QuikChange XL II kit. The pYDL dual luciferase reporter series of plasmids for monitoring programmed ribosomal frameshifting (PRF) were used as described earlier ([@B20]).
Assays for the killer phenotypes and measurement of frameshifting
-----------------------------------------------------------------
The killer virus assay was carried out as described earlier ([@B18]). Briefly, yeast colonies were replica plated to 4.7MB plates newly seeded at an optical density at 595 nm (OD~595~) of 0.5 of the 5 × 47 killer indicator strain per plate. After 2--3 days at 20°C, killer activity was scored as a zone of growth inhibition around the Killer^+^ colonies. To monitor programmed −1 frameshifting using the dual luciferase reporter plasmids, glass beads were used to prepare lysates from cells expressing the 0-frame, −1 (L-A derived), or +1 (Ty*1* derived) dual luciferase plasmids ([@B20]). After clarification of the lysates by centrifugation, typically 5 μl was used in a total volume of 100 μl of dual luciferase assay reagents (Promega, Madison WI, USA), and *Renilla* and firefly luciferase activities were quantitated using a TD20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). Frameshifting efficiencies were calculated by dividing the firefly/*Renilla* luminescence ratios from lysates of cells expressing the PRF test reporters by the same ratio obtained from lysates of cells expressing the zero-frame control reporter. All assays were replicated enough times to achieve \>95% confidence levels, and statistical analyses were performed as described earlier ([@B21]).
Synthesis of aminoacyl--tRNA and acetylated aminoacyl--tRNA
-----------------------------------------------------------
Aminoacyl--tRNA synthetases were purified as described earlier ([@B22],[@B23]).Yeast tRNA^Phe^ was aminoacylated with unlabeled phenylalanine or with \[^14^C\]Phe to make Phe-tRNA^Phe^ and \[^14^C\]Phe-tRNA^Phe^, respectively. \[^14^C\]Phe-tRNA^Phe^ was used to monitor enzymatic binding to the A site of poly(U) primed ribosomes, and acetylated-\[^14^C\]Phe-tRNA^Phe^ (Ac-\[^14^C\]Phe-tRNA^Phe^) was generated to monitor nonenzymatic P-site binding using poly(U) primed salt washed ribosomes. Phe-tRNA^Phe^ and Ac-Phe-tRNA^Phe^ were used in SHAPE structure probing experiments (see below). Yeast tRNA^Phe^ was aminoacylated as described earlier ([@B24]) with minor modifications. The reaction mix (1 ml) contained 100 mM HEPES--KOH, pH 7.6, 10 mM KCl, 20 mM MgCl~2~, 10 mM ATP, 100 μM \[^14^C\]Phe \[496 mCi/mM\], 1 mM DTT, 2000 U pyrophosphatase (Sigma), plus 0.2 mg of tRNA^Phe^ and 40 μl of aminoacyl--tRNA synthetases. Reaction mixtures were incubated for 30 min at 30°C, and proteins were removed by extraction with acid--phenol--chloroform. Charged tRNAs were ethanol precipitated and purified using G25 spin columns. \[^14^C\]Phe-tRNA^Phe^ was separated from uncharged tRNA by high-performance liquid chromatography (HPLC) as described earlier ([@B25]) with the following modifications. Samples were loaded onto a 4.6- by 250-mm JT Baker wide-pore butyl column equilibrated with buffer A (20 mM NH~4~Cl, 10 mM MgCl~2~, 400 mM NaCl; pH 5.0) at 1 ml/min. The column was washed with 10 ml of buffer A, conditions under which free phenylalanine and aminoacyladenylate are eluted from the column. Uncharged tRNAs and residual free \[^14^C\]Phe and nucleotides were eluted by isocratic elution of 19 ml at 15% of buffer B (20 mM NH~4~Cl, 10 mM MgCl~2~, 400 mM NaCl, 60% methanol; pH 5.0). \[^14^C\]Phe-tRNA^Phe^ was eluted using a programmed binary gradient of buffers A and B. Elution of aa-tRNA was monitored by OD~260~ readings, and \[^14^C\]Phe-tRNA^Phe^ concentrations and specific activities were determined. The presence of aa-tRNA in the eluted material was confirmed by TLC ([@B24]). Ac-\[^14^C\]Phe-tRNA^Phe^ was obtained in a similar manner. Yeast tRNA^Phe^ was charged with \[^14^C\]Phe as above, extracted with phenol and purified using G25 columns. Reaction mix (4 ml) contained 200 mM NaOAc, pH 5.2 and 7 nmol of \[^14^C\]Phe-tRNA^Phe^. Acetylation was carried out by addition of 64 μl of acetic anhydride at 1 h intervals for 2 h on ice. After incubation, NaOAc concentration was raised to 300 mM and Ac-\[^14^C\]Phe-tRNA^Phe^ was ethanol precipitated. Ac-\[^14^C\]Phe-tRNA^Phe^ was further purified by HPLC as described earlier.
Purification of yeast ribosomes
-------------------------------
Sulfolink resin was charged with cysteine as described earlier ([@B26]). Yeast cells were grown in YPAD media to mid log phase, collected and washed with binding buffer (10 mM Tris--HCl, pH 7.5; 5 mM MgCl~2;~ 60 mM NH~4~Cl; 2 mM DTT). Cells were suspended in binding buffer and disrupted using a Mini Bead Beater. Lysates were centrifuged at 30 000*g* for 30 min in Beckman MLS 50 rotor. Supernatant (2 ml) was removed and added to 2 ml of cystein-charged Sulfolink slurry (50% resin equilibrated with binding buffer) and incubated on ice for 15 min with mixing as resin sediments. After incubation resin was spun down at 1500*g* for 0.5 min. Supernatants were removed and resin washed five times with 5 ml of binding buffer. After washing, resin was suspended in 1 ml of elution buffer (10 mM Tris--HCl, pH 7.5; 10 mM MgCl~2;~ 0.5 M KCl; 1 mg/ml heparin; 2 mM DTT) and incubated for 5 min on ice with occasional mixing. The suspensions were centrifuged, supernatant collected, and elution was repeated two more times. Supernatants were combined (3 ml total volume) and GTP and pH neutralized puromycin were added to 1 mM each. After incubation at 30°C for 30 min, reaction mixtures were loaded on top of a 1 ml glycerol cushion (10 mM Tris--HCl, pH 7.5; 10 mM MgCl~2;~ 0.5 M KCl; 2 mM DTT; 25% glycerol) and centrifuged at 100 000*g* for 16 h. Ribosome pellets were suspended in 2 ml of elution buffer without heparin, loaded on top of 2 ml glycerol cushions and centrifuged at 100 000*g* for 16 h. Ribosome pellets were resuspended in storage buffer \[50 mM HEPES--KOH pH 7.6; 5 mM Mg(CH~3~COO)~2;~ 50 mM NH~4~Cl; 1 mM DTT; 25% glycerol\] at 5--10 pmol/µl (1 OD~260~ = 2 pmol) and stored at −80°C.
Characterization of peptidyltransferase activity
------------------------------------------------
Complex C \[ribosome-poly(U)-AcPhe-tRNA\] was formed in 400 μl of binding buffer (80 mM Tris--HCl, pH 7.4, 160 mM ammonium chloride, 11 mM magnesium acetate, 2 mM spermidine and 6 mM β-mercaptoethanol) containing 0.4 mM GTP, 500 pmol ribosomes, 0.4 mg/ml poly(U) and 700 pmol Ac-\[^14^C\]Phe-tRNA. Mixtures were incubated for 20 min at 30°C and then placed on ice. Complexes were purified from free Ac-\[^14^C\]Phe-tRNA^Phe^ by centrifugation through a glycerol cushion (0.5 ml; 20% glycerol in binding buffer by centrifugation at 50 000 rpm for 2 h in MLS 50 rotor). Ribosome pellets were rinsed twice with 1 ml of binding buffer and suspended in 1.15 ml of binding buffer. For puromycin reactions, 1.15 ml of complex C extract was pre-incubated at 30°C for 5 min, and reactions were initiated by adding pH neutralized puromycin (100 mM stock) to final concentrations of 10 mM. Aliquots of 100 μl were removed, and reactions were terminated at the indicated time intervals by addition of 100 μl of 1.0 N NaOH. Reaction products were extracted with 0.4 ml of ethyl acetate, 0.2 ml of organic phase was transferred to scintillation vials, and radioactivity was determined by scintillation counting. A 50-μl aliquot of initial reaction mixture was also transferred to scintillation vials, and total radioactivity (*N*~o~) was determined. Controls without puromycin were included in each experiment, and the values obtained were subtracted as background. The percent of the bound Ac-\[^14^C\]Phe-tRNA^Phe^ converted to Ac-\[^14^C\]Phe-puromycin was corrected with the extent factor α (determined if complex C were allowed to react for 1 h; *C*~o~ = α*N*~o~), as described earlier ([@B27],[@B28]). The reaction plots were fit to a first-order exponential equation, and values of *K*~obs~ (the apparent rate constant of entire course of reaction at a given concentration of puromycin) were calculated by using Graphpad Prism software.
aa-tRNA and Ac-aa-tRNA-binding studies
--------------------------------------
Soluble protein factors were prepared as described earlier ([@B28],[@B29]). aa-tRNA binding to the A-site of the ribosome was carried out as described earlier ([@B13]). Ribosome mixtures (50 μl) contained 80 mM Tris--HCl, pH 7.4, 160 mM NH~4~Cl, 11 mM Mg(CH~3~COO)~2~, 6 mM β-mercaptoethanol, 2 mM spermidine, 0.4 μg/ml of poly(U) and 25 pmol of ribosomes. For A-site-binding experiments, ribosome mixtures were preincubated with uncharged tRNA (4:1 tRNA/ribosomes) at 30°C for 15 min to ensure full occupation of P-sites by uncharged tRNA. \[^14^C\]Phe-tRNA^Phe^ /GTP/eEF1A ternary complex was formed by incubating (5 min at 30°C) \[^14^C\]Phe-tRNA^Phe^ in binding buffer with 0.4 mM GTP and 10 µg/ml soluble protein factors. The 2-fold dilutions of \[^14^C\]Phe-tRNA^Phe^ ternary complex were added to ribosome mix. Reaction mixtures were incubated at 30°C for an additional 20 min to allow formation of \[^14^C\]Phe-tRNA^Phe^-80S-poly(U) complexes. For P-site-binding experiments 2-fold dilutions of Ac-\[^14^C\]Phe-tRNA^Phe^ were added to ribosome mixtures and incubated for 20 min at 30°C. Aliquots were then applied onto nitrocellulose membranes, filters were washed with 6 ml of binding buffer, and radioactivity was measured by scintillation counting. Background levels of radioactivity were determined using a blank sample (without ribosomes) and subtracted from test samples. *K*~d~ values were determined assuming single binding sites using Graphpad Prism software.
eEF2 binding
------------
6xHis-tagged eEF2 was purified from TKY675 yeast cells (kindly provided by Dr T. Kinzy) as described earlier ([@B30]) with the following modifications. EDTA was added to 5 mM to eluted eEF2 just before dialysis to bind leached Ni^2+^ ions and prevent precipitate formation during dialysis due to aggregation of His-tagged protein. eEF2 concentration was determined by \[^14^C\]ADP-ribosylation with diphtheria toxin (see below). Each preparation of eEF2 showed linear concentration response curves in the range of eEF2 amounts used in binding experiments. For eEF2-binding experiments, reaction mixes (25 μl) containing 12.5 pmol of salt washed 80S ribosomes and various concentrations of 6xHis-tagged eEF2 in binding buffer (50 mM Tris--HCl, pH 7.5, 50 mM ammonium acetate, 10 mM magnesium acetate, 2 mM DTT, 100 μM GDPNP) were incubated for 20 min at room temperature. Estimation of bound eEF2 was carried out as follows by assuming that ribosome bound eEF2 is not susceptible to ADP-ribosylation by diphtheria toxin ([@B31; @B32; @B33]). Free (unbound) eEF2 was estimated by ADP-ribosylation of eEF2: 100 pmol \[^14^C\] NAD^+^ and 0.2 μg of diphtheria toxin were added to each reaction mix and incubated for 30 min at 30°C. Total eEF2 in each reaction mix was determined by ADP-ribosylation reaction after bound eEF2 was released by adding EDTA to 10 mM. Reaction mixes were precipitated with TCA, and amounts of \[^14^C\]ADP-ribosylated eEF2 were determined by liquid scintillation counting. Control values (lacking diphtheria toxin) were subtracted. Ribosome bound eEF2 was calculated by subtracting free values from total amount. *K*~d~ values were determined assuming single binding sites using Graphpad Prism software.
SHAPE structure probing of mutant ribosome--tRNA complexes
----------------------------------------------------------
To prime ribosomes with poly(U), reaction mix (100 µl) in binding buffer (80 mM Tris--HCl, pH 7.4, 100 mM NaCl, 15 mM Mg(CH~3~COOH)~2~, 6 mM β-mercaptoethanol) containing 55 pmol ribosomes and 50 µg poly(U), was incubated for 20 min at 30°C. Next, for P-site complex, 200 pmol of Ac-Phe-tRNA^Phe^ was added. For structure probing of ribosomes with occupied A sites, ribosomal P sites were blocked with 4× excess of deacylated tRNA^Phe^ and Phe-tRNA^Phe^ (200 pmol), GTP (0.5 mM) and 5 µl of crude elongation factor mix were added. Reaction mixes were incubated for 20 min at 30°C. Reactions were divided into two parts of 50 µl each (control and modification tubes) and 75 µl of binding buffer was added to each tube. 25 µl of 1M7 (130 mM in DMSO) was added to modification tubes. Control samples contained 25 µl of DMSO. After incubating for 85 min at 30°C ribosomes were precipitated with 450 µl ethanol. Ribosomal RNA was extracted using RNAqueous kit (Ambion). Pellets were dissolved in 100 µl of RNAqueous Lysis Buffer and processed according manufactures instructions. RNA was eluted in 50 µl volume and concentration adjusted to 1 µg/µl with elution buffer. Reverse transcriptase (RT) primer extension analyses of modified RNAs were performed as described ([@B34]). The site-specificity of charged tRNA binding was confirmed using the puromycin reaction ([@B35]).
Computational analysis of ribosome structure
--------------------------------------------
The cryo-electron microscopy (cryo-EM) reconstruction of *Thermomyces lanuginosus* modeled with Saccharomyces cerevisiae rRNA and ribosomal proteins ([@B36]) was visualized using PyMOL (DeLano Scientific LLC).
RESULTS
=======
The L3 basic thumb mutants affect cell growth, programmed −1 ribosomal frameshifting and yeast Killer virus maintenance
-----------------------------------------------------------------------------------------------------------------------
*rpd3Δ*cells harboring wild-type pRPL3-Ura were transformed with mutant *rpl3* alleles expressed from a low copy *TRP1* vector, and the viability of mutants was assessed by their ability to grow in the presence of 5-FOA. All of the L3 basic thumb single mutants (R232A, K236A, K237A, K241A, R244A, R247A and R248A) were viable as the sole forms of L3 with the exception of R240, which was lethal ([Figure 2](#F2){ref-type="fig"}A, summarized in [Table 1](#T1){ref-type="table"}). Among the viable single mutants, only R232A and R247A conferred noticeable growth defects. A series of double mutants were constructed based on the viable mutants and their physical locations relative to one another. This analysis revealed that the K236A/R247A and K241A/R244A mutants significantly affected cell growth, while the R247A/R248A mutant was inviable. The K236A/K237A double mutant did not grossly affect cell growth. Table 1.Summary of L3 Basic Thumb mutantsL3^a^Growth^b^Killer^c^% --1 PRF^d^A-site (nM)^e^rRNA SHAPE^f^% +1 PRF^g^P-site (nM)^h^eEF2 (nM)^i^Puro *K*~obs~ (min^−1^)^j^WTWT+7.6 ± 0.492.4 ± 10.7WT7.4 ± 0.672.6 ± 8.3382.8 ± 93.30.34 ± 0.05R232A↓↓w5.7 ± \<0.1126.3 ± 7.1PTC6.6 ± 0.374.2 ± 7.1341.0 ± 115.30.22 ± 0.02K236AWT--4.9 ± 0.2163.3 ± 11.7H73, H90, H91, PTC6.7 ± 0.178.4 ± 5.1312.4 ± 74.40.20 ± 0.03K237AWT--4.9 ± 0.1167.7 ± 24.8H73, H91, H92, H946.5 ± 0.381.2 ± 4.4341.7 ± 55.90.61 ± 0.08R240ALethalNDNDNDNDNDNDNDNDR241A↓+NDNDNDNDNDNDNDR244A↓wNDNDNDNDNDNDNDR247A↓↓↓--4.2 ± \<0.1305.7 ± 41.7H73, H94, H91, PTC, H94, H956.0 ± 0.773.9 ± 9.446.6 ± 26.90.23 ± 0.03K248AWTwNDNDNDNDNDNDNDK236A/K237AWT--4.9 ± 0.1244.9 ± 23.3ND7.1 ± 0.1794.9 ± 10.684.0 ± 42.30.17 ± 0.02K236A/R247A↓↓--4.9 ± 0.1272.4 ± 34.4ND7.0 ± \<0.171.3 ± 6.9110.2 ± 46.20.18 ± 0.03K241A/R244A↓↓↓--4.3 ± 0.3340.3 ± 39.2ND6.5 ± 0.184.4 ± 14.8229.4 ± 71.30.14 ± 0.04R247A/R248ALethalNDNDNDNDNDNDNDND[^1][^2][^3][^4][^5][^6][^7][^8][^9][^10][^11]
Most strains of *S. cerevisiae* harbor a symbiotic virus called 'Killer', a bipartite dsRNA viral system composed of the L-A helper virus and the M~1~ satellite \[reviewed in ([@B37])\]. The 4.6-kb dsRNA L-A viral genome encodes the viral coat protein (Gag), and the Gag-pol replicase that is encoded by a programmed −1 ribosomal frameshift (−1 PRF) ([@B38]). The M~1~ satellite dsRNA is encapsidated and replicated inside of L-A encoded viral particles. The M~1~ encoded preprotoxin is processed by the Kex1p and Kex2p cellular proteases into the mature secreted toxin. Cells infected by L-A and M~1~ can kill uninfected cells, but are themselves immune, hence the name 'Killer'. The presence of Killer can be easily assayed by replica plating test cells onto a lawn of diploid uninfected (indicator) cells: Killer^+^ cells will kill the nearby indicator cells, resulting in a ring of growth inhibition around the test cells. One of the first yeast mutants cloned, *mak8-1*, was first identified by its inability to maintain the [K]{.smallcaps}iller phenotype (Mak^−^ phenotype) and encodes an allele of *rpl3* ([@B39]). Analyses of the L3 basic thumb mutants revealed that three of the single mutants (K236A, K237A and R247A) were completely unable to maintain the Killer virus, while R232A and R244A had weak Killer phenotypes. None of the double mutants were able to maintain the virus ([Figure 2](#F2){ref-type="fig"}B, summarized in [Table 1](#T1){ref-type="table"}).
The efficiency of −1 PRF determines the relative ratio of structural Gag to enzymatic Gag-pol available for viral particle self-assembly, and changing −1 PRF strongly inhibits virus maintenance ([@B18],[@B40]). Previous studies have identified numerous L3 mutants that promoted altered rates of L-A promoted programmed −1 ribosomal frameshifting (−1 PRF), but did not affect Ty*1* mediated programmed +1 ribosomal frameshifting (+1 PRF) ([@B13],[@B17],[@B19],[@B23]). However, while all previous L3 mutants analyzed to date enhanced −1 PRF efficiency, all of the viable L3 basic thumb mutants promoted decreased −1 PRF, ranging between ∼55% and ∼75% of wild-type rates ([Figure 2](#F2){ref-type="fig"}C, summarized in [Table 1](#T1){ref-type="table"}). The significance of these changes in −1 PRF is confirmed by loss of the Killer virus, maintenance of which is known to be sensitive to even small decreases in −1 PRF rates ([@B18],[@B41]). Consistent with prior studies none of the basic thumb mutants affected +1 PRF (summarized in [Table 1](#T1){ref-type="table"}).
The Killer^−^ L3 basic thumb mutants affect binding of ligands to the ribosomal A-site and peptidyltransfer
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The simultaneous slippage model of −1 PRF requires that both the aa-tRNA in the ribosomal A-site, and the peptidyl--tRNA in the P-site must shift on the mRNA ([@B42]), while in Ty*1* mediated +1 PRF, only the peptidyl--tRNA slips ([@B43]). Consistent with the frameshifting data, all of the L3 basic thumb mutants promoted decreased affinity for aa-tRNA to the A-site ([Figure 3](#F3){ref-type="fig"}A and B, and summarized in [Table 1](#T1){ref-type="table"}), but did not affect binding of Ac-aa-tRNA to the P-site ([Figure 3](#F3){ref-type="fig"}C and D, summarized in [Table 1](#T1){ref-type="table"}). Specifically, R232A, which does not directly contact any rRNA bases, had the smallest effect on aa-tRNA binding (*K*~d~ values ∼126 nM compared to ∼94 nM for wild-type, i.e. 1.4-fold increase), while the K236A and K237A mutants, which participate in only a few rRNA contacts ([Figure 4](#F4){ref-type="fig"}B), had moderate effects (∼165 nM each, 1.8-fold wild-type). In contrast, R247A, which contacts both H61 and H90 had a very strong effect on aa-tRNA binding (∼300 nM, 3.3-fold wild-type). The double mutants, which also affected multiple rRNA contacts had comparable effects on aa-tRNA binding (from ∼245 nM to ∼340 nM). Figure 3.Biochemical characterization of ribosomes expressing L3 basic thumb mutants. (**A**) Single site isotherms of eEF1A stimulated binding of \[^14^C\]Phe-tRNA^Phe^ to A-sites of poly(U) primed ribosomes pre-loaded with tRNA^Phe^ in their P-sites. (**B**) Dissociation constants calculated from data shown in (A). (**C**) Single site isotherms of Ac-\[^14^C\]Phe-tRNA^Phe^ to P-sites of poly(U) primed ribosomes. (**D**) Dissociation constants calculated from data shown in (C). (**E**) eEF2-binding isotherms for wild-type and mutant ribosomes. (**F**) Dissociation constants calculated from data shown in (E). (**G**) Ac-\[^14^C\]Phe-puromycin formation is plotted as percent of bound donor reacted. (**H**) Rates of peptidylpuromycin formation (*K*~obs~) were calculated from data shown in (G). Figure 4.rRNA structure probing of wild-type and L3 basic thumb mutant ribosomes. (**A**) Poly(U) primed salt-washed ribosomes were either loaded with Ac-Phe-tRNA^Phe^ (P-site occupied) or tRNA^Phe^+ Phe-tRNA^Phe^ (A + P-site occupied). Ribosomes were unmodified (control) or treated with 1M7 as indicated. Reverse transcriptase primer extension reactions spanned sequences from the 3′ half of Helix 73 through Helix 96 (left panel), or from Helix 91 through the 3′ half of Helix 73 (right panel). Sequencing reactions (left sides of panels) are labeled corresponding to the rRNA sense strand. Nucleotides whose 2′OH riboses were protected from 1M7 modification in mutant relative to wild-type ribosomes of are indicated by white arrowheads, and those deprotected relative to wild-type are indicated by black arrowheads. Bases marked in gray (A2926 and A2971) were deprotected when the A-site is unoccupied relative to when it contains aa-tRNA. **(B)** rRNA protection patterns of the L3 basic thumb mutants mapped onto the 2D diagram of 25S rRNA. Arrowheads indicate relatively protected and deprotected bases as above. Colored boxes indicate bases that interact with specified L3 basic thumb amino acid side chains. A2926 and A2971 are circled in gray, and C2925, which is the first gate in the aa-tRNA accommodation corridor, is circled in purple. The three bases participating the Type II A-minor motif that stabilizes the PTC are boxed and indicated. **(C)** Data from panels A and B mapped onto the 3D structure of the yeast ribosome. Indicated bases colored black correspond to bases deprotected in the mutants, while those colored gray are hyperprotected. Bases participating in the Type II A-minor motif (A^m^) are colored purple. Helical structures and the PTC are color coded as indicated. Note that the loop formed between H61--H64 was removed from this figure because it obscures the L3 basic thumb.
Eukaryotic elongation factor 2 (eEF2) catalyzes translocation and binds to the same site as the aa-tRNA--eEF1A--GTP ternary complex. Given the effects of the mutants on aa-tRNA binding, the effects of seven mutants on eEF2 binding were assayed ([Figure 3](#F3){ref-type="fig"}E and F, summarized in [Table 1](#T1){ref-type="table"}). For wild-type ribosomes, the dissociation constant for eEF2 was ∼383 ± 93 nM. Similar values were observed for R232A, K236A and K237A mutant ribosomes, but the R247A mutant promoted an ∼8-fold increase in affinity for eEF2 (*K*~d~ ∼ 47 nM). Among the double mutants assayed, the K237A/K236A mutant promoted the largest increase in affinity for eEF2 (∼84 nM, ∼4.5-fold increase), followed by K236A/R247A (∼110 nM, ∼3.5-fold increase), and K241A/R244A (∼229 nM, ∼1.7-fold increase).
Single round assays of peptidyltransferase activity were performed on puromycin treated salt washed ribosomes pre-loaded with Ac- \[^14^C\]Phe-tRNA^Phe^ and purified through glycerol cushions (Complex C) as described in the 'Materials and Methods' section. The observed *K*~obs~ = 0.34 min^−1^ in wild-type ribosomes ([Figure 3](#F3){ref-type="fig"}G, [Table 1](#T1){ref-type="table"}) is comparable to similar reactions using *E. coli* ribosomes and the Ac-Phe-tRNA substrate ([@B44]), confirming that these relatively low rates are determined by Ac-Phe-tRNA as a poor substrate for the peptidyltransferase reaction, and thus represent true measurements of peptidyltransferase activity, as opposed to peptidyl-tRNA turnover or other artifacts. The R232A, K236A and R247A mutants all promoted decreased rates of peptidyltransfer to approximately two--thirds of wild-type levels, while the double mutants (K236A/K237A, K236A/R1247A, and R247A/K248A) had stronger effects, decreasing rates to ∼50% of wild-type ([Figure 3](#F3){ref-type="fig"}G and H, [Table 1](#T1){ref-type="table"}). Unexpectedly, the K237A mutant enhanced the rate of peptidyltransfer by almost 2-fold above wild-type levels.
The L3 basic thumb mutants promote changes in 25S rRNA structure both locally and in elements associated with aa-tRNA and eEF2 related functions
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Inspection of atomic resolution ribosome structures reveals that the L3 basic thumb participates in a highly conserved set of interactions with the PTC proximal bases of Helix 73, and Helix 90, with bases on both sides of Helix 61, and with bases in a complex loop structure connecting Helices 61--64 of the LSU rRNA ([Figures 1](#F1){ref-type="fig"}C, [4](#F4){ref-type="fig"}B and D) ([@B6],[@B36],[@B45; @B46; @B47]). Strikingly, while 2D maps of the LSU suggest that these structural elements are in physically separate domains from one another in ([Figure 4](#F4){ref-type="fig"}B), the 3D structures reveal that the peptidyltransferase center, Helices 73, 90 and 94 are complexly folded to form a roughly Y-shaped topology, while the Helix 61--64 loop forms a cagelike structure opposite of this Y, with the L3 basic thumb positioned in the center where all of the basic side chains seem to glue all of the rRNA pieces together ([Figures 1](#F1){ref-type="fig"}C, [4](#F4){ref-type="fig"}B and C). Importantly, the distal tip of Helix 92 is folded back upon itself where G2922 in the A-loop participates with C2876 and G2951 at the base of H90 to form an A-minor motif in the A-site of the peptidyltransferase center ([@B48]). For ease of comparison, yeast 25S rRNA bases are listed with their *E. coli* homologs in [Table 2](#T2){ref-type="table"}. Table 2.Homologous yeast 25S rRNA and *E. coli* 23S rRNA bases pertinent to this studyYeast 25S rRNA base*E. coli* 23S rRNA baseU1887^2′O--Me^, G1888 (R244, R247, K248)C1656, U1657C1904, G1905 (R240)A1671, 11672G2325, U2326, C2327^2′O--Me^ (K237, K241)G1992, U1993, C1994C2328 (R247)U1995C2392 (R240)C2050A2820A2451 (PTC)C2876C2507 (A^m^ motif)G2877 (R240)C2508C2878 (R240)G2509Ψ2880, C2881 (R247)U2511, C2512G2912G2543G2922^2′O--Me^G2553 (A^m^ motif)U2924U2555C2925C2556 (Gate 1)C2929A2560A2934A2564A2946^2′O--Me^ (R244)A2577G2947 (R244, R240)G2578C2848^2′O--Me^, U2949 (R240)C2579, Ψ2580G2951G2582U2954U2584U2956U2586A2971A2602G2977G2608U2980^2′O--Me^ (K236)C2611C2985C2616A2987G2618A2995C2626G3003C2626A3006A2639G3009G2642U3019C2652U3023U2656A3033C2666[^12]
To assess the effects of the viable mutants on rRNA structure, SHAPE \[Selective 2′-Hydroxyl Acylation and Primer Extension, ([@B34],[@B49])\] using 1M7 \[1-methyl-7-nitroisatoic anhydride, ([@B50])\] was used to probe wild-type and selected mutant ribosomes containing either Ac-aa-tRNA^Phe^ at the P-site alone, or both tRNA^Phe^ and aa-tRNA^Phe^ at the P-and A-sites respectively ([Figure 4](#F4){ref-type="fig"}). Inspection of the results revealed two general trends. First, that the mutants promoted significant changes in rRNA structure in the peptidyltransferase center (U2953, U2955, G2977), along the path taken by aa-tRNA as it accommodates into the LSU (accommodation corridor, G2912, U2924, C2929, A2934), in Helix 94 where it interacts with the globular domain of L3 (G3003, A3006, G3009), and in Helix 95 (U3019, U3023, A3033). Secondly, while the mutants only altered the Helix 73--Helix 95 region when only the P-site was occupied by Ac-aa-tRNA, the majority of changes in the Helix 91--93 region were observed when both A- and P-sites were occupied. Detailed analyses reveal that the R247A mutant conferred the largest number of changes in rRNA structure, promoting deprotection of C2985 in Helix 91, G3003, A3006, G3009 in Helix 94, and U3019, U3023 and A3033 in Helix 95 when only the P-site was occupied ([Figure 4](#F4){ref-type="fig"}A and B). In contrast, this mutant promoted increased deprotection of G2912 in Helix 92, U2924 in the A-loop, A2934 in the bulge between Helix 92 and Helix 90, and U2955 in the peptidyltransferase center when the A-site was occupied by aa-tRNA. This mutant also caused hyperprotection of A2987 in Helix 73 when the A-site was occupied. Some of the other mutants had similar effects on some but not all of the same bases, e.g. K237A caused deprotection of G2912 and U2955, and enhanced protection of A2987 when the A-site was occupied. Other mutant specific effects were observed. For example, when only the P-site was occupied K247A promoted enhanced protection of G2977, while both K247A and K236A promoted deprotection of A2995 and U2953 under these conditions ([Figure 4](#F4){ref-type="fig"}A and B). K237A also promoted increased protection from 1M7 at G2977 when both the A- and P-sites were occupied. R232A had significant effects in the 3′ loop between H90 and H92, and in the peptidyltransferase center (U2955). The observation that the bulged A2971 in Helix 93 was generally deprotected when only the P-site was occupied by Ac-aa-tRNA^Phe^, but became protected from chemical attack upon loading of aa-tRNA^Phe^ into the A-site ([Figure 4](#F4){ref-type="fig"}) serves as an important control, as this site occupancy-specific conformational change has also been observed for bacterial ribosomes ([@B51]). Interestingly, this pattern was also observed for A2926 in the A-loop, the possible significance of which is discussed below.
DISCUSSION
==========
How is information flow coordinated through the ribosome to ensure the directionality of protein synthesis? Although the bulk of the ribosome is comprised of rRNA, and indeed, many of its critical functions are mediated through RNA--RNA interactions, it is clear that protrusions of ribosomal proteins, which can be thought of as loops, hooks and fingers, function to help 'switch' the ribosome between different conformational/functional states. For example, the C-terminal extension of *E. coli* S13 is thought to help coordinate movement of the peptidyl--tRNA with structural rearrangements at the ribosome interface that are critical for translocation by sampling the tRNA occupancy status at the decoding center ([@B52],[@B53]). The N-terminal 'hook' of yeast L10 (*E. coli* L16) is believed to play an active role to coordinate switching of the ribosome between the pre- and post-translocational states ([@B16]). Ribosomal protein L2, which is intimately intertwined with multiple domains of the LSU, is thought to coordinate long range interactions between tRNAs and the ribosome ([@B14]). Ribosomal protein L3 is of particular interest because of its function as a 'gatekeeper' to the ribosomal A-site ([@B13]). A follow-up study suggested that two critical structures of L3, the W-finger and the N-terminal extension, function together as a 'rocker switch' to coordinate LSU associated functions ([@B17]). The L3 basic thumb is of interest because it appears to provide the structural link in this rocker switch mechanism. Protruding roughly perpendicular from the L3 W-finger toward the intersubunit face of the LSU, it is surrounded by a cagelike structure formed by a large bulge framed between Helices H61--64, Helix 73, Helix 90 and Helix 94. It is in the center of a nexus connecting the W-finger with the L3 globular domain, the peptidyltransferase center, the aa-tRNA accommodation corridor, and the SRL. Furthermore, it is proximal to the B5 intersubunit bridge, which involves multiple contacts involving bases in Helices 62 and 64 ([@B54]).
Unlike the small subunit (SSU), where the four rRNA domains are largely physically distinct, the six rRNA domains of the LSU are highly intertwined ([@B54]). With regard to the current study, the loop bounded by Helices 61--64 lie in domain IV, the PTC and Helices 90--93 and Helix 73 are in domain V, and Helices 94 and 95 are in domain IV. Previously, we demonstrated that the conformationally dynamic nature of the W-finger enables the central extension of L3 to function like a lever, and as such contribute to allosteric repositioning of rRNA structural elements ([@B13],[@B17]). However, it was not clear how a small radial movement of thin, essentially planer element, could have such large and long ranging effects on rRNA structure and ribosome function. The perpendicular orientation of the basic thumb may answer this: we propose that it amplifies the action of this lever by adding three-dimensionality to the central extension in the form of a platform upon which structural elements from three different domains of 25S rRNA are anchored. In support of this, comparison of the L3 structures between EF-Tu and EF-G bound *Thermus thermophilus* ribosomes ([@B55]) reveals displacement of the α-carbon backbone of the W-finger and basic thumb structures by ∼2--3 Å, and of some individual sidechains by as much as 5 Å ([Supplementary Figure S1](http://nar.oxfordjournals.org/cgi/content/full/gkq641/DC1)). This model explains how the L3 central extension can have significant effects on coordinating the flow of information among the critical functional centers of the LSU, as well as to the SSU.
The large number of basically charged amino acids in the basic thumb enables it to participate in numerous hydrogen bonding/electrostatic interactions with rRNA bases, phosphates and riboses, functioning as a 'molecular clamp' to bridge these three domains. The current study focusing on eight amino acids in the L3 basic thumb neutralized their positive charges with alanine substitutions both singly and in selected pairs. The lethality of the R240A mutant as the sole form of L3 indicates that it may function to 'glue' the PTC proximal stem of H90 with the H61-H64 loop, and may well aid in coordinating formation of the C2876-G2922-G2951 A-minor motif that is critical for the 'induced fit' function of the peptidyltransferase center ([@B5],[@B6]). Similarly, R247, which bridges Helix 61 with Helix 90 had profound effects on rRNA structure, ribosome biochemistry, translational fidelity, and cell growth, suggesting that it too is a critical bridging component between these two domains. Interestingly, the R247A/R248A double mutant was lethal, while R248A had a wild-type phenotype. This confirms the role of R247 and also indicates that the molecular defect conferred by the R247A single mutant might be partially complemented by the positively charged R248 adjacent to it. R240A and R247A appear to be the exception rather than the rule however, as the other single mutants had significantly lesser effects on these parameters. This is consistent with mutagenesis studies on other residues of L3, and with other LSU proteins and rRNA bases ([@B11; @B12; @B13; @B14; @B15],[@B17],[@B23],[@B56; @B57]), suggesting that the ribosome is an elegantly evolved molecular machine containing multiple levels of functional redundancy. Furthermore, all of the double mutants tested had strong effects on binding of aa-tRNA and eEF2, and on peptidyltransfer ([Figure 3](#F3){ref-type="fig"}), suggesting that it multiple defects are generally required to disrupt the functionally redundant interactions between the basic thumb and the LSU rRNA. As an aside, the observation that A2926 (base paired to U2920) was protected from 1M7 modification in wild-type ribosomes when both A- and P-sites were occupied by tRNAs but deprotected when only the P-site was occupied ([Figure 4](#F4){ref-type="fig"}A) is unique to yeast. To our knowledge, this has not been observed in *E. coli* ribosomes ([@B51]), which contains a G-C base pair (C2551-G2557) at this position, a conformational difference that suggests a potentially novel antibiotic target. Interestingly, *Haloarcula marismortui* appears to split the difference with a G-U base pair (U2576-G2582).
Some specific changes in rRNA structure are particularly telling. The base stack of aa-tRNA C74 with *H. marismortui* U2590 (*E. coli* U2555, yeast U2924) is thought to promote the induced fit of peptidyltransfer ([@B6]). The enhanced deprotection of this base in R247A mutant ribosomes when both A- and P-sites were occupied by tRNAs is consistent with its strong effect on peptidyltransferase activity by this mutation ([Figure 3](#F3){ref-type="fig"} H). Interestingly, this base was also strongly deprotected under the same conditions in K237A mutant ribosomes, but in this case peptidyltransferase activity as monitored using the puromycin reaction was actually enhanced ([Figure 3](#F3){ref-type="fig"}H). The rates of peptidyltransfer observed in the current study (∼0.3 min^−1^) and in similar analyses using *E. coli* ribosomes are significantly lower than naturally occurring rates \[estimated to be \>300 s^−1^, ([@B58])\] because Ac-Phe-tRNA is a poor substrate for this reaction. However, this property actually enables us to tease out the effects of local structural changes on PTC activity. Recent molecular dynamics simulations of portions of the ribosome reveal that individual bases can undergo a conservable degree of structural mobility due to local Brownian movements \[reviewed in ([@B59])\], suggesting that specific bases in the PTC are relatively free to assume either the induced or uninduced conformations in the absence of tRNAs, and that the equilibrium between these two states is influenced by the presence or absence of aa-tRNA in the A-site. Thus, we suggest that R247A mutant drives this equilibrium toward the uninduced conformation, while the K237A mutant favors the induced arrangement. The observation of distinctly different patterns of rRNA protection/deprotection (e.g. compare G2912, C2929, U2953, G2977, C2985, A2995, G3003, A3006, G3009, U3019, U3023 and A3033 between the K237A and R247A mutants in [Figure 4](#F4){ref-type="fig"}A) is consistent with the idea that they also have opposing effects on PTC conformation and functionality. In addition, early studies demonstrated that while empty ribosomes are heterogeneous in their affinity for eEF2, consisting of two sub-populations having *K*~d~'s for eEF2 ranging from subnanomolar to hundreds of nanomoles ([@B60]), the affinity for eEF2 strongly depends on the functional status of the ribosome as determined by the occupancy status of the A- and P-sites ([@B61],[@B62]). This suggests that the R247A and the double mutants that increased eEF2 affinity shift this equilibrium as well, possibly stabilizing ribosomes in the pre-translocation state, and that the interactions between the 25S rRNA bases and L3 amino acid residues investigated here are involved in transitions between the pre- and post-translocational states.
As discussed above, the effects of the K237A mutant are locally confined to the PTC, while those conferred by R247A are more global. The latter is reflected in their different effects on ligand binding to the A-site: R247A promoted very strong effects on both aa-tRNA and eEF2 binding as compared to the much weaker effects conferred by K237A. Examination of the ligand binding data reveals a reciprocal relationship between affinities for aa-tRNA and eEF2, i.e. increased aa-tRNA *K*~d~ correlates with decreased eEF2 *K*~d~ ([Figure 3](#F3){ref-type="fig"}). This is consistent with the model that L3 plays a central role as an allosteric switch to coordinate binding of elongation factors, opening and closing of the accommodation corridor, and PTC activity to ensure the unidirectionality of protein synthesis ([@B13],[@B17]). Interestingly, all of the mutants affected peptidyltransfer, consistent with observations that this process is highly sensitive to even minor structural changes in the ribosome \[reviewed in ([@B63])\].
In the end, the most important parameter is life; i.e. how does the L3 basic thumb contribute to the fitness of the organism? While only some of the mutants had gross effects on cell growth, they all affected translational fidelity as monitored by decreased rates of −1 PRF ([Figure 2](#F2){ref-type="fig"}). In fact, the effects on −1 PRF correlated well with the A-site aa-tRNA-binding data. For example, R247A and K241A/R244A, which had the most pronounced effects on −1 PRF, also promoted \>3-fold decreases in affinity for aa-tRNA. In contrast, R232A and K236A, which had the smallest effects on aa-tRNA binding, also promoted the smallest decreases in −1 PRF. As noted above, the mutants investigated in this report are unique in that they are the first examples that promoted decreased −1 PRF. This trend had only previously been observed with anisomycin, a competitive inhibitor for aa-tRNA 3′ binding to the PTC ([@B41]). We have suggested that the majority of −1 PRF occurs after aa-tRNA accommodation into the A-site, and prior to peptidyltransfer, while a smaller fraction can occur during translocation \[reviewed in ([@B64])\], a view that is supported by a recent study coupling kinetic modeling of −1 PRF within the translation elongation cycle with mass spectroscopic analyses of frameshifted peptide products (P.-Y. Liao *et al.*, submitted for publication). By this model, decreased rates of aa-tRNA accommodation into the A-site should decrease the steady state abundance of substrate for −1 PRF, thus inhibiting this reaction. Changes in −1 PRF in turn alter the relative amounts of viral protein products available for viral particle self assembly, a ratio that is critical for virus propagation ([@B18],[@B40]). This illustrates how minute changes in ribosome structure at the atomic scale can propagate outward, affecting ribosome biochemistry, translational fidelity, and the ability of cells to replicate viruses.
SUPPLEMENTARY DATA
==================
[Supplementary Data](http://nar.oxfordjournals.org/cgi/content/full/gkq641/DC1) are available at NAR Online.
FUNDING
=======
National Institutes of Health (5 R01 GM058859; to J.D.D.); American Heart Association (AHA 0630163N; to A.M). Funding for open access charge: National Institutes of Health (5 R01 GM058859); American Heart Association (AHA 0630163N).
*Conflict of interest statement*. None declared.
Supplementary Material
======================
###### Supplementary Data
The authors would like to thank members of the Dinman laboratory for their critical input and helpful suggestions. We would also like to thank Kevin Weeks for the kind gift of 1M7.
[^1]: WT denotes wild-type, and ND represents Not Determined.
[^2]: ^a^L3: Version of L3 assayed.
[^3]: ^b^Effects of mutants on cell growth from [Figure 1](#F1){ref-type="fig"}A. Down arrows indicate relative effects observed using 10-fold dilution spot assay.
[^4]: ^c^Data summarized from [Figure 1](#F1){ref-type="fig"}B. + = Wild-type Killer phenotype; w = weak Killer phenotype; -- = unable to maintain Killer virus.
[^5]: ^d^% --1 PRF data from [Figure 1](#F1){ref-type="fig"}C with standard error indicated.
[^6]: ^e^Mean and standard deviation *K*~d~ values of aa-tRNAs bound to the A-site from [Figure 2](#F2){ref-type="fig"}B.
[^7]: ^f^Summary from [Figure 4](#F4){ref-type="fig"}A of regions in 25S rRNA where changes were observed using SHAPE.
[^8]: ^g^% +1 PRF data, standard error indicated.
[^9]: ^h^Mean and standard deviation *K*~d~ values of Ac-aa-tRNAs bound to the P-site from [Figure 2](#F2){ref-type="fig"}D.
[^10]: ^i^Mean and standard deviation *K*~d~ values of eEF2 bound to ribosomes from [Figure 2](#F2){ref-type="fig"}F.
[^11]: ^j^Mean and standard deviations of rates of peptidylpuromycin formation from [Figure 2](#F2){ref-type="fig"}H.
[^12]: Contacts with specific L3 basic thumb amino acid side chains are indicated in parentheses. Superscript 2′O-Me indicates that 2′O-methylated bases. Ψ indicates pseudouridine.
| {
"pile_set_name": "PubMed Central"
} |
Libraries of cells with defined genetic alterations have proven transformative for connecting poorly understood genes to biological pathways and uncovering novel roles for previously characterized genes. However, in eukaryotes these libraries have been difficult to generate, and even in some widely used collections, such as the yeast knockout library, a majority of the members contain undesired secondary mutations^[@R1]^ and suffer from the presence of selection markers^[@R2]^.
In this work, we present a Cas9-based strategy for the simultaneous, seamless creation of hundreds of genetic variants without integrated selection markers in wild-type yeast cells that express the Cas9 protein along with a donor repair template. Our system is built upon CRISPR/Cas9 and its ability to stimulate homology-directed recombination (HDR) repair of a double-stranded break at a given target locus^[@R3]^. Each isogenic mutant is generated by a plasmid containing a single guide RNA (sgRNA) paired with a corresponding donor template that carries a programmed mutation (hereon referred to as the guide+donor strategy) ([Figure 1a](#F1){ref-type="fig"}). The advantages of our concatenated guide+donor design are threefold; it enables: a) rapid cloning of all library members within one reaction, b) simultaneous delivery of both the guide and the donor in one contiguous unit thus preventing uncoupling that may result in inefficient repair and unproductive repair outcomes, and c) high-throughput molecular phenotyping using next generation sequencing (NGS) with guide+donor-containing plasmids serving as unique barcodes for tracking edited cells. A similar concept of *in cis* delivery of guide+donor was recently demonstrated in bacteria^[@R4]^.
In our initial test, we integrated a copy of the *cas9* gene into the neutral *HO* locus and performed individual transformations of 34 guide+donor plasmids ([Supplementary Figure 1](#SD1){ref-type="supplementary-material"}). Upon selecting for cells with the guide+donor, however, we found that the number of colonies with the desired genetic alteration was low (0--30%), consistent with earlier attempts at *in cis* guide+donor delivery in yeast^[@R5]^ ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}). We sought to increase the percentage of correctly edited cells in order to enable efficient genome-scale measurements via NGS.
To test if linearization of our guide+donor plasmid would increase the efficiency of our system^[@R6]--[@R10]^, we introduced our guide+donor substrate as two linear pieces of DNA. The larger DNA fragment contained the guide+donor portion of the plasmid with an internal portion of the selection marker removed. The smaller DNA fragment consisted of the missing segment of the selection marker with \~150bp of flanking homology such that HDR was required to reconstitute the full circular plasmid ([Supplementary Figure 1a](#SD1){ref-type="supplementary-material"}). With the modified approach, we observed a 6--14 fold increase in transformation efficiency ([Supplementary Figure 1b](#SD1){ref-type="supplementary-material"}) with 80--100% of the transformants containing the desired repair event, which is in stark contrast to the 0--30% proper editing observed with the unmodified method^[@R4],[@R5]^ ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}). No programmed edits were observed in the absence of Cas9 ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}).
To begin characterizing the limitations of our system, we tested a series of vectors designed to introduce either targeted point mutations, short deletions, or sequence replacements within the *ADE2* locus. For programmed point mutations, we obtained a genome modification efficiency close to 100% for changes that occurred proximal to the Cas9-generated cut site ([Supplementary Figure 2](#SD1){ref-type="supplementary-material"}). In contrast, when the desired mutation was positioned further away from the Cas9 cut site, we noted a decrease in efficiency, with mutations 12--15bp away showing rates of editing of \~40%. While longer homology length ([Supplementary Figure 3a](#SD1){ref-type="supplementary-material"}) increases the number of colonies obtained per transformation ([Supplementary Figure 3b](#SD1){ref-type="supplementary-material"}), it does not substantially improve the proportion of correct edits ([Supplementary Table 2](#SD1){ref-type="supplementary-material"}). Of clones that fail to obtain the desired point mutation, the majority mutate the protospacer adjacent motif (PAM) as designated on the provided guide+donor to escape Cas9 cutting. Further characterization of our method for generating programmed deletions revealed that our design allows efficient removal of up to 61 contiguous bases (\>90% of colonies with the desired change) but experiences a sharp decline in efficiency in creating larger deletions ((≥121bp) ([Supplementary Figure 4a](#SD1){ref-type="supplementary-material"}). Similarly, our strategy enables efficient replacement of 61bp of endogenous sequence with up to 60bp of user defined sequence ([Supplementary Figure 4b](#SD1){ref-type="supplementary-material"}).
Having gained insight into the limitation of our guide+donor strategy, we next sought to determine the generality of our method by targeting three additional loci (*SGS1, SRS2*, and *ARS214*) with a series of point mutations, deletions, and sequence replacements. Similar to our initial results, we obtained a high efficiency of genome modification (90--100%), across all targets and mutation types ([Figure 1b](#F1){ref-type="fig"}).
To examine the targeting specificity of our Cas9-based platform, we performed whole genome sequencing on three mutant strains (*ade2Δ61bp*, *sgs1Δ60bp*, and *sgs1Δatg*) generated via our guide+donor method and observed the expected genomic edits ([Supplementary Figure 5](#SD1){ref-type="supplementary-material"}). Upon surveying all the regions in the genome that have up to 2 mismatches within the N20 guide sequence, we did not find off-target sites. Off-target effects due to Cas9 are known to result in indels. When the N20 matching parameter was further relaxed to N15+PAM, we did not observe any indels indicative of off-target Cas9 effects.
The strong correlation between the presence of a particular guide+donor plasmid and the presence of the desired genetic alteration should allow us to infer the fitness effects of these modifications by sequencing the abundance of different guide+donor pairs within a mixed pool. To test this hypothesis, we built a small library containing a mixture of guide+donor plasmids designed to modify either the non-essential *ARS214* locus or the DNA damage repair helicase *SGS1* ([Figure 1c](#F1){ref-type="fig"}). Cells arose from the pooled transformation were grown in media with or without the genotoxic agent hydroxyurea (HU) and the abundance of various guide+donor plasmids within the population was determined by NGS. As expected, we observed a marked depletion of guide+donor pairs encoding modifications that disrupted the ATPase domain of Sgs1, which is known to play a critical role in its function ([Figure 1d](#F1){ref-type="fig"})^[@R11]--[@R15]^, whereas mutating the less essential C terminus^[@R11]^ lead to less depletion ([Supplementary Figure 6](#SD1){ref-type="supplementary-material"}). Introducing synonymous changes within the ATPase domain or C-terminus of *SGS1* did not show depletion, suggesting that the effects were not due to non-specific disruption of the *SGS1* locus by Cas9. Furthermore, when each of the generated strains was tested individually, the results correlated well with our pooled analysis, lending additional support for the validity of our method ([Supplementary Figure 7](#SD1){ref-type="supplementary-material"}).
In addition to exposing the mutant library to environmental perturbations, we also examined if our system could be used to observe gene-gene interactions by transforming our small library into cells defective in the structural endonuclease Mms4. In an *mms4Δ* genetic background, all *SGS1* mutants in the library exhibited \~5-fold depletion, consistent with known synthetic sickness between *SGS1* and *MMS4* ([Figure 1e](#F1){ref-type="fig"})^[@R16],[@R17]^.
We subsequently applied our method to perform systematic characterization of a single protein and targeted *SGS1*, a gene that encodes the yeast homologue of the human DNA helicase BLM with known roles in mitotic stability, cancer, and aging^[@R18]^. To map the critical domains within Sgs1 that provide cellular resistance to the genotoxic stressor HU, we designed a set of guide+donor constructs that generate 20 amino acid deletions with 5 amino acid sliding windows across the majority of the *SGS1* gene (see Materials and Methods for details). Among the regions showing strongest depletion within edited cells were guide+donors deleting amino acid stretches 1--85, 686--1090, and 1116--1225, which correspond to the Sgs1-Top3-binding domain, Sgs1-helicase, and RQC domains, respectively (2-tailed t-test, P\<0.0001) ([Figure 2a](#F2){ref-type="fig"}, [Supplementary File 1](#SD1){ref-type="supplementary-material"})^[@R19]--[@R22]^. These results are consistent with the known mechanism by which Sgs1 functions through the recruitment of accessory proteins (through N-terminal residues)^[@R12],[@R14],[@R15],[@R19],[@R23]--[@R27]^ and by resolution of DNA structural intermediates via its helicase and RecQ domains^[@R12],[@R28]^. We performed biological replicates of our library experiments to assess reproducibility and observed a correlation of 0.86 between the log~2~FC observed in the two independent yeast transformations ([Figure 2b](#F2){ref-type="fig"}). Furthermore, we performed individual phenotypic validation of seven hits from the library screen via spot assay and observed similar results ([Figure 2c](#F2){ref-type="fig"}).
Next, we created a series of precise point mutations within Sgs1. Towards this goal, we selected a set of 9 evolutionarily conserved amino acid residues within the Sgs1 helicase domain and attempted to change them to all other possible amino acids using our guide+donor strategy. This library was exposed to increasing concentrations of HU to assay for mutant drug sensitivity. Despite targeting highly conserved residues within Sgs1, all but one tolerated alanine substitution without causing an obvious loss in resistance to our highest concentration of HU at 40mM ([Figure 3a](#F3){ref-type="fig"}, [Supplementary File 2](#SD1){ref-type="supplementary-material"}). In the case where activity was lost, alanine was used to replace the essential helicase catalytic residue K706. Selecting one representative pair of biological replicates (40mM), we observed a correlation of 0.88 between the first and second biological replicate ([Figure 3b](#F3){ref-type="fig"}). We individually validated six variant hits from the library screen and observed concordant results ([Supplementary Figure 8](#SD1){ref-type="supplementary-material"}). Overall, we captured expected trends of amino acid substitutions of similar charge and size being well tolerated while those with opposite properties being more detrimental to Sgs1 function.
To determine the capacity of our method to perform targeted editing across the entire yeast genome, we designed and built a guide+donor library for generating small deletions around the initiating ATG for a set of 307 randomly chosen canonical ORFs (including both essential and non-essential genes) along with 315 poorly characterized smORFs. Unlike canonical ORFs, smORFs remain largely ignored and are often missing in modern genome annotations due to their size, low conservation scores, and lack of similarity to known proteins and protein domains.
Using our genome-scale deletion library, we first performed an essentiality screen. We observed strong depletion (\~8--100 fold) for all targeted essential ORFs (2-tailed t-test, P\<0.0001) compared to \<3-fold depletion for nearly all nonessential ORFs (2-tailed t-test, P=0.01), thus highlighting the specificity and sensitivity of our method ([Figure 4a](#F4){ref-type="fig"}, [Supplementary File 3](#SD1){ref-type="supplementary-material"}). Out of the smORFs that were examined, 19 smORFs showed similar levels of depletion as our essential controls (2-tailed Z test, P\<0.001), in line with previous results^[@R29]^. When we repeated our screen, we observed a correlation of 0.71 between the two independent biological replicates ([Supplementary Figure 9](#SD1){ref-type="supplementary-material"}).
Although a number of our smORF library members are located in close proximity to essential ORFs (in some cases within 132bp), our screen did not identify any of them as essential, emphasizing the specificity of our targeting method. To further demonstrate the ability of our guide+donor strategy to characterize a large number of proteins in parallel, we subjected our smORF mutant library to a series of environmental stressors including growth: at 37°C ([Figure 4b](#F4){ref-type="fig"}), in the presence of HU ([Figure 4c](#F4){ref-type="fig"}), or with the antifungal drug fluconazole ([Figure 4d](#F4){ref-type="fig"}). For each of our screens, we identified nearly all of the previously known smORFs with tolerance towards each of the tested conditions, along with uncovering previously unreported roles for a large number of additional smORFs^[@R30]^. We individually validated thirteen of the hits from our library screens and observed phenotypes in agreement with the screen results ([Supplementary Figure 10](#SD1){ref-type="supplementary-material"}).
Of the 315 smORFs examined, 68 were found to play a role in cellular fitness under test conditions. This is in contrast to conventional ORFs for which 104 of 307 tested ORFs were found to be involved in growth under the same environmental conditions (Chi-squared test, P\<0.0001). Next, we examined features (including amino acid size, gene expression level, secondary structure formation and evolutionary conservation) that could be shared by the smORFs or the ORFs exhibiting biological activity. Although smORFs show a range of sizes across the yeast genome (smallest smORF hit was 28 amino acids), we found that longer smORFs with elevated levels of RNA expression exhibited a trend of being more likely to come up as hits in our screen ([Supplementary Table 3](#SD1){ref-type="supplementary-material"}). Notably, ORFs showed no such correlation with regard to length, but maintained a similar trend with respect to expression ([Supplementary Table 4](#SD1){ref-type="supplementary-material"}). Moreover, we did not observe any difference in the prevalence of structural elements (e.g. alpha-helices and beta-sheets) within smORF hits as compared to non-hits. We did, however, observe an increased propensity for beta-sheets and a decrease in unstructured loops when smORFs as a whole were compared to the set of ORFs that were also examined in our screens ([Supplementary Table 5](#SD1){ref-type="supplementary-material"}). Finally, a large difference in the rate of gene conservation was found with 32 of the 68 smORF hits being conserved in humans as compared to only 43 of the 247 smORFs that showed no effect upon the examined conditions (Chi-squared test, P\<0.0001) ([Supplementary Table 6](#SD1){ref-type="supplementary-material"}).
Here, we present a high-throughput method for the rapid generation and phenotypic characterization of hundreds of mutants and illustrate its potential in domain/residue mapping and functional interrogation of nearly any user-defined genomic target by introducing deletions, amino acid substitutions, and sequence replacements. This enables the creation of specific user-defined loss-of-function, gain-of-function, and altered regulation mutants *en masse*.
By editing the locus within its native context without the need for exogenous markers, we avoid artifacts from using surrogate reporter systems and false positive and negative results due to selection marker-driven positional effects^[@R31]^. The high library editing efficiency of our system (85%-95%) ([Supplementary Table 7](#SD1){ref-type="supplementary-material"}) allows users to read the guide+donor sequence on the plasmid delivered to each cell and use the sequence to identify the cell's genotype. Ultimately, this feature enables the fitness of hundreds, potentially thousands, of mutants to be tracked by sequencing the abundance of each guide+donor sequence within a population. While our method employs a similar gap-repair mechanism as reported by Horwitz *et al*. ^[@R32]^, our design is unique in that each guide is concatenated to a corresponding donor repair template, enabling simultaneous delivery of guide+donor.
Our tiling deletion experiment on SGS1 demonstrated our technology's ability to rapidly hone in on the critical domains required for protein function. A similar CRISPR-based protein perturbation concept to identify critical functional domains in mammalian cells^[@R33],[@R34]^ and in yeast^[@R35]^ was reported previously. Of note, the underlying mechanisms of functional perturbation between these aforementioned two systems and our guide+donor platform are different in which the former ones rely on unpredictable CRISPR-induced indel and random transposase-induced insertion mutagenesis, respectively, while the variants created by our method are through programmed genetic alterations.
Deep mutational scanning (DMS) methods provided a framework for generating point mutations in a single protein of interest and functionally annotating a large fraction of these amino acid substitutions^[@R35]--[@R39]^. However, these methods are only meant to interrogate a single gene at a time, which hinders the scale of functional genomics experiments one can perform. In addition, many deep mutational scanning methods are carried out on a plasmid, thus taking the examined protein variant out of its native context^[@R37]^. Although our amino acid substitution library was not as exhaustive in its targeting scope as DMS, we are able to target hundreds of genes at a time and perform all of our genetic alterations within the native genomic locus. Previous work by Kastenmayer *et al.* ^[@R29]^ used labor-intensive conventional techniques to make specific gene deletions of 140 smORF mutants. In contrast, we demonstrated the ease of our guide+donor method in rapidly covering over \~79% of the 299 putative smORFs within the yeast genome, including many that had previously been neglected^[@R29]^. Given the degree of conservation between yeast and human genomes and the conservation between several smORFs and higher eukaryotes^[@R30]^, it will be interesting to see if the smORFs identified in our work with roles in stress tolerance have similar functions in humans.
Our method employs the commonly used *Streptococcus pyogenes* Cas9 (SpCas9) which limits the potential target sites because of its PAM-specific requirement. Using Cas9 variants recognizing alternative PAMs^[@R40]^ could greatly broaden the range of sequences that can be modified by our approach.
Although we have focused on the usage of our technology for high-throughput characterization of coding elements, we envision a broad range of additional applications such as: directed evolution, metabolic engineering, and functional interrogation of non-coding elements. Moreover, given that most clinically relevant mutations are point mutations and the high degree of gene conservation between yeast and humans, our guide+donor editing platform provides an easy way to engineer and test the effects of hundreds of currently uncharacterized single nucleotide polymorphisms that exist within human populations via their nearest yeast orthologue.
MATERIALS AND METHODS {#S1}
=====================
Yeast strains and growth conditions {#S2}
-----------------------------------
All strains were derived from YAC2370 (BY4741 derivative; *MATa his3Δ leu2Δ met15Δ ura3Δ*). YAC2563 was constructed by one-step integration of a PmlI-linearized plasmid carrying human codon-optimized Cas9 under the expression of *NOP1* promoter along with a linked NatMX drug selection marker (AC6218) into the *HO* locus. *MMS4* was deleted in YAC2563 background by one-step gene replacement using PCR-generated deletion cassettes (*mms4Δ::KanMX*).
Cells were grown non-selectively in YPAD (1% Bacto-yeast extract, 2% Bacto peptone, 2% dextrose; 1.5% agar for plates) supplemented with 500μg/ml adenine hemisulfate. Ura^+^ colonies were selected on synthetic complete (SC) medium deficient in uracil (SC-Ura). All growth was at 30°C. For the experiments with SGS1 mutants, hydroxyurea (Sigma-Aldrich) was added to final concentrations of 5mM, 10mM, 20mM and 40mM. For the smORF library drug conditions, fluconazole (Sigma-Aldrich) and HU (Sigma-Aldrich) were added to final concentrations of 25μg/ml and 100mM, respectively.
Plasmids {#S3}
--------
Guide+donor plasmids were built in the yeast pRS426 2μm backbone containing the *URA3* selection marker^[@R41]^. The guide RNA expression cassette contained *SNR52* promoter, guide RNA sequence, chimeric single-guide RNA structural tail (sgtail), and SUP4 terminator. The donor sequence carrying the desired modification was placed immediately downstream of the terminator sequence. Individual guide+donor fragments were generated from three overlapping PCR fragments using 90-mer oligos from IDT designed to create the guide sequence and its corresponding donor sequence. The ends of the stitched PCR amplicon were designed such that they contained overlapping regions for Gibson assembly. These fragments were then assembled in combination with the plasmid backbone that was digested with NgoMIV and NheI to prepare it to accept the incoming guide+donor sequence. For library cloning described below, the plasmid backbone was further modified to remove BsmBI and SapI sites.
Guide+donor library design {#S4}
--------------------------
Custom python scripts were used to design the libraries. Oligos were synthesized by CustomArray Inc. For the *SGS1* tiling deletion library with a sliding window of 15bp, we generated donor sequences with 80bp total homology flanking each 60bp deletion region then coupled a 20bp guide RNA that was present in each deletion region closest to the middle of the section being removed. For the Sgs1 amino acid library, we targeted the conserved residues previously reported by Kusano *et al*. (1999)^[@R42]^ and also included the known catalytic residue lysine 706 (K706) as a positive control. The N20 was positioned closest to the target residue and 80bp donors were designed to change the conserved target residue to every other amino acid. Finally, the smORF deletion library was designed to delete 60bp from the 5′ terminus of each target, including the initiating ATG when possible. SapI sites were added between the guide and the donor sequence that was synthesized by CustomArray to enable downstream cloning of the sgtail and an RNA polIII terminator between these two elements. Finally, all synthesized oligos had BsmBI sites added to each end to enable the first stage of cloning in which the oligo library members were inserted into the pRS426 backbone. Library members containing restriction sites including BsmBI, SapI, NcoI and StuI were excluded from the sequence file and were not synthesized.
Cloning of the library {#S5}
----------------------
The CustomArray-synthesized oligo library was diluted to 1 ng/μl and 1μl of the library was amplified with Kapa SYBR FAST qPCR Kit Master Mix (Kapa Biosystems) using unique primer pairs specific to each desired library (e.g. *SGS1* tiling deletion, smORF library, etc.). Primers used for oligo library amplification were further modified to contain the necessary overlaps to enable the library to be inserted into our vector backbone via Golden Gate cloning. The PCR products were run on a gel to confirm amplicons are of the expected length. After PCR purification (Zymo Research), the amplicon is cloned into the BsmBI-containing library vector (XG128) using a standard Golden Gate protocol with BsmBI (NEB R0580S) and T4 ligase (NEB M0202S) then electroporated into 5-alpha Electrocompetent *E.coli* cells (NEB C2989). This ensuing library now contained the guide and donor sequences adjacent to the *SNR52* promoter but was still missing the sgtail and an RNA polIII terminator. To clone in the additional functional components between the guide and donor, we amplified and cloned in the sgtail and terminator sequences following the same Golden Gate cloning method as described above, but this time using SapI (NEB R0569S) and T4 ligase. The resulting Golden Gate reactions were then PCR purified and electroporated into 5-alpha Electrocompetent *E.coli* cells to create a final guide+donor library.
Transformation into yeast {#S6}
-------------------------
Prior to transformation into yeast, each guide+donor library was double-digested with NcoI (NEB R0193T) and StuI (NEB R0187L), resulting in a linearized vector with a gap within the *URA3* selection marker. Linearized DNA containing the majority of the vector backbone, but lacking a portion of the *URA3* selection marker, was then gel extracted and purified (Zymo Research). To enable the reconstruction of the guide+donor vector within yeast via homologous recombination, a second linear fragment was generated by PCR using primers that annealed to regions flanking the NcoI and StuI restriction sites, creating a PCR fragment with \>100bp of overlap homology to the region removed from the guide+donor backbone. Digested DNA and PCR amplicons (1μg each per transformation) were co-transformed into yeast using standard lithium acetate transformation protocol with the addition of dimethyl sulfoxide (DMSO, 10% final concentration) before heat shock and grown on SC-URA plates for 3 days to obtain Ura^+^ colonies.
For our initial library pilot experiments ([Figures 1d b 1e](#F1){ref-type="fig"}, and [Supplementary Figures 6b and 6c](#SD1){ref-type="supplementary-material"}), 500ng of each indicated guide+donor plasmid were pooled together and double-digested with NcoI and StuI. 1μg of the linearized plasmid mix was co-transformed with 1μg of Ura3 PCR fragment (as described above) into Cas9-expressing wildtype and Mms4-inactivated strains in parallel and selected on SC-URA. Ura+ colonies were scraped off plates after 3 days. For HU sensitivity screen, cells were further diluted 1:100 in liquid media that contains no HU or 40mM HU and grown for 2 days. Cells were collected and genomic DNA was extracted for NGS. Two rounds of independent yeast library transformations were performed.
For the HU condition test of the *SGS1* mutant libraries, each library was first transformed into no-Cas9 and Cas9-expressing cells in parallel using the yeast transformation procedures as described above and selected on SC-URA. After 3 days, colonies were scraped off the plates, diluted 1:100 in liquid media that contains no HU or 40mM HU, and grown for 2 days. Cells were then collected and genomic DNA was extracted for NGS. Experiments were done in duplicates.
For the essentiality/non-essentiality test of smORF library, the library was transformed into no-Cas9 expressing cells and Cas9-expressing cells in parallel. Colonies were scraped and diluted 1:100 in liquid media and grown for 2 days. In addition, transformants from the Cas9-expressing cells were also grown in liquid media containing either 100mM HU, 25μg/ml fluconazole, or subject to 37°C for 2 days. Subsequently, cells were collected, genomic DNA extracted, and NGS was performed. All experiments were done in duplicate.
Guide+donor library preparation and sequencing {#S7}
----------------------------------------------
Genomic DNA was isolated from each yeast sample. Two rounds of PCR were performed using Q5^®^ Hot Start High-Fidelity polymerase (New England Biolabs). The first round amplified each guide+donor with forward (CTTTCCCTACACGACGCTCTTCCGATCTNNNNNNAGTGAAAGATAAATGATC) and reverse primers (GGAGTTCAGACGTGTGCTCTTCCGATCTGCGAATTGGGTACCATGT) hybridizing to common flanking regions. Subsequently, standard Illumina Truseq and/or Nextera barcodes were attached through a second round of PCR amplification. Gel purification was performed on all amplicons to confirm the amplicon size and quality before extracting and purifying the sample using the QIAquick gel extraction kit (Qiagen). DNA libraries for NGS were quantified using the KAPA Library Quantification Kit (Kapa Biosystems). Samples were pooled in equimolar amount. The final library was prepared using standard MiSeq Reagent Kit v2 (2 × 150bp) protocol with 12pM diluted DNA libraries with 15% to 25% PhiX spiked into the mixture and run on an Illumina MiSeq or NextSeq 500 Systems, respectively.
Preprocessing of library sequences and count generation {#S8}
-------------------------------------------------------
Guide RNA and donor sequences were extracted from R1 and R2 reads, respectively, and mapped to reference library members containing each guide+donor pair using a custom python script. Sequences that do not match to any of the library members were discarded from the analysis. Only sequences that contain the perfectly matched N20, sgtail (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGGTGCTTTTTTTGTTTTTTATGTCT) and donor sequences were included in count generation. We first sequenced the plasmid libraries to determine the distribution of sequences. Reads that were severely underrepresented, i.e. less than 30 reads mapped to the guide+donor, were removed from further analysis.
Data analysis and fitness calculation {#S9}
-------------------------------------
For all the conditions, the mapped reads were compared against the corresponding control experiment. The control experiment for each *SGS1* library (tiling deletion and amino acid substitutions) was the experiment performed in the absence of HU. A fold change (FC) for each guide+donor is calculated as follows: $$FC_{i} = \frac{\frac{\mathit{test}_{i}}{\mathit{test}\mathit{total}_{i}}}{\frac{\mathit{control}_{i}}{\mathit{control}\mathit{total}_{i}}}$$ where *test~i~* and *control~i~* are the number of reads that mapped to guide+donor *i* in all the test conditions and control, respectively. The *test total~i~* represents the total number of reads in the test conditions and *control total~i~* is the number of reads in the control. If the guide+donor is enriched in the test condition, FC would be \>1. If the guide+donor is depleted in the tested condition, FC would be \<1. The average log~2~FC values of the duplicates and p-values (2-tailed Z-test) corresponding to each tested guide+donor for each library are provided in [Supplementary Files 1 and 2](#SD1){ref-type="supplementary-material"}.
The smORF library was subjected to four screens: essentiality, heat, HU, and fluconazole. While the control experiment for the last 3 test conditions was conducted in the absence of the environmental stress, the control experiment for essentiality screen was performed in a yeast strain lacking Cas9. The same FC calculation described above was carried out for each guide+donor in the smORF library. [Supplementary File 3](#SD1){ref-type="supplementary-material"} lists the average log~2~FC value and p-value for each tested guide+donor.
Validation of mutants from the three libraries {#S10}
----------------------------------------------
Individual Ura+ transformants were picked from each library and grown overnight in 96-well plates. DNA extraction was performed followed by PCR amplification (forward primer TTCGGCGTTCGAAACTTCTCCGCA and reverse primer TAGACCGAGATAGGGTTGAGTG) and sequencing of the guide+donor on the plasmid (TTCGGCGTTCGAAACTTCTCCGCA) to determine programmed edits intended for each transformant. Individual primer pairs specific to the corresponding endogenous site were designed. Each endogenous site was amplified and sequenced with the forward primer to determine if the programmed edits as specified by the donor had successfully occurred.
Phenotypic validation of library hits {#S11}
-------------------------------------
To validate the hits exhibiting phenotypic sensitivity and lack of sensitivity in each library screen, we picked 2--4 sensitive and 2--4 non-sensitive targets from each screen, constructed the corresponding guide+donor plasmids, and performed similar transformation experiment as described above. Individual transformants were genotyped followed by phenotyping onto the corresponding test conditions to confirm our NGS screening results. For the phenotypic growth assay, cells were grown to log phase. 3μl of each undiluted and 5-fold serially diluted culture were spotted onto SC-URA or SC-URA under tested conditions. All plates were incubated at 30°C for 48 hours and photographed.
Feature examination of hit versus non-hit between smORFs and ORFs {#S12}
-----------------------------------------------------------------
### Comparison of target length {#S13}
The amino acid length for each target in the library was obtained from YeastMine^[@R30]^. The distributions of protein sizes between the different groups, namely smORF hits versus non-hits and ORF hits versus non-hits, were compared. To determine if there is a significant difference in amino acid lengths between groups, we performed a 2-tailed t-test (summarized in [Supplementary Table 3](#SD1){ref-type="supplementary-material"}, [Supplementary File 4](#SD1){ref-type="supplementary-material"}).
### Comparison of gene expression {#S14}
The FPKM values for the targets were generated as follows. Raw RNA-seq data for BY4741 yeast strain was obtained from SRA (SRR3126113)^[@R43]^. FASTX Toolkit (<http://www.bioinformatics>. babraham.ac.uk/projects/fastqc/) was used to remove the adapters (fastx_trimmer) and trim the ends of base pairs with a quality score lower than 30 (fastq_quality_trimmer). After quality trimming, the read pairs were intersected using an in-house pipeline. Subsequently, the reads were aligned to the S288C genome (Bioproject) using Tophat^[@R44]^ and the FPKM vales were generated using Cufflinks^[@R45]^. To determine if the expression levels between the different groups were significant, we performed a 2-tailed t-test between the log~10~ FPKM values between the hits and non-hits of the smORF class and ORF class (summarized in [Supplementary Table 4](#SD1){ref-type="supplementary-material"}, [Supplementary File 4](#SD1){ref-type="supplementary-material"}).
### Comparison of secondary structure {#S15}
We mapped the possible presence of secondary structures (alpha-helices and beta-sheets) in each amino acid sequence using PredictProtein^[@R46]^. Several comparisons with regards to the overall distribution of secondary structures between different groups were made (summarized in [Supplementary Table 5](#SD1){ref-type="supplementary-material"}, [Supplementary File 4](#SD1){ref-type="supplementary-material"}) and examined for significant difference through Kolgomorov-Smirnov test.
### Comparison of homolog conservation in human {#S16}
The corresponding human homologues for the targets in the smORF library were obtained from YeastMine^[@R30]^. The number of targets containing a human homologue were counted and compared among different groups (summarized in [Supplementary Table 6](#SD1){ref-type="supplementary-material"}, [Supplementary File 4](#SD1){ref-type="supplementary-material"}). A Chi-squared test was used to test for significant difference between the different groups.
Comparison of transformation and editing efficiencies between unmodified and engineered approaches {#S17}
--------------------------------------------------------------------------------------------------
Thirty-four guide+donor contigs were selected from the smORF library screen and were individually constructed followed by Gibson-cloning into pRS426 backbone as described above. Each smORF-targeting guide+donor plasmid construct was introduced into a Cas9-expressing yeast strain in either the unmodified or the engineered configurations. A similar transformation was also carried out side-by-side in a non-Cas9-expressing yeast strain. Colony counts for each guide+donor transformation were obtained 3 days post-transformation. A fold change in transformation efficiency was calculated based on the colony count generated from the engineered approach divided by the colony count obtained from the unmodified approach. In addition, 5 random colonies from each guide+donor transformation were PCR amplified and Sanger sequenced at the corresponding endogenous site to determine if the correct genomic edit took place. Editing efficiency was determined by the proportion of sequenced transformants with the correct genomic edit over the total number of sequenced transformants. This whole experiment was performed twice. All Sanger sequencing was performed by Genewiz, Inc.
Effect of homology length on more distant SNPs editing {#S18}
------------------------------------------------------
*ADE2*-targeting guide+donor plasmids with various homology lengths (60bp, 70bp, 80bp, 90bp and 100bp) on the donor sequence to introduce genomic edits of SNPs at different PAM-distant positions ([Supplementary Figure 3a](#SD1){ref-type="supplementary-material"}) were constructed via Gibson assembly. Each guide+donor construct was transformed in the engineered configuration into both yeast strains expressing and not expressing Cas9 to examine effect of homology length on transformation efficiency. Transformation efficiency was represented by the number of transformants obtained in the presence of Cas9 over the number of transformants obtained in the absence of Cas9. Each transformation experiment was performed twice. A few colonies from each transformation were PCR amplified and Sanger sequenced at the *ADE2* target site to determine the proportion of correct genomic edits.
Whole genome sequencing to detect off-target effects of guide+donor system {#S19}
--------------------------------------------------------------------------
### Sample preparation {#S20}
A Cas9-expressing parental yeast strain (YAC2563) and three yeast strains (YXG231, YXG232, YXG234) modified by guide+donor plasmids, ADE2Δ61bp, SGS1Δ60bp, and SGS1ΔATG, respectively, were grown overnight in 5ml YPAD. Genomic DNA was isolated from these cells followed by a PCR purification (Zymo Research) step to clean up the DNA. For library preparation, we used Nextera (Illumina) to fragment the genome. Roughly 35ng of genomic DNA was used for each sample, equivalent to 3 million haploid yeast genomes. After the tagmentation reaction (20μL reaction system, 55°C 15min, 70°C 30min), fragmented DNA was purified with DNA Clean-up & Concentrator-5 (Zymo Research) and used as PCR template (NEBNext® High-Fidelity 2X PCR Master Mix, NEB, 72°C 3min for Tn5 gap filling and end repair, 98°C 30s, 4 cycles of 98°C 10s, 63°C 30s, 72°C 40s, and 72°C 2min for a final extension) to add sequencing adaptors. Amplified library was cleaned up with 0.8× Ampure beads and sequenced with 2x 150bp NextSeq500/550 for a total of 28M paired-end reads.
### Computational analysis {#S21}
The quality of the fastq files was first evaluated using the FASTQC tool (<https://www.bioinformatics.babraham.ac.uk/projects/fastqc/>) followed by end trimming using FASTX Toolkit (<http://hannonlab.cshl.edu/fastx_toolkit/>) to obtain base pairs with a quality score lower than 30 (fastq_quality_trimmer). After quality trimming, an in-house algorithm was used to intersect the read-pairs. Subsequently, BWA (version 0.6.1-r104) was utilized to align the reads to the S288C genome downloaded from Genbank as assembly GCA_000146045.2. SNPs were detected by SAMtools mpileup and bcftools. A hard filter removing all SNPs/indels below 25% of the median depth was chosen as cutoff. The median depth was deduced using genomeCoverageBed from BEDTools (version 2.16.2) as described by Kaas *et al*^[@R47]^. Off-target analysis was carried out using Bowtie (version 0.12.7) to search the yeast genome for the guide RNA sequences corresponding to the guide+donor constructs for up to 2 mismatches. A region of 500bp surrounding each of the potential off-target sites were manually cross-referenced with the list of detected SNP/indels as previously described^[@R48]^. The expected genomic changes were manually evaluated from the aligned BAM file in Geneious (Biomatter Ltd).
Statistical analysis {#S22}
--------------------
Each figure description indicates the number of independent experiments. A 2-tailed Z-test was used to examine significance of depletion in each library screen in [Figures 2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}. A 2-tailed Z-test was applied to examine depletion and enrichment in [Figure 4](#F4){ref-type="fig"}. A Chi-squared test was used to assess statistical differences between groups in [Supplementary tables 1 and 2](#SD1){ref-type="supplementary-material"}. A 2-tailed t-test was to examine statistical significance in [Supplementary tables 3 and 4](#SD1){ref-type="supplementary-material"}. Kolgomorov-Smirnov and Chi-squared tests were used to assess the statistical differences between groups in [Supplementary Tables 5 and 6](#SD1){ref-type="supplementary-material"}, respectively.
Data availability {#S23}
-----------------
All NGS data generated in this study are available through NCBI SRA. Data used for amino acid length, gene expression, and human conservation comparisons are presented on [Supplementary File 4](#SD1){ref-type="supplementary-material"} and summarized in [Supplementary Tables 3--6](#SD1){ref-type="supplementary-material"}.
Code availability {#S24}
-----------------
All custom scripts are available upon request.
Supplementary Material {#S25}
======================
G.M.C was supported by NIH grants RM1 HG008525 and P50 HG005550. A.C. was funded by the National Cancer Institute grant no. 5T32CA009216-34. J.J.C was funded by the Defense Threat Reduction Agency grant HDTRA1-14-1-0006, the Paul G. Allen Frontiers Group. Y.Y. was supported by the Damon Runyon Research Foundation grant DRG-2248-16. X.G. and A.C. conceived the idea, led the study, and designed all experiments. A.C. and R.C. with input from J.E.D. demonstrated the initial feasibility of the guide+donor approach. X.G. performed majority of the experiments, including the oligo library design, library construction and analysis with significant technical contribution from A.T. Y.C. provided expertise in statistical analysis. Y.Y performed the whole genome sequencing experiment for off-target analysis. C.K. generated the RNA-seq data for BY4741 yeast strain, provided the FPKM values and analyzed the whole genome data from yeast isolates modified by guide+donor for off-target effects. S.L.G. assisted with oligo library design. E.K. provided insight with regard to library construction methods and analysis. M.S. provided technical expertise with regard to methods to increase guide+donor efficiency. J.J.C and G.M.C. oversaw the study. X.G. and A.C. wrote the manuscript with input from all authors.
COMPETING FINANCIAL INTERESTS
G.M.C. is the founder and holds leadership positions of many companies (<http://arep.med.harvard.edu/gmc/tech.html>). X.G., A.C., M.S. and E.K. have filed a patent application (U.S. Patent Application 62/348,438) relating to this work.
{#F1}
{#F2}
{#F3}
{#F4}
[^1]: Co-first authors
[^2]: Current address: Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York, USA
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Pediatric Sarcoidosis is a rare multisystem granulomatous disorder and series from the Asian subcontinent are few.
Aim
===
We describe our experience to date with an inceptional cohort.
Methods
=======
Retrospective chart review of the demographic, clinical, diagnostic and genetic characteristics were studied.
Results
=======
Over seven years, 12 of 1214 new cases seen in the Pediatric Rheumatology Clinic (1%) (M: F = 1:1), were diagnosed as Sarcoidosis. 11/12(91.7%) had an onset ≤4 years of age, 8/12(67%) maintained a cumulative follow-up of 33.7 years ( range 1-9 years). 7/12(58.3%) had received anti-tuberculous therapy prior to referral.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Cohort features Characteristics at Onset/ 1^st^ visit Follow-up ( ≥ 1 year)
--------------------------------------------- -------------------------------------------------------------- ------------------------------------------------------------
Number(N)\ 12\ 8
Median age (years) \[IQR\] Onset:1.25\[0.9-1.7\]Diagnosis:7.8\[4.9-9.9\]
Fever; median duration 10/12(83.3%);18 months Resolution
Arthritis 7/12(58.3%) Resolution
Skin manifestations 7/12(58.3%) Resolved 6/8(75%)
Ocular abnormalities Onset: 6/12(50%), 1st visit: 5/6(83.3%) 2/5(40%)
Triad-Arthritis,Rash,Uveitis Onset: 5/12(41.7%), 1st visit: 3/5(60%) None
Systemic features Sicca(1),Adenopathy(4),GIT(4), Pulmonary(5), Organomegaly(9) Aortoarteritis(1/8)\
Interstitial lung(1/8)
Growth retardation 12/12(100%) 5/8(62.5%)
Median Steroid dose(mg/kg/day) \[IQR\]\ 0.6\[0.2-0.8\]\ 0.3\[0.1-0.3\]\
Median Methotrexate dose(mg/m2BSA) \[IQR\]\ 10.1\[0-10.4\] 10.9\[9.7-15.3\]\
Others: Azathioprine(vasculitic rash)(1),Mycophenolate(uveitis)(1)
Treatment side-effects \- Hepatotoxicity(1/8) Osteoporosis(2/8) Cataracts(3/8)
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Diagnosis was by clinical presentation \'plus\': ACE (4/12), biopsy (1/12), biopsy and ACE (3/12), biopsy and mutation (1/12), mutation (2/12). 3/9(33.3%) are positive for CARD15 mutation (Blau Syndrome). 2 have sporadic mutations at R334W while 1 with a mutation at G464W, developed cardiomyopathy and aortoarteritis and has a symptomatic parent with the identical mutation. None of the 8 patients following up are off therapy. 5/8(62.5%) achieved clinical improvement in a median duration of 6.9 months\[5.6-9.6 IQR\].
Conclusions
===========
In our setting, Pediatric Sarcoidosis had a significant time lag to diagnosis, being often initially diagnosed as tuberculosis owing to similar clinical picture and histology. Morbidity is considerable, with arthritis, fever and rash responding to therapy while eye changes and organ damage are relatively refractory. All children show significant growth retardation at diagnosis and follow up inspite of control of constitutional features. Amongst the 3 Blau Syndrome patients, one had an atypical presentation and an autosomal dominant inheritance.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction
===============
The cytolethal distending toxins (CDTs) comprise a family of bacterial proteins toxins produced by a variety of Gram negative bacteria, such as *Escherichia coli*, *Aggregatibacter actinomycetemcomitans*, *Haemophilus ducreyi*, *Shigella dysenteriae*, *Campylobacter* sp., *Helicobacter* sp., and *Salmonella enterica*.
CDT and Colibactin, a putative hybrid peptide-polyketide genotoxin produced by commensal *E. coli* strains \[[@B1-toxins-03-00172]\], are the first bacterial genotoxins described, having the unique characteristic to cause DNA damage in the target cells.
In this review, we will focus on the molecular mode of action, the internalization pathway and the cellular responses induced by CDT intoxication. We will further discuss the role of these toxins as virulence factors in bacterial pathogenesis.
To facilitate the reading, we have adopted the nomenclature proposed by Thelestam *et al.*, where each CDT is specified by indicating the initials of the producing bacterium before CDT and, if necessary, the strain number or other common designation after CDT (e.g., HdCDT: *H. ducreyi* CDT or EcCDT-I: *E.* *coli* CDT type I) \[[@B2-toxins-03-00172]\].
2. CDT Structure and Enzymatic Activity
=======================================
CDT is the product of an operon encoding three proteins: CdtA, CdtB and CdtC. All three subunits are essential to confer full activity of the holotoxin (reviewed in \[[@B3-toxins-03-00172]\]).
The crystal structure of the *H. ducreyi* CDT (HdCDT) has been solved by Nesic and collaborators, and revealed that the holotoxin is a tripartite complex. The CdtA and CdtC subunits are lectin-type molecules, sharing structural homology with the B-chain repeats of the plant toxin ricin. The CdtB subunit adopts the canonical four-layered fold of the DNase I family: a central 12-stranded β-sandwich packed between outer α-helices and loops on each side of the sandwich \[[@B4-toxins-03-00172]\]. The crystal structure confirms previous data, demonstrating that CdtB shares five conserved residues with the active site of the mammalian DNase I, and possesses DNase capacity *in vitro* and when ectopically expressed or microinjected in eukaryotic cells. Mutation in any conserved residue important for the catalytic activity or the Mg^2+^ binding abolishes the ability of CdtB to cleave DNA *in vitro* and to induce DNA damage responses *in vivo* \[[@B5-toxins-03-00172],[@B6-toxins-03-00172],[@B7-toxins-03-00172],[@B8-toxins-03-00172]\].
The three subunits form a complex with three globular protein-protein interfaces (CdtA-CdtB, CdtA-CdtC and CdtB-CdtC). Furthermore, the CdtA and CdtC subunits present non-globular amino acid extensions at the amino- and carboxyl-termini, which interact with each other and with the CdtB subunit. Two very conserved structures can be observed within the surface formed by the CdtA and CdtC subunits: (1) a large aromatic cluster of eight bulky side-chains in CdtA; (2) a deep groove formed by the juxtaposition of these subunits. Mutations of the aromatic patch do not change the stability of the ternary complex, but completely abolished the ability of the toxin to cause cell cycle arrest in the human cell line HeLa, suggesting that it plays a relevant role in modulating toxin binding to its receptor \[[@B4-toxins-03-00172]\].
The CdtB subunit is the most conserved component of the holotoxin amongst all the CDT-producing bacteria. The overall sequence identities of CdtA and CdtC homologs are generally less than 30%. However modeling studies based on the HdCDT crystal structure showed that a number of structural features are remarkably conserved, such as the close interplay of the CdtA and CdtC subunits in the formation of the groove and aromatic patch, and the similarity in their positioning with the two lectin repeats in the ricin B-chain. This suggests that these two components of CDT work together to mediate cell surface binding and internalization of the holotoxin \[[@B9-toxins-03-00172]\].
Based on these data, CDT can be regarded as an A-B~2~ toxin, where CdtA and CdtC are required for binding the holotoxin to the plasma membrane of the target cells, allowing entry of the active CdtB, which can translocate to the nucleus and induce DNA lesions.
There are still several open questions regarding the interaction of the holotoxin with the target cells. Little information is available on the biogenesis of CDT holotoxin. Furthermore, it is still not clear how CdtA and CdtC contribute to the binding on the plasma membrane, and the nature of the CDT receptor still remains unknown.
To study the biogenesis of A. *actinomycetemcomitans* (AaCDT), Ueno and co-workers have used an *E. coli* strain carrying the *A. actinomycetemcomitans cdtABC* genes. They have shown that membrane-associated CdtA is a lipoprotein. In the periplasm, CDT is a complex composed of CdtA, CdtB, and CdtC, whereas CDT in the culture supernatant contains an *N*-terminally truncated CdtA. This suggests that CdtA undergoes lipid modification during the export process and subsequent *N*-terminal processing after forming a complex with CdtB and CdtC in the periplasm \[[@B10-toxins-03-00172]\].
Using enzyme-linked immunosorbent assays or FACS analysis it was shown that the CdtA and CdtC subunit from the *Campylobacter jejuni* (CjCDT), *E. coli* (EcCDT-II), and AaCDT bind with specificity to the surface of the human cell lines HeLa \[[@B11-toxins-03-00172],[@B12-toxins-03-00172]\] or U937 \[[@B13-toxins-03-00172]\], whereas CdtB does not. Conversely, Di Rienzo and colleagues showed that the *A. actinomycetemcomitans* CdtA, but not the CdtC subunit, binds to the surface of Chinese hamster ovary (CHO) cells \[[@B14-toxins-03-00172]\]. This discrepancy could be related to the different profile of surface molecules expressed on the cell types used. Alternatively, it is possible that the CdtA subunit is responsible to bind on the surface of the plasma cell, while CdtC may assist the trafficking of the CdtB subunit toward the nuclear compartment, as demonstrated for the AaCDT \[[@B15-toxins-03-00172]\]. This hypothesis is supported by the fact that the CdtC, but not the CdtA subunit, transits via the Golgi complex similarly to the active CdtB component of the holotoxin \[[@B16-toxins-03-00172]\].
What is the nature of the CDT receptor? Several studies has been conducted, and yielded to contrasting results.
EcCdtA-II and CjCdtA are able to compete with the binding of the corresponding CdtC subunits, indicating that they interact with the same structure on the target cell, which was identified as *N*-linked glycoproteins \[[@B12-toxins-03-00172]\]. Moreover, EcCDT binds fucose *in vitro*, and fucose-specific lectins block Ec-CDT-mediated cell cycle arrest, presumably by preventing binding of toxin to its receptor. These findings suggested that fucose is a binding determinant for Ec-CDT.
Another study indicated that, similarly to other bacterial proteins toxins, the CDT holotoxin binds to surface glycosphingolipids. Inhibitors of glycosphingolipids synthesis prevents intoxication of the human monocytic U937 cell line by AaCDT, and thin layer chromatography demonstrates a strong binding of the CdtA with GM1, GM2, GM3, and Gb4, while CdtC reacts strongly with GM1 and GM2. Intoxication of the U937 cell line is blocked by preincubation of toxin with liposomes that contain GM3 \[[@B13-toxins-03-00172]\].
Using laser confocal microscopy Shenker *et al.*, have demonstrated that the AaCDT colocalizes with GM1-enriched membrane regions of the plasma membrane, which are characteristic of membrane rafts \[[@B8-toxins-03-00172],[@B17-toxins-03-00172]\]. Cholesterol depletion by methyl β-cyclodextrin reduces the ability of the AaCDT or HdCDT to associate with Jurkat or HeLa cell lines, respectively, and prevents the toxic activity \[[@B8-toxins-03-00172],[@B17-toxins-03-00172]\].
Site directed mutagenesis of a human cell line haploid for all chromosomes except chromosome 8 identified SGMS1 and TMEM181 mutants as resistant to EcCDT. The SGMS1 mutation reduces levels of sphingomyelin, a key component of lipid rafts, confirming the previous data obtained for HdCDT and AaCDT. TMEM181 is present at the cell surface. Flag-tagged CDT is able to bind HA-tagged TMEM181, suggesting that this protein may be the EcCDT receptor, however it could not be ruled out that TMEM181 plays a role in the trafficking of the receptor-toxin complex \[[@B18-toxins-03-00172]\].
The divergent results on factors that mediate the toxin binding on the cell surface may depend on the different receptor specificity exhibited by each CDT. It is noteworthy that several CDT are species specific: HdCDT and AaCDT cannot intoxicate cells of rodent origin. Furthermore, Eshraghi and co-workers have demonstrated that CDTs from *H. ducreyi*, *A. actinomycetemcomitans*, *E. coli*, and *C. jejuni* differ in their abilities to intoxicate host cells. Binding of Aa, Hd, and EcCDT-III, but not CjCDT is dependent on the presence of cholesterol. Surprisingly, mutant CHO cells that lack *N*-linked complex and hybrid carbohydrates, cells that lack glycosphingolipids or are deficient in fucose biosynthesis are similarly sensitive as the wild type to intoxication by all four CDTs tested, indicating that *N*- and *O*-glycan, or fucosylated structures are dispensable to mediate toxin binding \[[@B19-toxins-03-00172]\].
An exception to the general structure of the CDT holotoxin is the CDT produced by *S. enterica*, serovar Typhi CT18. The *cdtB* gene is located within a region of the chromosome, however there are no homologues of the *cdtA* or *cdtC* genes \[[@B20-toxins-03-00172]\]. Ectopic expression of the StCdtB in Cos-2 is sufficient to cause DNA damage, and mutation of the key conserved residues of the DNase activity abrogates toxicity, indicating that this protein acts as a CDT active subunit \[[@B21-toxins-03-00172]\]. CDT intoxication in *S. typhi* infected cells requires the products of two genes, named *pltB* (persussis-like toxin B) and *pltA* (persussis-like toxin A), and similarly to *cdtB*, their expression is induced after bacteria uptake by the host cells. The role of these components is most likely to transport CdtB from its site of production to the extracellular medium, from where StCDT can also intoxicate cells that have not been infected with the bacterium, in a paracrine manner \[[@B22-toxins-03-00172]\].
3. Internalization
==================
CDT is the first bacterial protein toxin known to act in the nucleus of the target cell. As discussed in the previous paragraph, binding to the plasma membrane is a pre-requisite for the intoxication, and as many other bacterial protein toxins, CDT has to cross the plasma membrane to reach the nucleus. The internalization pathway has been mainly studied using HdCDT, AaCDT and EcCDT-II as a model. HdCDT is internalized in HeLa cells by dynamin-dependent endocytosis \[[@B23-toxins-03-00172]\], but it does not require clathrin, since conditional knock down of this protein by RNA interference does not prevent intoxication \[[@B16-toxins-03-00172]\]. Induction of CDT-mediated DNA damage is completely inhibited under conditions that block the fusion of the endosomal compartment with downstream compartments, indicating that the toxin further transits through the endosomal compartment \[[@B23-toxins-03-00172]\]. Using a holotoxin with a modified CdtB subunit, carrying either a sulfation site, or a sulfation and three partially overlapping *N*-linked glycosylation sites at the *C*-terminus, it was possible to demonstrate that the active subunit transits via the trans-Golgi, where sulfation occurs, and it is retrograde translocated via the endoplasmic reticulum (ER), where it is glycosylated \[[@B8-toxins-03-00172]\]. Many ER-translocating toxins exploit the ER-associated degradation (ERAD) pathway to transit from the ER to the cytosol. However, CDT intoxication occurs in cells carrying an altered ERAD system, and its translocation does not require protein unfolding \[[@B8-toxins-03-00172],[@B24-toxins-03-00172]\]. Furthermore, using a combination of confocal microscopy analysis with ER specific markers and biochemical assays, Guerra *et al.* have failed to detect the presence of the CdtB subunit in the cytosol of the intoxicated cells. All these data indicate that most likely the active subunit of CDT is directly translocated from the ER to the nucleus, where it exerts its genotoxic activity \[[@B24-toxins-03-00172]\].
[Figure 1](#toxins-03-00172-f001){ref-type="fig"} summarizes the key steps in CDT internalization.
{#toxins-03-00172-f001}
How the nuclear translocation of the CdtB subunit takes place is still an open question. Nishikubo and co-workers have identified an atypical nuclear localization signal (NLS) within the amino-terminal region of the AaCdtB, which is essential for nuclear translocation of the recombinant His-tagged CdtB-GFP. Deletion of 11 amino acids within the NLS sequence of CdtB abolishes intoxication \[[@B25-toxins-03-00172]\]. On the other hand, two NLS sequences, designated as NLS1 and NLS2, have been identified in the carboxy-terminal region of the EcCdtB-II. Deletion of these two regions prevents induction of cell cycle arrest and nuclear localization of the holotoxin, without affecting its DNase activity, as tested in *in vitro* assays \[[@B26-toxins-03-00172]\]. Interestingly, deletion of each of these sequences produces a differential localization of the active toxin subunit. Cells intoxicated with a holotoxin containing the EcCdtB-II-ΔNLS1 display a perinuclear distribution, which is consistent with trapping of the active toxin component in the late endosome and/or ER compartment. A diffuse cytoplasmic staining is observed in cells exposed to the EcCdtB-II-ΔNLS2 containing toxin. It is possible that this second NLS may act as scavenger motif to retrieve CdtB molecules, which have escaped to the cytosol, similar to the suggested ER retention function of the KDEL sequence in cholera toxin \[[@B27-toxins-03-00172]\].
The presence of NLSs in entirely different parts of the CdtB molecule in the AaCdtB and EcCdtB-II is still puzzling, considering the high degree of conservation of this subunit amongst different bacteria species \[[@B28-toxins-03-00172]\].
Another important point of discussion is the observation that the majority of the studies on CDT internalization have been performed using recombinant purified soluble holotoxin. However, it has been reported that both EcCDT-II and CjCDT are secreted within outer membrane vesicles (OMVs) \[[@B29-toxins-03-00172],[@B30-toxins-03-00172]\]. It will be interesting to assess whether the delivery of the OMV-associated holotoxin follows the same route as the soluble complex.
4. Cellular Responses
=====================
4.1. Induction of DNA Damage
----------------------------
The first demonstration that CDT is a genotoxin stems from the work of Elwell *et al.* and Lara-Tejero *et al.* who identified position-specific homology between the CdtB subunit from EcCDT-II and CjCDT, respectively, and the mammalian DNase I. The sequence conservation is associated with a functional homology, since preparation of EcCDT-II completely degrades a DNA plasmid substrate in 12 h, and ectopic expression of CjCdtB induces nuclear fragmentation and a marked chromatin disruption in HeLa cells \[[@B5-toxins-03-00172],[@B6-toxins-03-00172]\]. Mutations of the conserved residues required for catalysis or for magnesium binding abolish both the DNase activity as well as the cytotoxic effect.
Direct demonstration of CDT-induced DNA fragmentation was subsequently shown using pulsed field gel electrophoresis (PFGE) analysis both in mammalian cells intoxicated with HdCDT and in budding yeast carrying a conditional CjCdtB expressing plasmid \[[@B7-toxins-03-00172],[@B31-toxins-03-00172]\].
It is noteworthy that the efficiency of CDT to degrade DNA is much lower than that of purified bovine DNase I, as seen both in *in vitro* assays with EcCDT-II and upon microinjection of HdCDT in HeLa cells \[[@B32-toxins-03-00172],[@B33-toxins-03-00172]\].
4.2. Activation of DNA Damage Responses
---------------------------------------
To protect the genome from DNA damage induced by endogenous or exogenous sources, cells activate a series of complex mechanisms. These mechanisms are referred to as DNA damage responses (DDRs) that act to repair the genome and minimize the probability of lethal or permanent genetic damage. The cellular response to DNA damage encompasses multiple repair mechanisms and checkpoint responses that can delay cell cycle progression or modulate DNA replication, and are coordinated primarily by two distinct kinase signaling cascades: the ATM-Chk2 and ATR-Chk1 pathways. The ATM-Chk2 and ATR-Chk1 pathways respond to different types of DNA damage: ATM is recruited to and activated primarily at DNA double-strand breaks (DSBs) in conjunction with the MRE11:RAD50:NBS1 (MRN) sensor complex, while ATR is recruited at sites of single-stranded DNA (ssDNA) in association with its partner protein, ATRIP. Recent evidence indicates that the ATR--Chk1 repair axis is also activated in an ATM-dependent manner in response to DSBs. Prolonged exposure to DNA damage will chronically activate the DDR machinery, including the ATM-Chk2-p53 axis, resulting in either cell death or a long-term cell cycle arrest state known as cellular senescence (reviewed in \[[@B34-toxins-03-00172]\]). This response represents an inducible barrier against acquisition of genomic instability and tumor initiation and/or progression (reviewed in \[[@B35-toxins-03-00172]\]).
Based on the DNase activity of CDT, it is not surprising that intoxicated cells activate the full pattern of DDRs. The cells exposed to CDTs have been shown to arrest in the G1 and/or G2 phases of the cell cycle, or undergo apoptosis, depending on the cell type \[[@B36-toxins-03-00172],[@B37-toxins-03-00172],[@B38-toxins-03-00172],[@B39-toxins-03-00172]\]. The CDT-induced cell cycle arrest resembles the checkpoint response to ionizing radiation (IR), characterized by activation of the ATM kinase and ATM-dependent induction of the tumor suppressor p53 and its transcriptional target, the cyclin-dependent kinase inhibitor p21, phosphorylation of histone H2AX and re-localization of the DNA repair proteins, such as MRE11 and RAD50, to the sites of DNA DSBs \[[@B33-toxins-03-00172],[@B39-toxins-03-00172],[@B40-toxins-03-00172],[@B41-toxins-03-00172],[@B42-toxins-03-00172]\].
Guerra *et al.* recently demonstrated that the proto-oncogene MYC plays an important role in response to CDT- and IR-induced DNA damage, since MYC is required for prompt activation of the ATM-dependent DNA damage pathway in irradiated or intoxicated cells \[[@B43-toxins-03-00172]\].
Normal and cancer cells that survive the acute phase of intoxication by HdCDT possess the hallmarks of cellular senescence. This characteristic phenotype includes persistently activated DNA damage signaling (detected as 53BP1/γH2AX-positive foci), enhanced senescence-associated β-galactosidase activity, expansion of PML nuclear compartments, and expression of IL-6, IL-8, IL-20 and IL-24 \[[@B44-toxins-03-00172]\].
A genome wide analysis performed in budding yeast has identified homologous recombination (HR), activation of the DNA damage checkpoint, and S-phase checkpoint as essential mechanisms for the response to CdtB. This study could not conclusively demonstrate a contribution of the non homologous-end joining (NHEJ) repair pathway to the CdtB-induced DNA lesion. Interestingly, the genes identified in this analysis indicate that there are specific features of the CdtB response that do notfully overlap with the components required for direct DSBs \[[@B45-toxins-03-00172]\]. Conversely, as reviewed above, the majority of the responses activated in mammalian cells fully support a relevant role of the ATM-Chk2 axis in response to CDT-induced DNA damage.
4.3. Survival Signals Activated in Intoxicated Cells
----------------------------------------------------
The survival of cells with damaged DNA may promote genomic instability and favor tumor initiation and/or progression (reviewed in \[[@B46-toxins-03-00172],[@B47-toxins-03-00172]\]). Characterization of the survival signals in response to CDT intoxication is therefore relevant to understand whether infection with CDT-producing bacteria can contribute to genomic instability and therefore tumor progression.
In adherent cells, irradiation or CDT intoxication is associated with formation of actin stress fibers \[[@B48-toxins-03-00172],[@B49-toxins-03-00172]\]. This effect is regulated by the activation of the small GTPase RhoA, and promotes cell survival in intoxicated cells \[[@B31-toxins-03-00172]\]. Activation of RhoA and actin stress fiber formation in response to CDT is dependent on the RhoA-specific [g]{.ul}uanine nucleotide exchange factor (GEF) Net1, which is dephosphorylated at a critical inhibitory site \[[@B50-toxins-03-00172]\].
The DNA damage-dependent Net1/RhoA signaling diverges into two different effector cascades: one dependent on the RhoA kinases ROCKI and ROCKII, which controls the formation of actin stress fibers; and one regulated by the mitogen-activated protein kinase (MAPK) p38 and its downstream target MAPK-activated protein kinase 2, which promotes cell survival \[[@B50-toxins-03-00172]\].
The characteristic cell distension observed in intoxicated cells of epithelial and mesenchymal origin is RhoA-independent, and requires a functional PI3-kinase and its downstream effector mTOR \[[@B31-toxins-03-00172]\].
The cellular responses to CDT are summarized in [Figure 2](#toxins-03-00172-f002){ref-type="fig"}.
{#toxins-03-00172-f002}
To identify novel CDT-induced survival signals, we have screened a yeast deletion library in cells expressing the CdtB active subunit under the control of a galactose-inducible promoter. This screen identified 78 deletion mutants with reduced growth rate following inducible expression of CdtB. Bioinformatics analysis revealed that 12 human orthologs of these genes interacts with the RhoA signaling pathway. Functional studies in mammalian cells showed that *FEN1* (yeast ortholog *RAD27*) promotes RhoA activation, MAPK p38 phosphorylation and ultimately cell survival in response to CDT intoxication \[[@B51-toxins-03-00172]\].
4.4. CDT-Induced Apoptosis
--------------------------
While the majority of cells of epithelial and mesenchymal origin are arrested in different phases of the cell cycle, and cell death is a very late event observed after more than 96 h post-intoxication, B and T lymphocytes exposed to HdCDT or AaCDT, respectively, are more susceptible to apoptosis \[[@B39-toxins-03-00172],[@B52-toxins-03-00172]\].
Most of the studies aimed at understanding the molecular mechanisms associated with CDT-induced cell death have been performed using as model AaCDT.
Treatment of activated human T cells with AaCDT induces activation of caspases 8, 9 and 3, and DNA fragmentation is detectable 72--96 h after intoxication \[[@B52-toxins-03-00172]\]. These effects are associated with mitochondrial changes, such as decreased transmembrane potential and elevated levels of reactive oxygen species. Overexpression of Bcl-2 decreases the CDT-induced apoptosis, without inhibiting the G2 arrest \[[@B52-toxins-03-00172]\]. AaCDT intoxication also induces cell death in a biphasic manner in two T cell leukemia cell lines, Jurkat and MOLT-4 \[[@B53-toxins-03-00172]\]. In the presence of the caspase inhibitor z-VAD-fmk, CDT-inducedapoptosis is completely blocked for 16 h in Jurkat cells, suggesting that CDT-induced cell death is dependent on caspase activation. However, a subpopulation of cells dies at a later stage of intoxication (more than 24 h post-intoxication) by a caspase-independent cell death \[[@B54-toxins-03-00172]\].
Also cells of the myeloid linage are sensitive to CDT mediated cell death. AaCDT intoxication induces apoptosis in both proliferating and non-proliferating U937 monocytic cells. The induction of apoptosis in proliferating U937 cells is caspase-dependent and requires the DNase activity of CdtB. In contrast, apoptosis in non-proliferating cells is caspase independent \[[@B55-toxins-03-00172]\]. Immature dendritic cells exposed to HdCDT show signs of cell death 24 h to 48 h after intoxication \[[@B33-toxins-03-00172]\]. Interestingly the effect of HdCDT on DCs is dependent on their stage of differentiation, since LPS-treated DCs are resistant to HdCDT-induced cell death \[[@B33-toxins-03-00172]\]. It is possible that upon DC maturation, the repertoire of surface molecules is modified in such a way that HdCDT is no longer able to bind and be internalized. This hypothesis is supported by the lack of activation of DNA damage sensor complex MRN and stabilization of p53 in LPS-treated DCs exposed to HdCDT \[[@B33-toxins-03-00172]\].
4.5. CDT as Phosphatase
-----------------------
Shenker and colleagues have reported that the CdtB subunit from *A. actinomycetemcomitans* exhibits PI-3,4,5-triphosphate (PI-3,4,5-P~3~) phosphatase activity similar to that of the tumor suppressor phosphatases PTEN and SHIP1. Mutation analysis indicates that CDT toxicity correlates with the phosphatase activity, and lymphocytes treated with the toxin exhibit reduced PI-3,4,5-P~3~ levels. Finally, lymphocyte sensitivity to CDT-induced G2 arrest correlates with intracellular levels of PI-3,4,5-P~3~ \[[@B56-toxins-03-00172]\].
Conversely, specific CdtB mutations that inhibit the phosphatase activity but retain the DNase activity, are sufficient to induce cell death in proliferating U937 monocytes \[[@B55-toxins-03-00172]\]. Furthermore, the G2 arrest and cell death induced by conditional expression of CdtB in *Saccharomyces cerevisiae* depend exclusively on its DNase-catalytic residue, since yeast does not harbor the substrate for the CdtB phosphatidylinositol-3,4,5-triphosphate phosphatase activity. These results suggest that the DNA damaging activity alone is sufficient to confer the CdtB toxicity \[[@B57-toxins-03-00172]\].
The discrepancy between the requirements of the different enzymatic activities of CDT may depend on the cell type used as model. It is conceivable that T lymphocytes are more susceptible than all the other cell lines tested to the phosphatase activity of CDT.
5. Role of CDT as Virulence Factor
==================================
In this paragraph, we will briefly review the possible role of CDT in bacterial pathogenesis, and we will focus on three bacterial-associated diseases: inflammatory bowel disease and CDT-producing enteric bacteria; periodontitis and *A. actinomicetemcomitans*; delayed wound healing in chancroid and *H. ducreyi* ([Figure 3](#toxins-03-00172-f003){ref-type="fig"}).
{#toxins-03-00172-f003}
5.1. Role of CDT in Colitis and Inflammation-Associated Carcinogenesis
----------------------------------------------------------------------
Several CDT-producing bacteria are associated with gastro-enteritis, including *C. jejuni* and *Helicobacter hepaticus*.
In the past ten years, it has been clearly shown that chronic inflammation is associated with enhanced risk of tumor development, and the best-studied model is colorectal carcinoma (CRC) \[[@B58-toxins-03-00172],[@B59-toxins-03-00172]\]. It is likely that in the case of bacterial infection several events, such as the establishment of persistent inflammation, the production of toxins that interfere with regulation of cell cycle progression and apoptosis in association with host genetic factors contribute to the acquisition of genomic instability, and consequently tumor initiation and/or progression.
Epidemiological evidence has linked chronic bacterial infections with increased risk of tumor development. *Helicobacter pylori* is associated with gastric cancers and has been classified as a type I carcinogen by the World Health Organization \[[@B60-toxins-03-00172]\]. There is growing evidence that other gastric *Helicobacter* species may be associated with chronic liver diseases in humans, including chronic hepatitis, liver carcinoma, chronic cholecystitis, and cholangiocarcinoma \[[@B61-toxins-03-00172],[@B62-toxins-03-00172]\].
To study the role of CDT in chronic infection and inflammation, several animal models have been developed. Adult severe combined immunodeficient (SCID) mice are susceptible to infection by *Campylobacter* sp. To test the role of CDT in the establishment of persistent infection, these mice were infected by intragastric inoculation either with the wild type strain CJ81-176, or with an isogenic strain harboring an inactivating insertion within the *cdtB* gene (Cj81-176 mutant cdtB). Blood, liver and spleen samples were taken 2 h, 6 h and 24 h post-infection (p.i.) to assess for the presence of invasive bacteria. More bacteria-positive samples were detected in mice infected with the wild type CDT producing strain compared to the CdtB deficient *C. jejuni*, indicating that CDT promotes bacteria invasion \[[@B63-toxins-03-00172]\]. However, this was not associated with enhanced levels of intestinal colonization by 7 days p.i. in mice infected with the wild type or the mutant strains \[[@B63-toxins-03-00172]\].
Conversely, gastric colonization of mucin deficient 129/SvJ mice by *C. jejuni* was enhanced if the bacteria produced a functional CDT \[[@B64-toxins-03-00172]\], and *C. jejuni* carrying a wild type CDT were able to colonize the gastro-intestinal tract in 50% of C57BL/129 mice 4 months p.i., while an isogenic strain deficient for CDT expression was not detected in any of the infected mice \[[@B65-toxins-03-00172]\]. Bacterial clearance was dependent on a functional NF-κB complex, since mice homozygously deficient for p50 and heterozygous for p65 (p50^−/−^ p65^+/−^), referred to as 3× mice, showed persistent colonization of stomachs and lower bowels with both *C. jejuni* strains at 2 and 4 months p.i. \[[@B65-toxins-03-00172]\]. 3× mice colonized with the *cdtB* mutant strain had significantly less gastritis and hyperplasia at 4 months post-infection than 3× mice colonized with wild-type *C. jejuni*, indicating that CDT promotes proinflammatory responses \[[@B65-toxins-03-00172]\]. In line with this hypothesis, Hickey and colleagues demonstrated that CjCDT elicits secretion of IL-8, an important mediator of inflammation in INT407 (human embryo intestinal epithelial) cells \[[@B66-toxins-03-00172]\].
CDT is also produced by *H. hepaticus*, an enteropathogenic species, which naturally infects the distal gastrointestinal tract of mice \[[@B67-toxins-03-00172]\]. HhCDT was shown to play a crucial role in persistent bacterial colonization of the small intestine of Swiss Webster mice \[[@B68-toxins-03-00172]\], which was associated with significantly higher production of Th1-associated immunoglobulin G2a (IgG2a), Th2-associated IgG1 and mucosal IgA in the mice infected with wild type *H. hepaticus* when compared to animals exposed to the isogenic strain HhcdtBm7, where the *cdtB* gene was inactivated by transposon mutagenesis \[[@B68-toxins-03-00172]\]. Challenge of C57BL/6 interleukin 10 deficient mice with isogenic *H. hepaticus* mutants revealed that CDT expression is not required for colonization of the murine gut. However, a CDT-negative *H. hepaticus* mutant had a significantly diminished capacity to induce lesions in this murine model of inflammatory bowel disease \[[@B69-toxins-03-00172]\].
Infection of A/JCr mice with wild type or an isogenic mutant of *H. hepaticus* lacking CDT activity induced comparable levels and severity of chronic hepatitis at both 4 and 10 months p.i. The presence of CDT was instead necessary for the development of hepatic dysplasic nodules 10 months after infection. This effect was associated with enhanced hepatic transcription of proinflammatory (*TNF*-*α*, *IFN*-*γ* and *Cox*-*2*, *IL*-*6* and *TGF*-*α*) and anti-apoptotic (*Bcl*-*2* and *Bcl*-*X~L~*) genes, upregulation of hepatic mRNA levels of components of the NF-κB pathway (p65 and p50), and increased hepatocyte proliferation compared with the control or the CDT mutant-infected mice \[[@B70-toxins-03-00172]\].
Collectively, these data demonstrate that CDT is an important virulence factor to promote invasion of enteric bacteria and activate proinflammatory responses that can be associated with chronic inflammation, and its possible progression toward malignant transformation.
5.2. Chancroid and Wound Healing
--------------------------------
*H. ducreyi* is the causative agent of chancroid, a sexually transmitted disease, characterized by soft and slowly healing genital ulcers (reviewed in \[[@B71-toxins-03-00172]\]).
Cutaneous wound healing is a complex process involving blood clotting, inflammation, new tissue formation, and tissue remodeling. Neutrophils normally begin arriving at the wound site within minutes of injury, to control bacterial infection and produce proinflammatory cytokines, which probably serve as some of the earliest signals to activate local fibroblasts and keratinocytes. Neutrophils recruitment is followed by accumulation of macrophages, which are essential for effective wound healing, since healing is severely impaired if macrophage infiltration is prevented. Re-epithelialization of the skin requires both migration and rapid proliferation of keratinocytes, fibroblasts and epithelial cells (reviewed in \[[@B72-toxins-03-00172]\]). Adaptive immunity, specifically T lymphocytes are essential for a normal wound healing outcome (reviewed in \[[@B73-toxins-03-00172]\]).
Involvement of CDT in ulcer formation was showed in a rabbit model of chancroid, where intradermal inoculation of *H. ducreyi* co-administered with purified HdCDT resulted in significant aggravation of the bacteria-induced inflammatory lesions and in ulcer development \[[@B74-toxins-03-00172]\].
How can CDT contribute to the delayed would healing observed in chancroid? It has been shown that HdCDT affects cell proliferation and survival of many cell types involved in wound healing, such as primary human fibroblasts, primary keratinocytes and cells of epithelial origin \[[@B39-toxins-03-00172],[@B75-toxins-03-00172]\]. Furthermore, HdCDT may interfere with angiogenesis, since it inhibits proliferation of normal human microvascular endothelial cells from adult dermal tissue (HMVEC-d) and human umbilical vein endothelial cells (HUVEC), preventing new blood vessel formation in an *in vitro* angiogenesis model \[[@B76-toxins-03-00172]\].
HdCDT affects also effector cells of the innate and adaptive immune system: intoxication inhibits proliferation and IFN-γ secretion of T lymphocytes and induces apoptosis of B lymphocytes \[[@B39-toxins-03-00172],[@B48-toxins-03-00172],[@B77-toxins-03-00172]\], and monocyte-derived DCs, the key activators of the adaptive immune responses \[[@B33-toxins-03-00172],[@B78-toxins-03-00172]\]. Upon phagocytosis of heat-inactivated *H. ducreyi*, DCs produces the proinflammatory cytokines, IL-1β, IL-6, IL-8, and TNF-α. Preincubation of DCs with purified HdCDT results in an approximate 50% reduction in cytokine secretion \[[@B78-toxins-03-00172]\], suggesting an immuno-inhibitory effect of the toxin.
Targeting DCs may represent a strategy to avoid/delay the onset of immune responses, while inhibition of cellular proliferation may impair the wound healing process, thus increasing pathogen spread from host to host and/or favoring the establishment of a life-long latent infection.
5.3. Periodontitis and *A. actinomycetemcomitans*
-------------------------------------------------
Periodontitis is a chronic inflammatory disease associated with loss of the supporting connective tissue and alveolar bone around teeth. *A. actinomycetemcomitans* has been described as a member of the indigenous oral microbiota of humans \[[@B79-toxins-03-00172]\].
Does AaCDT contribute to periodontitis? The lack of suitable animal models does not allow answering this question directly. However, AaCDT may interfere with the normal periodontal connective tissue remodeling equilibrium, by over-stimulating osteoclast-dependent bone resorption. Production of mature osteoclasts is regulated by interaction between the receptor activator of NF-κB ligand (RANKL) present on the surface of hematopoietic bone marrow stromal cells, periosteal tissue osteoblasts, as well as T lymphocytes, and its receptor (RANK) expressed on osteclast progenitor cells. This process is negatively regulated by Osteoprotegerin (OPG), which acts as a decoy receptor for RANKL, preventing its interaction with RANK and osteoclast maturation (reviewed in \[[@B80-toxins-03-00172]\]). Therefore, the ratio of RANKL/OPG expression determines the amount of osteoclasts formed and controls the degree of bone resorption. It has been shown that AaCDT is sufficient to induce RANKL expression and downregulate OPG mRNA expression in gingival fibroblasts (GF) and periodontal ligament cells (PDLC), obtained from healthy individuals, and in the T lymphocyte cell line Jurkat \[[@B81-toxins-03-00172],[@B82-toxins-03-00172]\].
Furthermore, AaCDT blocks proliferation and causes apoptosis of mitogen activated human CD8^+^ and CD4^+^ T lymphocytes \[[@B37-toxins-03-00172],[@B52-toxins-03-00172],[@B53-toxins-03-00172],[@B54-toxins-03-00172]\], and peripheral blood mononuclear cells (PBMC) exposed to AaCDT are able to produce a wide range of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, and IFN-γ, while no secretion of IL-10, IL-12 and TNF-α was detected \[[@B83-toxins-03-00172]\]. It is conceivable that AaCDT stimulates the innate host immune response, promoting production of a specific set of cytokines, leading to an inflammatory pathology, inhibiting T cell functions and possibly creating a suitable niche for bacterial survival and proliferation.
6. Conclusions
==============
In spite of the exponential progress in understanding the CDT mode of action and the cellular responses induced by intoxication, there are still many questions that need to be answered regarding the biology of CDT and its role in disease. We still do not know: (1) how the toxin is translocated from the ER to the nucleus where it exerts its genotoxic activity, and which cellular partners regulate the intracellular trafficking of the toxin; (2) why evolution has positively selected a bacterial genotoxin; (3) what is the contribution of CDT in promoting genomic instability in chronic infections. We are looking forward to an even more exciting and productive period of research in the field of the cytolethal distending toxins.
This work has been supported by the Swedish Research Council, the Swedish Cancer Society, the Åke-Wiberg Foundation, the Magnus Bergvall Foundation, and the Karolinska Institutet to TF, Robert Lundberg Memorial Foundation to LG. XCB is a NETROPICA fellow. TF is supported by the Swedish Cancer Society.
| {
"pile_set_name": "PubMed Central"
} |
T cell activation initiates the adaptive immune response and requires extracellular ligation of the TCR and the subsequent formation of dynamic signaling complexes. After TCR engagement, Lck phosphorylates its TCRζ subunit, enabling the recruitment and activation of ZAP70, which in turn phosphorylates the adapter LAT. Phosphorylated LAT acts as a scaffold, recruiting other adapters and effectors into multiprotein complexes driving downstream signal amplification and diversification, leading to T cell activation ([@bib1]). TCR signaling is sustained and regulated within a specialized cellular interface formed between a T cell and an antigen-presenting cell, the immunological synapse. Immunological synapse settings and function depend on both spatial cues and on the active transport of molecules to and within the synapse ([@bib2]; [@bib25]).
Compartmentalization in cells of the immune system facilitates the spatiotemporal organization of cellular responses essential for specialized immune functions. In T cells, TCR signal transduction relies on the compartmentalization of signaling molecules into plasma membrane nanodomains ([@bib11]; [@bib27]; [@bib36]). However, some molecules involved in TCR signaling do not just move on the plasma membrane, but must be transported across the T cell and delivered to the immunological synapse. Namely, the TCR, LAT, and Lck localize to vesicular compartments that are targeted to the immunological synapse upon TCR engagement ([@bib14]; [@bib6]; [@bib9]; [@bib15]). In resting T cells, Lck is constitutively active and distributes between the plasma membrane and a vesicular compartment. Curiously, TCR triggering has no impact on the extent of Lck activity ([@bib30]). This implies that Lck relocalization from its vesicular compartment to the immunological synapse may be responsible for TCR signal propagation. One important question raised by these findings concerns how the traffic of signaling molecules to specific regions of the plasma membrane is regulated to execute spatially restricted signaling.
Previous works put forward several traffic regulators involved in cytokine secretion and lytic granule release at CD4 ([@bib22]) and CD8 ([@bib10]) T cell synapses, respectively. However, it is unknown how the vesicular traffic of signaling molecules to the immunological synapse is regulated. TCR signal transduction might rely on endosomal traffic regulators and their specific subcellular localization. Validation of this concept requires the identification of Rab proteins and their effectors, which coordinate the transport and delivery of Lck, LAT, and TCRζ vesicles to the immunological synapse.
Here, we show that the regulated fusion of Lck, LAT, and TCRζ distinct vesicular compartments at the synapse determines the spatial organization, number, density, and molecular composition of its signaling nanoclusters, as well as the presence of signaling nanoterritories within phosphorylated LAT and SLP76 clusters. Lck acts as the signal switch and calcium acts as the mediator of a vesicle fusion positive feedback loop that builds a functional immunological synapse capable of driving T cell activation and cytokine production.
RESULTS
=======
Lck, TCRζ, and LAT reside in distinct exocytic vesicular compartments
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We assessed Lck, TCRζ, and LAT subcellular localization and traffic regulators to establish whether they trafficked in distinctly regulated vesicular compartments. Primary CD4 T and Jurkat cells (unpublished data) displayed a Lck intracellular compartment finely intermingled with those of LAT and TCRζ; however, co-localization was minimal (\<3%), whereas TCRζ and LAT compartments co-localized to a higher, yet still feeble, extent (unpublished data).
The specification of the intracellular traffic route and the vesicular compartment identity rely on the Rab family GTPases ([@bib17]). In turn, vesicle fusion is mediated by SNARE proteins whose function is restricted to precise subcellular microenvironments ([@bib39]). To ascertain the identity of Lck, LAT, and TCRζ intracellular compartments, we looked into 19 Rabs and 1 SNARE that have both been linked either to regulated exocytosis or to the transport of endosomes to plasma membrane ([@bib17]), and to a previously described protein for specialized transport, myelin and lymphocyte protein (MAL), which mediates Lck transport to the plasma membrane ([@bib3]). We transiently expressed each Rab-GFP fusion protein in Jurkat cells and assessed their co-localization with respect to Lck, LAT, and TCRζ vesicular compartments. These signaling molecules localized in highly individualized compartments, with little to no overlap in their Rab specification ([Fig. 1](#fig1){ref-type="fig"}). Lck vesicular compartment co-localized (∼35%) with the recycling endosomal marker Rab11b and with MAL (∼23%; [@bib3]; [Fig. 1, D and H](#fig1){ref-type="fig"}). LAT vesicular compartment co-localized with Rab27a (∼35%) and Rab37 (∼30%; [Fig. 1, M and N](#fig1){ref-type="fig"}), two Rab molecules known to regulate cytokine and cytotoxic granule exocytosis in CD4^+^ and CD8^+^ T cells, respectively ([@bib38]; [@bib22]). TCRζ peripheral vesicles co-localized to the fast recycling Rab4b compartment (∼8%), whereas its core co-localized to Rab3d- (∼25%) and Rab8b- regulated (∼30%) exocytic compartments ([@bib22]; [@bib17]; [Fig. 1, Q--S](#fig1){ref-type="fig"}). Noteworthy, the secretory v-SNARE protein Ti-VAMP ([@bib7]) co-localized with both LAT (∼30%) and TCRζ (∼30%) vesicular compartments ([Fig. 1, O and W](#fig1){ref-type="fig"}).
{#fig1}
LAT and TCRζ vesicular release is regulated by calcium and synaptotagmin-7
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The traffic regulators present in LAT and TCRζ vesicles ([Fig. 1](#fig1){ref-type="fig"}) have been reported to be involved in stimulus-induced exocytosis at cytotoxic synapses ([@bib29]; [@bib28]). To pinpoint the molecular mechanism regulating Lck, LAT, and TCRζ release from vesicular compartments, we assessed the role of calcium, a known regulator of stimulus-dependent exocytosis ([@bib39]; [@bib31]). To increase cytosolic calcium levels, we treated CD4 T cells either with thapsigargin or ionomycin. To accurately quantify Lck, LAT, and TCRζ distribution between intracellular vesicles and the plasma membrane, we stained for surface CD2 and then used it to automatically segment the plasma membrane. The resulting automated mask delimiting the internal and external outlines of the plasma membrane allowed us to discriminate and quantify the subcellular distribution (plasma membrane versus vesicular compartment) of our proteins of interest (materials and methods; unpublished data). We found LAT and TCRζ distribution to the vesicular compartment to be higher than anticipated by others ([@bib6]), ∼75% ([Fig. 2, A, B, H, and I](#fig2){ref-type="fig"}). In fact, through plasma membrane segmentation, we were able to distinguish plasma membrane--resident LAT and TCRζ from their intracellular compartments and subcortical vesicles, within the limits of the confocal microscopy resolution (unpublished data). Lck displayed a higher plasma membrane distribution with ∼25% in intracellular vesicles ([Fig. 2, C and J](#fig2){ref-type="fig"}; and not depicted).
{#fig2}
Consistent with LAT localization in Rab27a and Rab37 exocytic compartments, increased cytosolic calcium led to a clear release of its vesicular compartment into the plasma membrane ([Fig. 2, A, I, and O](#fig2){ref-type="fig"}; and not depicted). In agreement with TCRζ-segregated distribution into a Rab3d and Rab8b exocytic compartment and also into Rab4b rapid recycling endosomes, cytosolic calcium increase induced an incomplete release of TCRζ vesicular compartment ([Fig. 2, D, K, and O](#fig2){ref-type="fig"}; and not depicted). As expected from their presence in Rab11b and MAL compartments, neither thapsigargin nor ionomycin induced Lck vesicle release ([Fig. 2, F, M, and O](#fig2){ref-type="fig"}; and not depicted).
Thapsigargin effects on LAT and TCRζ exocytosis seem specific, rather than the consequence of the redistribution of multiple compartments, as neither the trans-Golgi network (TGN38^+^) nor the endosomal-recycling compartment (transferrin receptor^+^) were released by a cytosolic calcium raise ([Fig. 2, C and H](#fig2){ref-type="fig"}; and not depicted). There was no significant overlap of LAT with the biosynthetic compartment labeled by TGN38 (∼5% co-localization; unpublished data). LAT's fast traffic through the secretory pathway might preclude its concentration required for detection.
Calcium-regulated exocytosis relies on the presence of vesicle-associated fusion proteins (v-SNARES) and calcium sensors (synaptotagmins; [@bib31]). LAT and TCRζ vesicular compartments displayed Ti-VAMP ([Fig. 1, O and W](#fig1){ref-type="fig"}), a v-SNARE known to interact with the calcium sensor synaptotagmin-7 (Syt7; [@bib33]), suggesting Syt7 involvement in this process. Syt7-silencing in primary CD4 T cells did not alter LAT and TCRζ steady-state distribution ([Fig. 2, J and L](#fig2){ref-type="fig"}), or their distribution into the TGN38 compartment (not depicted), obviating any effects of Syt7 silencing on the constitutive fusion of the biosynthetic and endocytic pools of LAT and TCRζ to the plasma membrane. However, Syt7 silencing impaired TCRζ and LAT release from their vesicular compartments in response to increased cytosolic calcium levels ([Fig. 2, B, E, J, and L](#fig2){ref-type="fig"}). Single Syt7 siRNA oligonucleotides directed to distinct RNA sequences inhibited calcium-induced vesicular LAT release, albeit at a lesser extent than their combination, ruling out siRNA off-target effects (unpublished data).
The egress of Lck from vesicles into the plasma membrane requires MAL ([@bib3]). Consistent with its specific distribution in Lck vesicles ([Fig. 1, H, P, and X](#fig1){ref-type="fig"}), MAL silencing led to an accumulation of Lck in the vesicular compartment (approximately threefold signal increase), without altering LAT or TCRζ steady-state distribution ([Fig. 2, P and Q](#fig2){ref-type="fig"}).
Therefore, Lck, LAT, and TCRζ vesicle traffic is regulated by distinct molecular mechanisms. Lck depends on MAL and is calcium-insensitive, whereas TCRζ and LAT vesicle release is induced by calcium and mediated by Syt7. Still, a fraction of the TCRζ compartment is not released in response to calcium. This may reflect the presence of TCRζ in the Rab4b rapid recycling compartment ([Fig. 1 R](#fig1){ref-type="fig"}).
Lck controls the release of TCRζ and LAT vesicular compartments to the immunological synapse
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We next investigated how the differential regulation of Lck, LAT, and TCRζ vesicular compartments conditioned their targeting to the immunological synapse. We inhibited Lck traffic by silencing MAL expression, or TCRζ and LAT vesicle fusion by silencing Syt7, and analyzed 30-min synapses. Strikingly, MAL silencing prevented not only Lck, but also LAT and TCRζ vesicle release, and their full clustering at the synapse, although compartment polarization and an incipient TCRζ cluster were observed ([Fig. 3, A--G](#fig3){ref-type="fig"}). In contrast, Syt7 silencing inhibited LAT and TCRζ compartment delivery and synaptic clustering, without effecting Lck clustering. A residual TCRζ clustering was observed in Syt7-silenced cells ([Fig. 3, L--O](#fig3){ref-type="fig"}, arrowheads and arrows). Consistent with a calcium-dependent triggering of TCRζ and LAT vesicle fusion, intracellular calcium chelation by BAPTA abrogated TCRζ and LAT compartment release and clustering without significantly perturbing Lck synaptic clustering (unpublished data).
{#fig3}
These data show that Lck, TCRζ, and LAT vesicle release at the synaptic membrane is differentially regulated. The inhibitory effect of MAL silencing on LAT and TCRζ vesicle fusion at the synapse, and the fact that it doesn't affect LAT and TCRζ steady-state traffic, suggests that previous Lck synaptic delivery is necessary for LAT and TCRζ vesicle fusion.
Lck-, TCRζ-, and LAT-regulated traffic is pivotal to TCR signal transduction at the immunological synapse
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TCR signaling is mediated by formation of transient, heterogeneous clusters of signaling molecules whose structure and subcellular origin remain uncertain ([@bib12]). Our experimental build-up provided us with the means to disentangle the contributions of regulated signaling molecule exocytosis for TCR signaling.
We inhibited Lck traffic through MAL-silencing, and TCRζ and LAT vesicle fusion through Syt7-silencing. We monitored specific phosphorylated tyrosine residues (pY), hallmarks of TCR signal transduction: the activatory pY^394^ of Lck, pY^142^ of TCRζ and pY^191^ of LAT. From now on, phosphorylated proteins will be designated by pLck, pTCRζ, and pLAT.
Consistent with their synaptic clustering patterns ([Fig. 3, A--F](#fig3){ref-type="fig"}), at 30 min of conjugate formation, the extent of pLck and pLAT clustered at synapses formed by MAL-silenced cells was strongly diminished ([Fig. 4, A--H](#fig4){ref-type="fig"}), although a partial pTCRζ cluster was observed ([Fig. 4, F and I](#fig4){ref-type="fig"}, arrows). Conversely, Syt7-silenced cells displayed normal pLck, very low pLAT, and residual pTCRζ clustering at the synapse ([Fig. 4, J--R](#fig4){ref-type="fig"}).
{#fig4}
These data indicate that the juxtaposition of vesicular LAT to an immunological synapse displaying fully clustered pLck is not enough for LAT phosphorylation to occur. Instead, LAT synaptic phosphorylation requires the prior clustering of Lck at the synapse, as well as the calcium and Syt7-dependent synaptic fusion of vesicular LAT.
MAL silencing differentially affects Lck, TCRζ, ZAP70, LAT, and SLP76 phosphorylation
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Our previous findings raised the question of how the regulated traffic and fusion of vesicle-associated Lck, LAT, and TCRζ determine TCR signaling. To further investigate the effect of hindering Lck traffic on early TCR signaling, MAL-depleted cells were activated for 30 min with superantigen-pulsed Raji cells in the presence or absence of thapsigargin. The presence of specific phosphotyrosine residues was assessed by intracellular flow cytometry.
Consistent with a previous report ([@bib30]), Lck phosphorylation was not affected either by MAL silencing or TCR engagement ([Fig. 5 A](#fig5){ref-type="fig"}). In contrast, LAT and SLP76 phosphorylation were fully inhibited in MAL-silenced cells ([Fig. 5, D and E](#fig5){ref-type="fig"}), whereas TCRζ and ZAP70 phosphorylation were partially inhibited ([Fig. 5, C and E](#fig5){ref-type="fig"}). Thapsigargin treatment of MAL-silenced cells restored TCRζ, ZAP70, LAT, and SLP76 phosphorylation levels.
{#fig5}
These data indicate that synaptic Lck sets the threshold for the calcium and Syt7-mediated TCRζ and LAT vesicle release that controls pTCRζ and pLAT clustering and the subsequent recruitment of pZAP70 and pSLP76.
TCRζ and ZAP70 signaling organization is mildly affected by MAL silencing
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Our aforementioned findings raised the question of how the regulated traffic and fusion of vesicle-associated Lck, TCRζ, and LAT determine TCR signal organization at the synaptic membrane. Hence, we inhibited vesicle traffic or fusion and analyzed, at high spatial resolution, the distribution of early (i.e., pTCRζ, pZAP70) and amplification (i.e., pLAT, pSLP76) signaling complexes, at high spatial resolution.
We performed two-color direct stochastic optical reconstruction microscopy (dSTORM) combined with total internal reflection fluorescence (TIRF; [@bib18]) on fixed cells. dSTORM super-resolution microscopy accurately localizes single molecules (see Materials and methods) and allowed us to quantify different parameters of the plasma membrane signaling organization, including the presence of protein clusters, their size, relative density, and abundance. In addition, we coupled 3D wide-field imaging to our dSTORM imaging apparatus to monitor for the presence of LAT vesicular compartment, by expressing a GFP-tagged LAT ([Fig. 6](#fig6){ref-type="fig"}; boxes, top left).
{#fig6}
MAL silencing lead to mild differences in pTCRζ and pZAP70 nanocluster organization, with a small reduction in pTCRζ numbers and cluster area ([Fig. 6, B, F, and H](#fig6){ref-type="fig"}) and to an enrichment in small pZAP70 clusters (dimers and trimers; [Fig. 6, B and I](#fig6){ref-type="fig"}). No changes were observed in nanocluster density or shape ([Fig. 6, J--M](#fig6){ref-type="fig"}).
TCRζ and LAT vesicle fusion determine pLAT nanocluster organization and their ability to recruit pSLP76
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Current models for TCR signaling issued from biochemical approaches and standard light microscopy propose the generation of an amplification signalosome formed by direct association of pLAT and pSLP76--Gads complexes ([@bib1]). Thus, we monitored the spatial distribution of pLAT and pSLP76 at the immunological synapse. We observed important differences in pLAT and pSLP76 nanocluster distribution under different activation conditions. Anti-CD3 stimulation (αCD3) led to a significant increase in the number and relative density of both pLAT and pSLP76 clusters, compared with nonstimulated cells spread on anti-CD45 (αCD45; [Fig. 7, A and E](#fig7){ref-type="fig"}; and [Fig. 8, A--D](#fig8){ref-type="fig"}). Inhibition of TCRζ and LAT compartments synaptic fusion, directly through Syt7-, or indirectly through MAL-silencing, caused a twofold reduction in pLAT and pSLP76 cluster number, size and relative density to levels of nonstimulated cells (αCD45). This was accompanied by a significant shift toward smaller clusters and a concomitant decrease in the number and particle density of pSLP76 clusters ([Fig. 7](#fig7){ref-type="fig"}; and [Fig. 8, A--E](#fig8){ref-type="fig"}).
{#fig7}
{ref-type="fig"}. (A) Number of clusters per square micrometer is as follows: siCtr, *n* = 24; siSyt7, *n* = 21; siMAL, *n* = 14; siMAL TPS, *n* = 11; αCD45, *n* = 20; and αCD45 TPS, *n* = 22. (B) Number of clusters per square micrometer is as follows: siCtr, *n* = 18; siSyt7, *n* = 21; siMAL, *n* = 14; siMAL TPS, *n* = 11; αCD45, *n* = 19; and αCD45 TPS, *n* = 22. (C and D) Number of pLAT (C) and pSLP76 (D) detections per individual cluster for each cell analyzed for *n* ≥ 11 cells. (E) Mean pLAT cluster area per cell, *n* ≥ 11 cells. (F) Percentage of clusters whose circularity is equal to 1 per cell. Circularity value is given by the ratio between the large and the small feret diameters for all the clusters detected *n* ≥ 11 cells. (G) Mean area of pLAT clusters per cell according to circularity, measured as in F (*n* = 24). \*\*\*, P ≤ 0.0001; \*\*, P ≤ 0.01; \*, P ≤ 0.05; Mann-Whitney test. Each circle represents a cell. Representative of three experiments.](JEM_20130150R_Fig8){#fig8}
To disentangle the effect of blocking Lck traffic on LAT phosphorylation, from its effect on vesicular LAT synaptic fusion, we blocked the calcium-insensitive Lck traffic through MAL silencing and provoked the calcium-sensitive fusion of vesicular TCRζ and LAT. Thapsigargin treatment of MAL-silenced cells lead to the release of LAT vesicular compartment and a concomitant increase in the number, relative density and area of pLAT clusters which displayed increased ability to recruit pSLP76 clusters when compared with MAL-silenced cells. Interestingly, promoting TCRζ and LAT vesicular fusion through thapsigargin treatment of MAL-silenced cells is sufficient to recover a number and density of pLAT clusters similar to the one observed in activatory synapses ([Fig. 7](#fig7){ref-type="fig"}; and [Fig. 8, A and C](#fig8){ref-type="fig"}).
These results indicate that, first, Lck-deficient clustering at the synapse creates a signaling bottleneck caused by the impaired synaptic delivery of vesicular TCRζ and LAT, and second, once vesicular LAT is delivered at the synapse, even reduced amounts of synaptic Lck are apt to extensively phosphorylate LAT clusters, if not as comprehensively ([Fig. 8, C and E](#fig8){ref-type="fig"}).
The contribution of vesicular TCRζ and LAT to the synapse nanoscale organization went beyond an effect in the number and density of pLAT clusters, it also affected their morphology. αCD3-stimulated control cells displayed a population of larger and elongated pLAT nanoclusters, which were adjacent to pSLP76 clusters ([Fig. 7 A](#fig7){ref-type="fig"}; and [Fig. 8 F](#fig8){ref-type="fig"}). In contrast, upon LAT compartment retention, the majority of pLAT nanoclusters were circular, as in nonstimulated cells ([Fig. 7, B, C, and E](#fig7){ref-type="fig"}). Elongated pLAT clusters (∼20 µm^2^) are the main contributors to the increase in pLAT cluster size in αCD3-stimulated cells ([Fig. 8 G](#fig8){ref-type="fig"}). Reinforcing the role of LAT vesicle fusion in the spatial organization of signaling complexes, thapsigargin treatment of MAL-silenced cells elicited the appearance of the elongated pLAT clusters ([Fig. 7 D](#fig7){ref-type="fig"}; and [Fig. 8 F](#fig8){ref-type="fig"}). Thapsigargin-induced LAT vesicle fusion in αCD45-adhered cells did not recover pLAT nanoscluter organization, indicating that it depends on TCR stimulation ([Fig. 7 F](#fig7){ref-type="fig"}; and [Fig. 8, A-F](#fig8){ref-type="fig"}).
Our live imaging ([Video 1](http://www.jem.org/cgi/content/full/jem.20130150/DC1){#supp1}) is consistent with a model whereby LAT vesicle delivery at the synapse occurs as soon as the cell contacts the activatory surface (15--45 s). The likely fusion of vesicle LAT at discrete synaptic locations (highlighted by arrows) originates the elongated LAT clusters (demarked by polygons) that are fed by LAT vesicles during the entire length of the acquisition (7 min mark). Due to spatial resolution limitations inherent to live imaging, our video microscopy interpretation was performed in light of the aforementioned super-resolution data.
These results suggest that pLAT clusters result from two subpopulations with distinct subcellular origins: the plasma membrane and the vesicular compartment. Moreover, the fact that pTCRζ and pZAP70 were mildly affected by Lck vesicle traffic inhibition indicates that TCR stimulation modulates the synapse nanoscale organization in a TCRζ and LAT vesicle fusion-dependent manner.
LAT vesicle fusion promotes pLAT and pSLP76 intermolecular proximity
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Our data indicate that TCRζ and LAT vesicle fusion is key to generate the spatial organization of pLAT and pSLP76 nanoclusters in TCR-stimulated synapses. We investigated whether this nanocluster organization could correlate with increased interactions between these two adapters, and thus with enhanced TCR signal amplification ([@bib1]).
In agreement with previous super-resolution studies ([@bib27]; [@bib36]), we found that pLAT and pSLP76 clusters appeared contiguous, but only mildly overlapped ([Fig. 7 A](#fig7){ref-type="fig"}), indicating that pLAT and pSLP76 do not interact extensively. This raises the possibility that pLAT and pSLP76 interactions could be occurring in spatially discrete locations. To address this hypothesis, we developed a method to identify possible pLAT and pSLP76 interactions, independent of their molecular concentration. To do so, we mapped the locations where pLAT and pSLP76 molecules were less than 20 nm apart and attributed to each event a nonzero pixel value, whose intensity is inversely related to the distance ([Fig. 9](#fig9){ref-type="fig"}; see Materials and methods). In our analysis, the pixel corresponding to a pLAT and pSLP76 protein pair at distances \<20 nm depicts a signaling nanoterritory. In this case, nano defines the spacing between molecules at possible interacting distances (\<20 nm) rather than the size of the domain.
{#fig9}
Syt7 and MAL silencing reduced the frequency and extension of pLAT and pSLP76 signaling nanoterritories down to those of nonstimulated cells (αCD45; [Fig. 9, A--I](#fig9){ref-type="fig"}). Under stimulatory conditions, the majority of synaptic pLAT forms signaling nanoterritories with pSLP76 ([Fig. 9, G and H](#fig9){ref-type="fig"}). Signaling nanoterritories were present both at the fringes and, notably, within clusters, spanning ∼50% of pLAT cluster area ([Fig. 9 I](#fig9){ref-type="fig"}). Reinforcing the notion that vesicular LAT synaptic fusion promotes the formation of pLAT signaling hubs, signaling nanoterritories were present more extensively (∼75%) in elongated pLAT clusters (circularity \<0.4) than in rounder ones ([Fig. 9 J](#fig9){ref-type="fig"}). Once more, provoking LAT vesicular fusion through thapsigargin treatment of MAL-silenced cells spread on αCD3-coated, but not on αCD45-coated, coverslips is sufficient to recover the frequency, extension, and morphology of signaling nanoterritories containing pLAT and pSLP76 to stimulatory levels (αCD3; [Fig. 9, C--J and L--O](#fig9){ref-type="fig"}).
These data indicate that there are two populations of functionally distinct pLAT clusters at the synapse: the preexisting round pLAT clusters, whose capacity for forming signaling nanoterritories with pSLP76 is low, and elongated pLAT clusters generated upon the synaptic fusion of vesicular LAT, which favors their molecular association with pSLP76 both at the rims and within pLAT clusters.
Lck-, TCRζ-, and LAT-regulated synaptic fusion is crucial for T cell activation leading to cytokine production
--------------------------------------------------------------------------------------------------------------
TCR-driven LAT phosphorylation leads to MAP kinase activation, CD69 up-regulation, and cytokine production ([@bib1]). To determine the contribution of regulated vesicular traffic to TCR-triggered T cell activation, we interrupted the Lck, TCRζ, and LAT vesicle traffic cascade by silencing either MAL or Syt7.
In primary CD4 T cells, activation events downstream of LAT, such as Erk1/2 activation, CD69 surface up-regulation, and IL-2 and IFN-γ cytokine production were all significantly inhibited in MAL- and Syt7-silenced cells ([Fig. 10, A--G](#fig10){ref-type="fig"}). Importantly, the increase in cytosolic calcium levels in MAL-silenced cells by thapsigargin or ionomycin restored an IL-2 and IFN-γ production comparable to control cells ([Fig. 9, C--F](#fig9){ref-type="fig"}, right). These results underscore the role of Lck-, TCRζ-, and LAT-regulated vesicle traffic in TCR signal integration conducive to T cell function.
{#fig10}
DISCUSSION
==========
It is increasingly clear that vesicle traffic achieves a variety of effects on signal transduction and, conversely, that receptor signaling regulates the exocytic machinery. Here, we sought to determine how the highly specific compartmentalization of Lck, LAT, and TCRζ contributes to the functional nanoscale organization of the immunological synapse and to its capacity to transduce downstream activation signals leading to cytokine production.
The molecular machinery that regulates the vesicular delivery of signaling molecules at the immunological synapse is largely unknown. We have analyzed this machinery by detailing its traffic regulators: Rab GTPases and SNARE proteins. We found that although Lck, LAT, and TCRζ traffic through exocytic compartments, there is no overlap in their traffic regulators ([@bib17]). Lck localizes to the calcium-insensitive Rab11b/MAL compartment, whereas LAT and TCRζ traffic through a calcium-sensitive Rab27a-Rab37-Ti-VAMP and Rab3d-Rab8b-Ti-VAMP vesicular compartments, respectively. Interestingly, except for Rab8b, the exocytic traffic regulators present in Lck, LAT, or TCRζ vesicles have been also implicated in either cytokine secretion ([@bib22]), or cytotoxic granules release ([@bib29]; [@bib28]), at the CD4 and CD8 T cell synapses, respectively.
In cytotoxic T cell granule exocytosis ([@bib10]) and in lymphocyte migration ([@bib8]), stimulus-induced vesicle fusion is regulated by synaptotagmins (Syt), which couple calcium influx to the formation of SNARE complexes. Previously, we have shown ([@bib9]) that syntaxin-4 and SNAP-23, two plasma membrane t-SNARE proteins, accumulate at the CD4 T cell--APC interface, which provides a molecular basis for selective vesicle fusion at the immunological synapse. However, SNARE complexes required for calcium-dependent exocytic vesicle fusion at the synapse were not identified. Here, we found that LAT and TCRζ vesicular compartments display Ti-VAMP/VAMP7. This v-SNARE mediates lysosomal exocytosis through the interaction with syntaxin-4 and SNAP-23, the two t-SNARES that cluster at the immunological synapse ([@bib9]), and with the vesicle fusion regulator Syt7 ([@bib33]; [@bib8]). Similarly, we show that Syt7 regulates the calcium-induced exocytosis of the LAT and TCRζ vesicular compartments, in response to TCR triggering. In contrast, Lck synaptic recruitment is regulated by MAL and might be fulfilled by the Rab11b ability to traffic in response to activatory signals ([@bib29]; [@bib40]). We found that Lck vesicular compartment is released faster than the ones of LAT and TCRζ in response αCD3 stimulation (unpublished data). In a concerted manner, Lck vesicular delivery to the immunological synapse might trigger local calcium fluxes, which in turn would induce TCRζ and LAT vesicle release. Therefore, Lck is the signal switch and calcium the mediator of a vesicular traffic and fusion amplification loop that delivers TCRζ and LAT, building up a fully competent signaling synapse.
Our results are consistent with the observation that Lck is constitutively active in resting CD4 T cells and its total activity is not increased upon TCR triggering ([@bib30]). We propose that rather than controlling its phosphoactive status, TCR engagement triggers the delivery of vesicular Lck to the immunological synapse concentrating it locally and rapidly.
The relevance of LAT localization (plasma membrane versus vesicles) for TCR signal transduction is a subject of active debate. Previous studies ([@bib32]; [@bib42]) put forward a role for LAT vesicles in TCR signaling, challenging previous studies that demonstrated the requirement of LAT plasma membrane oligomerization and cis phosphorylation ([@bib21]) for TCR signal transduction. Moreover, [@bib24] very recently proposed that a VAMP7-mediated LAT vesicle docking to the synapse was responsible for TCR signaling. In contrast, another recent work ([@bib4]), using a CD4-LAT chimeric protein, proposed that it is mainly plasma membrane LAT that is phosphorylated and incorporated into microclusters, with minor influence from its vesicular pool. Although the phosphorylation of plasma membrane LAT appears clear, it is not clear whether CD4-LAT chimeras are properly targeted to stimulus-regulated exocytic compartments. Therefore, the mechanistic aspects of this process (plasma membrane, versus vesicle transient approach, docking, or fusion), its regulation and the relative relevance of both LAT origins for TCR signaling remain ill defined.
By combining molecular control over TCRζ and LAT vesicle fusion with dual-color dSTORM and TIRF microscopy approaches, we unveil here that the nanoscale organization of pLAT and pSLP76 upon TCR stimulation depends on TCRζ and LAT vesicle fusion at the synaptic membrane. In contrast, pTCRζ and pZAP70 nanocluster organization was only mildly affected by these fusion events. To our knowledge, this is the first observation in which the fusion of vesicles carrying signaling molecule impacts the functional nanoscale organization of the immunological synapse. First, inhibition of TCRζ and LAT vesicular fusion resulted in a pLAT and pSLP76 patterns similar to nonstimulated cells, suggesting that plasma membrane resident pLAT contributes poorly to early TCR signaling. Second, the fusion of vesicular LAT is accountable for the pLAT clusters increase in size, number, and density. Third, LAT compartment fusion originates a subpopulation of elongated clusters that act as signaling hubs through extensive formation of signaling nanoterritories with pSLP76. Fourth, the activatory synapse nanoscale organization was rescued when LAT vesicular retention due to MAL silencing was bypassed by thapsigargin, which causes a calcium flux that mediates LAT and TCRζ synaptic fusion. Finally, TCR stimulation was needed for thapsigargin-induced synapse nanoscale organization, as it did not occur in αCD45-plated cells.
We detected a wide range of clustering, from a few detections to larger spatial domains, as described previously ([@bib27]; [@bib35]; [@bib42]). The elongated pLAT clusters, observed upon vesicular LAT fusion at the synapse, are consistent with high resolution imaging studies that described that LAT signaling clusters may represent discrete interdigitating domains in T cells ([@bib11]; [@bib26]) and compatible with plasma membrane confinement zones ([@bib23]). Super-resolution imaging has not been able to resolve the pLAT cluster size obtained by different studies with [@bib36] reporting a predominance of very small LAT clusters. This apparent discrepancy might result from the terminology used and/or the different statistical methods applied to segment clusters. The inability of other studies ([@bib32]; [@bib42]) to detect elongated pLAT clusters at later time-points (10 min) might be caused by the endocytosis of LAT clusters, which could also explain the presence of the late endocytic marker Rab7 in LAT vesicles ([@bib32]).
The fact that elongated pLAT clusters preferentially recruit pSLP76 indicates that they are especially adept at conveying TCR signaling, when compared with isolated molecules or to clusters formed by 3--4 LAT molecules. This may occur by various means: (1) elongated clusters may offer a higher contact area for a given cluster volume; (2) they may increase the duration and affinity of interaction allowing for the slow phosphorylation reactions to occur; (3) they may favor the assembly of different signaling pathways. Accordingly, we found that single pLAT and pSLP76 molecules associate in signaling nanoterritories both at the fringes and within clusters. This discrete molecular association of pLAT and pSLP76 within clusters has not been previously appreciated ([@bib27]; [@bib36]) and might result from hop diffusion. Our data are compatible with a model whereby specialized intracellular calcium microdomains ([@bib41]) and the presence of SNAREs in particular plasma membrane subdomains would locally target LAT vesicle fusion (Video 1). Within those subdomains, delivered LAT would rapidly cluster, as a direct consequence of LAT oligomerization ([@bib21]). It remains to be addressed whether elongated pLAT clusters originate directly from vesicular LAT, or if they originate because of lateral movement of the plasma membrane after vesicle fusion. The rounder clusters observed in the absence of LAT vesicle fusion may represent more static plasma membrane--resident nanostructures with other possible functions. Novel approaches combining super-resolution microscopy with live cell imaging will help test this proposition.
Finally, MAL or Syt7 silencing blocked TCR signal integration through the inhibition of Erk activation, CD69 up-regulation and IL-2 and IFN-γ production. The extensive restoration of IL-2 and IFN-γ release induced by thapsigargin or ionomycin treatment in MAL-silenced cells, or by PMA-ionomycin in Syt7-silenced cells, shows that neither MAL nor Syt7 silencing affected cytokine secretion. Thus, we conclude that TCRζ- and LAT-regulated fusion at the synapse determine the signaling nanoterritories required for TCR signal amplification and T cell effector function.
In conclusion, this work shows that the spatiotemporal regulation of TCR signaling results from the regulated fusion of vesicles carrying signaling molecules. TCR signaling is not merely a function of which signaling molecules are present at the synaptic membrane but also a function of their subcellular origin and traffic regulation.
MATERIALS AND METHODS
=====================
### Cell cultures and transfections.
Jurkat cells clone J77cl20 and the APC Raji B cells were grown in complete RPMI medium containing 10% (vol/vol) fetal bovine serum, nonessential amino acids, and [l]{.smallcaps}-glutamine ([@bib9]). DNA constructs were inserted into Jurkat cells using the Invitrogen Neon Transfection system. Transiently transfected cells were imaged 24--48 h after transfection. Transfection efficiency was evaluated by flow cytometry.
### Primary CD4 T cell culture and transfections.
Peripheral blood cells were grown in the presence of staphylococcal enterotoxin B (SEB; 5 ng/ml) and TSST-1 (5 ng/ml) for 48-- 72 h, washed, and cultured in IL-2--containing medium (20--50 U/ml) for 7 d, at which time they were restimulated with SEB (5 ng/ml), TSST-1 (5 ng/ml), and PHA (0.4 µg/ml) for 24 h. Cells were resuspended for another 24 h in IL-2--containing medium, and then negatively selected through magnetic bead purification (MACS; Miltenyi Biotec) according to the manufacturer's instructions. CD4 T cells were cultured for 5--6 d in complete RPMI medium enriched with 20 U/ml of human IL-2. DNA constructs were electroporated into primary CD4 T cells using the Invitrogen Neon Transfection system (PBMC program). Primary CD4 T cells were imaged 24--48 h after electroporation. Transfection efficiency was evaluated by flow cytometry.
### Cell stimulation for confocal microscopy.
Immunological synapses formation were promoted by incubating Raji cells unpulsed or pulsed with superantigen (10 µg/ml, 20 min), either SEE for Jurkat or SEB+TSST1 for primary CD4 T cells at a 1:1 T cell/APC ratio for 30 min.
### Confocal imaging.
Cells plated onto poly-L-lysine--coated coverslips were fixed in 4% paraformaldehyde for 15 min, rinsed, and treated with 50 mM NH~4~Cl in PBS for 10 min to quench the aldehyde groups. After PBS wash, nonspecific binding was prevented by 15-min incubation with 1% BSA (wt/vol) and 0.05% saponin in PBS, used throughout the procedure as staining and washing buffer. After 1-h incubation with the indicated primary antibodies, cells were rinsed and incubated with the corresponding secondary antibodies. The anti-Lck mAb (3A5) and the anti-TCRζ mAb were obtained from Santa Cruz Biotechnology, Inc.; the anti-LAT polyclonal antibody was purchased from EMD Millipore; the anti-pY^142^TCRζ mAb was purchased from BD; the anti-pY^191^LAT polyclonal antibody was obtained from BioSource, the anti-pY^394^Src polyclonal antibody was from MBL. The cyanine 3 (Cy3)-coupled anti--mouse IgG2b, the fluorescein-coupled anti--mouse IgG2b, the fluorescein-coupled anti--mouse IgG2a and the fluorescein-coupled goat anti--rabbit Ig were from Jackson ImmunoResearch Laboratories. Alexa Fluor 488--coupled goat antifluorescein antibody was obtained from Molecular Probes. Cell grade thapsigargin was purchased from Sigma-Aldrich. Confocal images were obtained using a LSM 700 confocal microscope (Carl Zeiss) over a 63× objective. Z stack optical sections were acquired at 0.2 µm depth increments, and both green and red laser excitation were intercalated to minimize cross-talk between the acquired fluorescence channels.
### Confocal image post-treatment.
Complete image stack deconvolution was performed with Huygens Essential (version 3.0, Scientific Volume Imaging), and 2D images were generated from a maximum intensity projection over a 3D volume cut of 1 µm depth, centered either on the vesicular compartment when visible or on the cell center.
### Quantification of Lck, LAT, and TCRζ subcellular distribution.
The plasma membrane of CD4 T cells was surface labeled with anti-CD2 mAbs (clone TS2/18; American Type Culture Collection \[ATCC\]). Vesicular compartments were intracellularly stained for the proteins of interest. Confocal images were acquired at 2-µm increments in the z-axis. Two to three contiguous optical sections contained the whole-cell fluorescence information. Plasma membrane segmentation was implemented through the Weka supervised machine-learning segmentation algorithm ([@bib16]), using the Fiji image analysis software ([@bib34]). To teach the machine-learning algorithm, we have manually segmented at least three datasets for each imaging condition. 2D probability maps generated by the Weka algorithm were then converted into binary masks and used to delimitate the internal and external outlines of the plasma membrane. Our automated method provides accurate segmentation of the plasma membrane in the stained tracts and reconstructs the approximate location of the unstained tracts resulting from plasma membrane labeling gaps. % vesicular compartment FI = Total Fl − Plasma membrane Fl/Total Fl × 100.
### Manipulation of intracellular calcium levels.
Jurkat and primary CD4 T cells were treated with medium alone or medium containing either 5 µM of the endoplasmic reticulum calcium ATPase inhibitor thapsigargin or 5 µM of the calcium ionophore ionomycin for 30 min, washed, and processed for microscopy.
### siRNA-silencing experiments and Western blot analysis.
Jurkat or primary CD4 T cells were transfected once with siRNA oligonucleotide pools (Invitrogen) targeting either Syt7, MAL, or with a nontargeting siRNA pool, as negative control (siCtr; Thermo Fisher Scientific). Shutdown efficiency was assessed 72 h after transfection. Cellular extracts were prepared with lysis buffer (containing β-mercaptoethanol) and complete protease inhibitors (Roche). After separation by PAGE, proteins were transferred to nitrocellulose membranes. Anti-Syt7 antibody (rabbit polyclonal; Sigma-Aldrich) was added at 1:200 dilution, whereas anti-MAL antibody (provided by M.A. Alonso) was added at 1:200 dilution in a solution of 1% (wt/vol) dehydrated milk in Tris HCl-buffered saline with 0.05% (vol/vol) Tween-20. Analysis of β-actin served as a loading control; anti--β-actin was added to the blots at a dilution of 1:100,000 in a solution of 1% (wt/vol) dehydrated milk in Tris HCl-buffered saline with 0.05% (vol/vol) Tween-20. Blots were developed with ECL Plus reagents (GE Healthcare).
### dSTORM sample preparation.
Glass coverslips were washed 2--3 times in optical grade acetone and soaked overnight in 0.1 M KOH. Coverslips were then thoroughly rinsed in deionized water and dried. The glass coverslips were coated overnight at room temperature with 0.001% poly-L-Lysine (Sigma-Aldrich) diluted in PBS. Dried coverslips were subsequently incubated with stimulatory (αCD3 mAb MEM 92; EXBIO) or nonstimulatory (αCD45 mAb GAP 8.3; ATCC) antibodies at concentration of 10 µg/ml overnight at 4°C. Cells were resuspended in imaging buffer ([@bib18]), and 500,000 cells were dropped onto the coverslips and incubated for 3 min at 37°C. In some cases thapsigargin (5 µM) was added during the incubation period. Cells were then fixed with 4% paraformaldehyde for 30 min at room temperature. After 15-min incubation with blocking buffer, fixed cells were incubated for 1 h with primary antibodies. The anti-pY^191^LAT polyclonal antibody was obtained from BioSource; the anti-pY^118^SLP-76 was purchased from BD and the anti-pY^319^ZAP-70 was obtained from Cell Signaling Technology. Cells were washed and incubated for 45 min, with secondary antibodies conjugated to Alexa Fluor 568 or to Cy5 (Molecular Probes). To maximize the number of detected molecules, care was taken to minimize photobleaching: cells were embedded in oxygen scavenger buffer as previously described ([@bib18]), imaged on the same day of labeling, and kept in the dark until imaged. Tetraspeck beads (100 nm diameter; Invitrogen) were mounted with the sample as fiduciary landmarks and later tracked for computational drift correction and chromatic realignment.
### Widefield and dSTORM acquisition procedure.
For each coverslip, 8--10 area fields (55 × 55 µm^2^ per field, 107 × 107 nm^2^ per pixel) were preselected on the microscope for imaging; each field typically contained between 1 and 6 cells. We developed the automated acquisition sequence based on the open-source μManager microscopy acquisition software ([@bib13]). First, the field-of-view was centered in the preselected coverslip region. The cells were then imaged by triple-color epifluorescence (GFP, Alexa Fluor 568, and Cy5). The z-stack acquisition was performed at 200 nm z-steps encompassing the size of the Jurkat cell. This enabled to determine the proper localization and expression levels of the proteins of interest in the analyzed cells. XZ and YZ wide-field projections were taken to monitor LAT vesicular compartment delivery at the synapse. Finally, the objective was refocused on the surface of the coverslip, TIRF illumination was switched on into and a dSTORM image-stream acquisition was performed for the Cy5 channel (635 nm laser-excitation at 1.7 kW/cm^2^, 662--690 nm emission) and then the Alexa Fluor 568 channel (561 nm laser-excitation at 2.4 kW/cm^2^, 589--625 nm emission), each composed of 20.000 images acquired at 30 Hz. Imaging parameters were set using the μManager freeware running on a desktop PC. Laser control was achieved with custom software ([@bib19]). A detailed description of the imaging apparatus can be found in ([@bib19]). Custom algorithms were applied to determine the position map of the identified molecules, as well as their distance from each other, as described in the next section.
### Single-molecule detection and localization.
Particle detection and localization was carried by a custom algorithm using the Fiji-Jython scripting interface ([@bib34]). The algorithm uses remote procedure calls to connect to a Python virtual machine implementing the DAOSTORM algorithm ([@bib20]). This method uses DAOSTORM to detect and localize particles in both unprocessed and treated frames combining time averaging with frame subtraction in a process similar to a gSHRImP analysis ([@bib37]). Multiple detections caused by the same particle appearing in adjacent frames (distances smaller than 0.25 pixels) are merged and detections with low fitting accuracy are discarded. For our acquisition settings we have observed that combining the DAOSTORM engine with gSHRImP analysis allows us to decrease the localization error by up to 20% when compared with simple DAOSTORM analysis while supporting up to a threefold increase in particle detection.
### Super-resolution drift correction and chromatic realignment.
Before particle detection and localization, fiduciary beads were identified and marked, typically more than five. Sample drift was then calculated by tracking the group displacement of selected beads throughout the acquired image sequence. After particle detection and localization, the position of each particle was readjusted by subtracting the drift identified at the corresponding time-point. Chromatic realignment was generated after drift correction, the channel displacement of beads was calculated, a position correction for each particle on the Cy5 channel was then computed and applied by subtracting the average channel-displacement of the closest beads, weighted by their distance to the particle. To estimate the chromatic realignment error, beads were randomly selected and the corresponding chromatic realignment vector was calculated not using their channel displacement information, an ∼3 nm error was observed by comparing the chromatic realignment vector and channel-displacement vector.
### Super-resolution estimation and image reconstruction.
The localization error was calculated by measuring the full-width-half-maximum of manually selected small detection clusters (5--21 particles per cluster; [@bib5]). The average localization error for Alexa Fluor 568 was 19 nm (7 clusters) and Cy5 was 16 nm (6 clusters). Final super-resolution reconstructions were then generated in Fiji ([@bib34]) by creating images with 10-nm pixel-size and additively superimposing a Gaussian kernel of 20-nm full-width-half-maximum to each particle.
### Super-resolution protein cluster segmentation and characterization.
Protein cluster segmentation was performed through the automatic triangle threshold implementation of the Fiji software ([@bib34]). The triangle threshold method was applied individually to regions-of-interest demarking a single-cell, allowing us to discriminate clustered particle detections at the immunological synapse. Background detections (false-detection) caused by noise fluctuations or motile unbound labeled antibodies were minimally present in the thresholded images. For each segmented particle cluster in the rendered image, the following properties were measured: particles density, area, circularity, and diameter. We verified that cell illumination was considerably homogenous for the individual cell regions-of-interest, but slightly nonhomogeneous for the full field-of-view due to the Gaussian profile of the laser illumination in our microscopy setup. Additionally, we quantified the percentage of overlap between the segmented clusters and corresponding interdistance maps (see below).
### Interdistance maps calculation.
Interdistance maps are artificially generated super-resolution images where pixel values identify the presence of a pair of pLAT and pSLP76 proteins at distances \<20 nm away from each other (detailed below). These maps are insensitive to particle concentration. The presence of either a single or more pairs in a region of space represented by a pixel will lead to a pixel value equal to the shortest distance between any of the pairs. This feature allowed us to map and easily visualize regions of space where proteins are closely packed or potentially interact, but not necessarily in large numbers. Concentration insensitivity has the potential to augment false detections. To minimize any unwanted false contributions, we created a prefiltering process described below that severely diminishes the presence of erroneous detections.
### Pre-filtering.
Throughout dSTORM image acquisition, an individual fluorophore typically undergoes multiple cycles of photoswitching, leading to several detections of the same fluorophore in a region whose extent is determined by the localization accuracy ([@bib18]). We have verified that for both Alexa Fluor 568 and Cy5 imaging conditions, small detection clusters featured 5 or more particles typically generated by individual fluorophore photoswitching. Based on this feature, we constructed a prefiltering algorithm that eliminates any detected particle that does not have at least 5 neighboring particles of the same color at distances smaller than 10 nm. We verified that these threshold values reduced spatially random false detections within the cells while severely removing detections outside and keeping the morphology of the labeled ultrastructures.
### Interdistance map generation.
For a specific cell, the interdistance map is generated by creating an image with the same pixel size (10 nm) and image size as the corresponding super-resolution reconstructions. Detected particles for the Cy5 and Alexa568 channels are prefiltered as described above. Subsequently, for each particle identified in the Cy5 channel the closest particle in the Alexa568 channel is searched for. For each found pair, if the Euclidian distance between both particles is smaller than 20 nm and there is no other pair of particles with a smaller distance within the same pixel region of space, then the corresponding pixel value is set to be equal to the pair distance, or a null value otherwise. For convenience, we then apply an inverted grayscale lookup table to the image, giving pixels a bright value where pairs are found at very close distances, dim value for short distances and black value where no pairs have been found at distances smaller than 20 nm.
### Flow cytometry.
Primary CD4 T cells were knocked down for either Syt7 or MAL expression 3 d before the experiment using siRNA oligonucleotides or scramble oligo as control, as described above. For Lck, LAT, TCRζ, SLP76, and ZAP70 phosphorylation analysis, 5 × 10^5^ CD4 T cells were co-cultured at 1:1 ratio with Raji cells for 30 min in the presence or absence of SEE (10 µg/ml). Thapsigargin (5 µM) was added to some of the samples. The cells were fixed and permeabilized (Cytofix/Cytoperm Plus; BD) and stained with the antibodies of interest at room temperature. For Erk phosphorylation analysis, 5 × 10^5^ primary CD4 T cells prepared as described above were co-cultured at 1:1 ratio with Raji cells for 10 min in the presence or absence of SEB and TSST1 superantigens (10 µg/ml), and then fixed with 4% paraformaldehyde and permeabilized with saponin. For CD69 up-regulation analysis, 5 × 10^5^ cells primary CD4 T cells prepared as described above were co-cultured at 1:1 ratio with Raji cells in the presence or absence of SEB and TSST1 for 4 h. For cytokine production analysis, 10^10^ cells primary CD4 T cells prepared as described above were stimulated with either Raji cells loaded with SEB and TSST1 (10 µg/ml) or medium at 1:10 ratio for 16 h. Thapsigargin (5 µM), ionomycin (500 ng/ml), or PMA (20 ng/ml) plus ionomycin (500 ng/ml) were added to some of the samples. Brefeldin A (2 mg/ml; Sigma-Aldrich) was added for the last 15 h. Cells were washed and stained with FITC anti-CD4 (RPA-T4; BD) for 20 min at 4°C. The cells were permeabilized (Cytofix/Cytoperm Plus; BD) and stained with PE anti--IFN-γ (4S.B3; BD) and APC anti--IL-2 (MQ1-17H12; BD) at room temperature. Erk, CD69 up-regulation, and IL-2/IFN-γ production were determined gating on primary CD4 T cells. We used a FACSORT with data analysis in FlowJo (Tree Star).
### Single-cell ELISPOT assay for IFN-g-secreting cells.
Primary CD4 T cells were electroporated with siRNA oligonucleotides for Syt7, MAL, or scramble control 3 d before the experiment, as described above. 96-well filtration plates (Millipore) were coated with anti--human IFN-γ antibody (1-DIK; Mabtek). 2 × 10^5^ cells primary CD4 T cells prepared as described above were plated per well and stimulated with either Raji cells loaded with SEB or medium at 1:10 ratio, or PMA (20 ng/ml) and ionomycin (500 ng/ml) for 16 h. After culture, the plates were washed, followed by incubation with biotinylated anti--IFN-γ antibody (7B6-1; Mabtek). Spots were developed using freshly prepared substrate buffer (0.3 mg/ml of 3-amino-9-ethyl-carbazole and 0.015% H~2~0~2~ in 0.1 M sodium acetate, pH 5.0). The frequency of IFN-γ--secreting CD4 T cells was calculated based on the percentage of CD4 T cells present in the well.
### 3D time-lapse microscopy.
GFP epifluorescence imaging was performed by continuous acquisition of sequences of z-stacks with 0.5-µm z-steps, 7--20-µm z-range, for 3--10 min. These values were selected to optimize the visualization of the cell adherence to the coverslip and formation of the immune synapse, and to capture a sufficient cell volume and minimize photodamage. While imaging, cells were kept at 37°C in an incubator encompassing most of the microscope body. For simplicity of visualization, time-lapse movies were generated in Fiji ([@bib34]) by creating a horizontal montage where each lateral element corresponds to a maximum intensity projection over a 2.5-µm z-range. Before creation of the time-lapse movies, datasets were denoised by a 3D Gaussian blur of 0.5 × 0.5 × 0.5 pixels (x sigma, y sigma, z sigma).
### Statistical analysis.
Were performed by Mann--Whitney nonparametric test using Prism software (GraphPad).
### Online supplemental material.
Video 1 shows LAT-GFP expressing cells deposited on an αCD3 stimulatory surface. Online supplemental material is available at <http://www.jem.org/cgi/content/full/jem.20130150/DC1>.
Supplementary Material
======================
###### Supplemental Material
We are thankful for the technical assistance of Imagopole, C. Cuche, and M. Lelek. We thank A. Echard, J. Enninga, T. Galli, and B Goud for reagents and O. Schwartz for critical reading of the manuscript.
This work was funded by grants from Agence National de Recherche sur le SIDA (ANRS), Agence National de Recherche (ANR-07-MIME-030; ANR-2011-Blanc-SVSE3-025), Institut Pasteur PTR-214, Ile de France DIM-MALINF (to A. Alcover, M.-I. Thoulouze, and C. Zimer) and BFU2009-07886 and CSD2009-00016 from the Spanish Ministry of Science and Innovation (to M.A. Alonso). H. Soares was funded by the Institut Pasteur PTR-214, EMBO long-term fellowship, ANR, Sidaction, and ANRS. R. Henriques was funded by an Institut Pasteur-Roux fellowship.
The authors declare to have no financial conflict of interest.
Abbreviations used:dSTORMdirect stochastic optical reconstruction microscopyMALmyelin and lymphocyteSyt7synaptotagmin-7TIRFtotal internal reflection fluorescence
[^1]: M.-I. Thoulouze and A. Alcover contributed equally to this paper.
| {
"pile_set_name": "PubMed Central"
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Introduction
============
Terminal 11q deletion syndrome, also known as Jacobsen Syndrome (JBS), is a rare genetic disorder associated with multiple dysmorphic features and occurs in 1 in 100,000 live births with a female predominance of 2:1.[@b1-cmped-8-2014-045]--[@b4-cmped-8-2014-045] JBS occurs because of the loss of contiguous set of genes present at 11q23 with deletion size varying from 7 to 20 Mb in size but could be as small as 2.9 Mb in some cases.[@b1-cmped-8-2014-045],[@b3-cmped-8-2014-045],[@b5-cmped-8-2014-045] A "de novo" deletion is seen in 85% JBS patients, and 15% cases arise as a result of parental translocations.[@b6-cmped-8-2014-045] Hallmark clinical features of JBS include pre- and post-natal physical growth retardation, intellectual disability, and characteristic facial dysmorphism. We present two cases of JBS who presented milder phenotypes than those described in literature despite of larger deletion size.
Case 1
======
A 4-year-old girl was born to non-consanguineous young parents. No family history of physical and intellectual disability was recorded. Antenatal period was uneventful except suspected intrauterine growth retardation. She was born through Caesarian section with a birth weight of 2.25 kg. She initially had developmental delay but by the age of referral (ie, 4 years), she achieved all motor milestones but was failing to thrive. Short stature and facial dysmorphism were also noted. Her height was 90 cm (\<third centile) and was also presented with microbrachycephaly. The facial profile showed sparse eyebrows, telecanthus, deep set eyes, strabismus, prominent epicanthal folds, low set dysmorphic ears, depressed nasal bridge, and a bulbous upturned nasal tip. The vermillion of upper lip was thin, and retrognathia along with a high arched palate is noted. No major limb dysmorphism was found except for bilateral short and clinodactyly. The neck was short, broad, and had slight webbing. No neurocutaneous markers were noted, and the genitalia was normal ([Fig. 1a](#f1-cmped-8-2014-045){ref-type="fig"}). Central nervous system examination revealed hypotonia. The child had no meaningful speech despite normal hearing potential, but her receptive language had developed marginally. The child had a low IQ of 30 suggesting severe intellectual disability. 2D echocardiogram showed ventricular septal defect and tricuspid valve tags. The skeletal survey and ultrasonography of the abdomen were normal. Magnetic resonance imaging revealed marginal white matter affection and a thin corpus callosum. Hearing and ophthalmic evaluation including fundus was normal. There were no symptoms of platelet dysfunction on clinical examination in the form of superficial bleeding (which may reflect qualitative and quantitative platelet abnormalities). The platelet count at presentation was normal. Platelet morphology with peripheral smear did not reveal giant platelets or any other abnormality. Karyotyping showed heterozygous segmental distal deletion of 11 with breakpoint at q24 region ie, 46,XX,der(11)del(11) (q24) of "de novo" origin. Oligonucelotide array comparative genomic hybridization \[aCGH\] characterized the large deletion of 11.8--11.9 Mb size at 11q24.1q25 \[arr cgh 11q24.1q25 (123,045,174--134,868,407)x1 (hg19-GRCh37)\] ([Fig. 2a](#f2-cmped-8-2014-045){ref-type="fig"}).
Case 2
======
A girl was referred at an age of 6½ years for chromosomal analysis because of multiple congenital anomalies and developmental delay. She was born to young and non-consanguineous parents. The birth history was uneventful. She presented a global developmental delay during examination. At the time of the referral, her height was 107 cm (at the third centile). Physical examination showed microcephaly, flat occiput, epicanthal folds, hypertelorism, mild ptosis, strabismus, low and posteriorly set ears, depressed and low nasal bridge, prominent and bulbous nasal tip, choanal atresia, flat alae nasi, smooth and long philtrum, large open mouth, thin vermillion of upper lip, everted lower lip, retrognathia, and high arched palate. Her neck was short and broad. Arachnodactyly was noted in the upper limb. She had no genital abnormalities ([Fig. 1b](#f1-cmped-8-2014-045){ref-type="fig"}). Her cardiac evaluations and ultrasound of the abdomen were unremarkable, and skeletal survey showed mild lordosis. She had an affected cognition with an IQ of 37, but did have some meaningful words to her vocabulary. Iron deficiency anemia was detected on the hemogram and later on confirmed by serum iron profiles. The patient neither had abnormal bleeding time nor bleeding manifestation. There was no thrombocytopenia as evident by normal platelet count or platelet dysfunction as evidenced by the clinical examination and platelet morphology on smear examination.
Cytogenetic study exhibited heterozygous deletion of the \#11q distal region at the site q24 ie, 46,XX,der(11)del(11)(q24). Oligonucleotide aCGH further confirmed a 13.9--14 Mb deletion at 11q23.3q25, but a relatively large 7.3--7.6 Mb duplication at 12q24.32q24.33 was also observed \[arr cgh 11q23.3q25(121,000,318--134,868,407)x1, 12q24.32q24.33(126,482,69 8--133,767,986)x3 (hg19-GRCh37)\] ([Fig. 2b](#f2-cmped-8-2014-045){ref-type="fig"}). This structural rearrangement was missed by the conventional cytogenetic analysis. The aberrant chromosome 11 was inherited from the phenotypically normal father who was the carrier of a balanced translocation 46,XY,t(11;12)(q23.3; q24.32).
Discussion
==========
JBS is a rare chromosomal disorder involving terminal deletion of chromosome 11q. The implementation of array technology in the clinics has permitted precise characterization of the deletions and detailed genotype--phenotype correlation in cases with JBS. We present two cases of JBS with deletions spanning 11.9 and 14 Mb, respectively. Despite the difference in the deletion size, both patients had nearly similar phenotypes as shown in [Table 1](#t1-cmped-8-2014-045){ref-type="table"}. The critical region underlying intellectual disability[@b7-cmped-8-2014-045],[@b8-cmped-8-2014-045] is located within the 4.11 Mb of the 11q telomere encompassing genes *SNX19*, *THYN1*, *OPCML*, *VPS26B*, *NCAPD3*, and *NTM*, which were deleted in our patients. Deletion of the *NTM* (neurotrimin) gene hampers the normal expression of the NTM protein in the developing nervous system that may promote neurite outgrowth that has been associated with cognitive function.[@b7-cmped-8-2014-045],[@b8-cmped-8-2014-045] Larger deletion encompassing all four genes associated with JBS-related thrombocytopenia (*FLI1*, *ETS1*, *NFRKB*, and *JAM3*) did not manifest a phenotype in both of our patients. Thrombocytopenia is a usual feature in \>88% JBS patients.[@b9-cmped-8-2014-045] We could not see any correlation between the size of deletion and manifestation of thrombocytopenia; this could be because of the heterogeneous nature of the deletion. Recently, it has been shown that second hit may be a prerequisite for the full blown expression of the thrombocytopenia.[@b10-cmped-8-2014-045] *JAM3* is suggested as the candidate gene for JBS cardiac phenotype[@b11-cmped-8-2014-045] that was deleted in both these patients. However, ventricular septal defect and tricuspid valve tags were observed only in case 1. Absence of heart disease in case 2 even with *ETS1* deletion is consistent with the hypothesis of a more centromeric localization of the cardiac critical region.[@b12-cmped-8-2014-045],[@b13-cmped-8-2014-045] A crucial role of the *ETS1* gene in heart development is reported.[@b13-cmped-8-2014-045] *ETS1* gene is also proposed as a candidate gene for involvement in limb anomalies, which was not observed in our cases.[@b14-cmped-8-2014-045] Although most features of JBS may be caused by deletion of different sets of contiguous genes, relative difference in the phenotypic expression may depend on the genetic background and gene--gene interactions in individual patient. The absence of typical JBS features in our patients may exclude an impact of genes in the deleted region on these features, but indicate a possible proximal localization of critical genes for JBS phenotype suggestive of a reduced penetrance. The overlap of phenotypic features between JBS and Noonan syndrome was not characteristically discernable in our patient other than a triangular facial appearance.[@b15-cmped-8-2014-045] The current study suggests that the detailed analysis is necessary in cases with milder phenotype. This can help to understand the underlying genetic etiology in 11q deletion phenotypes.
Our thanks to Dr. Sunil Trivedi for giving critical comments and to both the families for their kind co-operation.
**ACADEMIC EDITOR:** Praveen Kumar, Associate Editor
**FUNDING:** This work is partly supported by Foundation for Research in Genetics and Endocrinology (FRIGE). The authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal.
**COMPETING INTERESTS:** Authors disclose no potential conflicts of interest.
This paper was subject to independent, expert peer review by a minimum of two blind peer reviewers. All editorial decisions were made by the independent academic editor. All authors have provided signed confirmation of their compliance with ethical and legal obligations including (but not limited to) use of any copyrighted material, compliance with ICMJE authorship and competing interests disclosure guidelines and, where applicable, compliance with legal and ethical guidelines on human and animal research participants.
**Author Contributions**
Conceived and designed the experiments: FJS, JA. Wrote the first draft of the manuscript: FJS, JJS, CD, MR. Contributed to the writing of the manuscript: FJS, MR, CD, JA, AP, DN, JJS. Agree with manuscript results and conclusions: FJS, MR, CD, JA, AP, DN, JJS. Jointly developed the structure and arguments for the paper: FJS, MR, CD, JA, AP, DN, JJS. Made critical revisions and approved final version: FJS, MR, CD, JA, AP, DN, JJS. All authors reviewed and approved of the final manuscript.
{#f1-cmped-8-2014-045}
{#f2-cmped-8-2014-045}
######
Comparison of clinical features observed.
CLINICAL FEATURES IN JBS REPORTED PHENOTYPE CASE 1 CASE 2
------------------------------------- --------------------------------- --------- ----------
Pregnancy and related complications Intrauterine growth retardation \+ −
First signs and symptoms Intellectual disability \+ \+
Age at diagnosis 4 years 6½ years
Musculoskeletal problems Hypotonia \+ \+
Macrocrania − −
Facial asymmetry \+ \+
Microcephaly \+ \+
Brachycephaly (flat occiput) \+ \+
High prominent forehead \+ \+
Trigonocephaly − −
Ophthalmic problems Sparse eyebrows \+ \+
Hypertelorism \+ \+
Down slanting palpebral fissures \+ −
Cataract − −
Strabismus \+ \+
Epicanthal folds \+ \+
Palpebral ptosis \+ \+
Nasal problems Short nose − −
Flat nasal bridge \+ \+
Prominent nasal bridge \+ \+
Anteverted nares − −
Choanal atresia − \+
Flat alae nasi − \+
Prominent columella − −
Broad nasal bridge − −
Oral problems Smooth and long philtrum \+ \+
Thick lower lip − −
V-shaped mouth \+ \+
High arched palate \+ \+
Retrognathia \+ −
Problems with ear Small Low set ears \+ \+
Posteriorly rotated ears − −
Malformed external ears − −
Neck problems Short and broad neck \+ \+
Neck webbing \+ \+
Anomalies of upper limbs Thin fingers − −
Hypoplastic hypothenar region − −
Abnormal palmar creases − −
Cardiac anomalies Cardiac involvement (VSD) \+ −
Anomalies of lower limbs Clynodactylous toes \+ \+
Cutaneous syndactyly \+ −
Stubby and flat feet − −
Large and long first toe − −
Brachydactyly − −
Hematological abnormalities Thrombo-/Pancytopenia − −
| {
"pile_set_name": "PubMed Central"
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Facet joint (FJ) osteoarthritis (OA) is an important component of degenerative changes in the lumbar spine, and is a major contributing factor to low back pain[@b1]. However, not all individuals with FJ OA develops back pain, and patients with back pain may be radiologically negative of FJ OA[@b2][@b3][@b4]. Early detection of FJ OA before the onset of back pain-associated symptoms may help to prevent the disease progression. At present, FJ OA is diagnosed using computed tomography (CT) or magnetic resonance imaging (MRI). Due to the poorly understood mechanisms of FJ OA, there is no available marker for the early detection of this condition.
Indian Hedgehog (Hh), sonic Hh, and desert Hh constitute the Hh family, which plays critical role in growth, patterning, and morphogenesis[@b5][@b6][@b7][@b8][@b9]. Indian Hh has been found to play roles in osteoarthritis[@b10][@b11][@b12][@b13][@b14]. It has been shown that during endochondral bone growth, Indian Hh is mainly produced and secreted by pre-hypertrophic chondrocytes and regulates chondrocyte hypertrophic differentiation. Patched1 (*PTCH1*) and Smoothened (*SMO*) are two transmembrane receptors that respond to Indian Hh signaling. When Indian Hh is not present, Patched1 inhibits Smoothened, and represses the downstream gene expression by suppressing the Gli zinc finger transcription factors (*GLI1*, *GLI2*, and *GLI3*)[@b15][@b16]. When Indian Hh is present, it binds to Patched and releases Smoothened, activating the Hh signaling pathway and allowing the active Gli transcription factors to enter the nucleus and enhance the transcription level of downstream targets[@b17]. Gli1 mainly functions as a transcription factor, and increased Gli1 level indicates activation of the Hh signaling pathway[@b18][@b19]. The vertebrate cell surface protein Hh-interacting protein (*HHIP*) have also been shown to bind vertebrate Hh proteins and negatively modulate Hh signaling[@b20].
In this study, we examined the pathological changes in the FJ OA in humans and analyzed the expression levels of Hh signaling-associated *PTCH1, GLI1,* and *HHIP*. The prospective relationship between the pathology of FJ OA and Hh signaling is discussed.
Material and Methods
====================
Ethical Statement
-----------------
All experimental protocol were approved by the Ethical Committee of The First Affiliated Hospital of Chinese PLA General Hospital. Written informed consent was obtained from all enrolled patients. All the subsequent research analyses were carried out in accordance with the approved guidelines.
Patients
--------
This study included 24 consecutive patients (mean age of 61.7 (range 42--75 years)), including 15 males and 9 females, diagnosed with lumbar intervertebral disk herniation or degenerative spinal stenosis. These patients were treated with FJ resection *en bloc* at our hospital between October 2012 and April 2013.
MRI
---
Preoperative T2-weighted MRI images of the diseased FJs were obtained and independently evaluated by two experts in this field. Discrepancy between the two evaluators were solved by discussion. The morphological MRI grading system of Weishaupt *et al.* was used to evaluate the FJs[@b21][@b22]. This grading system utilizes joint space width, osteophytes, facet hypertrophy, bone erosions and subchondral cysts to assign grades 0 to III, where 0 and III represents normal and the most severe changes, respectively.
Pathological analysis
---------------------
The diseased FJs were resected *en bloc* and dissected into two parts through a plane perpendicular to the joint surface. For pathological analysis, half of the FJs was fixed in 4% paraformaldehyde for 48 hours, decalcified, and embedded in paraffin. The other half of the FJs were immediately put into liquid nitrogen and used for quantitative real time polymerase chain reaction (qRT-PCR). The block was cut into 4-μm sections on the coronal plane. The sections were stained with toluidine blue and hematoxylin-eosin (HE), respectively. Osteoarthritis Research Society International (OARSI) histopathology grading system was used to evaluate the sections[@b23][@b24]. In this system, the grade of damage from 0 to 6 is defined as the depth of progression of OA into the cartilage and the stage of damage is defined as the horizontal extent of cartilage involvement from 0 to 4. The final score is the combined value of grade and stage (score range 0--24).
qRT-PCR
-------
Total RNA from the facet joints (inclusive of both the cartilage and subchondral bone) was extracted using the Trizol method (Invitrogen, US). The RNA was reversely transcribed into cDNA using random hexamers. The cDNAs were subsequently used to template qRT-PCR reactions using KAPA SYBR® FAST Universal 2X qPCR Master Mix (KAPA BIOSYSTEMS, Wilmington, MA, USA) using primers mentioned in [Table 1](#t1){ref-type="table"} and following manufacturer recommended reaction conditions. The data were analyzed with 2^−ΔΔCt^ method and normalized to *ACTB* expression.
Statistical analysis
--------------------
Continuous data were expressed as mean ± SD. Correlation analysis was performed using Spearman rank coefficient with SPSS 17.0 (SPSS, US). A *P*-value less than 0.05 was considered statistically significant.
Results
=======
MRI image grades
----------------
The Weishaupt grades included grade 0 in 5 cases, showing normal FJ space of 2--4 mm; grade I in 8 cases, showing narrowed FJ space of \<2 mm, small osteophytes, and mild articular hypertrophy; grade II in 7 cases, showing narrowed FJ space, moderate osteophytes and articular hypertrophy, mild subchondral erosion; grade III in 4 cases, showing narrowed FJ space, large osteophytes, severe articular hypertrophy, subchondral erosion and cysts ([Table 2](#t2){ref-type="table"}).
### Pathological examination
The toluidine blue and HE staining results were scored with OARSI grading system ([Table 2](#t2){ref-type="table"}). Typical pathological images and corresponding patient information are shown in [Fig. 1](#f1){ref-type="fig"}. Grade 0 showed intact articular surface, clear layer structures, and normal matrix. Grade 1 showed fibrosis in the articular surface and chondrocyte hypertrophy (relative increase of chondrocyte cytoplasm compared to other chondrocytes in the histologic cartilage layer). Grade 2 showed matrix discontinuity and chondrocyte hypertrophy. Grade 3 showed cracks in the matrix and chondrocyte necrosis. Grade 4 showed matrix loss and cyst formation. Grade 5 showed exposure of the subchondral bone and fibrocartilage repair. Grade 6 showed bone reconstruction and microfracture in the fibrocartilage.
### Correlation analysis
OARSI scores were positively correlated with GLI1 (r = 0. 646), PTCH1 (r = 0. 518), and HHIP (r = 0.762) mRNA expression levels (*P* \< 0.01 in each case) ([Fig. 2A--C](#f2){ref-type="fig"}). MRI Weishaupt grades were positively correlated with GLI1 (r = 0.491), PTCH1 (r = 0.444), and HHIP (r = 0.654) mRNA expression levels (*P* \< 0.05) ([Fig. 2D--F](#f2){ref-type="fig"}). Furthermore, pathological OARSI scores were positively correlated with MRI Weishaupt grades (r = 0.894, *P* \< 0.01) ([Fig. 3](#f3){ref-type="fig"}).
Discussion
==========
In this study, we collected FJs with OA from 24 patients and examined the mRNA levels of Hh signaling-associated genes. It was found that the mRNA levels of GLI1, PTCH1, and HHIP are positively associated with the MRI image and pathological scores of the FJs, suggesting a role of the Indian Hh pathway in the development of FJ OA.
It has been shown that ablation of Indian Hh from postnatal chondrocytes leads to closure of the growth plate, loss of trabecular bone, and defective skeletal growth[@b25][@b26], suggesting that Indian Hh plays important roles in postnatal growth. In addition, Indian Hh is induced after femoral fracture in adults, suggesting that Indian Hh might be involved in the repair after bone injury[@b27][@b28], Indian Hh plays a critical role in endochondral bone formation in embryo and limb development as well as postnatal bone formation. However, its expression starts to decrease with aging under physiological conditions[@b29]. In a human study, Indian Hh was almost undetectable in healthy adult cartilage but it increased in early cartilage damage[@b30].
In human OA cartilage and synovial fluid, Indian Hh is upregulated and is correlated with OA progression[@b12]. Changes in gene expression and chondrocyte morphology that are consistent with chondrocyte hypertrophy and cartilage degeneration were also found in OA cartilage. These findings provide strong evidence that Indian Hh plays a critical role in OA pathology.
In addition, genetically modified mice that have elevated Hh signaling in chondrocytes showed more severe OA phenotype, and OA-caused cartilage damage can be alleviated by inhibiting Hh signaling in mice or human cartilage explants[@b31]. Lin *et al.* had reported that *PTCH1*, *GLI1*, and *HHIP* were all overexpressed in human osteoarthritic samples, as well as in the articular cartilage from a mouse model of osteoarthritis. In addition, mice with aberrant activation of the Hh pathway showed cartilage degradation and an increased tendency to develop osteoarthritis. The severity of the disease correlated with the degree of Hh pathway activation. Furthermore, genetic or pharmacologic inhibition of the Hh pathway in a mouse model of osteoarthritis significantly decreased collagen X deposition, cartilage degradation and osteoarthritis severity. Remarkably, inhibition of Hh in explant cultures of human osteoarthritic cartilage samples blocked expression of key genetic markers of osteoarthritis, including the metalloproteinases *ADAMTS5* and *MMP13*, the transcription factor *RUNX2*, and *COL10A1*[@b31]. Conditional deletion of Indian Hh in chondrocytes can attenuate OA progression in a mouse model[@b14]. Given all this information and our findings it would be rational to presume that IHH signaling is increased in OA and is perhaps a key component in OA pathogenesis and progression. However, our future endeavors would be geared towards specifically testing the same.
In our study, the mRNA expression levels of three Indian Hh-associated genes, *GLI1*, *PTCH1*, and *HHIP*, were found to be positively correlated with FJ OA pathological scores. This is the first study demonstrating a role of Indian Hh in the pathology of FJ OA. Future endeavors will deal with defining the precise role and underlying molecular mechanism by which Indian Hh signaling promote FJ OA.
Author Contributions
====================
F.S. and S.X.H. wrote the main manuscript text, J.L.Z., Y.L. and Y.Z. collected data and carried out the experiments, C.L.Z. analyzed the data, J.G.T. designed the experiments. All author has approved the manuscript.
Additional Information
======================
**How to cite this article**: Shuang, F. *et al.* Indian Hedgehog signaling pathway members are associated with magnetic resonance imaging manifestations and pathological scores in lumbar facet joint osteoarthritis. *Sci. Rep.* **5**, 10290; doi: 10.1038/srep10290 (2015).
{#f1}
{#f2}
{#f3}
###### Primers used for RT-PCR.
**Primer** **Sequence** **Product (bp)**
------------ ----------------------------- ------------------
*GLI1*-F 5′-GGTGGTTCACATGCGCAG-3′ 170
*GLI1*-R 5′-CATTGCTGAAGGCTTTACTGC-3′
*PTCH1*-F 5′-CCACCAAGTGATCGTGGAAG-3′ 244
*PTCH1*-R 5′-GCCAGAATGCCCTTCAGTAGA-3′
*HHIP*-F 5′-TCCGGTCACATCTTGGGATT-3′ 167
*HHIP*-R 5′-GTCTGTGCAGGTTGTACCGTG-3′
*ACTB*-F 5′-TCCCTGGAGAAGAGCTACG-3′ 131
*ACTB*-R 5′-GTAGTTTCGTGGATGCCACA-3′
###### Pathological scores and mRNA levels of GLI1, PTCH1, and HHIP.
**Patient number** **Weishaupt grades** **OARSI scores** ***GLI1*** **(10**^**−6**^) ***PTCH1*****(10**^**−4**^) ***HHIIP*** **(10**^**−5**^)
-------------------- ---------------------- ------------------ ----------------------------- ----------------------------- ------------------------------
1 3 18 1.893 16.736 7.104
2 1 9 5.258 11.200 5.909
3 1 8 2.014 8.564 1.909
4 2 6 1.439 14.970 2.909
5 0 3 1.262 9.103 2.844
6 2 12 1.042 25.754 6.730
7 1 6 0.859 16.726 1.026
8 0 3 1.293 16.052 1.907
9 1 4 1.563 17.253 1.167
10 3 24 5.887 25.300 7.179
11 3 20 4.482 20.690 3.502
12 2 12 5.377 19.448 5.650
13 1 4 1.357 9.137 2.462
14 0 0 0.986 8.245 0.890
15 1 3 0.748 10.172 2.610
16 3 20 2.914 12.450 4.990
17 2 9 1.056 15.470 2.844
18 0 2 1.096 9.640 1.730
19 2 6 2.7212 14.600 1.026
20 0 3 2.7199 21.550 1.907
21 2 12 3.6874 22.680 5.282
22 1 8 2.6554 21.220 3.554
23 2 12 4.5783 23.045 4.113
24 1 8 1.9477 21.954 1.292
[^1]: These authors contributed equally to this work.
| {
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INTRODUCTION {#S1}
============
Cholecystectomies are one of the most common surgical procedures performed in the United States, with over 300,000 laparoscopic cholecystectomies performed every year.^\[[@ref1]\]^ The complication rates of cholecystectomies range from 1.56% to 6%. Common complications include bleeding from adjacent tissues or the cystic artery, iatrogenic perforations of the gallbladder, injuries to the bile duct, and infection, including abscess formation.^\[[@ref2]\]^ We submit a case of a post- cholecystectomy patient who presented with a ruptured hepatic artery mycotic aneurysm from an abscess within the porta hepatis ultimately requiring endovascular treatment.
Topical thrombin is a common tool utilized by physicians in the treatment to achieve hemostasis. Its use has even expanded to the treatment of superficial pseudoaneurysm. However, there have been only a few cases of catheter-directed endovascular use of thrombin. This case presentation describes the transarterial use of thrombin in the treatment of the aneurysm.
PATIENT CASE {#S2}
============
This case involves a 71-year-old male with a past medical history of depression, complex regional pain syndrome, and non-insulin dependent diabetes mellitus Type 2 who presented with abdominal pain and found to have symptomatic cholelithiasis requiring laparoscopic cholecystectomy followed by a complex course, including acute blood loss of approximately 350 cc intraoperatively.
He returned to the emergency department (ED) approximately 2 weeks post-discharge with complaints of worsening abdominal pain, nausea, and vomiting. Contrast-enhanced CT of the abdomen and pelvis (CT A/P) demonstrated multiple large subscapular hepatic abscesses \[[Figure 1a](#F1){ref-type="fig"} and [b](#F1){ref-type="fig"}\]. Drainage catheters were subsequently placed in two abscesses \[[Figure 2a](#F2){ref-type="fig"} and [b](#F1){ref-type="fig"}\]. On repeat, contrast-enhanced CT A/P demonstrated new focal dilatation in the right hepatic artery concerning the development of a mycotic pseudoaneurysm \[[Figure 3](#F3){ref-type="fig"}\].
{#F1}
{#F2}
{#F3}
A visceral angiogram was performed, which demonstrated aneurysmal dilation at the distal portion of the proper hepatic artery measuring approximately 1.5 cm \[[Figure 4](#F4){ref-type="fig"}\]. A collaborative decision was made to conservatively treat the aneurysm with medical management due to the high risk of inducing liver failure with coil embolization. The patient was later discharged on oral antibiotics.
{#F4}
He returned to the ED approximately a week later with worsening of his symptoms, and a repeat CT A/P demonstrated a new fluid collection within the porta hepatis with interval enlargement of the pseudoaneurysm \[[Figure 5](#F5){ref-type="fig"}\]. The patient acutely decompensated and suffered cardiac arrest requiring multiple rounds of cardiopulmonary resuscitation. It was presumed that there was active bleeding from the known hepatic artery aneurysm. Bleeding was presumed secondary to a rupture of the known mycotic aneurysm which required massive transfusion protocol with approximately 10 units of PRBC and vasopressors. The patient was immediately taken to the interventional radiology suite.
{#F5}
The celiac artery was cannulated with an SOS catheter. An angiogram was performed demonstrating frank extravasation just after the origin of the proper hepatic artery \[[Figure 6](#F6){ref-type="fig"}\]. No distal flow into the liver parenchyma was identified past the rupture. A microcatheter and microwire were used to obtain access past the gastroduodenal artery (GDA) into the stump of the proper hepatic artery, which was then embolized with coils up to the confluence of the proper hepatic artery and GDA branches. Despite the placement of multiple coils, there was continued visualization of active hemorrhage \[[Figure 7](#F7){ref-type="fig"}\]. The continued hemorrhage was presumed to be from evolving DIC a transfusion-related coagulopathy.
{#F6}
{#F7}
Given lack of other suitable embolic agents available at the time, at a regional community hospital, a decision was made to inject thrombin into the coil mass. The microcatheter was advanced into the proximal coils, positioning was verified with an injection of contrast, and subsequently, 1000 units of reconstituted thrombin were injected. The microcatheter was then removed, the 5 French catheters was aspirated and flushed, and a repeat angiogram through the 5 French catheters demonstrated successful embolization \[[Figure 8](#F8){ref-type="fig"}\]. Unfortunately, due to multiorgan failure and persistent shock-like physiology from bacteremia, the patient was pronounced dead 3 days after the procedure.
{#F8}
DISCUSSION {#S3}
==========
Mycotic aneurysms, commonly referred to as infected aneurysms, are rare in their frequency. However, they can be life-threatening with a high incidence of arterial rupture and recurrence even after treatment. The term "mycotic," which usually means fungus, was coined by physician Sir William Osler in 1885 when he described the gross pathological appearance of two small saccular aortic aneurysms and not the underlying pathological organism responsible for its development.^\[[@ref3],[@ref4]\]^
Mycotic aneurysms normally arise from bacterial invasion into the arterial wall through hematogenous spread usually in immunocompromised patients such as those with diabetes mellitus, liver cirrhosis, and acquired immune deficiency syndrome or with patient switch arterial luminal defects such as atherosclerotic plaque, ulceration, and preexisting aneurysm. Common locations for the development of mycotic aneurysms include the thoracic and abdominal aorta, abdominal visceral arteries, lower extremity arteries, and intracranial arteries. In the case of our patient, the presence of the abscess within the porta hepatis allowed for the proliferation of the mycotic aneurysm.
Aneurysms of the hepatic artery are uncommon. They tend to be saccular and multiple. Causes typically include infection, arteriosclerosis, trauma, and vasculitis. Post-liver transplantation patients can have pseudoaneurysm formation of the hepatic artery and its branch vessels, these usually occur within the 1^st^ month of transplantation. A vasculitis such as polyarteritis nodosa has been reported to result in the formation of aneurysms.^\[[@ref5]\]^ Pseudoaneurysm formation can occur after trauma to the hepatic artery; furthermore, there have even been cases of pseudoaneurysm formation trauma from a percutaneous biopsy.^\[[@ref6]\]^
Diagnostic testing {#S3_1}
------------------
Before definitive treatment, most patients with known or suspected mycotic aneurysms undergo essential diagnostic testing, including complete blood count, comprehensive metabolic panel, gram stain, blood cultures, and sensitivities, as well as contrast-enhanced CT or magnetic resonance imaging. The two most essential components in the treatment of mycotic aneurysm involve culture-specific antibiotic therapy and physical treatment of the pseudoaneurysm either with surgical excision or with endovascular coiling depending on the patient's clinical condition.^\[[@ref7]\]^
Treatment {#S3_2}
---------
Surgical intervention is the first-line treatment in patients who are sta ble once antibiotic therapy which is initiated. Surgical options such as excision and ligation can be performed in smaller distal arteries where the chance of end- organ ischemia is low. Excision with reconstruction is another option when there is the involvement of larger arteries such as the thoracic aorta or suprarenal aorta. The reconstruction can be performed immediately but can result in recurrent infection or breakdown of the anastomoses. Interval reconstruction can be performed as well but with the risk of end-organ ischemia.^\[[@ref3]\]^ Due to the hemodynamic instability of our patient, he was not a candidate for immediate surgical intervention and immediate endovascular treatment was needed.
Endovascular treatment is a viable option when surgical treatment is contraindicated or unavailable. The two main available endovascular therapies involve the placement of endovascular stents in the case of an intact aneurysm or coil embolization. Endovascular stent placement is a suitable option in situations involving high-risk patients where surgery is prohibitive due to high mortality risk or during emergent cases involving the rupture of a mycotic aneurysm. Coil embolization can be performed as a temporizing measure until surgical debridement can be performed in more controlled circumstances or in situations where the likelihood of end-organ ischemia is considered low, such as with smaller visceral organ branches.^\[[@ref3]\]^ In the case of our patient, the placement of an endovascular stent was not feasible due to the acuity of the patient's condition and lack of availability of stents at the regional community hospital.
Thrombin {#S3_3}
--------
Thrombin acts within the last step of the coagulation cascade by converting fibrinogen into fibrin to ultimately form a clot. Thrombin also assists in causing vasoconstriction and platelet activation. Thrombin was initially used clinically in the early 1900s as an emulsion on incision sites to promote healing.^\[[@ref7]\]^ Human thrombin together with fibrin was used in burn injuries during World War II. However, due to the risk of transmission of viral hepatitis, bovine and recombinant thrombin formulations were developed.^\[[@ref8]\]^
Thrombin is only approved by the Food and Drug Administration for topical application. The most common formulations of thrombin involve a solution or reconstituted powder. Thrombin is commonly used in conjunction with fibrin-based products or hemostatic sponges. Thrombin is available in three topical formulations: Bovine plasma- derived (b-thrombin), human plasma-derived (h-thrombin), and recombinant (r-thrombin).^\[[@ref8],[@ref9]\]^ Even though thrombin is typically used for topical formulations due to the potential risk of intravascular thrombus formation, off label uses for percutaneous injections in the treatment of superficial angiography access site pseudoaneurysms has been documented and has demonstrated a high success rate upward of 95%. In our review of the literature for Cather directed endovascular use of thrombin, only 28 cases were reported from 1987.^\[[@ref8]\]^
CONCLUSION {#S4}
==========
While the complications of cholecystectomy are well known, some of the adverse outcomes may result in complex and life-threatening complications, often requiring a multidisciplinary approach in treatment such as in the case of our patient which required medical, surgical, and interventional collaboration.
The treatment of our patient's ruptured aneurysm required the use of thrombin, an agent which is rarely used endovascularly as a means of hemostasis, allowing for hemodynamic stability. Post-surgical complications after cholecystectomy and other similar procedures may necessitate the use of endovascular intervention to aid in the patient's care, often in highly complex and life-threatening conditions. Depending on the clinical situation and availability of resources, the use of novel or atypical agents such as thrombin may be the difference between life or death for patients.
**How to cite this article:** Patel AP, Cantos A, Butani D. Mycotic aneurysm of the hepatic artery: A case report and its management. J Clin Imaging Sci 2020;10:41.
Declaration of patient consent {#S5}
==============================
Patient's consent not required as patients identity is not disclosed or compromised.
Financial support and sponsorship {#S6}
=================================
Nil.
Conflicts of interest {#S7}
=====================
There are no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
} |
Dear Editor,
On February 20, 2020 a 38-year-old man was admitted with acute respiratory insufficiency to the intensive care unit (ICU) of the Codogno Hospital, marking the beginning of the SARS-CoV2 epidemic in Italy. Codogno is a small town in Lombardy, the Italian region most affected by SARS-CoV2, with currently the 37.8% of nationwide cases \[[@bib1]\]. The sudden massive inflow of patients overwhelmed existing protocols in place to handle rapidly evolving emergency situations.
In order to adequately respond to the wide-spreading of the SARS-CoV2 infection, hospitals needed new strategic organizational planning.
Here we summarize the experience of ASST Fatebenefratelli-Sacco, a tertiary center for infectious disease in Milan (Lombardy). This dynamic situation, indeed, required unprecedented modifications of both logistics and organizational hospital structure: the first week of April 2020 (maximum opening time) 477 out of 894 beds of the Hospital were dedicated to COVID patients.
We also present what are, in our opinion, the main priorities and issues that any hospital advisory board must consider to effectively deal with this kind of emergency.
First and foremost, it is essential to establish an Internal Crisis Unit for emergency management focused on the analysis of epidemiologic data and related information. The Unit receives information from other institutions (regional, national, and international) and where appropriate, applies this data to their own organization and infrastructure.
This Unit will be the top of the chain of command and should enlist: representatives from the hospital advisory board, infectious diseases and microbiology units, emergency medicine, ICU, occupational medicine. The chosen profiles guarantee the necessary epidemiological skills. It may also be appropriate to involve non-medical staff including administrative personnel.
Policy updates should be readily available and distributed to the hospital staff. Additionally, short documents enlisting clear and critical operational instructions may be drafted and spread internally through corporate communication channels.
The current emergency requires a great deal of flexibility for facilities usage/purpose and staff allocation.
The increasing need for ICU beds and non-invasive ventilation devices used outside ICU \[[@bib2]\] may require a thorough overhaul of the hospital floorplan. It is important to accurately estimate the number of ICU and inpatients ward units with non-invasive ventilation devices that can be realistically created after a proper reorganization of the infrastructure.
To minimize virus transmission, it is crucial to avoid cross contaminations between COVID and non-COVID areas; consequently it should be considered to dedicate an entire building to COVID cases.
The presence of proper filter areas is important for creation of routes specifically assigned to the passage of either infected or contaminated (dirty) or non-infected (clean) materials and patients. The routes should be separated to avoid cross contamination not only within the inpatient units, the radiology, laboratory, and emergency room department, but also for the food, laundry, environmental services, and morgue units.
The frontline departments in this disease are the ICU, the intermediate care unit, the infectious disease units, and the pulmonary units. Those should be the first to be expanded from either the existent space or re-purposing of other areas.
Next, staff utilization undergoes profound changes by organizing novel teams of experts with different priorities/tasks. The two main kinds of team should be: one dedicated to care for positive COVID patients and the other providing conventional assistance (non COVID). For all staff members proper training must be developed and continuously delivered on the use of the personal protective equipment (PPE); in particular the correct dressing and undressing manoeuvres \[[@bib3]\]. Teams should include experienced providers to pass on critical knowledge to *in-training* staff members.
The physical and emotional workload to which care providers and non-medical healthcare operators experience cannot be ignored; therefore, psychological support services should be available from the early stages of the emergency \[[@bib4]\].
Although all elective care activities will be suspended \[[@bib5]\] to increase resources and decrease risk of infection, it is necessary to foresee the occurrence of medical and surgical emergencies, together with non-deferrable health care services. The genesis of a collaborative network between local and regional hospitals based on their expertise and space/staff availabilities is an effective strategy for the efficient use of limited resources.
An eventual lack of proper PPE has negative repercussions on the ability to assist patients because of increased occupational exposure and infection. A centralized stocking and distribution of PPE should be established to avoid waste or misuse.
Another area where limited supply affects quality of care is the lack of devices for assisted ventilation. A close collaboration between hospital management and distribution centers is essential for drafting a practical supply plan to ensure availability of those materials/devices.
Moreover, a computerized, user friendly, Health Information System for the management of beds is essential to guarantee the availability of real-time, thorough and complete data.
Admission to the hospital for new patients and outpatient activities should be limited as much as possible. Emergency departments and COVID units should prohibit any visitors from entering or accompanying the patients. In order to respect hospital restrictions, a video call system should be instituted to facilitate the communication of medical staff with patients and patients\' family members.
In summary, based on our experience, key points for any given hospital to properly manage the on-going SARS-CoV-2 pandemic are: a) early establishment of the Internal Crisis Unit; b) careful evaluation of hospital infrastructure and potential re-purpose of areas to create new care units; c) training staff in the use of PPE; e) providing psychological support to the staff; f) providing proper communication between care providers, patients, and their families.
Authors\' contributions {#sec1}
=======================
GDF and FA conceived the study. LC wrote the manuscript. All authors collaborated on content development, revised the manuscript and, approved the final version of the manuscript.
Declaration of competing interest
=================================
The authors of this manuscript declare no relationships with any companies, whose products or services may be related to the subject matter of this manuscript.
This work was not supported by any funding. No medical writer or editor service was involved in the drafting of the manuscript.
| {
"pile_set_name": "PubMed Central"
} |
**Session:** 233. Antibiotic Stewardship: Antifungals
*Saturday, October 5, 2019: 12:15 PM*
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1}
============
Prostate cancer (PCa) is one of the most commonly diagnosed solid neoplasms in the male population \[[@cit0001]\]. In the United States, prostate cancer is the second leading cause of carcinoma deaths among males, with an estimated 192,000 new cases and 27,000 deaths in 2009 \[[@cit0002]\]. In European countries, it is recognized as the most common solid tumor, with an incidence rate of 214 cases in every thousand men, outnumbering lung and colorectal cancer \[[@cit0003]\]. Although epidemiological research indicated that the incidence of prostate cancer in Asians is much lower than that in the USA and European countries, the incidence and mortality rate of this disease have rapidly increased among Chinese men \[[@cit0004], [@cit0005]\]. Data from the Global Estimates of Cancer indicated that the age standardized incidence rate of PCa in China was 4.3 per 10,000 males in 2008 \[[@cit0006]\]. With the improvement of diagnostic techniques and the aging of the population, morbidity and mortality of PCa show an increasing trend. So far, many factors including lifestyle, environment and race have been demonstrated as possible contributors to the risk of PCa \[[@cit0001]\]. However, the etiology of PCa remains unclear for the reason that a complex interplay between genetic and environmental factors is involved in the development and occurrence of PCa.
The human vitamin D receptor (VDR), located on chromosome 12q12--q14, is a nuclear receptor gene with 75 kb and consists of 11 exons and 11 introns \[[@cit0007]\]. VDR acts as a ligand-dependent transcriptional factor found in various types of tissues (including the prostate) by the interaction with vitamin D \[[@cit0008]\]. It has been found that 1,25-dihydroxyvitamin D~3~ \[1,25(OH)~2~D~3~\], the active form of vitamin D, could enhance immune regulation, promote cell differentiation and inhibit tumor invasion and metastasis via the biological effects mediated by VDR in the prostate \[[@cit0009]--[@cit0011]\]. Therefore, a less active VDR could be associated with either a more aggressive disease or increased susceptibility to cancer risk \[[@cit0012]\]. Studies have shown that several single-nucleotide polymorphisms (SNPs) of VDR, which potentially affect the receptor binding of 1,25(OH)~2~D~3~, may influence vitamin D biological activity and confer susceptibility to prostate cancer \[[@cit0013]\]. Among them, the most commonly studied SNP is the restriction fragment length polymorphism of FokI, which is detected by the endonuclease FokI. The FokI located on the coding region (exon 2) of the VDR gene results in production of a VDR protein three amino acids longer \[[@cit0014]\].
A number of epidemiological studies have been carried out to explore the association between VDR FokI polymorphism and prostate cancer risk. However, the results of these studies remain controversial rather than conclusive, possibly because of conflicting results from different case-control studies. In 2006, Berndt and his group \[[@cit0015]\] performed a meta-analysis and found no statistically significant association between the FokI polymorphism and PCa risk. Ever since, new studies have provided additional data correlating with the VDR variants. Therefore, in this meta-analysis, the most up-to-date accumulated data from all eligible studies published were utilized to obtain a summary result of the association between the VDR FokI polymorphism and PCa susceptibility \[[@cit0016]--[@cit0036]\].
Material and methods {#sec2}
====================
Search strategy and identification of relevant studies {#sec2.1}
------------------------------------------------------
PubMed, Web of Science and Embase database searches were carried out using the following terms: 'vitamin D receptor' or 'VDR', 'prostate cancer' and 'polymorphism' or 'variant' (last search updated on August 25, 2015). References of the relevant paper and retrieved articles were also identified by a manual search. Eligible studies had to meet all the following inclusion criteria: (a) uses an unrelated case-control design; (b) includes available genotype frequencies; (c) research published in English; (d) provides sufficient data to calculate the odds ratio (OR) with 95% confidence interval (CI).
Data extraction and quality assessment {#sec2.2}
--------------------------------------
Data were collected on the genotype of VDR FokI according to prostate cancer. For each publication, the data extraction was carried out by two of the investigators independently according to the inclusion criteria above. Disagreement was to be resolved through a discussion between two authors. If they could not reach a consensus, the problem was to be discussed comprehensively by all investigators. Furthermore, eligible studies containing information about clinical stage of PCa were divided into two groups: localized and advanced (including cases with bone metastasis). Information from enrolled studies was extracted as below: first author's name, year of publication, ethnicity of subjects, sources of controls, sample size in cases and controls, number of cases and controls with wild type and variant allele, and *p*-value for Hardy-Weinberg Equilibrium (HWE).
Statistical analysis {#sec2.3}
--------------------
Odds ratios (ORs) with 95% CIs were used to assess the strength of association between the polymorphism in VDR FokI and prostate cancer risk. Five genetic contrasts were used to evaluate the association: allelic contrast (f allele vs. F allele), homozygote comparison (ff vs. FF), heterozygote comparison (Ff vs. FF), dominant genetic model (ff + Ff vs. FF) and recessive genetic model (ff vs. Ff + FF). Stratified analyses were performed by ethnicity and source of controls (hospital-based, population-based and benign prostatic hyperplasia (BPH) based). The pooled ORs for the risk were calculated using the random effects model and fixed effects model. Heterogeneity assumption was assessed by the χ^2^-based *Q* test among the studies. The data were evaluated using random-effects models (the DerSimonian and Laird method) \[[@cit0037]\] in the presence of heterogeneity (*p* \< 0.05) and fixed-effects (the Mantel-Haenszel method) models \[[@cit0038]\] were performed in the absence of heterogeneity (*p* \> 0.05). The HWE was calculated by the Pearson χ^2^ test for goodness of fit. The *Z*-test was performed to evaluate the statistical significance of the summary OR; a *p-*value of \< 0.05 was considered significant. The statistic of *I*^2^ was also used to test the heterogeneity, with *I*^2^ \> 75%, 25--75% and \< 25% to represent high, moderate and low degree of inconsistency, respectively. Significance of the intercept was determined by the *t*-test as suggested by Egger (*p* \< 0.01 represents a statistically significant publication bias) \[[@cit0039]\]. All the statistical analyses were conducted using STATA version 10.0 (Stata Corporation, College Station, TX).
Results {#sec3}
=======
Study characteristics {#sec3.1}
---------------------
A total of 21 articles (including 25 case-control studies) that met all the inclusion criteria were enrolled in our study ([Figure 1](#f0001){ref-type="fig"}). Characteristics of the eligible studies are summarized in [Table I](#t0001){ref-type="table"}. In general, 9,720 prostate cancer patients and 9,710 control subjects concerning the VDR FokI polymorphism were assessed. In the subgroup of ethnicity, 14 were carried out in Caucasian descendents, three were in Asian descendents, three studies were in Arabians and three in African-Americans. Only one article was in Spanish descendents. Hospital-based controls were carried out in 18 of these studies. The classical genotyping method called polymerase chain reaction-restriction fragment length polymorphism (RFLP) was used in 13 comparisons. Five studies performed TaqMan real-time polymerase chain reaction (PCR).
######
Characteristics of studies of the vitamin D receptor (VDR) FokI gene polymorphism included in this meta-analysis
First author Year Ethnicity Source of control Genotyping Sample size of case Sample size of control *P*~HWE~ Frequencyof f allele
--------------- ------ ------------------ ------------------- ---------------- --------------------- ------------------------ ---------- ---------------------- ----- ----- ----- ------ ------- -------
Correa 1999 Caucasian Population-based PCR-RFLP 10 58 50 118 9 42 38 89 0.598 0.337
Chokkalingam 2001 Asian Hospital-based PCR-RFLP 41 95 51 187 62 153 87 302 0.725 0.459
Tayeb 2004 Caucasian Population-based PCR-RFLP 2 10 16 28 11 24 21 56 0.391 0.411
Oakley-Girvan 2004 Caucasian Population-based PCR-RFLP 27 120 85 232 26 77 68 171 0.586 0.377
Oakley-Girvan 2004 African-American Population-based PCR-RFLP 4 25 84 113 5 42 74 121 0.752 0.215
Bodiwala 2004 Caucasian BPH-based PCR-RFLP 60 163 145 368 35 100 108 243 0.137 0.350
Cheteri 2004 Caucasian Population-based PCR-RFLP 101 247 204 552 80 234 207 521 0.305 0.378
Mishra 2005 Arabian Hospital-based PCR-RFLP 5 45 78 128 16 69 62 147 0.622 0.344
John 2005 Caucasian Population-based PCR-RFLP 69 203 153 425 57 209 171 437 0.581 0.370
Hayes 2005 Caucasian Population-based DGGE 112 359 340 811 98 322 293 713 0.526 0.363
Cicek 2006 Mixed Population-based PCR-RFLP 71 191 77 339 77 202 200 479 0.034 0.372
Huang 2006 Asian Hospital-based PCR-RFLP 87 204 125 416 119 248 135 502 0.806 0.484
Rukin 2007 Caucasian BPH-based Pyrosequencing 166 203 61 430 135 141 44 320 0.461 0.642
Mikhak 2007 Caucasian Population-based Taqman 101 337 232 670 108 310 255 673 0.398 0.391
Li 2007 Caucasian Population-based PCR-RFLP 152 475 383 1010 206 655 571 1432 0.413 0.373
Holick 2007 Caucasian Population-based SNPlex assay 100 261 222 583 85 245 222 552 0.204 0.376
Torkko 2008 Spanish Population-based Taqman 26 70 45 141 57 125 91 273 0.249 0.438
Torkko 2008 Caucasian Population-based Taqman 67 209 168 444 63 227 198 488 0.87 0.362
Holt 2009 African-American Population-based SNPlex assay 2 48 65 115 4 24 39 67 0.904 0.239
Holt 2009 Caucasian Population-based SNPlex assay 108 335 262 705 101 352 263 716 0.332 0.387
Bai 2009 Asian Hospital-based PCR-RFLP 26 63 33 122 37 55 38 130 0.08 0.496
Rowland 2013 African-American Population-based Taqman 24 161 236 421 10 90 134 234 0.287 0.235
Rowland 2013 Caucasian Population-based Taqman 193 598 406 1197 112 413 311 836 0.171 0.381
Yousaf 2014 Arabian Hospital-based PCR-RFLP 2 0 39 41 13 0 95 108 0 0.120
Atoum 2015 Arabian Population-based PCR 10 76 38 124 6 66 28 100 0 0.390
HWE -- Hardy-Weinberg equilibrium of controls, RFLP -- restriction fragment length polymorphism, BPH -- benign prostatic hyperplasia.
{#f0001}
Quantitative synthesis {#sec3.2}
----------------------
When all the eligible studies were pooled into the meta-analysis ([Table II](#t0002){ref-type="table"}), no obvious association was observed in the overall analysis between prostate cancer risk and the VDR FokI variant genotypes: allelic comparison (random-effects OR = 1.02, 95% CI: 0.96--1.10, *p*~heterogeneity~ \< 0.001, *p* = 0.498, *I*^2^ = 56.6) ([Figure 2](#f0002){ref-type="fig"}), homozygote comparison (random-effects OR = 1.08, 95% CI: 0.95--1.03, *p*~heterogeneity~ = 0.020, *p* = 0.216, *I*^2^ = 40.3), heterozygote comparison (random-effects OR = 1.06, 95% CI: 0.97--1.17, *p*~heterogeneity~ = 0.002, *p* = 0.211, *I*^2^ = 50.7), dominant genetic model (random-effects OR = 1.05, 95% CI: 0.95--1.16, *p*~heterogeneity~ \< 0.001, *p* = 0.326, *I*^2^ = 57.0) and the recessive genetic model (fixed-effects OR = 1.03, 95% CI: 0.96--1.11, *p*~heterogeneity~ = 0.280, *p* = 0.406, *I*^2^ = 12.8). However, in the subgroup analysis by ethnicity, positive associations between VDR FokI polymorphism and prostate cancer risk were found in Caucasian descendents for allelic contrast (fixed-OR = 1.03, 95% CI: 1.00--1.06, *P*~heterogeneity~ = 0.552, *p* = 0.026, *I*^2^ = 0) ([Figure 3](#f0003){ref-type="fig"}) and the dominant genetic model (fixed-effects OR = 1.03, 95% CI: 1.00--1.05, *p*~heterogeneity~ = 0.856, *p* = 0.032, *I*^2^ = 0), but not in Asian descendents (allelic comparison fixed-effects OR = 0.97, 95% CI: 0.90--1.04, *p*~heterogeneity~ = 0.536, *p* = 0.352, *I*^2^ = 0), Arabians (allelic comparison random-effects OR = 0.64, 95% CI: 0.37--1.11, *p*~heterogeneity~ = 0.036, *p* = 0.114, *I*^2^ = 69.8) or African-Americans (allelic comparison fixed-effects OR = 0.92, 95% CI: 0.71--1.18, *p*~heterogeneity~ = 0.151, *p* = 0.500, *I*^2^ = 47.2). In the subgroup analysis by tumor stage, there was a significant association between this variant and advanced prostate cancer under the recessive genetic model (fixed-effects OR = 1.15, 95% CI: 1.01--1.32, *p*~heterogeneity~ = 0.469, *p* = 0.032, *I*^2^ = 0) ([Figure 4](#f0004){ref-type="fig"}). Furthermore, a significant association between the VDR FokI polymorphism and prostate cancer was also found in population-based studies under homozygote comparison (random-effects OR = 1.12, 95% CI: 1.03--1.23, *p*~heterogeneity~ = 0.143, *p* = 0.010, *I*^2^ = 26.7) ([Figure 5](#f0005){ref-type="fig"}) and the recessive genetic model (fixed-effects OR = 1.09, 95% CI: 1.01--1.17, *p*~heterogeneity~ = 0.669, *p* = 0.034, *I*^2^ = 0), with no association among hospital-based studies (homozygote comparison: fixed-effects OR = 0.85, 95% CI: 0.65--1.11, *p*~heterogeneity~ = 0.090, *p* = 0.223, *I*^2^ = 50.3; recessive genetic model: fixed-effects OR = 0.83, 95% CI: 0.64--1.07, *p*~heterogeneity~ = 0.194, *p* = 0.155, *I*^2^ = 34.1) and BPH based studies in homozygote comparison (fixed-effects OR = 1.02, 95% CI: 0.84--1.23, *p*~heterogeneity~ = 0.226, *p* = 0.869, *I*^2^ = 31.9) and the recessive genetic model (fixed-effects OR = 0.95, 95% CI: 0.81--1.11, *p*~heterogeneity~ = 0.317, *p* = 0.522, *I*^2^ = 0.1).
######
Stratified analyses of the vitamin D receptor gene FokI polymorphism and prostate cancer risk
Variables *N*^[a](#tf2-1){ref-type="table-fn"}^ Cases/controls f allele vs. F allele ff vs. FF ff + Ff vs. FF ff vs. Ff + FF
-------------------- --------------------------------------- ---------------- ----------------------- ----------------------------------------- ---------------- ---------------- ------------------- ----------------------------------------- ---------- ------ ------------------- ----------------------------------------- ---------- ------ ------------------- ----------------------------------------- ------- ------
Total 25 9720/9710 1.02 (0.96--1.10) \< 0.001 0.498 56.6 1.08 (0.95--1.03) 0.020 0.216 40.3 1.05 (0.95--1.16) \< 0.001 0.326 57.0 1.03 (0.96--1.11) 0.280^[c](#tf2-3){ref-type="table-fn"}^ 0.406 12.8
Ethnicity:
Caucasian 14 7573/7247 1.03 (1.00--1.06) 0.552^[c](#tf2-3){ref-type="table-fn"}^ 0.026 0 1.06 (1.00--1.13) 0.544^[c](#tf2-3){ref-type="table-fn"}^ 0.065 0 1.03 (1.00--1.05) 0.856^[c](#tf2-3){ref-type="table-fn"}^ 0.032 0 1.05 (0.98--1.13) 0.571^[c](#tf2-3){ref-type="table-fn"}^ 0.183 0
Asian 3 725/934 0.97 (0.90--1.04) 0.536^[c](#tf2-3){ref-type="table-fn"}^ 0.352 0 0.93 (0.80--1.08) 0.540^[c](#tf2-3){ref-type="table-fn"}^ 0.344 0 0.99 (0.93--1.05) 0.548^[c](#tf2-3){ref-type="table-fn"}^ 0.668 0 0.90 (0.75--1.08) 0.444^[c](#tf2-3){ref-type="table-fn"}^ 0.271 0
Arabian 3 293/355 0.64 (0.37--1.11) 0.036 0.114 69.8 0.54 (0.21--1.39) 0.101^[c](#tf2-3){ref-type="table-fn"}^ 0.202 56.4 0.78 (0.55--1.13) 0.029 0.189 71.7 0.61 (0.25--1.50) 0.138^[c](#tf2-3){ref-type="table-fn"}^ 0.279 49.6
African-American 3 649/422 0.92 (0.71--1.18) 0.151^[c](#tf2-3){ref-type="table-fn"}^ 0.50 47.2 0.89 (0.42--1.86) 0.261^[c](#tf2-3){ref-type="table-fn"}^ 0.749 25.6 0.92 (0.71--1.19) 0.112^[c](#tf2-3){ref-type="table-fn"}^ 0.531 54.4 0.91 (0.42--1.95) 0.251^[c](#tf2-3){ref-type="table-fn"}^ 0.801 27.7
Spanish 1 141/273 0.98 (0.73--1.31) -- 0.888 -- 0.92 (0.51--1.16) -- 0.787 -- 1.07 (0.69--1.65) -- 0.771 -- 0.86 (0.51--1.44) -- 0.557 --
Mixed 1 339/479 1.64 (1.34--1.99) -- \< 0.001 -- 2.40 (1.58--3.63) -- \< 0.001 -- 2.44 (1.78--3.33) -- \< 0.001 -- 1.38 (0.97--1.98) -- 0.075 --
Source of control:
Population-based 17 8028/7958 1.03 (1.00--1.07) 0.017^[c](#tf2-3){ref-type="table-fn"}^ 0.051 46.3 1.12 (1.03--1.23) 0.143^[c](#tf2-3){ref-type="table-fn"}^ 0.010 26.7 1.10 (0.99--1.22) 0.002 0.090 56.0 1.09 (1.01--1.17) 0.669^[c](#tf2-3){ref-type="table-fn"}^ 0.034 0
Hospital-based 5 894/1189 0.81 (0.63--1.04) 0.022 0.100 65.1 0.85 (0.65--1.11) 0.090^[c](#tf2-3){ref-type="table-fn"}^ 0.223 50.3 0.94 (0.83--1.06) 0.026 0.311 63.9 0.83 (0.64--1.07) 0.194^[c](#tf2-3){ref-type="table-fn"}^ 0.155 34.1
BPH 2 798/563 1.02 (0.90--1.15) 0.133^[c](#tf2-3){ref-type="table-fn"}^ 0.811 55.7 1.02 (0.84--1.23) 0.226^[c](#tf2-3){ref-type="table-fn"}^ 0.869 31.9 1.02 (0.93--1.13) 0.171^[c](#tf2-3){ref-type="table-fn"}^ 0.629 46.8 0.95 (0.81--1.11) 0.317^[c](#tf2-3){ref-type="table-fn"}^ 0.522 0.1
Number of comparisons
p-value of Q-test for heterogeneity test (P~heter~).
A random-effects model was performed when P value for heterogeneity test \< 0.05; otherwise, a fixed-effects model was used.
{#f0002}
{#f0003}
{#f0004}
{#f0005}
Publication bias {#sec3.3}
----------------
Egger's test and Begg's funnel plot were carried out to evaluate the literature's publication bias. No obvious evidence of publication bias was found (f allele vs. F allele, *t* = --2.30, *p* = 0.031, [Figure 6](#f0006){ref-type="fig"}; ff vs. FF, *t* = --1.29, *p* = 0.209; ff vs. FF, *t* = --1.18, *p* = 0.251; ff + Ff vs. FF, *t* = --1.60, *p* = 0.251; ff vs. Ff + FF, *t* = --2.06, *p* = 0.051).
{#f0006}
Discussion {#sec4}
==========
Genetic susceptibility of solid tumors has led to growing attention to polymorphism studies of genes involved in the pathogenesis of carcinogenesis. Accumulating data have provided evidence that a low level of vitamin D is a risk factor for prostate cancer \[[@cit0040]--[@cit0042]\]. The vitamin D receptor, a significant regulator of the vitamin D pathway, could regulate conversion of serum 25(OH)D into the active hormone 1,25-dihydroxyvitamin D and mediate downstream transcription of a series of target genes \[[@cit0043]\]. Thus the FokI polymorphism of the VDR gene, encoding key proteins in vitamin D metabolism, has been chosen as a candidate polymorphism for prostate cancer susceptibility \[[@cit0044]\]. Nowadays, there is an increasing number of studies evaluating the FokI polymorphic variants of the VDR gene in prostate cancer susceptibility \[[@cit0045], [@cit0046]\]. Nevertheless, the association between this polymorphism and prostate cancer risk remains conflicting. Meta-analysis is used to combine the previous results to yield the most reliable and comprehensive conclusion because the individual research was too minor to achieve a valid conclusion \[[@cit0047], [@cit0048]\]. In this article, novel case-control studies from the last years were enrolled and a meta-analysis containing 9,720 prostate cancer patients and 9,710 controls from 25 independent case-control studies was performed.
Ethnicity is a significant biological factor which may affect the VDR functions via gene-gene interactions. When all the eligible studies were pooled into the meta-analysis, no obvious association of the VDR FokI polymorphism and prostate cancer risk was identified. However, in subgroup analysis by ethnicity, positive associations existed in Caucasian descendents (under the allelic contrast and dominant genetic model) but not in Asians, Arabians and African-Americans. Furthermore, in the analysis stratified by source of control, this VDR FokI variant was observed to increase the prostate cancer susceptibility in population-based studies (in homozygote comparison and the recessive genetic model), while no positive association was found in hospital-based and BPH-based controls. Nevertheless, some limitations of this pooled analysis ought to be addressed. The first limitation is the insufficient number of cases when specifying various ethnic backgrounds of prostate cancer. Only three studies were based on Asian \[[@cit0019], [@cit0034], [@cit0035]\] and Arabian descendents \[[@cit0016], [@cit0017], [@cit0028]\]. Second, some risk factors including age, smoking exposure, drinking, and family history were absent in this study. We tried to evaluate the gene and environment interaction effect on the susceptibility to prostate cancer. Unfortunately, the available data were not sufficient. Third, it has been suggested that positive results tend to be published faster than those with a 'negative' conclusion, which may need longer time to be accepted in time-lag bias \[[@cit0049]\]. In addition, combined interaction of multiple gene polymorphisms may have a stronger association with prostate cancer susceptibility, which is beyond the detection capacity of the present analysis.
On the other hand, the present meta-analysis has some key advantages compared with individual case-control studies. First, substantial numbers of cases and control subjects were enrolled from a variety of studies, which can significantly enhance the statistical power. Second, the quality of the case-control studies in this analysis was satisfactory and met the selection criteria with a wide representation. Third, no significant publication bias was detected through the qualitative funnel plot, which indicated that the conclusions are relatively stable and publication bias may not lead to an influence on the results of the present meta-analysis. In addition, studies using males with benign prostatic hyperplasia (BPH) as control subjects were enrolled. The reason for this inclusion was based on the assumption that BPH is a benign disease which has a similar probability for developing prostate cancer compared with normal prostate tissues. Previous epidemiological studies have provided no evidence concerning the association of increased BPH risk with VDR polymorphisms \[[@cit0050], [@cit0051]\]. Furthermore, the genotype distribution of the control population met the Hardy--Weinberg equilibrium in 23 of the studies.
In conclusion, this meta-analysis showed evidence that the VDR FokI variant may contribute to the risk for developing prostate cancer in a Caucasian population, but not with other descendents. Furthermore, there is a significant association between this variant and advanced prostate cancer. However, no association was detected in the overall analysis when all eligible studies were pooled into the analysis. Therefore, large, well-designed epidemiological studies, particularly referring to gene-environment interactions, are necessary to achieve a comprehensive conclusion of the association between VDR FokI polymorphism and prostate cancer risk.
Yuan-Yuan Mi, Yang-Zhi Chen and Jing Chen contributed equally to this work.
This study was supported by the foundation of High-Level Medical Talents Training Project (No. 2016CZBJ035).
Conflict of interest
====================
The authors declare no conflict of interest.
| {
"pile_set_name": "PubMed Central"
} |
Fig 1, "Classic typologies of kinship terminology," is missing several images. Please see [S1 Fig](#pone.0155170.s001){ref-type="supplementary-material"} for the corrected Fig 1. The publisher apologizes for the error.
Supporting Information {#sec001}
======================
###### Classic typologies of kinship terminology.
The six typologies created by Murdock, and how kin is referred to in relation to ego in each system.
(PDF)
######
Click here for additional data file.
| {
"pile_set_name": "PubMed Central"
} |
This article is part of the Thematic Series \"Organo photovoltaics\".
Introduction
============
Tremendous multidisciplinary research efforts have led to consistent increases in the efficiency of organic solar cells, making the technology a bright prospect in the quest for alternative energy \[[@R1]--[@R4]\]. In particular the rapid development of solution-processed small molecule materials over the last several years has led to leaps in state-of-the-art efficiencies and improved understanding of structure--property relationships. The well-defined molecular structures have inherent amenability to purification, batch-to-batch reproducibility and monodispersity, which make them an attractive alternative to their polymeric counterparts \[[@R5]--[@R6]\]. Also of import stands the fact that easily modified, modular structures lead to finely-tunable energy levels and optical properties through molecular design \[[@R7]--[@R8]\]. Most high-performing small molecule electron-donor materials are configured such that the conjugated backbone consists of alternating electron-rich donor (D) and the electron-deficient acceptor (A) moieties so as to facilitate efficient photo-induced charge transfer and harvest a broad spectral response \[[@R9]--[@R11]\]. One such molecular architecture introduced by Bazan and co-workers can be described as a D~1~--A--D~2~--A--D~1~ system \[[@R12]\], where D~1~ is an electron-rich unit such as bithiophene, A is a benzothiadiazole derivative and D~2~ can be different electron-rich planar cores such as dithienosilol or silanindacenodithiophene. Utilizing this push--pull molecular approach, efficiencies up to 9.0% have been achieved \[[@R13]\] due to deep highest occupied molecular orbitals (HOMO) and the corresponding large open circuit voltages (*V* ~OC~). There remains, however, room for improvement by further tuning the energetics of these materials so as to harvest photons from the widest possible spectral range while still maintaining high *V* ~OC~. Expressly, the optical bandgap must be further reduced by minimizing the energetic gap in the lowest occupied molecular orbitals (LUMO) between donor and acceptor \[[@R14]\].
One design approach towards this end involves adding electron-withdrawing end groups to existing central core chromophores. Chen and co-workers have successfully used cyanoacetate \[[@R15]\] and other electron-withdrawing end groups to create A--D--A type oligothiophene derivatives with tunable electronics, which are among the highest performing materials to date \[[@R16]--[@R19]\]. Starting with the previously reported molecule benzo\[1,2-*b*:4,5-*b*\]bis(4,4′-dihexyl-4*H*-silolo\[3,2-*b*\]thiophene-2,2′-diyl)bis(6-fluoro-4-(5′-hexyl-\[2,2′-bithiophene\]-5-yl)benzo\[*c*\]\[1,2,5\]thiadiazole, p-SIDT(FBTTh~2~)~2,~ ([Fig. 1](#F1){ref-type="fig"}), we have modified the conjugated backbone to include electron-withdrawing octyl cyanoacetate (CA8) end groups, essentially forming an "A~1~--D~1~--A~2~--D~2~--A~2~--D~1~--A~1~" molecular skeleton, benzo\[1,2-*b*:4,5-*b*\]bis(4,4′-dihexyl-4*H*-silolo\[3,2-*b*\]thiophene-2,2′-diyl)bis(6-fluoro-4-((*E*)-octyl-3-(5-thiophen-2-yl)-2-cyanoacrylate\]-5-yl)benzo\[*c*\]\[1,2,5\]thiadiazole, p-SIDT(FBTThCA8)~2~. We will show that this molecular substitution did indeed significantly reduce the bandgap while maintaining deep energy levels, as well as some of the other desirable properties of the parent material \[[@R20]--[@R22]\].
{#F1}
However, molecular design remains only the first step towards the development of high performance devices. Mismanagement of the organization and phase-separation processes or inappropriate device architecture choices can lead to non-ideal electronics at electrode interfaces and severely limit the performance of a materials system. In particular, the fill factor (*FF)*, which is simply a metric describing the field dependence of current, can be particularly sensitive to changes in morphology or interfacial effects. At the extreme, a strong field dependence near open circuit conditions can result in current vs voltage (*J--V*) curves adopting an "s-shape." Such curves have been observed experimentally in a number of diverse OPV systems, and have been ascribed to a host of morphological or interfacial issues. The s-shape arises from inefficient charge extraction of one charge carrier type at small electric fields. Mechanistically, a variety of underlying causes have been proposed, including large imbalances in charge carrier mobilities, energetic barriers to charge extraction at electrode interfaces, reduced surface recombination, and interfacial defects leading to traps; device simulations have shown that all of these could indeed result in the s-shape behavior \[[@R23]--[@R30]\].
Herein we describe the development of a novel small molecule system with nearly ideal optoelectronic properties, which unfortunately results in s-shaped *J--V* curves and poor performance. We show that this is due to non-ideal phase separation, specifically a preferential migration of the electron acceptor to the bottom anode interface. This can, however, be mitigated through appropriate processing, using a small amount of the solvent additive 1,8-diiodooctane (DIO).
Results and Discussion
======================
Synthesis and characterization
------------------------------
[Scheme 1](#C1){ref-type="fig"} depicts the synthesis toward p-SIDT(FBTThCA8)~2~. As opposed to using bottom-up synthetic procedures as reported in the literature in which ketone derivatives are converted to octyl cyanoactates in the final synthetic step via Knoevenagel condensation \[[@R15],[@R31]--[@R35]\], we chose to begin with (*E*)-octyl 3-(5-bromothiophen-2-yl)-2-cyanoacrylate as the starting material to ensure good solubility. Intermediate **1** was prepared by palladium-mediated stannylation \[[@R36]\] and then subjected to regioselective Stille conditions \[[@R37]\] in an oil bath (90 °C) to generate **2**. (Complete synthetic details and characterization of all compounds are provided in [Supporting Information File 1](#SD1){ref-type="supplementary-material"}). Its fluorine regiochemistry was confirmed by ^1^H-^1^H NOESY spectroscopy ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S1). Compound **3** was obtained through lithium--halogen exchange with *n*-BuLi followed by addition of trimethyltin chloride. Segments **2** and **3** were cross-coupled using a microwave-assisted Stille reaction to afford the target p-SIDT(FBTThCA8)~2~.
![Synthetic route towards p-SIDT(FBTThCA8)~2~. (i) Sn~2~Me~6~, Pd(PPh~3~)~4~, toluene, 85 °C; (ii) 4,7-dibromo-5-fluorobenzo\[*c*\]\[1,2,5\]thiadiazole, Pd(PPh~3~)~4~, toluene, 90 °C, 42.8%; (iii) *n*-BuLi, THF, −78 °C, 10 min; then Me~3~SnCl, 98.9%; (iv) Pd(PPh~3~)~4~, toluene, MW, 80.6%.](Beilstein_J_Org_Chem-12-2543-g008){#C1}
The thermal transitions of p-SIDT(FBTThCA8)~2~ were evaluated by differential scanning calorimetry (DSC) and compared to its predecessor, p-SIDT(FBTTh~2~)~2~. A significant impact on thermal behaviors was observed upon substituting 2-hexylthiophene with octyl cyanoacetate ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S4 and Table S1). As compared to p-SIDT(FBTTh~2~)~2~, the melting (*T* ~m~) and crystallization (*T* ~c~) temperatures of p-SIDT(FBTThCA8)~2~ are increased by 76.9 and 117.1 °C, respectively, which implies an enhancement of the intermolecular interaction in the solid state. This improved rigidity of p-SIDT(FBTThCA8)~2~ is correlated to a noticeable decrease in solubility, which was measured to be 32 mg/mL for p-SIDT(FBTThCA8)~2~ compared with over 50 mg/mL for p-SIDT(FBTTh~2~)~2~ in chloroform at room temperature.
Frontier molecular energy levels were estimated by cyclic voltammograms (CV) in dichloromethane and calculated theoretically by density functional theory (DFT) ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S5 and Table S2). In the CV measurement, energy levels of HOMO and LUMO were calculated from the onsets of oxidation and reduction potentials. The HOMO level (*E* ~HOMO,\ CV~: −5.27 eV, *E* ~HOMO,\ DFT~: −5.43 eV) is quite deep, even compared to that of p-SIDT(FBTTh~2~)~2~ (*E* ~HOMO,\ CV~: −5.21 eV, *E* ~HOMO,\ DFT~: −4.97 eV). We anticipate this should provide a high *V* ~OC~ when blended with PCBM. The band gap of p-SIDT(FBTThCA8)~2~ is also reduced with respect to p-SIDT(FBTTh~2~)~2~ as determined by CV (1.72 eV and 1.85 eV, respectively) and by DFT (1.90 eV and 2.01 eV, respectively) suggesting that substituting 2-hexylthiophene with octyl cyanoacetate on both wing-ends does noticeably reduce the bandgap while maintaining a deep HOMO level.
The normalized solid-state absorption profile of p-SIDT(FBTThCA8)~2~ is shown as the dotted line in [Fig. 2](#F2){ref-type="fig"} and the data are also summarized in Table S1 ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}). The film has strong absorption in the visible range, with an onset at 750 nm corresponding to an optical bandgap of 1.65 eV. This is consistent with the electrochemically determined bandgap. The primary absorption band shows vibronic progression, suggesting ordering in the solid state, with peak absorption at 650 nm. The red-shifted absorption of p-SIDT(FBTThCA8)~2~ with respect to p-SIDT(FBTTh~2~)~2~, whose absorption onset in the solid state occurs at 670 nm, is further confirmation that the addition of electron-withdrawing endgroups reduces the bandgap of the chromophore. Importantly, the shift in absorption onset represents about a 25% increase in the number of photons in the AM 1.5 solar spectrum available for absorption. If p-SIDT(FBTThCA8)~2~ maintains high internal quantum efficiencies and *FF* like its predecessor, and also achieves a high *V* ~OC~ as expected based on energy levels, the improved absorption imparts p-SIDT(FBTThCA8)~2~ with great potential.
{#F2}
Solar cell performance
----------------------
For initial photovoltaic device fabrication, conditions were chosen according to previously reported protocols of structurally similar small molecule systems \[[@R38]--[@R40]\]. Specifically, p-SIDT(FBTThCA8)~2~ was mixed with PC~71~BM and cast to form a bulk heterojunction (BHJ) atop poly(3,4-ethylenedioxythiophene) polystyrene sulfate (PEDOT) giving an architecture of ITO/PEDOT/p-SIDT(FBTThCA8)~2~:PC~71~BM/Ca/Al. The mass ratio of p-SIDT(FBTThCA8)~2~:PC~71~BM was held at 1:1 and cast from a chlorobenzene solution containing 40 mg/mL total solids, giving 120 nm thick active layers. Such devices show modest performance (*J* ~SC~ = 3.4 mA/cm^2^, *V* ~OC~ *=* 0.91 V, *FF* = 0.37, *PCE* = 1.1%). Though the performance is low, the efficiency is similar compared to other systems cast from pure chlorobenzene. Furthermore, the high *V* ~OC~ of 910 mV is encouraging, as it further confirms the advantage of the deep lying HOMO level of p-SIDT(FBTThCA8)~2~. However, an inflection point near *V* ~OC~, a clear kink in the *J--V* curve gives the curve a dramatic "s-shape" ([Fig. 2](#F2){ref-type="fig"}) limiting *FF* and *PCE* *~.~*
In the literature, it has been shown that incorporation of small amounts of the solvent additive DIO into the casting solvent can vastly improve small molecule device performance \[[@R38]--[@R43]\]. Accordingly, initial optimization required adjusting the concentration of DIO. It was found that at a concentration of 1.5% DIO (by volume) in chlorobenzene, the *PCE* was increased to 2.9% (*J* ~SC~ = 6.0 mA/cm^2^, *V* ~OC~ = 0.94 V, *FF* = 0.52); device characteristics are shown in [Table 1](#T1){ref-type="table"}. Though, the improvements in device performance are relatively modest compared to what has been observed in other systems, incorporation of the DIO into the solution noticeably reduces the s-shape of the curve leading to a greatly enhanced *FF*. While the use of additives has been shown to have a number of consequences on film formation and device operation \[[@R38]--[@R44]\], to the best of our knowledge, such a dramatic change in curve shape has not been demonstrated previously using solvent additives. And while these additive-processed devices still have not nearly reached the full potential of this materials system, and other possible processing changes may also affect the nature of the *J--V* curve, we have focused herein on understanding the mechanism leading to the change in curve shape to gain a better, fundamental understanding of the nature and operation of small-molecule solar cell devices and the role of solvent additives in film formation.
######
Device characteristics when cast with and without DIO, before and after treatment with MeOH in a standard architecture as well as in an inverted cell.
------------ ---------- ---------------------------- ----------- ------ --------
Solar cell characteristics
Conditions *J* ~SC~\ *V* ~OC~\ *FF* *PCE*\
(mA/cm^2^) (V) (%)
No DIO standard 3.4 0.91 0.37 1.1
w/MeOH 3.4 0.95 0.37 1.2
inverted 4.5 1.09 0.51 2.5
With DIO standard 6.0 0.94 0.52 2.9
w/MeOH 6.1 1.02 0.52 3.2
inverted 7.0 0.73 0.47 2.4
------------ ---------- ---------------------------- ----------- ------ --------
As a first insight into the difference in *J--V* behavior with and without DIO we examined the light intensity dependence of the two devices. Varying the intensity of the incident light serves to proportionally change the number of absorbed photons and thus generation of free charges. Of particular interest is the effect of light intensity on *V* ~OC~, since at the open circuit voltage carriers are created, but nearly none of the charges are extracted, *J =* 0; all charges must therefore recombine \[[@R45]\]. Thus, the relation of *V* ~OC~ with the incident light intensity for bimolecular (free charge) recombination has been shown to depend only on temperature and light intensity, given by
where *I* is light intensity, *k* is the Boltzman constant, *T* is temperature and *q* is the elementary charge. Thus, in a system dominated by bimolecular recombination, on a semi-log plot of *V* ~OC~ vs *I* we expect a linear relationship with a slope of *kT/q* \[[@R45]\]*.* It is worth noting that proper analysis of low light intensity data requires sufficiently low dark current, such that it does not constitute a significant fraction of the device current in the voltage regime close to *V* ~OC~. In both the devices cast with and without additive, even at only 0.02 suns, the dark current remains at least two orders of magnitude lower than the device current (see [Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S6). The *V* ~OC~ as a function of light intensity are shown in [Fig. 2](#F2){ref-type="fig"} for devices without and with DIO.
It is immediately clear that the *V* ~OC~ in devices without additive do not follow a single linear relationship across all light intensities. Instead it seems to follow a slope of *kT/q* closely at light intensities lower than 10 mW/cm^2^, but then has a shallower, seemingly linear dependence with a slope of ≈0.65 *kT/q* at higher intensities. The slope of 0.65 *kT/q* was fit empirically and does not fit the data unequivocally, but is displayed to show at the very least, that at higher light intensities the *V* ~OC~ has a dependency that is less than the expected *kT/q.* The suggestion is that at high charge densities, the dominant recombination mechanism may change. The device cast with DIO shows similar behavior but to a much lesser extent. The *V* ~OC~ only deviates from *s = kT/q* significantly at intensities close to 100 mW/cm^2^. Thus, even devices processed with DIO may, to some extent, suffer from the same problems as those cast from pure chlorobenzene albeit to a much lesser extent. Light intensity studies are thus a powerful tool to look at more nuanced details of current voltage characteristics.
To further inspect the effects of light intensity on device operation, the photocurrent, *J* ~Ph~ *,* defined as the current upon illumination with the dark current subtracted, was examined as a function effective voltage \[[@R46]--[@R49]\]. The effective voltage is the voltage difference between the applied voltage and the voltage at which no photocurrent is generated, *V* ~0~ *− V*, and determines the strength of the electric field within the device, the driving force for charge extraction. *J* ~Ph~ is shown as a function of light intensity for devices cast without and with DIO in [Fig. 3](#F3){ref-type="fig"} and 3b, respectively.
{ref-type="table"} for devices cast c) without DIO and d) with DIO.](Beilstein_J_Org_Chem-12-2543-g004){#F3}
At low effective voltages, (*V* ~0~ *− V* \< ≈0.1 V) implying a small electric field, the photocurrent of both devices linearly increases with voltage. This is due to the competition between drift and diffusion of photogenerated charges to the contacts \[[@R49]\]. In the device processed with DIO, beyond *V* ~0~ *− V* = 0.2 V the *J* ~Ph~ reaches a saturation regime, where it increases much less significantly with voltage. In this saturation regime, the larger electric field can effectively sweep out charges and bimolecular recombination does not play as significant a role. The voltage at which this rollover point occurs is independent of intensity. In these devices, there is not a true "saturation" as the photocurrent is always increasing, however, there is still a clear rollover point between two regimes. This increasing photocurrent could be due to field dependent charge generation \[[@R50]--[@R53]\].
As seen in [Fig. 3](#F3){ref-type="fig"}, *J* ~Ph~ has a much stronger dependence on voltage in devices processed without DIO. Even at high effective voltages, there remains a strong voltage effect and *J* ~Ph~ continues to increase without saturating. There are two clear regimes with two different voltage dependencies, but in contrast to devices processed with DIO, in this case the rollover voltage at which *J* ~Ph~ switches from one regime to the other does indeed depend on light intensity. At higher intensities, a higher voltage is required to reach the "saturation" regime. This has previously been associated with a build-up of space charge in the film \[[@R47]\].
It is expected that for devices not limited by charge extraction, *J* ~Ph~ at each and every effective voltage, should scale linearly with intensity, *J* ~Ph~  *I,* while devices limited by space charge build-up have been shown to characteristically have a sub-linear dependence, where *J* ~Ph~  *I* ^0.75^ \[[@R47]\]. At *V* ~0~ *− V* = 1.0 V, close to short-circuit conditions, in devices processed with and without additive, *J* ~Ph~ scales nearly linearly, following a power law where s = 0.95. This relation deviates from linearity when moving to lower fields particularly in the devices cast without DIO. As seen in [Table 2](#T2){ref-type="table"}, at an effective voltage of 0.3 V, s = 0.81 and at 0.2 V, s = 0.71. This is quite close to 0.75, the value one would expect for a device limited by space charge.
######
Power law dependences of photocurrent on light intensity at specific effective voltages for BHJ devices from [Fig. 3](#F3){ref-type="fig"}.
------------ ---------------------- ------- ------- ------
Conditions Power law dependence
0.2 V 0.3 V 0.5 V 1.0 V
no DIO 0.71 0.81 0.91 0.95
with DIO 0.88 0.91 0.94 0.95
------------ ---------------------- ------- ------- ------
In [Fig. 3](#F3){ref-type="fig"}, a pronounced uptick in photocurrent is seen at high reverse biases (\>1.5 V). This, however, is likely an artifact, as the "photocurrent" seems to follow the dark current which is not as low in the additive processed film as in the film without DIO. The dark current is plotted with the light intensity studies in Figure S6 ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}). While in the photocurrent the dark current is subtracted from, the illuminated it is likely that the linear leakage current may also change with light. This highlights the need for low levels of leakage current for reliable measurements at higher voltages.
At low fields, the device processed without DIO suffers from space charge build-up, while at higher fields, there is sufficient driving force to overcome these effects and extract the charges. A similar effect can be seen in the device processed with DIO, albeit to a lesser extent. At *V* ~0~ *− V* = 0.2 V in the optimized device, s = 0.88. This suggests again that while the DIO does not completely remove problems associated with charge extraction, it significantly reduces the magnitude of the effects, removing the dramatic s-shape of the curve.
Thin film X-ray diffraction
---------------------------
Changes in device performance upon addition of solvent additives are typically ascribed to improvements in the BHJ nanostructure by affecting the thermodynamics and kinetics of phase separation. In this class of small molecule systems, this is often attributed to asserting control over the crystallization and phase-separation processes within the blend; DIO helps induce crystallinity of the donor material \[[@R40]--[@R42] [@R54]--[@R56]\]. Grazing incidence wide-angle X-ray scattering (GIWAXS) was used to probe the crystallization behavior of the blend system with and without additive. The full 2-dimensional GIWAXS spectra from a film of the neat p-SIDT(FBTThCA8)~2~ and the two blends are shown in Figure S7 ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}) while line cuts showing *Q* ~z~ ("out-of-plane") and *Q* ~x-y~ ("in-plane") of the two blends are shown in the top and bottom plots respectively of [Fig. 2](#F2){ref-type="fig"}.
Looking first at the out-of-plane diffraction in the top panel of [Fig. 2](#F2){ref-type="fig"}, the BHJ film cast with no DIO shows a prominent peak at 0.37 Å^−1^. This corresponds to a real-space distance of 1.7 nm. While attempts to grow single crystals of this material have thus far been unsuccessful and thus the peaks cannot be indexed precisely, by convention we attribute this spacing to an "alkyl stacking peak", that is a spacing arising from molecules separated by alkyl chains analogous to the lamellae stacking in P3HT (i.e., (100) planes). In the film cast with DIO, this peak is more prominent suggesting a greater degree of crystallinity. There is also a peak at 0.74 Å^−1^, which corresponds to the second order reflection. There is even a small peak at 1.11 Å^−1^, which likely corresponds to a third-order reflection, suggesting a quite well-ordered film. Additionally, there is a small peak at 1.79 Å^−1^, corresponding to a spacing of 3.5 Å, which we attribute to π--π stacking. There is a broad feature centered at *Q* = 1.5 Å^−1^ which is seen in both films and at all orientations, which is the convolution of two peaks. In the neat p-SIDT(FBTThCA8)~2~ there is a relatively weak, broad feature at 1.52 Å^−1^ which convolves with the isotropic scattering peak of PC~71~BM which is typically found at 1.3--1.4 Å^−1^. These two peaks are nearly resolvable in the in-plane scattering of the film cast with DIO but are completely overlapping in the blend without additive, leading to a very broad peak.
Looking next at the traces from the *Q* ~x-y~ direction, that is, just from crystallites oriented in the plane of the substrate, there are no discernible features from p-SIDT(FBTThCA8)~2~ in the BHJ film cast without DIO. In the film processed with DIO, the alkyl stacking peak is again though is less prominent in-plane, while the π-stacking peak is more prominent. Assuming the alkyl and π-stacking directions are perpendicular, this suggests the material primarily adopts an edge-on orientation. This is in contrast with the preferential "face-on" orientation adopted by p-SIDT(FBTTh~2~)~2~ \[[@R40]\], demonstrating how sensitive molecular self-assembly can be to relatively small molecular design choices. However, consistent with previous reports of related molecules, DIO does seem to improve crystallinity.
Atomic force microscopy (AFM) topography images are shown in Figure S8 ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}). The film cast without DIO has a relatively smooth featureless surface while the film cast with DIO has a rougher surface with relatively large (\>100 nm diameter) features. This is consistent with phase separation and the crystallinity seen by GIWAXS.
Despite the differences in crystallization, this does not give a clear indication as to the root cause of why devices processed without DIO show signs of space charge and an s-shaped *J--V* curve. One might expect that the increase in crystallinity has a profound effect on the hole mobility in the blends, and space charge may occur due to imbalanced carrier mobilities in the device processed without DIO. However, the hole mobilities for blends processed without DIO and with 1.5% DIO are 5 × 10^−5^ and 9 × 10^−5^ cm^2^/Vs, respectively, each slightly lower than the neat hole mobility of p-SIDT(FBTThCA8)~2~, which is found to be 2 × 10^−4^ cm^2^/Vs ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S8). Although the mobility indeed increases with DIO processing, a mobility increase by a factor of two is not particularly significant and should not lead to such drastic changes in curve shape \[[@R24],[@R30],[@R57]\]. These mobilities are, however, somewhat lower than in related high-performance systems, which may always limit the system to a relatively low *FF* \[[@R40],[@R57]--[@R58]\]. Unfortunately attempts to measure electron mobilities in charge-selective diodes were unsuccessful due to poor film formation on aluminum bottom contacts.
Origin of the s-shape in *J--V* curves
--------------------------------------
Despite the relatively high *V* ~OC~, based on the CV data, one might expect to achieve voltages that are even higher compared with p-SIDT(FBTTh~2~)~2~, as p- SIDT(FBTThCA8)~2~ seems to have an even deeper HOMO level. However, a HOMO of −5.27 eV is close to the work function of the PEDOT interfacial layer, and thus there may be non-ohmic contacts between the PEDOT and active layer, limiting the voltage \[[@R59]\]. Such an extraction barrier may also explain the build-up of space charge at one contact, and the s-shape to the *J--V* curve \[[@R25],[@R30],[@R60]--[@R62]\]. It has recently been shown by Tan and co-workers that in some cases, when PEDOT limits the voltage in solar cells, casting methanol on top of the layer will improve efficiency \[[@R63]\]. The methanol has been shown to effectively deepen the work function of the anode layer while not significantly disrupting the morphology. Specifically, this improves the extraction rate of holes at the anode interface. An enhanced hole extraction rate at the semiconductor/anode interface will reduce the accumulation of holes near the electrode, thereby preventing the screening of the internal field and suppressing recombination. The reduction of charge recombination and improved transport enables a higher photocurrent collection yield across the forward bias regime and improved *V* ~OC~ \[[@R63]\]. We employed this processing method to improve the voltages in p-SIDT(FBTThCA8)~2~:PC~71~BM cells and look at the effects on curve shape ([Fig. 4](#F4){ref-type="fig"}).
{#F4}
After treatment with methanol, the *V* ~OC~ of devices processed with DIO increases to 1.01 V. A similar improvement in *V* ~OC~ is also seen for devices cast from chlorobenzene. The *J--V* characteristics are described in [Table 1](#T1){ref-type="table"} and shown in [Fig. 4](#F4){ref-type="fig"} along with the *J--V* curves replotted from [Fig. 2](#F2){ref-type="fig"} for comparison. Treatment with methanol has a little effect on *J* ~SC~ or *FF;* thus we suspect there is no significant change in morphology when methanol is cast. Rather, the treatments strictly improves electrical contact by deepening the work function as described previously \[[@R28],[@R34],[@R60]--[@R62]\]. Despite the improvement in *V* ~OC~ in both devices, for devices processed without additive, the s-shaped kink in the *J--V* curve near open circuit remains. Thus, a contact problem at the anode is ruled out as the underlying cause of the atypical curve shape.
Non-ideal vertical phase separation, that is to say, enrichment of donor material at the cathode or acceptor at the anode may also be a potential cause of s-kinks in *J--V* curves. The acceptor material at the PEDOT interface, for instance, can act as a barrier to hole extraction, leading to ineffective sweep out and a build-up of holes \[[@R23],[@R26],[@R64]\]. To examine the vertical separation behavior of the two blends dynamic secondary ion mass spectrometry (DSIMS) was employed. In DSIMS, a sample is bombarded with ions, ablating ionized material, which is analyzed using a mass spectrometer \[[@R65]\]. The composition of the ablated material is monitored as the beam mills through the thin film, resulting in a depth profile. To improve contrast between the two materials, deuterated fullerene PC~61~BM-*d* ~5~ was used as a surrogate for PC~71~BM to establish a unique mass signal for the fullerene component \[[@R12],[@R66]--[@R67]\]. Thus detection of deuterium in the mass spectrum implicitly signifies PC~61~BM-*d* ~5~ in the film. The implicit assumption made here is that blends with the surrogate PC~61~BM-*d* ~5~ behave phenomologically like those made with PC~71~BM, and thus the PC~61~BM-*d* ~5~ signal will be applied to analyze the PC~71~BM-containing blend. The amount of p-SIDT(FBTThCA8)~2~ was monitored as the occurrence of nitrogen atoms in the mass spectrum. Unique signatures for each material help to make discerning relative concentrations simple and accurate. The DSIMS profiles of the two systems are shown in [Fig. 5](#F5){ref-type="fig"}.
{#F5}
As the DSIMS profile is collected, time corresponds to film depth, as the beam ablates material at a constant rate. Thus the x-axis has been scaled for film thickness, where the turn-on of the nitrogen and deuterium signals at *x* = 0 nm corresponds to the top surface of the films, what would be the cathode interface in a complete device architecture. The turn-off of the signals thus corresponds to the BHJ/PEDOT interface. The absolute intensity of the two signals given by the instrument cannot be compared directly due to different instrumental sensitivity, thus each signal is scaled to an average composition of 50% based on the weight ratio used in the blend solutions. It is fair to monitor how the signals evolve relative to each other as the beam penetrates into the film.
Looking first at the BHJ processed without additive, when the signals first turn on, there is initially an enrichment of p-SIDT(FBTThCA8)~2~ immediately followed by a depletion of donor and an enrichment of the PC~61~BM-d~6~ signal. This corresponds to donor material preferentially accumulated on the top surface. Throughout the bulk of the trace, the concentration of the two materials remains nearly constant. At the PEDOT interface, *x* = 115--120 nm, the PC~61~BM-*d* ~6~ signal has a small peak while the p-SIDT(FBTThCA8)~2~ signal begins to drop off. This suggests that in the device there is an enrichment of PC~71~BM at the anode surface. Such an arrangement, with donor at the top surface and acceptor at the bottom, is non-ideal for the standard device architecture.
Processing with DIO has a significant effect on the vertical phase separation. At the top surface there is again an enrichment of the p-SIDT(FBTThCA8)~2~, evidenced by a faster turn on than the PC~61~BM-*d* ~6~ signal. There is then a slight depletion of the p-SIDT(FBTThCA8)~2~ through the bulk of the device. At the bottom surface, however, unlike in the film cast without DIO, the two material signals overlap, suggesting an even distribution of p-SIDT(FBTThCA8)~2~ and PC~71~BM in the better performing devices. The vertical phase separation is still not ideal in this additive processed film, as there remains an enrichment of p-SIDT(FBTThCA8)~2~ at the cathode interface, however, DIO helps to overcome the problem of PC~71~BM concentrated at the anode interface.
A high concentration of PC~71~BM at the anode helps to explain the s-shape behavior of the *J--V* curve for the devices processed without additive. The low concentration of p-SIDT(FBTThCA8)~2~ near that interface reduces the surface recombination velocity of holes within the device; reduced surface recombination results in a piling up of charges near the anode which creates a space charge effect in the device \[[@R64]\]. This helps to explain the anomalous *V* ~OC~ and *J* ~Ph~ light intensity data. The effect is most apparent at low fields and high carrier concentrations, i.e., near open circuit conditions and at high light intensities.
If the s-shape seen in devices cast from chlorobenzene is in fact due to an enrichment of PC~71~BM at the bottom interface, the use of an inverted device architecture should result in an improvement in curve shape. The inverted architecture has the cathode as the bottom contact and the anode on top; thus if the vertical separation in the BHJ remains, the PC~71~BM-rich phase will be at the cathode interface and p-SIDT(FBTThCA8)-rich phase at the anode interface \[[@R68]\]. However, it is not necessarily true that the phase separation observed in one architecture will occur in inverted devices, as fabrication requires casting atop different substrates with different surface energetics, which may play a role in determining film formation.
While the active layers were cast in the same way, for inverted devices we employed the architecture ITO/ZnO/PEIE/p-SIDT(FBTThCA8)~2~:PC~71~BM/MoO~3~/Al where PEIE refers to ethoxylated polyethylenimine. The cathode was cast from a sol--gel of zinc acetate, and thermally converted to ZnO in air as described in literature \[[@R69]\]. A thin (10 nm) layer of PEIE has been shown in the past to improve contact by reducing the work function of a ZnO surface, and was prepared as reported \[[@R70]\]. The *J--V* characteristics of the films cast with no DIO in the standard and inverted device architecture are shown in [Fig. 6](#F6){ref-type="fig"}.
{#F6}
Devices cast from pure chlorobenzene achieved much higher efficiency in the inverted architecture than in the standard architecture (*J* ~SC~ = 4.5 mA/cm^2^, *V* ~OC~ *=* 1.09 V, *FF* = 0.51, *PCE* = 2.5%). While the performance is still modest, there is a marked improvement in the shape of the *J--V* curve. The devices achieve high open circuit voltage with no sign of space charge build-up. All device parameters improve. While we cannot be completely sure the morphology of this film is identical as when it is in a standard architecture, this is a strong indication that the primary cause for the s-shape is indeed non-ideal vertical phase separation. Further SIMS analysis of the inverted cells and optimization of the film casting process in the inverted architecture may be the focus of future work.
Unfortunately, when the optimized 1.5% DIO condition was used to make inverted devices, the efficiency was lower than in a standard architecture (*J* ~SC~ = 7.0 mA/cm^2^, *V* ~OC~ *=*0.73 V, *FF* = 0.47, *PCE* = 2.4%). These devices showed very high dark (leakage) current ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S10), which is likely a result of a change in morphology when casting atop ZnO instead of PEDOT; the films show significantly rougher surfaces ([Supporting Information File 1](#SD1){ref-type="supplementary-material"}, Figure S11). It is possible that through further optimization using the inverted architecture, we may be able to improve the top efficiency, however, that is beyond the scope of this work. We were satisfied to demonstrate that changing architectures does indeed eradicate the s-shape of the curve for devices cast from pure chlorobenzene, helping to further prove the hypothesis that the root of s-shaped *J--V* curves was indeed non-ideal vertical phase separation.
Conclusion
==========
A new molecular donor material, p-SIDT(FBTThCA8)~2~, was developed based on the inclusion of electron-withdrawing endcaps within a previously reported high-performance molecular framework. The structural modification had the desired effect of reducing the band gap for extended absorption in the visible spectrum while maintaining a low-lying HOMO level to achieve high *V* ~OC~. The energy levels are nearly ideal match for incorporation into BHJ devices with the acceptor PC~71~BM, maximizing voltage and spectral coverage. Despite the structural similarity to previously reported materials, however, blends of p-SIDT(FBTThCA8)~2~ and PC~71~BM did not have device performance akin to its predecessors when processed in the same manner.
Specifically, when cast from chlorobenzene, the resulting *J--V* curve gives rise to a significant s-shape, resulting in extremely low *FF* and *PCE*. Through light intensity studies, the s-shape in the curve was attributed to the build-up of space charge. The use of DIO as a solvent additive helped to remove the s-shape character from the *J--V* curve and to improve the performance up to *PCE* = 3.2%. Analogous to what has been reported in literature, DIO helps to induce crystallinity of the p-SIDT(FBTThCA8)~2~ in the blend as evidenced by GIWAXS and a commensurate red shift in absorption. However, lack of crystallinity is not typically associated with the s-shape in the *J--V* curve seen when cast without additive.
Blends cast from chlorobenzene have reasonably high mobility, so a build-up of space charge simply due to an imbalance in carrier mobilities can likely be ruled out. Instead, the differences in curve shape are ascribed to changes in the vertical phase separation; when cast without additive there is a enrichment of PC~71~BM at the PEDOT:BHJ interface as evidenced by DSIMS. Subsequently, the low concentration of p-SIDT(FBTThCA8)~2~ at the anode likely leads to reduced surface recombination, a build-up of space charge and ultimately, and s-kink in the *J--V* curve. The inclusion of DIO helps to reduce the concentration of PC~71~BM at the anode improving surface recombination, and *J--V* characteristics. This is further evidenced by the elimination of the s-kink upon moving to an inverted structure. In fact, in the inverted structure the blend device gives a *V* ~OC~ of 1.09 V, which is quite remarkable, considering the absorption profile extends out to 730 nm (1.69 eV). Such a small voltage loss between absorption onset and V~OC~ demonstrates the tremendous potential of this blend system. Although without further device engineering the performance of this materials system is not yet on par with the state of the art, the drastic change in curve shape is important in understanding the nature of solvent additives and their effects on solution processed BHJ devices.
Supporting Information
======================
######
Detailed experimental procedures with physical and chemical analysis of compounds and additional device characterization data.
The authors thank the Office of Naval Research (Award No. N000141410076) for support of this work. T.-Q.N. thanks the Camille Dreyfus Teacher Scholar Award. The authors acknowledge the support of Stanford Synchrotron Radiation Light source (SSRL), which is supported by the U.S. Department of Energy.
| {
"pile_set_name": "PubMed Central"
} |
Despite a remarkable incidence rate (fourth most common malignancy in men in United States), the progress in the therapeutic paradigm of urothelial cancer (UC), particularly in advanced stages, was stagnant in the last few decades ([@bib10]). A paradigm shift is needed to advance the field. For patients with advanced disease and who have failed chemotherapy regimens, a variety of single-agent or combination therapies have yielded modest response rates and poor survival estimates. Although vinflunine is approved by the European Medicines Agency for progressive disease after platinum-based therapy, the US Food and Drug Administration has no approved agents ([@bib25]). A plethora of unsuccessful phase 2 trials of targeted compounds, either alone or combined with chemotherapy, was tested in UC at different clinical stages. With regards to the antiangiogenic setting, a compelling preclinical rationale fostered clinical research in the field, and results were reported with the use of compounds targeting the vascular-endothelial growth factor (VEGF) receptor axis, including sorafenib, aflibercept, sunitinib, everolimus, and bevacizumab ([@bib8]; [@bib11]; [@bib27]; [@bib2]; [@bib13]; [@bib5]; [@bib24]; [@bib1]; [@bib12]; [@bib19]). Despite the negative results achieved in a small study sponsored by the National Cancer Institute in United States ([@bib22]), pazopanib was active in our single-group, phase 2 study, whereby an objective response rate of 17.1% was achieved in heavily pretreated patients ([@bib20]). Taking together the results of these trials, an invariably uniform scenario can be drawn consisting of a rather low response rate ranging from 5 to 15%, and a small impact on expected progression-free (PFS) and overall survival (OS). Despite this, a small cohort of extreme responders could be identified by obtaining an incredibly long-term clinical benefit from antiangiogenic compounds. Paradigmatic examples are those observed in the sunitinib trial (one partial response (PR) lasting 24 months; [@bib11]) and in the everolimus trial (one response duration of 26 months; [@bib15]; [@bib19]).
This is the reason why further investigations on targeted agents should aim at identifying this class of long-term survivors for whom an antiangiogenic approach might have sense. In the absence of available tissue- and blood-based predictors, we aimed at evaluating circulating angiogenic factors (CAFs) over time in our phase 2 trial of pazopanib. Yet, the role of interleukin-8 (IL8) was anticipated ([@bib20]), and herein we present the full results of circulating biomarker analyses, matched with contextual computed tomography (CT)/positron emission tomography (PET) results. Refining the prognostic ability of recognised clinical factors could facilitate the proper selection of patients for conducting confirmatory trials with pazopanib in this disease as well as the interpretation of retrospective data from phase 2 studies with similar compounds.
Patients and methods
====================
Forty-one patients having failed at least one platinum-based chemotherapy regimen were enrolled in a single-group, phase 2 trial of Pazopanib 800 mg orally daily until disease progression/unacceptable toxicity. Ten millilitres of EDTA plasma samples were collected at baseline and every 4 weeks until drug discontinuation, together with CT and PET/CT restaging. Samples were centrifuged for 20 min at 2200 r.c.f./4 °C and immediately stored at ⩽−20 °C. The amount of cCAFs, such as VEGF, serum VEGF receptor (VEGFR)-1 and -2, stem-cell factor (cKIT), IL6, IL8, and IL12, hepatocyte growth factor, and transforming growth factor-*β* (TGF*β*), was determined at baseline (T0) and after 4 weeks of treatment (T1) using commercially available ELISA kits (R&D Systems Inc., Minneapolis, MN, USA), according to manufacturer\'s protocols. Samples and standards were added to the wells of a microtitre plate and the different CAFs were captured by the specific antibodies immobilised to the wells of the plate. Successively, a horseradish peroxidase-conjugated detection antibody was added to detect the bound protein. After incubation, the wells were washed and the antigen complex bound to the well was detected by addiction of tetramethylbenzidine substrate solution, and a blue colour developed in proportion to the amount of the biomarker present in the sample. Colour development was then stopped, turning the colour in the wells to yellow. The absorbance of the colour was measured at 450 nm, producing a signal that is proportional to the amount of the biomarker bound. Response Evaluation Criteria for Solid Tumors (RECIST) version 1.1 were used to evaluate objective response -- primary end point of the trial ([@bib9]). Patients were categorised as responders (complete response (CR), PR, including stable disease (SD)) or non-responders for study purposes. Metabolic responses were based on the European Organisation for Research and Treatment of Cancer criteria ([@bib30]). The PET CR was defined by the fluorodeoxyglucose (FDG) uptake disappearance in all lesions detected at baseline, whereas PR was defined as a decrease of standard uptake value (SUV)max⩾25%. Non-responders were considered as those patients with either a SUVmax decrease\<25% or any increase of FDG uptake, and the appearance of new focal FDG uptake(s) with anatomical confirmation. The metabolic response evaluation was assessed per patient by a blinded, referral nuclear medicine physician as the sum of SUVmax of the target lesions. Clinical protocol and the informed consents relative to clinical and biological study participation were approved by the Institutional Review Board of the Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. All patients provided written informed consent before study entry.
Statistical methods
-------------------
Statistical analyses focused on the investigation of CAFs as possible biomarkers. The association structure between biomarkers was investigated by estimating a Pearson\'s partial correlation matrix, whereby correlations between pairs of biomarkers were adjusted for their associations with others.
Changes in CAF levels between T0 (baseline) and T1 (after 4 weeks of treatment) were tested by means of paired Student\'s *t*-tests. To investigate whether T1 concentrations were associated with tumour response (either RECIST or PET), covariance analyses were adopted, which are known as the most efficient approach for the analysis of pre--post designs.
The prognostic effect of singly taken CAFs on OS was investigated using Cox proportional hazard regression models, with and without adjustment for clinical variables. For the sake of parsimony, such an adjustment was obtained by using the score of a separate Cox model including the following covariates, chosen on the basis of prior knowledge and previously used ([@bib20]): Eastern Cooperative Oncology Group performance status (0 versus ⩾1), presence of liver metastases, site of tumour primary (bladder versus upper tract), haemoglobin level at baseline (\<10 versus ⩾10 g dl^−1^), and number of disease sites (1--2 versus \>2). As regards the biomarkers, T0 levels for all CAFs and T1 levels only for those factors that changed significantly from baseline were modelled in the above analyses. Furthermore, for investigating CAF joint prognostic effect, the factors that achieved a Wald\'s test *P*-value⩽0.10 in either unadjusted or adjusted analyses were entered into a multivariable Cox model. A backward elimination procedure was then used to identify the strongest prognostic biomarkers. Net reclassification improvement (NRI) was calculated to measure the improvement in 6-month prediction yielded by selected CAFs when added to clinical variables in multivariable Cox models. This index is a novel measure of model predictive performance that has been specifically recommended for the assessment of biomarkers ([@bib21]; [@bib23]). The NRI is calculated distinctly for event and non-event patients, and the two estimates are then averaged. For completeness, we report all these figures, although the average is more meaningful for clinical interpretation: the closer to one, the better the prognostic improvement towards a reference model, which, in our case, relies on information provided by clinical variables only.
The analyses were carried out using the SAS (SAS Institute Inc., version 9.2) and R 2.15.2 software (<http://www.r-project.org/>, last access September 30th, 2013). Statistical significance was set at the conventional two-sided 5% level.
Results
=======
Updated clinical outcomes
-------------------------
Forty-one patients with UC and treated with at least one dose of pazopanib were enrolled in the study between February 2010 and July 2011. The majority of patients (51%) entered beyond the second line, 17.1% had a PR, and 51.2% had a clinical benefit. Median PFS and OS were 2.6 (95% CI, 1.7--3.7) and 4.7 months (95% CI, 4.2--7.3 months), respectively ([@bib20]).
There were two very long-term responders. The first patient (ID 04) had a PR lasting 32 months and an OS of 37.9 months, in spite of having poor prognostic features, namely, an upper tract UC in origin, third-line treatment for progressive bulky retroperitoneal disease, and the presence of isolated liver metastasis. The second patient (ID 12) had a bladder primary and was treated in second-line setting for disseminated nodal disease progressing after four cycles of cisplatin and gemcitabine. He obtained a durable SD of 19 months and the OS was 35.9 months.
Circulating biomarker assessment
--------------------------------
Although some significant results were achieved when investigating the association structure between CAFs ([Supplementary Table 1](#sup1){ref-type="supplementary-material"}), no strong correlations (as quantified by a coefficient *ρ*\>0.80) were detected. In particular, the highest levels of correlation were observed between VEGF and VEGFR2 (*ρ*=−0.48), VEGFR1 and IL8 (*ρ*=0.45), and VEGFR2 and TGF*β* (*ρ*=0.48).
[Table 1](#tbl1){ref-type="table"} and box plots of [Figure 1](#fig1){ref-type="fig"} provide a descriptive analysis of pre--post pazopanib treatment biomarker levels. Significant T1--T0 modulation was observed for VEGF (*P*\<0.001), VEGFR2 (*P*\<0.001), cKIT (*P*\<0.001), and IL8 (*P*=0.008). A 4-week increase was reported for VEGF and IL8 concentrations, whereas VEGFR2 and cKIT levels decreased. A separate description of T0--T1 CAFs changes in the two long-term responders is provided in [Supplementary Table 2](#sup1){ref-type="supplementary-material"}.
A significant association was detected between tumour RECIST response and IL8^T1^ levels (*P*=0.010 at covariance analysis). In detail, median IL8^T0^ levels were comparable between responders (67 pg ml^−1^) and non-responders (67.5 pg ml^−1^), whereas IL8^T1^ levels differed significantly between the two groups, being 69.8 pg ml^−1^ in responders ([Figure 2A](#fig2){ref-type="fig"}) and 97.6 pg ml^−1^ in non-responders ([Figure 2B](#fig2){ref-type="fig"}). As regards associations with PET response compared with baseline, significant results were achieved for IL12^T0^ levels (*P*=0.039 from covariance analysis) in contrast to IL8^T0^ and IL8^T1^ levels (*P*=0.111 each).
[Table 2](#tbl2){ref-type="table"} shows the results of Cox model analyses focusing on the prognostic effects of singly taken CAFs on OS. Significant results were obtained for IL8^T0^ (*P*=0.015), IL8^T1^ (*P*=0.012), VEGF^T1^ (*P*=0.007), and TGF*β*^T0^ (*P*=0.038). After adjustment for the clinical variables, only IL8^T0^ (*P*=0.019) and IL8^T1^ (*P*=0.004) remained significant.
By jointly modelling IL8^T1^, IL8^T0^, VEGF^T1^, cKIT^T1^, and TGF*β*^T0^, which are the biomarkers selected for the multivariable analysis, the average prognostic improvement over clinical variables (as quantified by NRI) was 60% ([Table 3](#tbl3){ref-type="table"}, model 1). Nevertheless, by applying a backward variable selection procedure, only IL8^T1^ retained its statistical significance. In this case, the average improvement in prediction was 39%.
By plotting the response probability and 6-month survival ([Figure 3A and B](#fig3){ref-type="fig"}) according to IL8^T1^ (and by adjusting for the fixed -- median -- value of IL8^T0^ at 67 pg ml^−1^), it turned out that a threshold of 80 pg ml^−1^ might be a reasonable cut-off value for prognostic discrimination; in particular, patients below the threshold show a relatively favourable prognosis for both outcomes.
Discussion
==========
Our study reports a prognostic improvement by adding a biological variable to clinical parameters in the context of an antiangiogenic treatment.
There are a number of preclinical evidences supporting a role for angiogenesis in UC. Angiogenesis and VEGF possess key roles in UC initiation, progression, and invasion. Moreover, investigators have demonstrated an association between VEGF expression and prognosis of UC, as well as an improved tumour control with platinum-based chemotherapy plus antiangiogenic therapy in preclinical models ([@bib7]; [@bib16]; [@bib29]). The present proof-of-principle trial showed a consistent pattern of cytokine reaction in UC patients treated with pazopanib and provided circumstantial evidences for the role of microenvironment as a framework of druggable targets in this disease.
To the best of our knowledge, this is the first time that the role of IL8 evaluated as dynamically in relation to response and outcome was obtained in the clinic. The most clinically sound observation was that patients with high baseline and 4-week levels of IL8, and, most importantly, those with rising serum levels of IL8 during pazopanib, particularly those with levels encompassing 80 pg ml^−1^ at 4 weeks of treatment, had a significantly lower chance of responding and 6-month survival probability. Interleukin-8 level at 4 weeks were then an independent prognostic factor for survival, together with recognised clinical variables.
Results should be taken with caution based on the limitations of the small sample size and the absence of a control arm. Moreover, when looking at the individual patient levels, it turned out that one of the two long-term survivors who achieved a prolonged SD with pazopanib had T1 levels rising to 100.0 pg ml^−1^ ([Supplementary Table 2](#sup1){ref-type="supplementary-material"}).
Rising levels of IL8 have been already associated with the development of resistance to antiangiogenic agent sunitinib in preclinical models of renal cell carcinoma, but this mechanistic association is hard to be unravelled based on present results ([@bib14]). In fact, IL8 is produced by tumour cells of different histologies and raising serum concentrations of this chemokine were associated with tumour burden and increasing stage in a variety of solid neoplasms ([@bib18]; [@bib28]; [@bib4]; [@bib17]). Hence, the question whether IL8 levels and their change over time might be solely a drug and tumour-induced epiphenomenon or rather a signal to allow selecting patients who are most likely to respond/survive remains unanswered. On the other hand, IL8 is a recognised mediator of tumour growth and metastatisation potential, and its role as a predictor of clinical benefit has been already reported in bladder cancer and renal cell carcinoma patients receiving sunitinib and pazopanib, respectively ([@bib2]; [@bib26]). Furthermore, interleukin-8, as well as IL6, represents an activation of the immunostimulatory system and has been associated with a worse prognosis in cancer, independent of the tumour heterogeneity ([@bib17]). Taken together, present observations and available knowledge might provide a rationale for the therapeutic role of agents targeting IL8 in UC, in combination or a sequence with a TKI. Among the available drugs, although the activity of the fully human anti-IL8 antibody ABX-IL8 was provided only in preclinical models ([@bib18]), another anti-IL8 compound, reparixin (Dompè s.p.a.), is currently in phase 2 development in early breast cancer (ClinicalTrials.gov number NCT01861054).
Investigation on the prognostic contribution of IL8 should be pursued further in this disease, particularly in trials with antiangiogenic TKIs, to validate a potential tool for a patient-enrichment design. This could apply to two ongoing phase 2 trials of pazopanib combined with paclitaxel and gemcitabine, respectively, in UC (registered with ClinicalTrials.gov, number NCT01108055 and NCT01622660, respectively).
Although aberrations resulting in sensitivity to VEGFR-directed TKI might exist in the microenvironment rather than the tumour itself, this theory has yet to be proven. Yet, another signal corroborating the role of microenvironment in this setting was relative to the association of baseline levels of IL12 and metabolic response at 4 weeks (lower levels associated with PET response). Again, this observation should be cautiously unravelled, but a possible explanation may be that IL12 is an essential pro-inflammatory cytokine that is decreased in several cancer types, particularly in later stages; hence, it might be in relation to inflammatory and FDG-avid peri-tumoural tissue, the first to be dampened by an active targeted compound ([@bib6]). Baseline TGF*β* provided signals of prognostic effect. TGF*β* is one of the principal immune-suppressive factors secreted by tumour cells and it possesses a huge spectrum of activity depending on the type of activated receptor ([@bib3]). A phase 2 trial is ongoing at our centre with the fully human monoclonal antibody directed against TGF*β* receptor ALK1, PF03446962 (Pfizer Inc, La Jolla, CA, USA), a compound endowed with distinct antivascular activity, as second-line therapy in UC (ClinicalTrials.gov, number NCT01620970). Combined results from our group and from other clinical trials worldwide underscored the clinical meaning of targeting angiogenesis thus far, but an improvement in trial design based on patient selection/enrichment is desperately needed. Thus far, the sobering realisation of clinical trials with this class of agents was that of a small activity followed by resistance developing in a few months. Observations are hampered by the class activity of these drugs, not corresponding to tumour shrinkage for the majority of cases. A discrepancy was usually observed between an overall modest survival improvement and the existence of small subset of patients achieving an incredibly long-term response-stabilisation or even CR, far beyond what could be reasonably expected *a priori*. Going forward, an international cooperation to validate the present findings is required. The design of a multicentre data set, including CAFs from multiple cohorts of patients receiving anti-VEGF(R) compounds in phase 2 trials has the potential to render these results broadly applicable to antiangiogenic drugs in future clinical trials.
In conclusion, a caveat of present series is that tumour biology is suboptimally captured by clinical and laboratory features, such as those evaluated and the discovery of molecular predictors linked to an aggressive phenotype, and treatment resistance still needs a paradigm change. This is the reason why we are now moving towards a genomic profiling that yields a number of theoretical advantages over the former approach to guide informed clinical trials ([@bib15]). Extensive genomic profiling of tumour samples, particularly of defined subsets of patients who achieve extreme responses to antiangiogenic drugs, such as pazopanib, may allow for the identification of a landscape of novel druggable biomarkers.
[Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License.
Presented in part at the 2012 Markers in Cancer Meeting, the 2012 ASCO Annual Meeting and the 2012 AACR Annual Meeting, 31 March--4 April 2012, Chicago, IL, USA.
The authors declare no conflict of interest.
Supplementary Material {#sup1}
======================
######
Click here for additional data file.
######
Click here for additional data file.
{#fig1}
{#fig2}
{#fig3}
###### Median (IQR) CAF circulating levels at T0, T1, and Δ
**T0** **T1** *Δ*[a](#t1-fn2){ref-type="fn"} ***P*****-value**[b](#t1-fn3){ref-type="fn"}
-------------- ------------------------- ------------------------- -------------------------------- ----------------------------------------------
**VEGF**
Median (IQR) 124.3 (79.3, 156.7) 183.3 (132.3, 250.7) 61.4 (18.6, 148.7) \<0.001
**VEGFR1**
Median (IQR) 136.6 (123.1, 168.1) 133.1 (114.9, 172.9) −3.2 (−25.5, 28.2) 0.971
**VEGFR2**
Median (IQR) 5489.3 (5059.3, 6190.3) 4234.8 (3488.8, 4564.3) −1385.0 (−1789.5, −955.0) \<0.001
**cKIT**
Median (IQR) 13.8 (9.2, 17.6) 10.7 (7.0, 13.1) −2.8 (−4.9, −1.1) \<0.001
**IL6**
Median (IQR) 12.8 (4.4--26.9) 16.7 (8.3--29.7) 3.8 (−4.7, 15.0) 0.067
**IL8**
Median (IQR) 67.0 (49.8−84.0) 87.3 (55.3−102.8) 10.5 (−6.0, 31.5) 0.008
**IL12**
Median (IQR) 44.2 (21.9--66.9) 37.1 (25.1--56.5) −4.4 (−11.4, 5.4) 0.193
**HGF**
Median (IQR) 481.5 (364.0−671.5) 532.0 (415.5−688.5) 82.0 (−38, 151) 0.257
**TGF*β***
Median (IQR) 29.0 (26.4−35.0) 29.0 (22.9−33.5) −2.5 (−5.8, 2.5) 0.070
Abbreviations: CAF=circulating angiogenic factor; HGF=hepatocyte growth factor; IL=interleukin; IQR=interquartile range; KIT=stem-cell factor; TGF*β*=transforming growth factor-*β*; VEGF(R)=vascular-endothelial growth factor (receptor).
T1--T0, values in pg ml^−1^ (VEGF, VEGFR1, VEGFR2, IL6, IL8, IL12, HGF and TGF*β*) or ng ml^−1^ (cKIT).
Paired Student\'s *t*-test, *P*-value.
###### Cox model analysis
**Unadjusted** **Adjusted**
------------ ---------------- -------------- ------- ------ ------------ -------
VEGF^T0^ 1.21 0.84--1.74 0.311 1.01 0.70--1.46 0.943
VEGF^T1^ 1.69 1.16--2.48 0.007 1.47 0.97--2.25 0.071
VEGFR1^T0^ 1.13 0.80--1.60 0.480 0.94 0.66--1.35 0.750
VEGFR2^T0^ 0.71 0.40--1.24 0.227 0.87 0.48--1.58 0.650
VEGFR2^T1^ 0.78 0.49--1.23 0.280 0.86 0.54--1.37 0.532
cKIT^T0^ 1.18 0.81--1.73 0.395 1.32 0.92--1.87 0.128
cKIT^T1^ 1.14 0.64--2.02 0.652 1.56 0.92--2.65 0.101
IL6^T0^ 1.07 0.89--1.29 0.482 1.05 0.83--1.33 0.680
IL8^T0^ 1.72 1.11--2.67 0.015 1.73 1.09--2.73 0.019
IL8^T1^ 1.87 1.15--3.05 0.012 2.09 1.27--3.43 0.004
IL12^T0^ 1.65 0.91--2.97 0.097 1.51 0.84--2.73 0.170
HGF^T0^ 1.00 0.95--1.06 0.857 0.98 0.93--1.04 0.535
TGF*β*^T0^ 1.28 1.01--1.63 0.038 1.10 0.85--1.43 0.471
Abbreviations: 95% CI=95% confidence interval; HGF=hepatocyte growth factor; IL=interleukin; KIT=stem-cell factor; TGF*β*=transforming growth factor-*β*; VEGF(R)=vascular-endothelial growth factor (receptor); HR=hazard ratio.
T1 values only for markers with significant modulation at 5% level.
HR for interquartile range
*P*=Wald\'s test *P*-value.
###### NRI of multivariable Cox models incorporating CAFs information
**NRI**
------------------------------------ --------- ------ ------
Model 1[a](#t3-fn2){ref-type="fn"} 0.44 0.75 0.60
Model 2[b](#t3-fn3){ref-type="fn"} 0.52 0.25 0.39
Abbreviations: NRI=Net reclassification improvement; CAF=circulating angiogenic factor; IL=interleukin; KIT=stem-cell factor; TGF*β*=transforming growth factor-*β*; VEGF=vascular-endothelial growth factor.
Model including IL8^T1^, IL8^T0^, VEGF^T1^, cKIT^T1^, TGF*β*^T0^.
Model including IL8^T1^ only, after applying a backward variable selection procedure.
[^1]: Conquer Cancer Foundation of ASCO Merit Award recipient, 11--13 October 2012, Hollywood, FL, USA and 1--5 June 2012, Chicago, IL, USA.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Uptake of measles-mumps-rubella (MMR) vaccine in the UK has remained below 85% for over a decade, and currently only 83% of five year-olds are adequately immunised in line with the recommended two-dose schedule [@pone.0019381-Department1]. This suboptimal vaccine coverage leaves the UK population at risk of a measles epidemic [@pone.0019381-Capital1], [@pone.0019381-McCauley1]. In response to this, an MMR catch-up campaign was launched in 2008 to improve MMR coverage among children who missed MMR doses at scheduled age (dose 1 at ∼13 months, dose 2 at ∼3 years and 4 months). From 1^st^ September 2008 Primary Care Trusts (PCTs) across the UK were instructed to offer catch-up MMR to children aged 13 months to 18 years [@pone.0019381-Department1]. Children were prioritised first by MMR doses received, then by age, such that younger children with no MMR doses on their General Practice (GP) or PCT record were the primary targets for the campaign. GPs/PCTs were advised to send postal invitations to parents/caregivers of eligible children asking them to bring their child to the GP surgery for catch-up MMR. Department of Health (DH) trial sentinel data for the first year of the catch-up campaign indicate a 5.1% increase in full MMR coverage among 5--18 year olds and a 2% decrease in the number who have no MMR doses recorded [@pone.0019381-Department2].
Catch-up campaigns providing a vaccine to those who missed it at scheduled age (for example, because their parents could not access it or chose to reject it) are typically only moderately successful, immunising less than 50% of their target populations [@pone.0019381-Capital1]--[@pone.0019381-Lashkari1], with only 20--25% uptake in some campaigns [@pone.0019381-Capital1], [@pone.0019381-McCauley1]. Most campaigns fail either to collect or report relevant evaluation data indicating why parents in their target populations accepted or rejected MMR within the catch-up, however withheld or missing parent consent (whilst some parents actively refuse the vaccine, often a greater number simply fail to respond to the invitation -- their consent may be consciously withheld or unconsciously omitted, but either way their children are not immunised by default in the absence of explicit consent) has been implicated in 45--62% of cases where an eligible child has not received catch-up MMR within school-based programmes [@pone.0019381-Roberts1], [@pone.0019381-Hadjikoumi1]. A number of attitudinal and demographic factors have been linked with MMR uptake within the routine schedule (see below) [@pone.0019381-Pearce1]--[@pone.0019381-Roberts2], and these factors may also relate to catch-up MMR uptake. The present work tests this hypothesis by identifying univariate and multivariate predictors of catch-up MMR uptake during the 2008--09 MMR catch-up campaign.
Factors related to routine MMR uptake {#s1a}
-------------------------------------
- Beliefs about and previous experience of MMR safety and efficacy
- Beliefs about severity, susceptibility and possible benefits (e.g. natural immunity) of measles
- Perceived social desirability and value of community benefit of MMR uptake
- Satisfaction with and trust in official (e.g. Department of Health, NHS) and unofficial (e.g. internet and lay advice) information around MMR and measles
- Practical barriers to clinic attendance (e.g. availability of appointments)
- Parent age and socioeconomic status (an inverted U curve is observed, with MMR uptake lower at the extreme ends of both parameters)
Methods {#s2}
=======
Ethics statement {#s2a}
----------------
The Health Protection Agency and PCTs involved classified the work as a service evaluation not requiring ethical approval as results were anonymised for analysis. Consent to participate was implied through questionnaire completion.
Participants {#s2b}
------------
Child Health Information Systems (CHIS) in three UK PCTs (two in London, one in north-west England) were used to identify all children aged 5--17 years and with suboptimal CHIS-recorded MMR status (\<2 doses) at 1^st^ September 2008 (the first day of the UK MMR catch-up campaign 2008--09). From this population, 2,300 children were randomly selected with stratification by child age. This sample size provided 80% power for hierarchical multiple regression to detect small to medium effects at the 0.05 significance level with a 20% response rate. PCTs provided postal and telephone contact details for the parent/guardian(s) of each child, plus the child\'s date of birth and MMR dose history.
Materials and procedure {#s2c}
-----------------------
The internal consistency, test-retest reliability, concurrent and predictive validity of the questionnaire ([Figure 1](#pone-0019381-g001){ref-type="fig"}) has been demonstrated previously [@pone.0019381-Brown2]. The questionnaire comprised 20 attitude items and seven demographic items all derived from the literature on factors underpinning parents\' routine schedule MMR decisions [@pone.0019381-Pearce1]--[@pone.0019381-Roberts2], and a single item assessing self-reported receipt of a postal MMR catch-up invitation. Attitude items took the form of statements with which the respondent indicated their level of agreement on five-point scales (1 = strongly disagree, 5 = strongly agree), for every attitude item a higher score indicated more 'pro-MMR' attitude. The attitude items (except item 19 assessing practical barriers) collapsed into four scales with adequate reliability (see [Table S1](#pone.0019381.s001){ref-type="supplementary-material"} for details of items in each scale. Cronbach\'s alphas = 0.59--0.76). Demographic data collected were parent (respondent) age, sex, highest educational qualification, ethnic group, number of children, marital status and job; responses were provided using tick-box options for all but the job item, which was free-text.
{#pone-0019381-g001}
Between April and September 2009, a copy of the questionnaire was posted to the parent/guardian of every child in the sample, along with a cover letter explaining the purpose and provenance of the survey, a freepost return envelope, and a notice advising in languages most commonly used in the PCTs that translations were available on request. A maximum of two postal and two telephone reminders were made, at approximately 3--4 and 6--7 weeks after the first copy was sent. Postal reminders contained replacement questionnaires, and telephone reminders comprised an invitation to respond verbally to the questionnaire during the call.
CHIS-recorded receipt of MMR dose(s) during the first year of the catch-up campaign (1^st^ September 2008--31^st^ August 2009), and postcode-level Indices of Multiple Deprivation 2007 data (IMD2007 [@pone.0019381-Department3]), were obtained for the entire sample including non-respondents. Where MMR dose history obtained at the end of the studied period differed from that which had been provided at the start of the period, the most up-to-date history was used. Free-text responses to the job item were coded by two independent analysts (very good agreement between analysts: Cohen\'s Kappa 0.91) to the 8-class version of the National Statistics Socio-Economic Classification (NS-SEC [@pone.0019381-Office1]), where code 1 is the highest socio-economic class (higher managerial/higher professional/large employer) and code 8 the lowest (never worked/long-term unemployed/student etc); respondents classifying themselves as 'mother', 'housewife' or similar were coded to category 8.
Analysis {#s2d}
--------
Data were analysed using SPSS v 17.0 (SPSS Inc. Chicago, IL). Participation rates and participant characteristics were assessed using all available data on the entire sample. Missing values were imputed using within-participant scale means for scales of 5 items or more where up to 2 items were missing. Scale scores were calculated by summing scores (including imputed values) for individual items comprising the scale then dividing by the number of items in the scale.
All further analyses were completed first for the sample as a whole, then with participants split into two groups: those whose child had received no MMR doses at the start of the catch-up period (henceforth referred to as 'unimmunised'), and those whose child had received one MMR dose at that time ('partially immunised'). Univariate (Chi-square tests and ordinal regression for nominal data, Mann-Whitney tests and ANCOVA for ordinal data) and multivariate (hierarchical logistic regression) comparisons were made within each group between those who gave MMR dose(s) during the catch-up period and those who didn\'t.
Results {#s3}
=======
Response rate and respondent characteristics {#s3a}
--------------------------------------------
365 of 2,300 (15.9%) identified cases returned a completed questionnaire. Achieved power differed minimally from planned power despite the response rate being 4% lower than expected. There was no difference in response rate by MMR status, but respondents had younger children (p\<0.01) and lived in less deprived postcode areas (p\<0.001) than did non-respondents ([Table 1](#pone-0019381-t001){ref-type="table"}).
10.1371/journal.pone.0019381.t001
###### Participation rates and representativeness.
{#pone-0019381-t001-1}
n n(%) / Mean(SD) p
----------------------------------------------------------------------------- ------ ----------------- --------------- ---------
MMR status at end of data collection[†](#nt101){ref-type="table-fn"}
0 doses 1166 182 (15.6) 984 (84.4) 0.31
1 dose 882 135 (15.3) 747 (84.7)
2 doses 252 48 (19.0) 204 (81.0)
Child age (years) at end of data collection[‡](#nt102){ref-type="table-fn"} \- 9.8 (3.6) 10.4 (3.8) \<0.01
IMD2007 score[‡](#nt102){ref-type="table-fn"} \- 26.28 (15.64) 31.43 (17.14) \<0.001
Total 2300 365 (15.9) 1935 (84.1) \-
: n(%), p values for Chi-square test;
: mean(SD), p values for independent samples t-test.
Factors associated with catch-up MMR uptake in univariate analyses {#s3b}
------------------------------------------------------------------
See [Tables 2](#pone-0019381-t002){ref-type="table"} and [3](#pone-0019381-t003){ref-type="table"}. In the sample as a whole, catch-up MMR uptake was associated with younger child age (p\<0.001), perceived social desirability/benefit of MMR uptake (p\<0.001, 7% variance), pro-MMR feelings (p\<0.001, 4% variance), satisfaction with available official information around MMR (p\<0.01, 3% variance), and concern about measles (p\<0.05, 1% variance). Less than one-third of parents reported having received an MMR catch-up invitation in the past year, and receipt of an invitation was not associated with catch-up MMR uptake, when child age and deprivation were taken into account. The individual items (see [Table S1](#pone.0019381.s001){ref-type="supplementary-material"}) explaining the most variance in catch-up MMR uptake for the whole sample were disbelieving serious MMR side effects, valuing community benefit of immunisation, and perceiving peers/family to be pro-MMR (all p\<0.001, 5% variance). With the sample split by MMR status at the start of the catch-up campaign (unimmunised versus partially immunised), univariate associations were largely as described above for both groups. However, measles beliefs showed no association with catch-up MMR uptake in these smaller subsamples (p\>0.05). In addition, catch-up MMR uptake was linked with younger parent age only among parents of unimmunised children (p\<0.05), and with lower educational attainment only among parents of partially immunised children (p\<0.01).
10.1371/journal.pone.0019381.t002
###### Demographic characteristics by catch-up MMR uptake.
{#pone-0019381-t002-2}
All cases Unimmunised Partially immunised
---------------------------------- ----------- ------------- --------------------- --------- ----- ---------
**Child age (years)†**
5--6 115 44 (38) 69 17 (25) 46 27 (59)
7--8 57 10 (18) 36 5 (14) 21 5 (24)
9--10 52 5 (10) 31 2 (6) 21 3 (14)
11--12 78 6 (8) 40 3 (8) 38 3 (8)
13--14 22 5 (23) 19 2 (11) 13 3 (23)
15--16 12 2 (17) 6 2 (33) 6 0 (0)
17--18 19 0 (0) 12 0 (0) 7 0 (0)
**IMD 2007 score‡**
\<sample mean (31.4) 240 42 (18) 145 22 (15) 95 20 (21)
≥sample mean 123 29 (24) 67 9 (13) 56 20 (36)
**Parent age (years)**
20--24 8 2 (25) 5 1 (3) 3 1 (3)
25--29 21 5 (24) 13 3 (10) 8 2 (5)
30--34 47 15 (32) 25 6 (20) 22 9 (23)
35--39 99 23 (23) 65 12 (40) 34 11 (28)
40+ 179 24 (13) 100 8 (27) 79 16 (41)
**Parent highest qualification**
None 26 7 (27) 17 2 (12) 9 5 (56)
GCSE/O-level 82 18 (22) 50 9 (18) 32 9 (28)
A/AS-level 45 11 (24) 25 3 (12) 20 8 (40)
Diploma 73 10 (14) 39 3 (8) 34 7 (21)
Degree 74 12 (16) 45 7 (16) 29 5 (17)
Postgraduate degree 40 9 (23) 28 6 (21) 12 3 (25)
Other 4 0 (0) 2 0 (0) 2 0 (0)
**Parent ethnicity**
White British 248 54 (22) 144 22 (15) 104 32 (31)
Black British 16 2 (13) 10 1 (10) 6 1 (17)
Asian British 24 2 (8) 14 0 (0) 10 2 (20)
Other British 3 1 (33) 2 1 (50) 1 0 (0)
White other 18 5 (28) 12 4 (33) 6 1 (17)
Black other 5 2 (40) 3 1 (33) 2 1 (50)
Asian other 26 3 (12) 18 2 (11) 8 1 (13)
Other or mixed 6 1 (17) 2 0 (0) 4 1 (25)
**Number of children**
1 56 14 (25) 32 4 (13) 24 10 (42)
2 172 36 (21) 99 17 (17) 73 19 (26)
3 75 10 (13) 42 4 (10) 33 6 (18)
4+ 46 10 (22) 30 5 (17) 16 5 (31)
**Parent marital status**
Single 63 13 (21) 42 7 (17) 21 6 (29)
Cohabiting 58 13 (22) 25 5 (20) 23 8 (35)
Married 222 37 (17) 127 14 (11) 95 23 (24)
Other 25 6 (24) 18 4 (22) 7 2 (29)
**Parent job (NS-SEC)**
1 39 7 (18) 26 2 (8) 13 5 (38)
2 31 8 (26) 17 4 (24) 14 4 (29)
3 21 7 (33) 11 2 (18) 10 5 (50)
4 12 1 (8) 10 1 (10) 2 0 (0)
5 32 5 (16) 24 3 (13) 8 2 (25)
6 54 7 (13) 30 2 (7) 24 5 (21)
7 49 14 (29) 24 5 (21) 25 9 (36)
8 91 17 (19) 51 9 (18) 40 8 (20)
10.1371/journal.pone.0019381.t003
###### Attitudes and catch-up invitation receipt by catch-up MMR uptake.
{#pone-0019381-t003-3}
All cases Unimmunised Partially immunised
----------------------------------------------- ------------ ------------- ------------------------------------------- ------------ ----------- --------------------------------------- ----------- ----------- -------------------------------------------
*n* *281--290* *65--70* *152--182* *27--31* *98--110* *27--41*
MMR beliefs 2.8 (0.7) 3.1 (0.7) 0.04[\*\*\*](#nt106){ref-type="table-fn"} 2.7 (0.7) 3.0 (0.6) 0.03[\*](#nt104){ref-type="table-fn"} 2.9 (0.7) 3.2 (0.7) 0.04[\*](#nt104){ref-type="table-fn"}
Measles beliefs 3.8 (0.7) 4.0 (0.6) 0.01[\*](#nt104){ref-type="table-fn"} 3.8 (0.7) 3.9 (0.6) 0.01 3.8 (0.6) 4.0 (0.6) 0.01
Social and parenting beliefs 3.2 (1.0) 3.7 (0.8) 0.07[\*\*\*](#nt106){ref-type="table-fn"} 3.1 (1.1) 3.4 (0.9) 0.02[\*](#nt104){ref-type="table-fn"} 3.4 (0.9) 4.0 (0.6) 0.10[\*\*\*](#nt106){ref-type="table-fn"}
Information source beliefs 3.0 (0.7) 3.2 (0.7) 0.03[\*](#nt104){ref-type="table-fn"} 2.9 (0.7) 3.1 (0.8) 0.02[\*](#nt104){ref-type="table-fn"} 3.2 (0.6) 3.3 (0.6) 0.03[\*](#nt104){ref-type="table-fn"}
Practicalities 4.3 (0.8) 4.4 (0.7) 0.002 4.3 (0.8) 4.3 (0.8) 0.003 4.2 (0.8) 4.6 (0.6) 0.03[\*](#nt104){ref-type="table-fn"}
MMR catch-up invitation received in past year 77 (26) 30 (42) \- 54 (30) 15 (48) \- 23 (21) 15 (37) \-
P values and effect size (partial Eta squared) from ANCOVA, adjusted for child age and IMD2007 score.
\* = p\<.05,
\*\* = p\<.01,
\*\*\* = p\<.001.
Multivariate predictors of catch-up MMR uptake {#s3c}
----------------------------------------------
See [Table 4](#pone-0019381-t004){ref-type="table"}. In the sample as a whole, catch-up MMR uptake was predicted by perceived social desirability/benefit of MMR uptake (OR = 1.76, 95% CI = 1.09--2.87) and younger child age (OR = 0.78, 95% CI = 0.68--0.89). However, the profile of multivariate predictors differed substantially between parents of previously unimmunised children and parents of previously partially immunised children. In the former, catch-up MMR uptake was predicted only by receipt of catch-up invitation (OR = 3.45, 95% CI = 1.18--10.05), younger parent age (OR = 0.58, 95% CI = 0.36--0.92), and residence in a less deprived postcode (OR = 0.96, 95% CI = 0.92--0.99). In the latter, catch-up MMR uptake was predicted only by perceived social desirability/benefit of MMR uptake (OR = 9.61, 95% CI = 2.57--35.97), lower parent educational attainment (OR = 0.08, 95% CI = 0.01--0.58), and younger child age (OR = 0.44, 95% CI = 0.29--0.66).
10.1371/journal.pone.0019381.t004
###### Independent predictors of catch-up MMR uptake.
{#pone-0019381-t004-4}
Predictor Odds ratios (95% CIs)
------------------------------ ----------------------- -------------------- --------------------
*n* *284* *174* *110*
*Nagelkerke R^2^* *0.27* *0.28* *0.61*
Child age 0.78 (0.68--0.89) 0.93 (0.79--1.10) 0.44 (0.29--0.66)
IMD2007 score 0.99 (0.96--1.01) 0.96 (0.92--0.99) 0.98 (0.94--1.02)
Parent age 0.79 (0.57--1.10) 0.58 (0.36--0.92) 1.41 (0.63--3.15)
Parent BME ethnicity 0.87 (0.38--1.99) 1.12 (0.33--3.77) 1.31 (0.23--7.33)
Parent married 0.51 (0.25--1.07) 0.41 (0.14--1.16) 0.20 (0.04--1.10)
Parent education≥degree 0.96 (0.43--2.17) 3.21 (0.98--10.47) 0.08 (0.01--0.58)
Number of children 0.98 (0.67--1.44) 1.35 (0.80--2.27) 0.58 (0.29--1.19)
Parent occupation 0.97 (0.83--1.12) 1.14 (0.92--1.41) 0.74 (0.53--1.05)
Catch-up invitation received 1.72 (0.83--3.57) 3.45 (1.18--10.05) 2.00 (0.44--9.09)
MMR beliefs 1.22 (0.64--2.31) 1.61 (0.63--4.11) 0.35 (0.09--1.36)
Measles beliefs 1.01 (0.55--1.86) 1.71 (0.74--3.97) 0.24 (0.05--1.06)
Social and parenting beliefs 1.76 (1.09--2.87) 0.82 (0.41--1.64) 9.61 (2.57--35.97)
Information source beliefs 1.18 (0.59--2.34) 1.34 (0.49--3.67) 5.12 (0.95--27.52)
Practicalities 0.94 (0.57--1.55) 0.76 (0.39--1.49) 1.93 (0.60--6.26)
Discussion {#s4}
==========
Summary of current findings and relation to previous work {#s4a}
---------------------------------------------------------
Perceiving MMR uptake to be socially desirable/beneficial, and having a younger child were the only independent predictors of MMR uptake during the catch-up campaign. Though univariate analyses indicated that catch-up MMR acceptors differed from catch-up MMR decliners also on MMR beliefs, measles beliefs, and information source beliefs, these factors were not independently responsible for variability in uptake behaviour. Independent predictors of catch-up MMR uptake differed by whether the dose in question was the first the child was to receive or the second. Acceptance of a first dose was primarily predicted by receipt of a catch-up invitation, and no attitudinal factors were implicated in this behaviour. Acceptance of a second dose was predicted most strongly by perceived social consequences of MMR immunisation, and invitation receipt had no bearing on this behaviour.
Whilst to our knowledge attitudinal and demographic predictors of MMR uptake during catch-up campaigns have not previously been modelled in multivariate analyses, the present findings may be usefully compared with the few relevant models predicting routine MMR uptake. Perceived social desirability/benefit of MMR uptake, a key predictor in this work, was unrelated to PCT-recorded routine MMR uptake in 1999--2000 [@pone.0019381-Flynn1], however perceived importance of eradicating rubella (similar to value placed on social benefit of MMR uptake) was a significant predictor of parent-reported MMR uptake in 2003--2004 [@pone.0019381-Gellatly1]. Other key predictors in these studies were previous immunisation behaviour, trust in information sources, and belief in MMR side effects, and whilst these factors were related to catch-up MMR uptake in our univariate analyses, their independent impacts on catch-up behaviour were not significant. These differences may reflect evolving views on MMR in society as the MMR controversy abates, or the different ages of children whose parents participated in the routine uptake studies versus our catch-up study. Our univariate findings generally correlate with results from relevant studies of routine MMR uptake [@pone.0019381-Cassell1], [@pone.0019381-Casiday1], with some interesting differences, again perhaps a function of study period or population. For example, in the present work most parents anticipated regret [@pone.0019381-Sevdalis1] as a consequence both of MMR reaction and of measles infection, and the extent of this regret did not vary by catch-up MMR uptake, however in 2004 [@pone.0019381-Cassell1] routine MMR rejectors were more likely than MMR acceptors to anticipate regret for MMR reaction, and vice-versa. In the same 2004 study [@pone.0019381-Cassell1], benefitting the community by immunising one\'s own child was one of the few factors on which routine MMR acceptors and rejectors did not differ, whilst in the present work this was one of the most polarising issues. In the only post-MMR controversy assessment of attitudinal factors underpinning catch-up MMR uptake (during the London 2004/5 primary school campaign) [@pone.0019381-Capital1], MMR safety concerns (particularly autism) were the most frequently cited reasons for catch-up MMR rejection. In our multivariate analyses, however, these factors did not figure, again perhaps a function of time elapsing since the controversy [@pone.0019381-Elliman1], [@pone.0019381-Smith1] and parents of older children being questioned.
Implications for policy, practice and further research {#s4b}
------------------------------------------------------
There are at least three possible explanations for the finding that attitudes, particularly those about the social aspects of MMR immunisation, were more predictive of uptake among parents who were to give a second dose of MMR than they were among parents who were to give a first dose: parents deciding about a second dose (a) had chosen not to give that second dose previously but the catch-up campaign changed their minds; (b) had always held 'pro-MMR' beliefs but had simply forgotten to obtain that second dose and the campaign reminded them; or (c) were more able to consider 'peripheral' factors like social benefits and norms since they were reassured about MMR risks following their child\'s earlier receipt of an MMR dose. These explanations require further investigation, perhaps most effectively with a qualitative methodology, but they offer some useful directions for future catch-up programmes or interventions within the routine schedule. This work also indicates that the attitudinal and demographic profile of parents who immunise during a catch-up campaign is different to that of parents who immunise within the routine schedule: key predictors of routine MMR receipt in this population are being of black/minority ethnicity and having positive MMR beliefs [@pone.0019381-Brown2], but those factors did not figure in the prediction of catch-up MMR receipt. Catch-up campaigns may therefore require different information materials, health professional approaches, and population targeting than do routine campaigns. Finally, the work demonstrates a clear relationship between younger child age and catch-up MMR receipt in the context of this PCT-based programme. School-based approaches may be more effective in reaching older children [@pone.0019381-Lashkari1].
It seems viable and desirable on the basis of the present findings to roll out the measurement instrument with a modified administration method in advance of the next catch-up campaign. This would allow collection of baseline attitudinal data, which can then be compared to post-campaign attitudes aiming to ascertain campaign efficacy in improving attitudes and beliefs. This strategy may be implemented over a large number of PCTs in a nationwide catch-up programme, or over individual PCTs running local programmes; the data can then be combined using meta-analytic techniques to obtain a comprehensive and reliable picture of predictors of MMR receipt during catch-up initiatives, thus contributing directly to rendering such campaigns more amenable to formal evaluation.
Strengths and limitations {#s4c}
-------------------------
This study is one of only a handful to explore factors underpinning catch-up MMR uptake [@pone.0019381-Roberts1], [@pone.0019381-Hadjikoumi1]. Despite the persistent disappointing performance [@pone.0019381-Capital1]--[@pone.0019381-Lashkari1] of catch-up immunisation campaigns, evaluation to date has been sparse and methodologically limited. The present work used a psychometrically robust, evidence-based instrument [@pone.0019381-Brown2] to assess a broad spectrum of predictors of MMR uptake, with a demographically diverse sample of catch-up MMR acceptors and rejectors, and an objective outcome measure. These methodological strengths are uncommon even in the much larger literature on routine schedule MMR decision-making [@pone.0019381-Brown1]. Importantly, these methodological advances allowed univariate and multivariate analyses which are, to our knowledge, unique contributions to the catch-up MMR uptake prediction knowledge base. Further, the work demonstrates the viability of evaluating future catch-up campaigns with the instrument used here. However, the work is not without limitations. Though the analysis was adequately powered for statistical comparisons between those who did and did not accept catch-up MMR within the sample, the modest response rate may have compromised the generalisability of the sample to the wider population from which it was drawn. The response rate was lower than has been obtained previously in catch-up and routine MMR populations [@pone.0019381-Roberts1], [@pone.0019381-Hadjikoumi1], [@pone.0019381-Cassell1], [@pone.0019381-Casiday1] -- likely due in part to poor PCT data quality [@pone.0019381-The1] inflating the denominator in our participation rate calculations (previous catch-up studies obtained more reliable denominators by sampling through schools or from subpopulations of parents who had already responded to an immunisation consent request) -- however our study is comparable to those with higher response rates with regard to ratio of MMR-acceptors to MMR-rejectors, and sample demographics, thus the differences we note above between predictors of catch-up and routine MMR uptake are unlikely to be explained by response rate or respondent characteristics alone. Though efforts were made to facilitate participation among hard-to-reach groups [@pone.0019381-Dormandy1], [@pone.0019381-Edwards1], and the sample was reasonably varied in educational attainment, occupation and ethnicity, those deprived, low literacy, non English-speaking populations who fail both to respond to questionnaires about immunisation and to attend for immunisation are perhaps not as well-represented here as their wealthier, more literate counterparts [@pone.0019381-Pearce1], [@pone.0019381-Brown1], [@pone.0019381-Falagas1]. We chose to assess the relationship between MMR invitation and MMR uptake via parent report of receipt rather than PCT/GP record of sending, because we sought to assess the impact of the invitation on the recipient, not the quality or success of PCT/GP efforts to send the invitation out; however, parent report is open to recall bias (parents may have received their invitation 6--12 months before they completed our questionnaire, and simply forgot about the invitation in this period) and experimenter bias (parents may have denied receiving an invitation in order to justify not obtaining MMR for their child). Some evidence suggests that receipt of immunisation invitation letters will be forgotten or denied by around 50% of parents [@pone.0019381-Lieu1], therefore our data may underestimate the number of parents who received invitations and thus overestimate the effect of invitation receipt on MMR uptake. However, to the extent that an invitation is only as useful as it is memorable or noticeable, novel invitation formats (for example, a personalised 'birthday card' to be displayed rather than a standard letter to be read and discarded) may have more of an effect on uptake [@pone.0019381-The1]. Finally, the cross-sectional design of the study means it is not possible to ascertain causality: we cannot infer whether positive attitudes and MMR invitation receipt caused catch-up MMR uptake, or whether catch-up uptake created more positive attitudes and heightened parents\' awareness of/memory for having received an invitation.
Conclusion {#s4d}
----------
Receipt of a first-ever MMR dose during the catch-up period was predicted most strongly by receipt of an invitation letter from the GP/PCT, whilst receipt of a second dose during the campaign was predicted most strongly by appreciation of the social benefits (for oneself and for the community) of accepting MMR. Future local and national catch-up programmes should be designed with these differential motivations in mind, and can be robustly evaluated using the attitude assessment tool employed here.
Supporting Information {#s5}
======================
######
Individual attitudes items by catch-up MMR uptake.
(DOC)
######
Click here for additional data file.
All authors had full access to all of the data (including statistical reports and tables) in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**The research reported here was funded by the UK Health Protection Agency (<http://www.hpa.org.uk/>) and Commissioning Support for London (<http://www.csl.nhs.uk/Pages/default.aspx>). Brown, Sevdalis, Shanley, Cowley and Vincent are affiliated with the Imperial College Centre for Patient Safety and Service Quality, which is funded by the National Institute for Health Research (<http://www.nihr.ac.uk/Pages/default.aspx>). The researchers are independent from the funders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: KB GF MR NS. Performed the experiments: KB RS NC. Analyzed the data: KB GF MR NS. Wrote the paper: KB GF MR NS. Contributed revisions to the methodology and manuscript: MH JG JSK CV. Negotiated access to and retrieved patient data: JvW PT MF.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1}
============
Congenital nephrotic syndrome is a disorder characterized by severe proteinuria, hypoalbuminemia, and anasarca presenting during the first 3 months of life. The disease is typically caused by genetic defects that disrupt the integrity of the glomerular filtration barrier, with mutations in the podocyte genes *NPHS1*, *NPHS2*, *WT1*, and *LAMB2* contributing to approximately 85% of cases of congenital nephrotic syndrome.[@bib1]
Recently, recessive mutations in the gene *sphingosine-1-phosphate lyase* (*SGPL1*) have been described in 16 families. Patients present with a syndromic form of congenital, steroid-resistant nephrotic syndrome characterized by a variable phenotype including adrenal insufficiency, neuronal dysfunction, muscular hypotonia, immunodeficiency, hypothyroidism, thrombocytopenia, and skin, skeletal, and genital abnormalities.[@bib2], [@bib3], [@bib4], [@bib5], [@bib6]
Sphingosine-1-phosphate (S1P) is a ubiquitously expressed sphingolipid molecule contributing to multiple cellular responses, including cell proliferation and survival, differentiation, migration, and immune response modulation.[@bib7] SGPL1 irreversibly degrades S1P, thus playing a key role in the regulation of this signaling molecule ([Figure 1](#fig1){ref-type="fig"}).[@bib8] Mutations in *SGPL1* lead to decreased cell survival through accumulation of S1P.[@bib9] Here, we describe the clinical course, renal histology, and lipidomic profile of a male infant with congenital nephrotic syndrome with immunodeficiency and neurodegeneration found to have novel *SGPL1* mutations.Figure 1Sphingolipid metabolism. Sphingosine-1-phosphate lyase (SGPL1) catalyzes the final step of the sphingolipid pathway by irreversibly converting sphingosine-1-phophate (S1P) to its by-products.
Case Presentation {#sec2}
=================
The patient was born as the second child of healthy, nonconsanguineous white parents after 39+3 weeks of pregnancy by spontaneous delivery (birth weight 3370 g). Prenatal ultrasound showed polyhydramnios and adrenal calcifications. He presented at 6 weeks of life with diffuse edema and was diagnosed with nephrotic syndrome. Infectious workup (cytomegalovirus, HIV, syphilis, and toxoplasmosis) was negative. He was managed at home on oral diuretics and was admitted twice for fluid management. Between 3 and 4 months of age he had rapid deterioration of kidney function with creatinine increasing from 0.32 mg/dl to 2.5 mg/dl and blood urea nitrogen increasing from 21 mg/dl to 80 mg/dl. In addition, he developed significant nephromegaly (right kidney 8.5 cm, left kidney 8.9 cm) leading to abdominal distention and feeding intolerance. The decision was made to perform bilateral nephrectomies and initiate peritoneal dialysis at 4 months of life, which remained the primary mode of renal replacement therapy.
At birth, he was found to have borderline micropenis, inguinal testes, and hypoplastic scrotum. Initial evaluation for endocrinologic abnormalities revealed normal serum electrolytes, fasting glucose, and adrenal steroids (sodium level 140 mmol/l, potassium level 5.2 mmol/l, testosterone-to-dihydrotestosterone ratio 1.1, 17-hydroxyprogestone level 30 ng/dl, and dehydroepiandrosterone level 47 ng/dl). A workup for hypogonadism revealed abnormally elevated gonadotropins (follicle-stimulating hormone 41 mIU/ml, luteinizing hormone 23 mIU/ml) and low serum testosterone (3 ng/dl). Postnatal imaging continued to show concern for calcification and enlargement of the adrenal glands. Low-dose adrenocorticotropic hormone stimulation test at 2 weeks of life was normal. Adrenocorticotropic hormone stimulation test was repeated at 14 months of age and confirmed adrenal insufficiency (baseline cortisol level 12.5 μg/dl and peak level 12.7 μg/dl). Stress dose hydrocortisone was given during illnesses and surgical procedures, but he did not require physiologic steroid administration. Additional endocrine issues included primary hypothyroidism (thyroid-stimulating hormone 13.3 mIU/l, free T4 1.4 ng/dl), for which he was started on levothyroxine at 1 month of life, and suboptimal growth with low-normal growth hormone level (4.7 ng/ml), low insulin-like growth factor--1 and insulin-like growth factor--binding protein 3 (15.7 ng/ml and 1011 ng/ml, respectively). Given his chronic kidney disease and poor growth velocity, he was started on growth hormone at 11 months of life. Dermatologic abnormalities included hyperpigmentation and hyperkeratosis.
He was found to have immunodeficiency consisting of persistent neutropenia (absolute neutrophil count \<500/mm^3^), lymphopenia (absolute lymphocyte count \<200/mm^3^) and hypogammaglobulinemia (IgA \<6 mg/dl, IgG \<120 mg/dl, IgM \<35 mg/dl). Inflammatory markers and cytokines, including C-reactive protein, procalcitonin, and interleukin-6, were elevated. Bone marrow aspirate and flow cytometry showed B-cell progenitor hyperplasia. He was treated with filgrastim and i.v. Ig infusions. The patient was admitted multiple times for presumed infections, including sepsis, osteomyelitis, and meningitis, all of which were culture negative.
Neurologically, he was found to have sensorineural hearing loss shortly after birth. Between 12 and 14 months of age, he experienced rapid loss of developmental milestones accompanied by seizures and choreiform movements requiring treatment with antiepileptics and benzodiazepines. Magnetic resonance imaging of the brain, which was initially normal at 2 months of life, showed abnormal T2 hyperintense signal and calcifications in the basal ganglia with abnormal signal extending into the surrounding white matter. Steroids were initiated but imaging continued to show worsening of brainstem edema.
Genetic and Pathologic Findings {#sec2.1}
-------------------------------
### Renal Pathology {#sec2.1.1}
Light microscopy showed dysplastic-appearing kidney tissue with abnormally developed glomeruli associated with progressive degenerative changes, including globally sclerotic glomeruli, pseudo-crescents, tubular atrophy, and interstitial fibrosis. Light microscopic findings were consistent with diffuse mesangial sclerosis characterized by increased mesangial matrix and mesangial cell expansion.
The electron microscopy showed proliferative lesions with abnormal podocytes and thin basement membranes. There was no evidence of immune complex--mediated glomerulonephritis.
Cryosections of kidney tissue were obtained from the patient and age/gender-matched control. Antibody staining was performed using goat anti-SGPL1 (R&D Systems, Minneapolis, MN), mouse anti-WT-1 (Santa Cruz Biotechnology, Dallas, TX), Ki-67 (Invitrogen, Carlsbad, CA), and 4′,6-diamidino-2-phenylindole nuclear stain. Imaging was obtained using a Nikon (Tokyo, Japan) A1 confocal microscope. Immunofluorescence intensity was measured using intensity profile on NIS Elements software version 5.20.00. Average intensity in the 561 nm was measured and a ratio of signal comparing patient with control was determined. Immunofluorescence of the patient's renal tissue showed an absence of SGPL1 staining and a 3-fold increase in S1P signal intensity in tubules compared with an age- and gender-matched control ([Figure 2](#fig2){ref-type="fig"}). Immunofluorescence staining by LC3A/B antibody was used to capture the degree of autophagy, and demonstrated reduced autophagy in the patient's renal tissue. In addition, patient kidney sections revealed increased Ki-67--positive cells, particularly in the glomeruli, suggesting increased proliferation.
### Exome Sequencing {#sec2.1.2}
Based on high suspicion for SGPL1 deficiency, targeted gene sequencing was performed at Cincinnati Children's Hospital Medical Center. DNA was isolated from peripheral blood. Polymerase chain reaction and bidirectional sequencing were used to analyze the entire coding region and exon/intron boundaries of the gene *SGPL1*. Targeted sequencing revealed 2 novel variants consisting of a paternally inherited c.868T\>C (p.Phe290Leu) and a maternally inherited c.993C\>G (p.Tyr331\*) variant. The first variant (c.868T\>C) was not found in any publicly available databases. Multiple *in silico* prediction tools (AlignGVGD, SIFT, MutationTaster, Polyphen-2, Grantham Distance, BLOSUM45, BLOSUM62, BLOSUM80) were used to determine pathogenicity. Based on all available information, the variant was classified by our laboratory as likely pathogenic. The latter mutation (c.993\>G) results in a premature stop codon in exon 11 of 15, which is thought to result in nonsense-mediated mRNA decay. This variant is not listed in the Human Gene Mutation Database or ClinVar. In gnomAD, it has a reported minor allele frequency of 0.00090% in the non-Finnish European population. Based on the available information, this variant was classified as pathogenic. As a result of these findings, we concluded that these variants were likely disease-causing in our patient.
Lipidomic Analysis {#sec2.2}
------------------
Serum and kidney tissue was obtained from our patient (nephrectomized specimen) and an age/gender-matched control. Sample preparation and lipid extraction were performed as previously described by Folch *et al*.[S1](#appsec1){ref-type="sec"} Following tissue homogenization and lipid extraction, highly sensitive ultraperformance liquid chromatography coupled to tandem mass spectrometry was used to quantify different sphingolipid classes in each sample. Given its hydrophilic nature, levels of S1P were unable to be assessed via lipidomic analysis. Analysis revealed elevated levels of SGPL1 precursor substrates sphingosine, ceramide, and glucosylceramide in the patient's kidney tissue ([Figure 3](#fig3){ref-type="fig"}a--c) and serum ([Figure 4](#fig4){ref-type="fig"}a--c) compared with control samples. Sphingomyelin levels were elevated in the patient's serum ([Figure 4](#fig4){ref-type="fig"}d), however, were decreased in kidney tissue ([Figure 3](#fig3){ref-type="fig"}d).Figure 2Immunofluorescence. Immunofluorescent microscopy of kidney tissue of a patient with sphingosine-1-phosphate lyase (SGPL1) deficiency showed absent SGPL1 staining, increased sphingosine-1-phosphate (S1P) staining, decreased autophagy (1A/1B-light chain 3 \[LC3 A/B\]), and increased proliferation (Ki-67 protein \[Ki67\]) compared with control tissue. Original magnification ×20. DAPI, 4′,6-diamidino-2-phenylindole; WT1, Wilms tumor 1.Figure 3Kidney tissue sphingolipids. Levels (a) sphingosine, (b) ceramide (Cer), (c) glucosylceramide (GlcCer), and (d) sphinomyelin (SM) in a patient with sphingosine-1-phosphate lyase deficiency compared with control tissue. Cer and GlcCer levels were increased in patient kidney tissue, whereas SM levels were elevated in normal kidney tissue.Figure 4Serum sphingolipids. Levels of (a) sphingosine, (b) ceramide (Cer), (c) glucosylceramide (GlcCer), and (d) sphinomyelin (SM) were elevated in patient serum with sphingosine-1-phosphate lyase deficiency compared with control.
Follow-up {#sec2.3}
---------
The patient was referred for transplantation; however, evaluation was put on hold to evaluate the etiology of the patient's neurodegeneration. The patient was diagnosed with SGPL1 deficiency at 12 months of age based on his clinical features and genetic testing results. In an effort to slow down the progression of the disease, he was started on soy formula containing phosphatidylethanolamine, an end product of SGPL1 metabolism. In an effort to stabilize the SGPL1 protein, he was started on high-dose pyridoxal phosphate, and later on i.v. pyridoxine when he developed feeding intolerance and concern for poor enteral absorption. Despite these efforts, his neurological status continued to worsen, and he died at 17 months of age secondary to cerebral edema.
Discussion {#sec3}
==========
In this study, we report a patient found to have 2 novel heterozygous variants in *SGPL1.* Including this study, 16 different variants of *SGPL1* in 16 families have been reported.[@bib2], [@bib3], [@bib4], [@bib5], [@bib6] Our patient with congenital nephrotic syndrome symptoms presented with similar findings to these reported cases, including adrenal insufficiency, neurologic abnormalities, muscular hypotonia, skin and genital abnormalities, immunodeficiency, and hypothyroidism. In addition, the patient presented here had rapid enlargement of his kidneys, significant decline in kidney function by 4 months of age, and neurologic deterioration starting at 12 months of age.
Defects of sphingolipid metabolism are known to result in renal disease through the accumulation of sphingolipids. In Fabry disease, accumulation of globotriaosylceramide leads to progressive podocyte injury.[S2](#appsec1){ref-type="sec"} *SGPL1* encodes for SGPL1, an enzyme that acts as a gatekeeper of sphingolipid metabolism by executing the only exit point for sphingolipid intermediates by converting the substrate S1P irreversibly into ethanolamine phosphate and hexadecenal ([Figure 1](#fig1){ref-type="fig"}).[@bib9] S1P, acting through plasma membrane G-protein--coupled receptors or directly on intracellular targets, commands a complex network of cellular responses including cell proliferation, survival, differentiation, and migration.[@bib7]
As previously identified, mutations in *SGPL1* have been shown to result in reduced or absent SGPL1 protein, enzymatic activity, or protein mislocalization. It has been hypothesized that disease may result from excess intracellular S1P or S1P receptor signaling, an imbalance of sphingoid bases, or lack of S1P by-products.[@bib2], [@bib3], [@bib4], [@bib5] We suspect our patient had a completely nonfunctional SGPL1 enzyme, given his genetic mutation, which resulted in a premature stop codon. Further supporting this theory, lipidomic analysis showed significantly elevated upstream products, and immunofluorescence showed absence of renal SGPL1 staining. The heterogeneity and severity of symptoms in reported subjects may reflect differing amounts of functional SGPL1 protein, with our case presenting with a more severe phenotype in the absence of a functional SGPL1 protein.
Prior research has shown that serum and fibroblasts isolated from patients with SGPL1 deficiency have increased levels of SGPL1 substrates in comparison with controls.[@bib2], [@bib3], [@bib4] SGPL1 has been shown to be localized to the endoplasmic reticulum of renal podocytes and mesangial and endothelial cells.[@bib2] Accumulation of S1P, a substrate for SGPL1, induces mesangial cell proliferation via activation of endothelial differentiation gene family of G-coupled receptors.[S3](#appsec1){ref-type="sec"} We propose that the complete absence of *SGPL1* expression coupled with increased S1P levels in renal tissue of our patient explains the extensive mesangial cell proliferation and nephromegaly. It has been shown that the balance between different sphingolipid levels, specifically S1P and ceramide, can regulate renal tubular apoptosis.[S4](#appsec1){ref-type="sec"} In our patient, levels of proteins upstream of SGPL1, including sphingosine, ceramide, and glucosylceramide, were increased in the kidney tissue. Increased levels of ceramides cause cells to exhibit proliferation arrest, apoptosis, and autophagy, and glycosylation of ceramides arrest these processes and has been shown to be responsible for resistance to cancer treatment in tumor cells.[S5](#appsec1){ref-type="sec"} We propose that increased glucosylceramide levels in patient kidney tissue limit apoptosis and tilt the balance toward cellular proliferation in mesangial cells. Increased levels of glucosylceramide are also seen in the lysosomal storage disease Gaucher disease, in which accumulation of this sphingolipid leads to cytopenia, splenomegaly, hepatomegaly, bone lesions, and neurological impairment.[S6](#appsec1){ref-type="sec"}
In response to cellular growth cues, autophagy manifests itself as a cellular mechanism to limit cell proliferation and promote cell survival. In stress cell signaling response through the endoplasmic reticulum causes increase in misfolded proteins. Mouse models have demonstrated that inhibition of sphingosine kinase-1, which converts sphingosine to S1P, leads to renal fibrosis through decreased autophagy in renal tubular epithelial cells.[S7](#appsec1){ref-type="sec"} Phosphatidylethanolamine, an end product of SGPL1 activity, modulates neuronal autophagy.[S8](#appsec1){ref-type="sec"} We speculate that decreased autophagy due to decreased phosphatidylethanolamine levels in the presence of proliferation inhibits elimination of misfolded proteins resulting in increased cellular damage and fibrosis in SGPL-1 deficiency. Further research is needed to investigate this. It also has been hypothesized that S1P buildup may contribute to renal fibrosis through its upregulation of cyclooxygenase-2 expression and subsequent prostaglandin E2 formation in renal mesangial cells.[S9](#appsec1){ref-type="sec"}
In addition, lipidomic analysis of patient kidney tissue revealed decreased sphingomyelin levels, whereas serum analysis showed increased sphingomyelin levels compared with control sample. This may represent a difference in the metabolism of ceramide to sphingomyelin in different tissue types and release into the circulation; however, further research is needed. Accumulation of S1P in SGPL1-deficient neurons has been shown to induce apoptosis indicating organ-specific effect of S1P on cell cycle.[S10](#appsec1){ref-type="sec"} Approximately half of the reported patients surviving for at least 1 month exhibit pathological neurological features ranging from mild neurodevelopmental delay, sensorineural hearing loss, and peripheral nerve paralysis to severe encephalopathic neurodegenerative disease.[@bib2], [@bib3], [@bib4], [@bib5], [@bib6] Our patient had rapid neurological decline with MRI abnormalities first presenting in the basal ganglia, which may have been secondary to accumulation of S1P and other sphingolipids. The importance of sphingolipid metabolism in neuronal function also has been demonstrated by researchers identifying an atypical form of Charcot-Marie-Tooth neuropathy caused by compound heterozygous mutations in *SGPL1*.[S11](#appsec1){ref-type="sec"} In addition, the accumulation of sphingolipid metabolites has been shown to cause neurodegenerative disease in other sphingolipidoses, including Gaucher, Fabry disease, and Niemann-Pick disease.[S6](#appsec1){ref-type="sec"}
Our patient also had immunodeficiency characterized by neutropenia, lymphopenia, and hypogammaglobinemia. SGPL1-deficient mice exhibit a heightened inflammatory response and have impaired neutrophil migration into inflamed tissue.[S12](#appsec1){ref-type="sec"} They also have lymphopenia from impaired egress from primary and secondary lymphoid organs, and enhanced apoptosis of developing T cells from accumulation of ceramides.[S13](#appsec1){ref-type="sec"} Our patient was hospitalized several times for culture-negative infection. Given his elevated cytokines and inflammatory markers, it was questioned whether these episodes represented true infections or the byproduct of excess inflammation. As patients with SGPL1 deficiency can have immunodeficiency, providers must be vigilant for signs of infection and also be judicious about antimicrobial use.
Patients with SGPL1 deficiency also demonstrate adrenal abnormalities, including insufficiency, calcifications, and enlargement.[@bib2], [@bib3], [@bib4], [@bib5], [@bib6] Recently, others have identified patients with primary adrenal insufficiency secondary to missense mutations in *SGPL1* without other clinical manifestations.[S14](#appsec1){ref-type="sec"} It has been suggested the adrenal issues seen in this disease may arise from disrupted adrenocortical zonation and the high presence of SGPL1 in the zona reticularis, which is responsible for adrenal androgen secretion.[@bib2]
Currently, there is no curative treatment for SGPL1 deficiency. It has been suggested that phosphatidylethanolamine supplementation may serve a therapeutic role in treatment of the neurodegenerative disease via modulation of neuronal atrophy.[S8](#appsec1){ref-type="sec"} Given the patient's rapid neurological decline, a trial of soy formula containing high amounts of phosphatidylethanolamine was initiated.[S15](#appsec1){ref-type="sec"} In addition, pyridoxal phosphate and pyridoxine were given in an effort to stabilize any SGPL1 protein present as SGPL1 is a pyridoxal 5ʹ-phosphate-dependent enzyme.[@bib9] These treatments may have been more beneficial if the patient had some degree of enzymatic activity or if initiated earlier. Studies have shown that bacterial SGPL1 delivery reduces circulating S1P levels by 70% in mice, and therefore enzyme replacement therapy may be a potential intervention in the future.[S16](#appsec1){ref-type="sec"}
This is the first study to examine renal sphingolipid metabolites using lipidomic analysis and immunofluorescence in a patient with SGPL1 deficiency. We showed sphingolipids upstream to the SGPL1 enzyme, including ceramide and glucosylceramide, were elevated in a patient with SGPL1 deficiency. However, we were unable to examine levels of S1P and SGPL1 by-products by lipidomic analysis. In addition, to our knowledge, we are the first to trial treatments as a way to slow disease progression. Evaluation of treatment response was limited as we were unable to assess enzymatic activity along with phosphatidylethanolamine and S1P levels before and after treatment. Further research is needed in these areas to improve patient outcomes.
Based on our patient's presentation and clinical course, we recommend that genetic testing directed to *SGPL1* mutations be considered in patients with congenital and steroid-resistant nephrotic syndrome presenting with extrarenal manifestations, particularly with immune and neurodevelopmental abnormalities. Although there is no treatment for SGPL1 deficiency, early diagnosis can allow early identification of other comorbidities of the disease and enable clinicians to counsel patients and families appropriately ([Table 1](#tbl1){ref-type="table"}).Table 1Teaching points1. Deficiency of the enzyme SGPL1 leads to increased accumulation of sphingolipids.2. Renal cells deficient in this enzyme show decreased autophagy and increased proliferation.3. Gene sequencing for mutations in *SGPL1* is recommended for patients with nephrotic syndrome presenting with extrarenal manifestations, immunodeficiency, and endocrinological and neurodevelopmental abnormalities.4. Early diagnosis of genetic causes of nephrotic syndrome can allow for appropriate counseling.[^1]
Disclosure {#sec4}
==========
All the authors declared no competing interests.
Supplementary Material {#appsec1}
======================
Supplementary File (Word)
This study was supported by the National Institutes of Health Research Training in Pediatric Nephrology grant 5T32DK007695--20.
[Supplementary File (Word)](10.1016/j.ekir.2019.07.018){#intref0010}
**Supplementary References.**
[^1]: SGPL1, sphingosine-1-phosphate lyase.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
The role of laparoscopy for major abdominal surgery has been well established, especially in the field of gynecology.^[@B1],[@B2]^ Multiple studies have shown that laparoscopic surgery results in lower morbidity, better visualization, decreased blood loss, decreased postoperative pain, and faster recovery.^[@B3][@B4][@B5]^ As endoscopic surgeons continue to advocate for minimally invasive surgery, a logical next step is to improve efficiency by using ambulatory care settings. There has been tremendous growth in the use of ambulatory surgery centers in the United States, which is likely due to the increased efficiency and decreased cost.^[@B6][@B7][@B9]^ Specialized surgical teams are routinely employed in ambulatory surgery centers, improving teamwork, communication, and readiness for the unanticipated needs of the surgeon. These factors increase productivity, which likely contributes to the decreased cost. However, there are limitations to free-standing surgery centers, such as decreased reimbursement, which can be slightly offset by instruments choice, limited external resources in certain geographies, and decreased availability for immediate intraoperative consultation.^[@B10]^
Although it has been shown that major gynecologic laparoscopic surgery is safe in a hospital ambulatory surgery site, there is limited data to suggest that this same safety and efficiency is also true in freestanding ambulatory surgery centers.^[@B11][@B12][@B17]^ This study attempts to evaluate the safety and efficacy of major gynecologic laparoscopic surgery in the ambulatory care setting and discusses the fast-track protocols at our ambulatory surgery center.
MATERIALS AND METHODS
=====================
This study is a retrospective, multicenter cohort analysis of consecutive major abdominal surgeries performed at freestanding surgery centers by a primary surgeon. Institutional review board approval was obtained. Data was collected from chart review of 134 consecutive patients who had major laparoscopic surgery at 3 ambulatory surgery centers from August 1^st^ 2010 to September 30^th^ 2011. Major gynecologic surgery was defined as fertility sparing-treatment of stage IV endometriosis, myomectomy, or hysterectomy. No patients were excluded. Data was collected from 3 freestanding ambulatory surgery centers, and the same primary surgeon performed all surgeries. In California, ambulatory surgery centers are not permitted to keep postoperative patients beyond 23 hours and 59 minutes after surgery. Therefore, the main outcome measures were discharge by 23 hours and 59 minutes and unplanned postoperative admissions.
Our surgical protocols are as follows. After patients have been identified as appropriate surgical candidates, they are scheduled for surgery at either an affiliated hospital or an ambulatory surgery center based on their current state of health. Patients who are American Society of Anesthesia category 1 or 2, defined as, healthy or with only mild systemic disease, are eligible for surgery in one of the ambulatory surgery centers. Prior abdominal surgery, chronic pain, size of fibroids, or stage of endometriosis are not exclusion criterion for surgery at an ambulatory surgery center. The ambulatory surgery centers are equipped with 2 to 4 operating rooms and 3 to 12 beds for preoperative preparation and postoperative recovery. The nurses are staffed 2:1 during the day and night and work in 8-hour shifts. The surgery centers remain open 24 hours if a patient remains overnight; otherwise, they will close after the last patient is discharged. The most common indications for continued observation include postoperative pain and/or nausea. The surgery centers have access to bedside hematocrit and blood chemistry labs and a complete laboratory service that picks up samples from 8 [am]{.smallcaps} to 10 [pm]{.smallcaps} daily. Each surgery center is located within 5 to 15 minutes of a full-service hospital, in the event of an emergency.
Prior to surgery, each patient undergoes a 1-hour preoperative appointment to prepare the patient on the details of the surgery and postoperative care. This includes the routine minor bowel preparation, which includes high-calorie clear liquid diet and 1 or 2 enemas the night prior to surgery, common postoperative discomforts, and routine postoperative care. They are given prescriptions to fill prior to surgery for 200 μg vaginal misoprostol for cervical softening, Motrin 600 mg, oxycodone 5 mg, Colace 100 mg, and omeprazole 20 mg and ondansetron 4 mg. They are given a detailed information packet with all of the information clearly described for reference. This visit is ideally completed with both the patient and their postoperative caregiver (eg, partner, parent, friend) so both parties can hear the information and ask questions.
On the day of surgery, patients arrive at the surgical center 1.5 hours prior to their scheduled surgery times. Once in the operating room, they are positioned on a beanbag in dorsal lithotomy position using Allen stirrups with their arms adducted. Care is taken to ensure that there are no pressure points. Regarding surgical technique, a uterine manipulator and Foley catheter are placed in all cases. A closed entry technique with the Veress needle and concurrent carbon dioxide gas insufflation is used, as previously described.^[@B18]^ Following the establishment of pneumoperitoneum, a 12-mm camera port is placed at the umbilicus. Thereafter, 3 additional 5-mm ports are placed, in the right lower quadrant, the left lower quadrants, and suprapubically. With a large myomatous uterus, placement of accessory trocars may vary in order to optimize triangulation or visualization.
The treatment of endometriosis is carried out per previously described protocols for fertility-sparing treatment of endometriosis.^[@B2],[@B19]^ These techniques involve precise and directed excision and ablation of all endometriotic implants and restoration of normal anatomy. Bowel and bladder surgery in the treatment of endometriosis is completed as previously described.^[@B2],[@B20][@B21][@B22]^ The laparoscopic procedures for fertility-sparing endometriosis include chromopertubation, hysteroscopy, cystoscopy, and proctoscopy. The approach to myomectomies is based on fibroid location, size, and quantity. A laparoscopic-assisted myomectomy is performed to decrease blood loss, operating time and iatrogenic spread of potential sarcoma if the fibroid is \>9 cm and non-pedunculated, there are multiple myomas totaling ≥15 cm^2^ or there are \>35 cm non-pedunculated myomas.^[@B23][@B24][@B25]^ The laparoscope is used to identify anatomy, treat concurrent endometriosis, and remove the smaller fibroids. A 4-cm suprapubic mini-laparotomy incision is made to morcellate the fibroids, remove the larger fibroids, and close the uterine defects. In cases of pedunculated myomas, even very large myomas are done laparoscopically if an electric morcellator is available. If not available, a 4-cm mini-laparotomy is made to morcellate the fibroid as described.
Total laparoscopic hysterectomies are performed in the usual fashion and are accompanied by cystoscopy in all cases. Twelve of the hysterectomies in this series were completed as modified radical hysterectomies for endometriosis and/or adhesions that included extensive dissection of the paravesical, pararectal, rectovaginal, and vesicovaginal spaces; desiccation of the round ligament as close to the pelvic sidewall as possible; ureterolysis; and desiccation of the uterine vessels at the origin.
RESULTS
=======
One hundred and thirty-four women underwent major laparoscopic gynecologic surgery. There were 160 total surgical procedures: 77 conservative stage IV endometriosis treatment including 7 disk excisions of bowel endometriosis, 3 ureteroneocystostomies, and 1 partial bladder resection; 38 myomectomies; and 34 hysterectomies **([Table 1](#T1){ref-type="table"}).** In the 38 women who underwent myomectomy, the average weight of the leiomyomas was 240 g (range 5 g--3400 g). In the hysterectomy group, the average uterine weight was 283 g (range 46 g--950 g).
######
Surgical Procedures
Surgery Patients, *n* (%) N = 134 Unplanned Transfer---Immediate Admission After Surgery Unplanned Admission After Discharge
------------------------------------------------------------- --------------------------- -------------------------------------------------------- -------------------------------------
Treatment of stage IV endometriosis 77 (57) 0 1 (1.3)
With large bowel disk excision 7 (4.1) 0 0
With ureteroneocystostomy 3 (2.2) 0 0
With partial bladder resection 1 (0.7) 0 0
Laparoscopic myomectomy or laparoscopic assisted myomectomy 38 (28) 2 (5.3) 0
Laparoscopic hysterectomy 34 (25) 1 (2.9) 2 (5.9)
The overall unplanned admission rate was 4.5%. One hundred and thirty-one patients (97.7%) were discharged within the 23 hours of surgery. Three patients (2.2%) were transferred to hospital immediately after the operation. One laparoscopic hysterectomy patient and 1 laparoscopic myomectomy patient developed a fever greater than 38.3°C so were transferred to the hospital. They subsequently had a negative infectious workup and were discharged home with uncomplicated follow-up. One laparoscopic myomectomy patient developed acute anemia immediately after the operation and was transferred to the hospital for continued observation and subsequent blood transfusion. She was discharged home without further event and had an uncomplicated follow-up.
Of the 131 patients who were discharged within 23 hours, 3 patients (2.2%) were later admitted to hospital. One hysterectomy patient developed a postoperative ileus, which resolved with conservative management. One hysterectomy patient developed a fever greater than 38.3°C and subsequently had a negative infectious workup. One fertility-sparing treatment of endometriosis patient developed a fever and pain postoperatively, which had a negative infectious workup and was responsive to heparin. She was diagnosed with septic pelvic thrombophlebitis. Each of these patients were discharged home with uneventful follow-up ([Table 1](#T1){ref-type="table"}).
DISCUSSION
==========
This study suggests that advanced laparoscopic gynecologic surgery can be safely performed using a fast-track model in freestanding ambulatory surgery centers with a low unplanned admission rate, a low complication rate, and high discharge rate within 23 hours. As the foundation for laparoscopic surgery in gynecology continues to grow and becomes the standard of care, the skill of individual surgeons and available instrumentation increases. Many surgeons, however, do not yet feel comfortable performing major gynecologic surgery as an outpatient surgery and certainly not in a surgery center. This study suggests that with a skilled surgeon and a trained operating room and recovery team, ambulatory surgery centers are safe and effective for major gynecologic surgery. There are several factors that contribute to the success of ambulatory surgery center surgeries: careful preoperative planning, meticulous intraoperative technique, and easy access to additional resources if needed.
Proper patient selection of low-risk surgical candidates is an important initial step to ensure safety. This includes correction of baseline anemia with iron supplementation or hormonal treatment, such as gonadotropin-releasing hormone injections, and preoperative assessment of American Society of Anesthesia category. Finally, extensive preoperative counseling prepares patients and their caregivers for common events and discomforts postoperatively. This helps alleviate the anxiety of being discharged home, which decreases postoperative admission rates. Each patient is given their medications prior to surgery so there are no lapses without pain medication or antiemetics. Additionally, patients are given the physician\'s pager number so that they may speak to a physician 24 hours a day.
The two most common indications for continued observation and admission to the hospital include persistent pain and postoperative nausea, which were not noted to be contributing factors in this study. The surgical techniques of using meticulous dissection, assurance of hemostasis, removal of all intra-abdominal fluid, and removal of the carbon dioxide gas appear to increase patient comfort postoperatively. Patients routinely receive intravenous Toradol postoperatively, which helps to decrease the need of narcotic drugs. This low incidence of postoperative pain is particularly impressive as a large proportion of this patient population is diagnosed with chronic pelvic pain and routinely takes narcotic pain medication. Postoperative nausea is controlled with intravenous odansetron and metoclopramide while recovering, but each patient is also prescribed oral odansetron to take at home. This significantly decreases nausea with narcotic pain medication and decreases the anxiety of developing nausea once discharged.^[@B26]^
Finally, it is necessary to have an ambulatory surgery center that has easy access to additional resources in the event they are needed. The surgery centers in this study have the availability for an overnight registered nurse if the patient remains longer than the usual 4 to 6 hours postoperatively. There is the availability to have typed and crossed blood delivered in case of emergencies, and there are several hospitals within the near vicinity. However, as shown in this study, these additional resources are rarely needed. The estimated rate of unplanned admission following ambulatory surgery center was 4.5% in our study, which aligns with the previously published rates 1.1% to 5.4%.^[@B27][@B28][@B29]^
As noted, ambulatory surgery centers have multiple benefits including increased efficiency, teamwork, and productivity. There is a growing body of evidence that ambulatory surgery centers are also a safe and acceptable location in which to have major surgery. Taylor^[@B11]^ was part of the first team in the United States to extol the feasibility of laparoscopic hysterectomy in an outpatient setting. Subsequently, continued support has gathered for the use of ambulatory surgery centers for laparoscopic hysterectomy.^[@B12][@B13][@B15]^ Alperin et al ^[@B16]^ recently published their data that supported the use of the outpatient setting for laparoscopic hysterectomy in patients with large uteri, with low subsequent morbidity. Despite these advantages, many procedures, which could be performed in an ambulatory surgery center, are still performed at hospitals with the possibility of decreased efficiency and unnecessary overnight stays. To our knowledge, this is the first study that shows by using proper patient selection and preparation, following strict surgical techniques, and having available resources, more advanced surgical procedures, such as treatment of stage IV endometriosis, bowel surgery, ureteroneocystostomies, and bladder resections, can be performed in fast-track ambulatory surgery centers with high patient safety and efficiency. However, it must be recognized that a very experienced surgeon with a high-volume practice performed these surgeries.^[@B30]^ This is a limitation to the generalizability of this study, as these results might not be applicable to the general gynecologic surgeon. Nonetheless, with the continued growth of minimally invasive surgery and more specialized surgeons, the use of ambulatory surgery centers will likely continue to expand as further studies support the safety of major gynecologic surgery in freestanding ambulatory surgery centers.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-materials-10-01267}
===============
Wood polymer composites (WPCs) are used for building materials, furniture, packaging, etc., because of its dimensional stability, high strength, hydrophobic properties, and corrosion resistance \[[@B1-materials-10-01267],[@B2-materials-10-01267],[@B3-materials-10-01267]\]. Generally, the components of a WPC are preblended in a mixer, extruded by a single twin-screw extruder, and finally molded by an injection molder \[[@B4-materials-10-01267]\]. The components of a WPC should be dried at a certain temperature to reach a constant weight, before processing. However, the increased cost of plastic and the limited availability of wood fibers have restricted WPCs' development \[[@B5-materials-10-01267]\]. Wood-fiber composites filled with plastic could be obtained using a mechanical grind and a hot-press. In order to solve the incompatibility between the fibers and the polymers, amide compounds could be used to form linking bridges \[[@B6-materials-10-01267]\].
In order to develop the potential commercial application of WPCs, nanomodification and interfacial coupling could be implemented \[[@B7-materials-10-01267],[@B8-materials-10-01267],[@B9-materials-10-01267],[@B10-materials-10-01267]\]. Inorganic nanoparticles used in WPCs could enhance the functionalized commercial applications of WPCs. The special properties of ZnO nanoparticles (NPs) were investigated, including catalysis, optics, magnetism, mechanics, etc. \[[@B1-materials-10-01267],[@B11-materials-10-01267],[@B12-materials-10-01267],[@B13-materials-10-01267]\]. It was concluded that ZnO NPs could be used for UV shielding materials, antibacterial agents, fluorescent materials, photocatalytic materials, and microwave absorbing materials \[[@B14-materials-10-01267],[@B15-materials-10-01267],[@B16-materials-10-01267],[@B17-materials-10-01267],[@B18-materials-10-01267]\]. Patterson et al. prepared aramid fiber composites through ZnO NPs coating; this treatment increased the interfacial strength and improved the resistance to UV irradiation \[[@B13-materials-10-01267]\]. Wang et al. fabricated the reduced graphene oxide/ZnO composites through self-assembly and in-situ photo-reduction. The product exhibited photocatalytic activity in degrading Rhodamine B (RhB) \[[@B15-materials-10-01267]\]. Wang et al. synthesized flower-like, ZnO-coated Ni nanoparticles via an atomic layer deposition method. The optimal reflection loss was −48 dB at 10.4 GHz \[[@B19-materials-10-01267]\].
In this study, we describe the fabrication of a PE/ZnO/wood-fiber composite through a grinding process and a hot-press. ZnO NPs were deposited on the fibers' surface during the grinding process. The results indicated that the ZnO NPs not only enhanced the mechanical strength of the product, but also provided it with multifunctional applications. The PE/ZnO/wood-fiber composite showed microwave absorption properties and UV light photocatalytic activity. The effects of ZnO on the storage modulus and damping properties of the PE/ZnO/wood-fiber composite were investigated.
2. Results and Discussion {#sec2-materials-10-01267}
=========================
[Figure 1](#materials-10-01267-f001){ref-type="fig"} shows the macromorphology, SEM, and energy-dispersive X-ray spectroscopy (EDS) elemental mapping of all composite samples. The macromorphology, SEM image, and EDS mapping of the PZW0 composite (PE/wood fiber composite without ZnO) is shown in [Figure 1](#materials-10-01267-f001){ref-type="fig"}a. The color of PZW0 was similar to that of the fibers. In the middle SEM image of [Figure 1](#materials-10-01267-f001){ref-type="fig"}a, the fibers were stacked together, and the PE powder was attached to the fibers. At 5000× magnification, the PE and microfiber sheets were visible on the fibers' surface. EDS mapping detected the main element composition of PZW0; the main elements were C, O, and N. [Figure 1](#materials-10-01267-f001){ref-type="fig"}b displays the appearance and micromorphology of PZW1 (PE/ZnO/wood fiber composite with 2% ZnO). The color of PZW1 faded slightly compared to that of PZW0. This is because the ZnO NPs that were dispersed in the composite became milky-white, causing PZW1 to fade slightly. In the inset of the SEM image, PE and few ZnO NPs are visible on the surface of the fibers. The ZnO NPs were deposited on the fibers by hydrogen bonding, which formed between the hydroxyl groups of cellulose and the water bound on the surface of the ZnO NPs. The PE powders were dispersed onto the fibers' surface by flocculation and physical adsorption of the cationic polyacrylamide (CPAM) suspension. The main elements of PZW1 were measured by EDS mapping and resulted to be C, N, O, and Zn. Zn was uniformly distributed on the fibers, which was beneficial to increase their mechanical strength. [Figure 1](#materials-10-01267-f001){ref-type="fig"}c--e display the morphology and EDS mapping of PZW2 (PE/ZnO/wood fiber composite with 4% ZnO), PZW3 (PE/ZnO/wood fiber composite with 6% ZnO), and PZW4 (PE/ZnO/wood fiber composite with 8% ZnO). As the concentration of ZnO increased, the nanoparticles on the surface of the PZW composites increased.
[Figure 2](#materials-10-01267-f002){ref-type="fig"} shows TEM images of the PE/wood-fiber composite and of the PE/ZnO/wood-fiber composite. As shown in [Figure 2](#materials-10-01267-f002){ref-type="fig"}a, the microfibers were connected to the fibers; the average diameter was 300 nm. Meanwhile, PE particles were distributed on the microfibers. As detected by EDS analysis shown in the inset, the main elements were C, derived from PE and wood fiber, with atomic percentage of 62%, and N, provided by the amino group of CPAM, with atomic percentage of 3%. ZnO NPs were distributed around the fibers, as seen at low resolution in [Figure 2](#materials-10-01267-f002){ref-type="fig"}b, and the presence of Zn was detected by EDS analysis, as shown in the inset. The average crystalline size of the ZnO NPs was 29 nm, which was calculated from the TEM images. The elements C, N, O, and Zn were detected in the EDS spectrum, with corresponding atomic percentages of 50%, 2%, 27%, and 21%, respectively. The presence of the ZnO NPs confirmed that ZnO NPs were loaded on the fibers by mechanical and chemical action. A high-resolution TEM image in [Figure 2](#materials-10-01267-f002){ref-type="fig"}c shows lattice fringes. The crystal lattice had a d-spacing (the spacing between planes in crystal) of 0.28 nm, which corresponded to the interspace of the (100) plane. The element diffraction pattern was obtained by fast Fourier transform of the blue line area of the high resolution TEM (HRTEM) image, as shown in the inset of [Figure 2](#materials-10-01267-f002){ref-type="fig"}c. The (100) and (002) lattices of ZnO are indicated in the inset. The observed diamond patterns indicated that ZnO was polycrystalline in nature \[[@B20-materials-10-01267]\]. [Figure 2](#materials-10-01267-f002){ref-type="fig"}d shows the electron diffraction (SAED) pattern of the PE/ZnO/wood-fiber composite. The bright diffraction spots confirmed the high crystallinity of ZnO, corresponding to wurtzite structure.
[Figure 3](#materials-10-01267-f003){ref-type="fig"}a shows the functional group structure of the PE/ZnO composite and of the PZW composite. In [Figure 3](#materials-10-01267-f003){ref-type="fig"}a, the absorption band at 3400 cm^−1^ was ascribed to the OH stretching vibration \[[@B21-materials-10-01267]\]. The peak at 1731 cm^−1^ was attributed to the C=O stretching vibration, which was a characteristic peak of hemicellulose \[[@B22-materials-10-01267],[@B23-materials-10-01267]\]. The absorption peak at 1639 cm^−1^ was assigned to the bending vibration of water absorption and to the C=O stretching vibration of the amide group \[[@B24-materials-10-01267]\]. The stretching vibration of the aromatic ring skeleton at 1513 cm^−1^ corresponded to the characteristic peak of lignin \[[@B25-materials-10-01267]\]. The absorption band at 1267 cm^−1^ was ascribed to the C--O stretching vibration of the ester and to the C--N stretching vibration \[[@B26-materials-10-01267]\]. The peak at 419 cm^−1^ was attributed to the metal-oxide bond \[[@B12-materials-10-01267]\]. [Figure 3](#materials-10-01267-f003){ref-type="fig"}b shows the changes of the hydroxyl groups in the range of 3200--3600 cm^−1^ of the FTIR spectrum. Compared to the FTIR spectra of the PZW0 composite without hot-pressing, the absorption peak of the hydroxyl group of the PZW4 composite without hot-pressing tended to widen and shift to lower wavenumbers. It was observed that the hydroxyl groups of the PZW4 composite without hot-pressing formed hydrogen bonds after loading the ZnO nanoparticles. After hot-pressing, the absorption peak of the hydroxyl group of the PZW composite widened, compared with the corresponding peak of the PZW composite without hot-pressing. It was observed that the hydroxyl groups bonded during hot-pressing. Meanwhile, the absorption peak of the PZW4 composite shifted to the low wavenumbers (3400 cm^−1^) as the ZnO NPs were loaded. This indicated that the hydroxyl groups on the ZnO NPs surface were linked via hydrogen bonding. [Figure 3](#materials-10-01267-f003){ref-type="fig"}c shows the changes of the different chemical groups in the range of 1100--1800 cm^−1^ of the FTIR spectra. Compared with the PZW composite without hot-pressing, the peak at 1731 cm^−1^ weakend and dispersed in the FTIR spectra of the PZW composite. It was described that part of the xylose released from hemicellulose could be degraded at high temperature. The reaction of the aromatic ring of lignin with the degradation products of xylose form an adhesive. The peak at 1267 cm^−1^ of the PZW composite widened, compared with that of the PZW composite without hot-pressing. The peak of the ester demonstrated that the adhesive was produced after hot-pressing. The ZnO NPs could also be attached to the composite via the adhesive.
[Figure 3](#materials-10-01267-f003){ref-type="fig"}d displays the crystalline structure of PE/ZnO, PZW0, and PZW4. The diffraction peaks at 16.2° and 22.5° were related to the (101) plane and the (002) plane of cellulose \[[@B1-materials-10-01267],[@B27-materials-10-01267]\]. The XRD peaks at 21.5° and 23.8° corresponded to the (100) and (200) planes of PE \[[@B28-materials-10-01267]\]. These peaks indicated that PE was attached to cellulose. For the PZW0 composite, there were only characteristic peaks of cellulose and PE on the XRD curve. The other diffraction peaks that appeared at 31.6°, 34.3°, 36.2°, 47.4°, 56.6°, 62.7°, 66.4°, 67.9°, and 69.3° were matched to the (100), (002), (101), (102), (110), (103), (200), (112), and (201) planes of the wurtzite structure (PDF No. 36-1451), respectively. \[[@B1-materials-10-01267]\] It was confirmed that the ZnO NPs were attached to cellulose. The lattice parameters of the hexagonal wurtzite phase were described as a = b = 0.325 nm, c = 0.521 nm, α = β = 90°, and γ = 120°. The crystalline structure of cellulose did not change as ZnO was added. The XRD patterns of the PE/ZnO composite showed the diffraction peaks of PE and ZnO. This indicated that the ZnO NPs were loaded onto the PE/ZnO composite via an extrusion procedure.
[Figure 4](#materials-10-01267-f004){ref-type="fig"} shows the X-ray photoelectron spectroscopy (XPS) spectra of the PZW0 and PZW4 composites; the atomic content and the C/O ratio, showing the elimination of carbon as a contaminant, are listed in [Table 1](#materials-10-01267-t001){ref-type="table"}. The binding energies obtained in the XPS analyses were corrected for specimen charging by referencing C 1s to 284.6 eV. After eliminating the contaminant carbon, the atomic percentage of Zn was 2.49%, 4.01%, 6.35%, and 8.81%, respectively. The survey XPS spectra of PZW0 displayed two binding-energy bands of C 1s and O 1s, as shown in [Figure 4](#materials-10-01267-f004){ref-type="fig"}a, while the XPS spectra of PZW4 showed four binding-energy bands of C 1s, O 1s, Zn 2p~3/2~, and Zn 2p~1/2~. These results further confirmed that ZnO was coated on the fibers. In [Figure 4](#materials-10-01267-f004){ref-type="fig"}a, the binding energy band at 401.17 eV was attributed to N 1s, which was derived from amide group of CPAM. The atomic content and C/O ratio were determined by XPS analysis, and the resulting data are listed in [Table 1](#materials-10-01267-t001){ref-type="table"}. As the concentration of the ZnO NPs increased, the relative atomic content of O increased and the C/O ratio decreased. [Figure 4](#materials-10-01267-f004){ref-type="fig"}b displays the C 1s spectra of PZW0 and PZW4. The binding energies of 284.87, 286.37, and 288.17 eV were ascribed to the C--C bond, the C--O bond, and the C=O bond, respectively \[[@B15-materials-10-01267]\]. Compared with the relative peak intensity of the C--C bond in PZW0 in [Figure 4](#materials-10-01267-f004){ref-type="fig"}b, the relative intensity of the C--C bond in PZW4 decreased. The O 1s spectrum is displayed in [Figure 4](#materials-10-01267-f004){ref-type="fig"}c; the peak with the binding energy of 532.81 eV was attributed to the O--H bond and to water absorption \[[@B29-materials-10-01267]\]. An additional peak with binding energy of 530.97 eV appeared, which was ascribed to the Zn--O bond \[[@B12-materials-10-01267]\]. The binding energies of 1022.57 and 1045.67 eV were assigned to Zn 2p~3/2~ and Zn 2p~1/2~, respectively, and the difference of the binding energies, corresponding to 23.1 eV, matched the standard value for ZnO \[[@B12-materials-10-01267]\].
[Figure 5](#materials-10-01267-f005){ref-type="fig"} shows the thermogravimetric (TG) and differential thermogravimetric (DTG) curves of PZW0, PZW1, PZW2, PZW3, and PZW4. All the TG curves could be divided into three stages, as shown in [Figure 5](#materials-10-01267-f005){ref-type="fig"}a. A weight loss of 5% occurred below 100 °C and was attributed to the evaporation of residual moisture. The second stage was due to the thermal decomposition of wood fibers, which occurred at temperatures in the range of 250--420 °C. The third stage was the decomposition process of PE and occurred at temperatures in the range of 450--550 °C \[[@B30-materials-10-01267]\]. The main reason of the third stage could be ascribed to the fact that the ZnO NPs could transfer the heat and disturb the spread of degradation components. DTG decomposition curves of all PZW composites are shown in [Figure 5](#materials-10-01267-f005){ref-type="fig"}b. The DTG curve of PZW0 shows a rapid decomposition of wood fibers occurring at a temperature of 368.5 °C. As the concentration of ZnO increased, the decomposition temperature of wood fibers was 365.8 °C, 371.9 °C, 374.6 °C, and 374.6 °C, respectively. These results showed that ZnO had a flame-retardant effect on the wood-fiber composites.
[Figure 6](#materials-10-01267-f006){ref-type="fig"}a shows the comparison of the reflection loss curves of the PE/ZnO composite, the PZW0 composite, and the PZW3 composite, with a thickness of 3.5 mm in the frequency range of 2--18 GHz. The minimum reflection loss (RL) value of PZW0 was −2 dB at 17.17 GHz, while the minimum RL value of PZW3 was −21.21 dB in the same frequency. The minimum RL value of the PE/ZnO composite was 19 dB in the frequency of 2--18 GHz. An RL value of −10 dB corresponded to a microwave absorption of 90% \[[@B19-materials-10-01267]\]. The RL value of PZW3 was lower than −10 dB in the frequency range of 17.04--17.20 GHz. Compared with the RL value of the PZW3 composite, the RL value of the PE/ZnO composite was lower. It was demonstrated that PE had no effect on the reflection loss. In [Figure 6](#materials-10-01267-f006){ref-type="fig"}b, the three-dimensional RL image of PZW3 shows the effects of frequency and thickness on microwave absorption. The RL value of PZW3 was lower than −10 dB in the frequency range of 17.04--17.20 GHz, with a thickness range of 3--5.5 mm. Moreover, the RL value below −20 dB appeared in the frequency of 17.12 GHz, with a thickness of 3.5 mm. Thus, the deposition of ZnO NPs on the wood-fiber composite could enhance the microwave absorption properties of the composite.
The photocatalytic abilities of the as-prepared composites were estimated by the decomposition of a methyl orange (MO) solution under UV irradiation. [Figure 7](#materials-10-01267-f007){ref-type="fig"} shows the photocatalytic curve of the MO solution. In [Figure 7](#materials-10-01267-f007){ref-type="fig"}a, the PZW0 composite showed a little photocatalytic ability of about 8% that increased as the exposure time increased, and that may be attributed to the physical absorption of cellulose \[[@B31-materials-10-01267]\]. Moreover, this result indicated that the effects of the wood fibers and of PE on the photocatalytic ability were eliminated. [Figure 7](#materials-10-01267-f007){ref-type="fig"}b shows the photocatalytic ability of the PZW4 composite. The characteristic absorption peak of the MO solution at around 464 nm changed after UV irradiation for 60 min. The intensity of the photodegration curve of PZW4 decreased strongly as the exposure time increased. It was obderved that the addition of the ZnO NPs enhanced the degradation efficiency of the PZW4 composite. The photodegration curves of the MO solutions of MO, PZW0, PZW4, and PE/ZnO composites under UV light irradiation are displayed in [Figure 7](#materials-10-01267-f007){ref-type="fig"}c. The photocatalytic activities of MO, PZW0, PZW4, and PE/ZnO were 3%, 8%, 84%, and 80%, respectively, after UV irradiation for 300 min. The photocatalytic activity of the PE/ZnO composite was lower than that of the PZW4 composite when the content of ZnO was the same. This indicated that ZnO plays a dominant role in regulating the photocatalytic activity. This was attributed to the fact that MO molecules were absorbed by physical adsorption on the fibers' surface. In addition, it was observed that the photocatalytic activity derived from the ZnO NPs. The images of MO, PZW0, PZW1, PZW2, PZW3, and PZW4 after UV irradiation for 300 min are shown in [Figure 7](#materials-10-01267-f007){ref-type="fig"}d. The experiment confirmed that the ZnO NPs enhanced the photocatalytic activity of the PE/ZnO/wood-fiber composite.
To investigate the reinforcing efficiency of the ZnO NPs on the PZW composites, the dynamic mechanical analysis (DMA) was used to measure the storage modulus and the loss factor. [Figure 8](#materials-10-01267-f008){ref-type="fig"} shows the dynamic mechanical properties of the PE/wood-fiber composite, the PE/ZnO composite, and the PE/ZnO/wood-fiber composite. [Figure 8](#materials-10-01267-f008){ref-type="fig"}a displays the effect of the ZnO NPs on the storage modulus of PE/ZnO, PZW0, PZW1, PZW2, PZW3, and PZW4. Compared with PZW0, the addition of the ZnO NPs enhanced the storage modulus of the PZW composites in the temperature range from −20 to 80 °C. The reinforcing effect of the ZnO NPs could result in the increase of stiffness. The storage modulus of the PZW composites decreased below the glass transition temperature (Tg) as the temperature raised. At temperatures near Tg, a rapid decrease of the storage modulus values was observed, indicating that the PZW composites would undergo a transition stage from glassy to rubbery. The storage modulus of the PE/ZnO composite decreased as the temperature reached 80 °C. This was ascribed to the fact that the heat distortion temperature of PE was 80 °C. The PE/ZnO composite showed a high storage modulus. In the presence of the same content of ZnO, the value of the PE/ZnO composite was slightly lower than that of the PZW4 composite. Therefore, PE had an enhancement effect on the dynamic mechanical properties. [Figure 8](#materials-10-01267-f008){ref-type="fig"}b displays the damping-factor (tan δ) curves of PE/ZnO, PZW0, PZW1, PZW2, PZW3, and PZW4. It was reported that the lower tan δ values associated with the glass transition temperature reflect the improved load-bearing properties of the system \[[@B1-materials-10-01267]\]. Compared with the tan δ of PZW0, the PZW composite had a low tan δ value. The peak height of tan δ decreased as the ZnO NPs increased, which indicated that the stress transfer and interface bonding between interface layers were increased \[[@B32-materials-10-01267]\]. The Tg value of the PZW composite increased by 4--5 °C as the ZnO NPs increased.
[Figure 9](#materials-10-01267-f009){ref-type="fig"} displays the flexural strength and modulus of elasticity (MOE) of the PZW composites. As shown in [Figure 9](#materials-10-01267-f009){ref-type="fig"}a, the strength of PZW0 was 43 MPa, and the MOE was 5205 MPa. The strength and MOE values of PZW0 were far higher than those found in other studies, which was attributed to the fact that the PE molecules had a reinforcing effect on fiber materials. With the addition of the ZnO NPs, the mechanical strength increased gradually. When the content of ZnO NPs was 8%, the mechanical strength reached the maximum value of 58 MPa. Compared with that of PZW0, the mechanical strength increased by 36%. The MOE reached the maximum value of 9625 MPa when the content of ZnO was 6%, thus increasing by 85% compared with that of PZW0. The PE/ZnO composite showed a high flexural strength and a low modulus of elasticity. It was demonstrated that PE had an enhancement effect on the flexural strength of the PZW composites. [Figure 9](#materials-10-01267-f009){ref-type="fig"}b shows the stress-strain curves of all composites with different ZnO contents. When the stress was lower than 6 MPa, the materials remained in a flexible stage (the stress was proportional to the strain). When the stress increased reaching a maximum limit, the materials started bending until they broke. [Figure 9](#materials-10-01267-f009){ref-type="fig"}c shows that the mechanical strength of the PZW composites was higher than that of other materials. [Figure 9](#materials-10-01267-f009){ref-type="fig"}d displays the internal bond strength of all PZW composites. The internal bonding (IB) strength of PZW0, corresponding to 0.88 MPa, reached the national standard (GB/T 11718-2009) requirements. With the addition of ZnO, the IB strength reached a maximum value of 0.90 MPa. [Figure 9](#materials-10-01267-f009){ref-type="fig"}e exhibits the thickness swelling properties of all PZW composites after soaking in water. The distribution of the ZnO NPs on the fibers' surface disturbed the water absorption capacity, resulting in the decrease of the thickness swelling (TS) values. The minimum TS value was 9% when the content of ZnO was 6%. These results demonstrated that the ZnO NPs had an enhancing effect on the mechanical properties of wood-fiber-polymer composites. [Figure 9](#materials-10-01267-f009){ref-type="fig"}f displays the comparison of the mechanical properties of the PZW composites with those of other peer biomass materials, showing that the properties of the PZW composites were better than those of the other materials \[[@B32-materials-10-01267],[@B33-materials-10-01267],[@B34-materials-10-01267],[@B35-materials-10-01267],[@B36-materials-10-01267],[@B37-materials-10-01267]\].
3. Materials and Methods {#sec3-materials-10-01267}
========================
3.1. Materials {#sec3dot1-materials-10-01267}
--------------
Wood fibers were provided by Zhejiang New Wood Material Technology Co., Ltd. (Ningbo, China). The wood fibers were derived from poplar wood, and had a length in the range of 300--1000 μm, with an average diameter of 40 μm. ZnO nanoparticles with an average diameter of 30 nm were supplied by Aladdin Industrial Corporation (Shanghai, China). Polyethylene (Grade: Ultra-high molecular weight Polyethylene, Mw = 5,000,000) was obtained from Mitsui Chemicals, Inc. (Shanghai, China). Cationic polyacrylamide (CPAM, Mw = 15,000,000) was acquired from Henan Huayang Water Treatment Materials Co., Ltd. (Zhengzhou, China).
3.2. Synthesis of the PE/ZnO/Wood-Fiber Composite {#sec3dot2-materials-10-01267}
-------------------------------------------------
A PE/wood-fiber mixture was soaked in 1000 mL of CPAM solution (0.01 wt %), and the mass ratio of PE to wood fiber was set as 1/10. The mixed liquid was transferred into a colloid grinder with a disc distance of 0.1 mm (Model: JM-L80, 2880 r/min, Shanghai Shen'ou General Valve Industry Co. Ltd., Shanghai, China), dispersed, and ground for 6 h. The mixed cylindrical model was obtained by vacuum filtration using a vacuum pump. The model was compressed by a hot-press at 200 °C under a pressure of 3 MPa for 25 min. In order to study the effect of ZnO content on the PZW composites, the mass ratio of ZnO to wood fiber was set as 0/50, 1/50, 2/50, 3/50, and 4/50, respectively. The as-prepared samples were designated PZW0, PZW1, PZW2, PZW3, and PZW4, respectively, according to the mass ratio of ZnO to wood fiber. The PE/ZnO composite was prepared via extrusion and injection molding, and its mass ratio of ZnO to PE was 4/50. The preparation process of the PZW composite is shown in [Figure 10](#materials-10-01267-f010){ref-type="fig"}.
3.3. Characterization {#sec3dot3-materials-10-01267}
---------------------
The morphologies and element distributions of the PE/ZnO/wood-fiber composites were investigated by scanning electron microscopy and energy dispersive spectrometer (SEM-EDS) analysis (FEI Quanta 200, EDS/EDX Genesis, FEI Inc., Hillsborough, OR, USA). The dispersion of the ZnO nanoparticles on the fibers' surface was investigated by transmission electron microscope (TEM, Tecnai G2 F20 S-TWIN, FEI Inc., Hillsborough, OR, USA). The X-ray diffraction (XRD) experiments were carried out in the 2θ range from 10° to 80° at a scan rate of 4°/min, using an X-ray diffractometer (Bruker D8 Advance, Bruker AXS GmbH, Karlsruhe, Germany) with Cu Kα radiation (λ = 1.5418 Å). The structure analysis based on chemical groups was performed by Fourier transform infrared spectroscopy (FTIR, Nicolet iN10 MX, Thermo Fisher Scientific, Waltham, MA, USA), with a spectrometer resolution of 4 cm^−1^, and 32 scans for signal on average. The chemical elements were determined by X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250XI, Thermo Fisher Scientific, Waltham, MA, USA) using an ESCALab MKII X-ray photoelectron spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with Mg-Kα X-rays as the excitation source. The thermal properties of the composites were measured using a thermo-gravimetric analyzer (TGA, STA449F3, NETZSCH GABO Instruments GmbH, Ahlden, Germany) with a heating rate of 20 °C/min, up to 800 °C, under a nitrogen atmosphere.
3.4. Electromagnetic Test {#sec3dot4-materials-10-01267}
-------------------------
The electromagnetic wave absorbing properties of the PE/ZnO/wood-fiber composites were measured by a vector network analyzer (Model: Keysight E5071C ENA, Agilent Technologies, Inc., Santa Clara, CA, USA) in the frequency range of 2--18 GHz. The reflection loss (RL) curves were calculated by relative complex permittivity and permeability at a given frequency and layer thickness, which can be calculated through the following equations \[[@B19-materials-10-01267]\]: where Z~in~ was the input impedance of the absorber, Z~0~ the impedance of free space, μ~γ~ the relative complex permeability, ε~γ~ the complex permittivity, f the frequency of microwaves, d the thickness of the absorber, and c the velocity of the light.
3.5. Photocatalytic Activity Test {#sec3dot5-materials-10-01267}
---------------------------------
The photocatalytic activities of the PE/ZnO/wood-fiber composites were evaluated through the decomposition of methyl orange (MO). The irradiation source was an ultraviolet 100 W lamp with a wavelength of 254 nm. Dried powders of the PZW composites were dispersed in 25 mL of MO aqueous solution with a concentration of 15 mg/L, and the PE/wood-fiber composite was used for comparison. The suspensions were stirred in the dark for 30 min to ensure the establishment of an adsorption-desorption equilibrium of the MO molecules. At set intervals (30 min), 2 mL of the suspension was extracted and then centrifuged to separate the catalysts from the supernatant. The degradation efficiency of MO was measured by UV-vis spectrophotometer (Pgeneral TU-1901 UV-Vis spectrophotometer, (Beijing Puxi General Instrument Co., Ltd., Beijing, China)); the maximum absorption wavelength of MO was 464 nm.
3.6. Dynamic Mechanical Analysis {#sec3dot6-materials-10-01267}
--------------------------------
The dynamic mechanical properties of the PE/ZnO/wood-fiber composites were determined using a dynamic mechanical thermal analyzer (DMTA, TA Q800, TA Instruments, New Castle, DE, USA) in a three-point bending system; the samples dimensions were 50 × 10 × 4 mm. The samples were measured in an air atmosphere at a fixed frequency mode of 5.0 Hz, and oscillation amplitude of 15 μm. The samples were evaluated in the temperature range of −20 to --200 °C with a heating rate of 2°/min.
3.7. Mechanical Studies {#sec3dot7-materials-10-01267}
-----------------------
The flexural properties of the PE/ZnO/wood-fiber composites were measured with the universal testing machine (Model: MWD-100, Jinan Asahi Instrument Equipment Co., Ltd, Jinan, China) using the three-point bending test at a loading rate of 5 mm/min. The experiment was carried out according to the Chinese Standard GB/T 11718-2009. The samples' dimensions were 100 × 20 × 5 mm, and the number of test pieces was 15. Fifteen test samples of 50 × 50 × 5 mm were prepared for the internal bonding (IB) strength test, which was performed using the universal testing machine (Model: MWD-100, Jinan Asahi Instrument Equipment Co., Ltd, Jinan, China) at a loading rate of 5 mm/min. Fifteen samples of the same size were examined in the thickness swelling (TS) test, after 24 h of immersion in water at 20 °C.
4. Conclusions {#sec4-materials-10-01267}
==============
ZnO NPs were deposited on PZW composites through the precipitation of CPAM during a grinding process, and the final PZW composites were obtained via hot-press. The PZW composites showed EM absorption properties and superior mechanical properties, and could degrade MO under UV irradiation. The minimum-reflection-loss value of the PZW composites was −21 dB, with a thickness of 3.5 mm in the frequency of 17.17 GHz. The degradation efficiency of the PZW composites was about 84% under UV light irradiation. The synergistic effects of the ZnO NPs and PE enhanced the mechanical strength of the composites. The value of the storage modulus of the PZW composites increased as the content of ZnO increased. It was found that the effectiveness of interfacial stress transfer was increased. The peak height of tan δ was lower in the PZW composites, which indicated lower damping and good adhesion. The flexural strength of PZW4 was 58 MPa, and the MOE of PZW3 was 9625 MPa; the IB and TS values of PZW3 were 0.88 MPa and 9%, respectively.
This research was supported by the Special Fund for Forest Scientific Research in the Public Welfare (Grant No. 201504501) and the Scientific Research Foundation of Zhejiang A&F University (Grant No. 2014FR077).
Q.S. and C.J. conceived the project and revised the whole manuscript. B.D. and Y.C. performed the experiments and wrote the paper. Y.C. and X.S. measured the characterizations and analyzed the data. B.C. contributed materials.
The authors declare no conflict of interest.
{#materials-10-01267-f001}
{#materials-10-01267-f002}
{#materials-10-01267-f003}
{#materials-10-01267-f004}
{#materials-10-01267-f005}
{#materials-10-01267-f006}
{#materials-10-01267-f007}
{#materials-10-01267-f008}
{#materials-10-01267-f009}
{#materials-10-01267-f010}
materials-10-01267-t001_Table 1
######
Element content and C/O ratios of the PE/ZnO/wood-fiber composite.
Sample C (at %) O (at %) N (at %) Zn (at %) C/O Ratio
-------- ---------- ---------- ---------- ----------- -----------
PZW0 61.35 34.54 4.11 \- 1.78
PZW1 57.95 35.46 4.10 2.49 1.63
PZW2 53.16 38.74 4.09 4.01 1.37
PZW3 49.67 39.95 4.03 6.35 1.24
PZW4 47.56 39.67 3.96 8.81 1.19
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{#sp1 .26}
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INTRODUCTION
============
Laparoscopic cholecystectomy is now the procedure of choice for treating uncomplicated symptomatic gallstones.^[@B1],[@B2]^ Even though laparoscopic cholecystectomy has been shown to be equivalent to open cholecystectomy with respect to morbidity and mortality, it offers the advantage of small scar size, shorter length of hospital stay, quicker recovery and return to full activity, and a relatively painless postoperative period. Mirizzi^[@B3]^ first popularized intraoperative cholangiography in 1932; however, its use has been controversial for years, and this controversy has continued into the laparoscopic era. Advocates ^[@B4]--[@B9]^ believe that routine cholangiography will not only help delineate biliary anatomy, detect common bile duct stones, facilitate dissection, and avoid injury to the biliary tract but also help identify other clinically silent diseases, such as cholangiocarcinoma, duodenal adenocarcinoma, and choledochal cyst. Those surgeons ^[@B10]--[@B12]^ who advocate selective use of intraoperative cholangiography cite the increased cost and the low yield. However, the time and the cost associated with intraoperative cholangiography are outweighed by the cost of managing bile duct injuries and working up retained stones postoperatively. Traditionally, intraoperative cholangiography has been performed by cannulating the cystic duct, which requires dissection and partial transection of the cystic duct and its cannulation, which can be tedious and time consuming. Daoud and McCallum^[@B13]^ reported a successful cystic duct cannulation rate of approximately 83%. In addition, the transcystic approach can also lead to ductotomy of other structures misidentified as the cystic duct.^[@B4]--[@B9]^ In preview cholangiography, the cholangiogram is obtained before any dissection is begun. It not only prevents erroneous ductotomies, but it is easier and quicker. In addition, it is more cost-effective because the operative time is shorter.
PATIENTS AND METHODS
====================
Methods
-------
We used the standard 4-cannula technique to perform laparoscopic cholecystectomy in all cases. A ratcheted grasper was passed through the midaxillary port. This was used to grasp the fundus of the gallbladder, which was then pushed in a cephalad direction to expose the gallbladder ampulla.
The traditional transcystic duct cannulation approach required dissection that began on the gallbladder and continued until the "cystic duct-gallblader junction" was identified. A single clip was then applied to the proximal "cystic duct." Endoscopic scissors were then used to partially transect the "cystic duct" just distal to the previously placed clip. The cholangiocatheter was then introduced through a separate 8 Fr introducer sheath into the cystic duct, and then cholangiogram was performed.
The specialized clamp used for the preview cholangiogram is a 5-mm grasper with a channel for the introduction of the cholangioneedlecatheter **([Figure 1](#F1){ref-type="fig"})**. Before any dissection is begun, this specialized clamp is passed through the midclavicular port and applied across the neck of the gallbladder. A disposable cholangiogram catheter with a 19-gauge needle is then introduced through the channel and advanced to puncture the gallbladder Hartman pouch. Biliary access is confirmed by aspiration of bile. Dye is injected, and the cholangiogram is performed. The clamp jaw can be opened and the gallbladder aspirated, or the grasper can be moved down to the puncture to prevent bile leakage and also to be used for traction on the gallbladder ampulla for the remainder of the operation. **[Figure 2](#F2){ref-type="fig"}** represents a typical cholangiogram obtained using the preview cholangiogram system.
{#F1}
{#F2}
Patients
--------
Of the 11 patients, 6 were males and 5 were females. The mean age of all the patients was 67 years (range, 34 to 80). Two patients were operated on for acute cholecystitis. In one patient, who had subacute cholecystitis, preview cholangiography revealed the presence of common bile duct stones, which were removed by laparoscopic common bile duct exploration. Six patients had chronic cholecystitis, and two of these patients were operated on with associated procedures (one had a laparoscopic sigmoid resection, and the other had a laparoscopic ventral hernia repair). One patient had biliary dyskenesia without stones. Her cholecystokinin (CCK) stimulated gallbladder ejection fraction was measured at 9%, and her symptoms consisted of postprandial right upper quadrant pain. One patient, an 80-year-old male, had a gangrenous gallbladder with pericholecystic abscess. He presented with diffuse abdominal pain. A computerized axial tomography (CAT) scan of the abdomen revealed a heterogeneous density within the gallbladder fossa with surrounding indurations suspicious for abscess involving the liver **([Figure 3](#F3){ref-type="fig"})**.
{#F3}
RESULTS
=======
Preview cholangiograms were successfully performed in all 11 patients. No complications occurred. In one patient, preview cholangiography revealed the presence of unsuspected common bile duct stones. In another patient, reflux of contrast into the gallbladder occurred due to incomplete exclusion of the gallbladder by the application of the specialized clamp. However, preview cholangiography was successfully performed with clear visualization of the cystic duct and the remainder of the biliary tree including flow of contrast into the duodenum. In the patient with the gangrenous gallbladder, extravasation of contrast and reflux of the contrast into the gallbladder occurred **([Figure 4](#F4){ref-type="fig"})**. Even in this patient, clear visualization of the cystic duct and the reminder of the biliary tree was obtained. The average time elapsed during cholangiography was 110 seconds (range 91 to 142 seconds). The mean operating time was 52 minutes (range 48 to 66 minutes). These times, even this early in our experience using this technique, are far better than our best accomplishments using the cystic duct cannulation method for performing cholangiography. The mean cholangiogram time was 22 minutes (range 12 to 38 minutes), and the mean operative time was 75 minutes (range 68 to 92 minutes) following the first 100 cases.^[@B4]^
{#F4}
DISCUSSION
==========
We have by previous experience and by this limited experience demonstrated that intraoperative cholangiography offers the advantage of visualization of the biliary anatomy, which we strongly believe will decrease the number and severity of bile duct injuries and will also lead to early detection of clinically silent diseases of the extrahepatic biliary tree that may be missed because of the limitations of the laparoscopic approach. The availability of preview cholangiography and the ease of its performance demonstrated even this early in our experience should eliminate the few remaining obstacles to making intraoperative cholangiography routine practice. Corbitt and Leonetti9 in their experience with 1060 consecutive laparoscopic intraoperative cholangiograms reported one case in which the cystic duct-common duct junction was misidentified and an incision was made at this junction leading to the need for T-tube placement. In our experience with 511 consecutive patients who underwent laparoscopic cholangiogram with the transcystic duct approach, we too, in two cases despite careful dissection, mistook the common hepatic duct as the cystic duct. This visual error led to the partial transection of the duct in both cases.^[@B4]^ Preview cholangiography should completely eliminate this potential problem.
CONCLUSION
==========
Preview cholangiography is the answer and the future. It has many advantages over the traditional transcystic duct cholangiography. It is a safe, relatively easy, and exceptionally quick method for outlining the anatomy of the extrahepatic biliary tree. It is safe because cholangiography is performed prior to any dissection. It is quick; therefore, the total cost is significantly reduced because operative time is decreased. It can be performed even on gangrenous gallbladders. Because of the ease of preview cholangiography and its ability to outline the extrahepatic biliary anatomy, intraoperative preview cholangiography should be routinely performed.
| {
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Zhang P, Wang W, Lu M, et al. Clinicopathological features and outcome for neuroendocrine neoplasms of gastroesophageal junction: A population‐based study. Cancer Med. 2018;7:4361--4370. 10.1002/cam4.1702
1. INTRODUCTION {#cam41702-sec-0005}
===============
Neuroendocrine neoplasm (NEN) presents a heterogeneous group of tumors arising from neuroendocrine cells of the diffuse neuroendocrine system.[1](#cam41702-bib-0001){ref-type="ref"} Multiple factors may influence the outcome of the NENs, and the tumor location is one factor determined the malignancy of the tumor. Furthermore, the various incidence and characteristics of the NENs between different populations suggested a racial disparity.[2](#cam41702-bib-0002){ref-type="ref"} According to SEER database, the rectum and small intestine were the most common sites for NENs, and those in the stomach were less frequent.[2](#cam41702-bib-0002){ref-type="ref"}, [3](#cam41702-bib-0003){ref-type="ref"} Epidemiological data from both Korea and Taiwan indicate that gastric NEN is the second common site of NENs in the digestive tract.[4](#cam41702-bib-0004){ref-type="ref"}, [5](#cam41702-bib-0005){ref-type="ref"} Studies from Norway[6](#cam41702-bib-0006){ref-type="ref"} and England[7](#cam41702-bib-0007){ref-type="ref"} also revealed the incidence of gastric NENs surpassed that of small intestinal and colorectal NENs. However, the epidemiologic pattern for NENs of gastroesophageal junction has not been fully described. Although classified as similar entities in both historical and classification schemes, GEJ‐NENs behave more aggressively than those located elsewhere in the stomach. The current understanding of GEJ‐NENs is based on case reports and limited single‐institution case series.[8](#cam41702-bib-0008){ref-type="ref"}, [9](#cam41702-bib-0009){ref-type="ref"}
Given the relative rarity of GEJ‐NENs, population‐based analyses are critical to provide an overview about the epidemiological and therapeutic trends for these subtypes. The primary aim of this study was to investigate the clinical and pathological features of Chinese patients with GEJ‐NEN by comparing with those from Surveillance, Epidemiology, and End Results (SEER) Cancer Registry, and to study the prognostic predictors for GEJ‐NENs using a multicenter cohort from China. A second aim was to characterize the GEJ‐NENs compared with other gastric NENs using a population‐based registry.
2. MATERIALS AND METHODS {#cam41702-sec-0006}
========================
Clinical data of patients with pathology confirmed GEJ‐NENs from 2000 to 2017 were retrieved from 10 hospitals in China. All of these hospitals were representative centers which located in different parts of China. This study was approved by the hospital institutional review board.
The study cohort included all patients registered in the SEER database from 2000 to 2013. Individual cases were retrieved with the SEER\*Stat software (version 8.1.5, 31 March 2014; Cancer Statistics Branch, NCI, Bethesda, MD). Because of the SEER database\'s inclusion of unidentifiable patient information, this study was exempted for approval by the Office of Human Subjects Research of the National Institutes of Health. We identified patients with NETs using the following ICD‐O‐3 codes: 8240‐8249. 8240, carcinoid tumor; 8241, enterochromaffin; 8242, enterochromaffin‐like; 8243, goblet; 8244, mixed adenoneuroendocrine carcinoma; 8245, adenocarcinoid; 8246, neuroendocrine carcinoma; and 8249, atypical carcinoid. We selected NENs with primary of stomach (site code: C16.0‐16.9). Exclusion criteria included age less than 18 years, NEN as the second primary malignancy, NENs diagnosed at autopsy or death, and diagnoses without microscopic confirmation.
The following variables were included in the analysis: age at diagnosis, race, sex, year of diagnosis, primary tumor location, tumor grade and differentiation, AJCC staging, nodal status, distant metastasis, type of surgery performed, and OS. Tumor grade according WHO 2010 classification based on Ki‐67 index and mitotic count was analyzed from Chinese cohort.[10](#cam41702-bib-0010){ref-type="ref"} Tumor stages were assigned according to the staging classification sequentially proposed by European Neuroendocrine Tumor Society (ENETS) and American Joint Committee on Cancer (AJCC)[11](#cam41702-bib-0011){ref-type="ref"}, [12](#cam41702-bib-0012){ref-type="ref"} which were identical in NENs of stomach.
2.1. Statistical analysis {#cam41702-sec-0007}
-------------------------
To investigate the clinicopathological characteristics of the study patients, Student\'s *t* test, χ^2^ test (or Fisher exact test) and Mann‐Whitney method were used. Overall survival (OS) time was measured from the date of initial diagnosis until the date of death or last follow‐up. Survival analysis was performed with OS as the primary outcome measure. Survival was evaluated using Kaplan‐Meier estimates and Cox proportional hazard regression. Statistical tests used two‐tailed *P* values, and *P* \< 0.05 was considered statistically significant. All statistical analyses were performed in SPSS (version 25; IBM, Chicago, IL).
3. RESULTS {#cam41702-sec-0008}
==========
3.1. Clinicopathological characteristics of Chinese patients with GEJ‐NENs {#cam41702-sec-0009}
--------------------------------------------------------------------------
We retrospectively analyzed clinical and pathologic features of 297 patients with histological confirmed GEJ‐NENs from 10 hospitals. The entire group had a median age of 63 (35‐85), and 87.2% were male (n = 259). Based on the WHO‐2010 grading classification, all the patients with GEJ‐NENs were grade 3, and the proportion of poorly‐differentiated NEC and MANEC was 67.7% and 32.3%. Regional lymph node metastasis was found in 155 (52.2%) patients and 78 (26.2%) had distant metastasis at diagnosis. According to the AJCC/UICC staging system, 2 (0.7%) patients were classified as stage I, 46 (15.5%) as stage II, 171 (57.6%) as stage III and the other 78 (26.2%) as stage IV, respectively.
Compared with their NEC counterparts, the MANECs were more highly associated with early stage (*P* = 0.002), curative operations (90.6% vs. 71.1%, *P* \< 0.000), and more lymphatic metastasis (60.4% vs. 48.3%, *P* \< 0.000), but were less associated with distant metastasis (14.6% vs. 31.8%, *P* \< 0.000). The MANEC and NEC groups were statistically similar in other clinicopathological characteristics, including gender, age, Ki67 index, and tumor size. The comparison of clinicopathological characteristics between NEC and MANEC of the gastroesophageal junction is shown in table [1](#cam41702-tbl-0001){ref-type="table"}.
######
Comparison of clinical features of NEC and MANEC in Chinese GEJ‐NENs
Characteristics NEC(n = 201) MANEC(n = 96) *P* value
----------------------------- -------------- --------------- -----------
Age median, years 63.1 ± 8.6 62.1 ± 9.1 0.297
Male, n (%) 175 (87.1%) 83 (86.5%) 0.885
Ki67 index, % 69.1 ± 17.1 66.7 ± 17.9 0.717
Tumor size, cm 4.7 ± 1.9 4.6 ± 1.8 0.907
Surgery, n (%) 143 (71.1%) 87 (90.6%) \<0.000
AJCC Stage
I‐II 25 (12.4%) 23 (24.0%) 0.002
III‐IV 176 (87.6%) 73 (76.0%)
Regional lymph nodes, n (%) 97 (48.3%) 58 (60.4%) \<0.000
Distant metastasis, n (%) 64 (31.8%) 14 (14.6%) \<0.000
John Wiley & Sons, Ltd
3.2. Comparison of the clinicopathological characteristics of GEJ‐NENs in different races {#cam41702-sec-0010}
-----------------------------------------------------------------------------------------
In total, 297 and 274 patients with GEJ‐NENs were included, respectively, from the Chinese cohort and SEER database. The clinicopathological characteristics of GEJ‐NENs among different races were distinct. The mean ages were 62.7, 64.5, 58.5, and 61.5, respectively, in Chinese, white, black patients, and Asian/Pacific Islander (AP) patients. Except for black patients, male patients were more frequent. In Chinese patients, tumor size was larger than that in other groups. Chinese patients were poorly differentiated NEC and MANEC. In SEER database, 70.8% of white, 62.5% of black and 87.5% of AP patients had poorly differentiated/undifferentiated tumors. Distant metastasis at the time of presentation was more frequent in white and black patients than Chinese patients (60.0% vs.55.6% vs.26.3%, *P* \< 0.000). Surgical of primary tumor was performed in most of the patients in different race groups. Table [2](#cam41702-tbl-0002){ref-type="table"} summarizes baseline characteristics of GEJ‐NENs among different races.
######
Comparison of the clinicopathological characteristics of GEJ‐NENs among different races
SEER database
------------------------------ ------------------ ------------------ ------------------ ------------------ ---------
Age, y
Median,95%CI 62.7 (61.7‐63.8) 64.5 (62.8‐66.2) 58.5 (53.5‐63.5) 61.5 (55.2‐67.8) \<0.000
Sex
Male, n (%) 258 (86.9) 137 (60.6) 15 (46.9) 9 (56.3) \<0.000
Female, n (%) 39 (13.1) 89 (39.4) 17 (53.1) 7 (43.8)
Size, cm
Mean,95%CI 4.7 (4.4‐4.9) 3.0 (2.5‐4.1) 3.1 (1.8‐4.4) 3.8 (1.7‐7.1) \<0.000
Range 0.3‐12 0.1‐15 0.4‐8.5 0.4‐10.6
Morphology
NEC 201 (67.7) ‐
MANEC 96 (32.3)
Grade
Well/Moderate differentiated 42 (29.2) 6 (27.5) 1 (12.5) \<0.000
Poorly/undifferentiated 102 (70.8) 10 (62.5) 7 (87.5)
AJCC Stage
I‐II 48 (16.2) 40 (27.6) 7 (38.9) 3 (37.5) \<0.000
III 171 (57.6) 18 (12.4) 1 (5.6) 3 (27.5)
IV 78 (26.3) 87 (60.0) 10 (55.6) 2 (25.0)
Surgery, n (%)
performed 230 (77.4) 81 (36.5) 16 (50.0) 9 (56.3) \<0.000
Unperformed 67 (22.6) 141 (63.5) 16 (50.0) 7 (43.8)
John Wiley & Sons, Ltd
3.3. Treatment and survival of Chinese patients with GEJ‐NENs {#cam41702-sec-0011}
-------------------------------------------------------------
Overall, treatment strategies were provided in all the 297 patients. Among these patients, 77.4% (n = 230) underwent surgical resection, of which 89.6% (n = 206) were curative and 10.4% (n = 24) were palliative. Surgical resection offered a survival advantage with HR 0.21 (95%CI 0.14‐0.33) and the median OS improved from 13.1 to 73.3 months (*P* \< 0.001) (Figure [1](#cam41702-fig-0001){ref-type="fig"}A). A total of 104 (50.5%) patients received adjuvant chemotherapy. There was no additional survival benefit to adjuvant chemotherapy in patients undergoing surgical resection (*P* = 0.141) (Figure [1](#cam41702-fig-0001){ref-type="fig"}B). Invasion depth, lymph node metastasis can predict the risk of postoperative recurrence (Figure [1](#cam41702-fig-0001){ref-type="fig"}C,D).
{#cam41702-fig-0001}
The median survival time of the entire GEJ‐NENs patients was 31.0 months (95% CI 16.6‐45.4mo), and the subgroup of MANEC had longer survival than NEC (73.3 vs. 25.2, *P* = 0.002). On multivariate analysis, NEC/MANEC (HR 2.09, 95%CI 1.24‐3.56; *P* = 0.006) (Figure [2](#cam41702-fig-0002){ref-type="fig"}A), stage (HR 3.29,95%CI 1.33‐8.12; *P* = 0.010) (Figure [2](#cam41702-fig-0002){ref-type="fig"}B), lymph nodes metastasis (HR 3.52,95%CI 1.68‐7.34; *P* = 0.001) (Figure [2](#cam41702-fig-0002){ref-type="fig"}C), and distant metastases (HR 3.90,95%CI 2.50‐6.08; *P* \< 0.001) (Figure [2](#cam41702-fig-0002){ref-type="fig"}D) were independent predictors of overall survival (Table [3](#cam41702-tbl-0003){ref-type="table"}).
{#cam41702-fig-0002}
######
Univariate and multivariate analysis of characteristics predicting overall survival
Univariate cox regression Multivariate cox regression
------------- --------------------------- ----------------------------- ----------- ------- ------ -----------
Age
\<65y ‐ 1
≥65y 0.36 1.21 0.80‐1.81
Size
\<4.5 cm ‐ 1
≥4.5 cm 0.007 2.37 1.85‐3.92
Sex
Female ‐ 1
Male 0.31 1.32 0.77‐2.26
Ki67 index
\<70% ‐ 1
≥70% 0.48 1.17 0.74‐1.83
Morphology
Large cell ‐ 1
Small cell 0.75 1.12 0.56‐2.23
MANEC/NEC
MANEC ‐ 1 ‐
NEC 0.002 2.25 1.33‐3.81 0.006 2.09 1.24‐3.56
Stage
I‐II ‐ 1 ‐ 1
III‐IV 0.005 3.61 1.46‐8.89 0.010 3.29 1.33‐8.12
Lymph nodes
No ‐ 1 1
Yes 0.000 4.15 2.01‐8.61 0.001 3.52 1.68‐7.34
Metastasis
No ‐ 1 ‐ 1
Yes 0.000 4.89 3.21‐7.46 0.000 3.90 2.50‐6.08
John Wiley & Sons, Ltd
3.4. Comparison of GEJ‐NEN and non‐GEJ NENs of SEER database {#cam41702-sec-0012}
------------------------------------------------------------
A total of 3152 patients with gastric NENs were identified in the SEER database from 2000 to 2013, including 274 GEJ‐NEN patients and 2878 non‐GEJ NEN patients. GEJ‐NENs were more commonly diagnosed at an older age (63.6 vs. 62.9 years, *P* \< 0.000) and in white patients (82.5% vs. 78.4%, *P* \< 0.000). Patients with GEJ‐NEN were more frequently male than female (58.5% vs. 40.1%, *P* \< 0.001). Tumors of GEJ‐NENs were larger (3.0 vs. 1.9 cm, *P* \< 0.000) and predominantly poorly differentiated and undifferentiated tumors (70.8% vs.19.5%, *P* \< 0.001). GEJ‐NENs were highly invasive with more distant metastases (41.5% vs.11.9%, *P* \< 0.001), whereas non‐GEJ NENs had more localized lesions (79.5% vs. 43.6%, *P* \< 0.000). Patients with GEJ‐NENs were less likely to be treated with resection (39.3% vs. 59.9%, *P* \< 0.000), but more likely to receive radiotherapy (6.2% vs.2.1%, *P* \< 0.001). Table [4](#cam41702-tbl-0004){ref-type="table"} contains patient characteristics that were assessed between GEJ‐NENs and other gastric NENs.
######
Clinical and pathological features of GEJ and other gastric NEN in the SEER database
Variable GEJ‐NEN(n = 274) Non‐GEJ NEN(n = 2878) *P* value
---------------------------------- ------------------ ----------------------- -----------
Age, median, years 63.6 ± 13.1 62.9 ± 13.6 \<0.000
Male, n (%) 161 (58.5%) 1155 (40.1%) \<0.000
Race
White 226 (82.5%) 2257 (78.4%) \<0.000
Black 32 (11.7%) 385 (13.4%)
Asian 15 (5.5%) 174 (6.0%)
Other 1 (0.4%) 62 (2.2%)
Grade
Well/Moderate differentiated 49 (29.25%) 1022 (80.5%) \<0.000
Poorly/undifferentiated 119 (70.8%) 248 (19.5%)
ICD‐O‐3 Code
Carcinoid (8240‐8243,8249) 99 (36.1%) 2061 (71.6%) \<0.000
Neuroendocrine carcinoma (8246) 172 (62.8%) 777 (27.0%)
Mixed adenoneuroendocrine (8244) 2 (0.7%) 25 (0.9%)
Adenocarcinoma (8245) 1 (0.4%) 15 (0.5%)
Tumor size, cm 3.0 (0.1‐15.0) 1.9 (0.1‐12.0) \<0.000
Surgery, n (%) 106 (39.3%) 1697 (59.9%) \<0.000
Radiation, n (%) 44 (6.2%) 60 (2.1%) \<0.000
AJCC Stage
I‐II 50 (29.1%) 1274 (75.6%) \<0.000
III‐IV 121 (70.8%) 411 (24.4%)
Extent of disease
Local 105 (43.6%) 1878 (79.5%) \<0.000
Regional 236 (14.9%) 204 (7.6%)
Distant 100 (41.5%) 281 (11.9%)
ICD‐O‐3, International Classification of Diseases for Oncology, 3rd Edition.
John Wiley & Sons, Ltd
Median OS was significantly worse for patients with GEJ‐NENs than those with non‐GEJ NENs (*P* \< 0.000) (Figure [3](#cam41702-fig-0003){ref-type="fig"}A). Stratified analysis by stage showed that: in localized disease, median survival showed no difference (Figure [3](#cam41702-fig-0003){ref-type="fig"}B); in regional or metastatic disease, median survival of GEJ‐NENs showed worse survival (*P* \< 0.001) (Figure [3](#cam41702-fig-0003){ref-type="fig"}C&D).
{#cam41702-fig-0003}
4. DISCUSSION {#cam41702-sec-0013}
=============
Gastroesophageal NENs account for a relatively small and heterogeneous population with an aggressive course; however, limited information is available regarding characteristics because of its rarity.[13](#cam41702-bib-0013){ref-type="ref"}, [14](#cam41702-bib-0014){ref-type="ref"} In this study, we investigated the clinical characteristics and outcomes of patients diagnosed with GEJ‐NENs using a population‐based registry from 10 Chinese hospitals in China and the SEER database. This study represents one of the largest and most detailed cohort analyses of the epidemiology and outcomes of GEJ‐NENs. We found the clinicopathologic features of GEJ‐NENs differ among different races, and Chinese patients mostly had poorly differentiated disease, including relatively high‐frequency of MANEC. Additionally, we showed that the GEJ‐NENs were particularly more aggressive than other gastric NENs.
The current study indicated that all the Chinese patients with GEJ‐NENs were poorly differentiated carcinomas, and 96 cases (32.3%) were MANEC with both adenocarcinoma and neuroendocrine differentiation. According to gastric NENs clinical classification,[15](#cam41702-bib-0015){ref-type="ref"} GEJ‐NENs are prone to be sporadic and poorly differentiated and thus classified as the fourth subtype. GEJ‐NENs were male dominated and gross appearance of the tumor was large in size. Due to aggressive biological behavior, GEJ‐NENs frequently metastasized to regional lymph nodes and distant organs and thus had a poor prognosis. Half of the patients had regional lymph node metastasis and one‐third of patients showed distant metastasis. For such heterogeneous carcinoma with aggressive behavior, multidisciplinary team is recommended during the process of clinical management and medical care.
Because inadequate understanding of GEJ‐NENs, it is controversial of the prognostic value of their histologic classification. Compared with the adenocarcinoma, NEC was more aggressive with poorly differentiated morphology.[16](#cam41702-bib-0016){ref-type="ref"} Shia et al reported[17](#cam41702-bib-0017){ref-type="ref"} the absence of an associated adenocarcinoma component was predictive of a worse outcome; however, previous studies about gastric or colorectal MANEC reported that there was no statistically significant difference in survival between MANECs and NEC.[18](#cam41702-bib-0018){ref-type="ref"}, [19](#cam41702-bib-0019){ref-type="ref"} In our cohort, a number of GEJ‐NECs were mixed with high grade adenocarcinoma, the outcome of which was better than pure NECs. Then we compared the clinicopathological features between MANEC and NEC, and there was no difference in age, sex, Ki67 index, and tumor size. However, metastatic patterns of the two entities were different: The regional lymph node metastasis of MANEC was more common, and distant metastasis frequently occurred in NEC, indicating that the behavior of NEC may be more aggressive.
The importance of surgical resection of gastric NENs on survival has been reported in the previous literature.[20](#cam41702-bib-0020){ref-type="ref"}, [21](#cam41702-bib-0021){ref-type="ref"} Accordingly, a wide spectrum of therapeutic options has been provided, from endoscopic follow‐up to curative partial or total gastrectomy.[22](#cam41702-bib-0022){ref-type="ref"} In our study, GEJ‐NENs mostly were diagnosed at stage III‐IV, and after surgery recurrence occurred in a relatively large number of patients. But it remains controversial whether adjuvant therapy reduces the risk of recurrence or prolongs the overall survival of GEJ‐NENs. In Chinese cohort, 104 (50.5%) patients received platinum‐based (cisplatin or oxaliplatin) adjuvant chemotherapy after radical surgery, but the cohort received adjuvant therapy showed no additional survival advantage. We should be cautious about drawing the conclusion as the adjuvant regimens included protocols referring to gastric adenocarcinoma and neuroendocrine carcinoma. Therefore, the role of adjuvant chemotherapy after radical resection and the specific regimen to choose need further investigation.
Although classified as similar entities in classification schemes, tumors of gastroesophageal have distinct characteristics compared with those located elsewhere in the stomach, which has been observed in gastric adenocarcinoma.[23](#cam41702-bib-0023){ref-type="ref"} Previous studies indicated that GEJ‐NENs were more similar to esophageal NENs than to those of the stomach, with more aggressive behavior.[24](#cam41702-bib-0024){ref-type="ref"} We compared the characteristics and outcome of GEJ‐NENs with other gastric NENs using SEER database, and the result showed that patients with GEJ‐NENs had tumors larger in size. The majority of the cohort was diagnosed at more advanced stage and tends to be poorly differentiated compared with non‐GEJ NENs entities. The unfavorable prognosis also indicated that GEJ‐NENs were more aggressive than their counterparties located in other sites of the stomach. Therefore, the morphology and histology may be a potential cause for the worse survival of GEJ‐NENs.
It has been reported that the biological behavior and clinical outcome of patients with gastric carcinoma varies among different human races.[25](#cam41702-bib-0025){ref-type="ref"} In our study, by comparing the clinicopathologic characteristics of GEJ‐NENs among different races, large disparities were found in terms of histology grade and clinical stage; The Chinese cohort of GEJ‐NENs is high‐grade MANE/NEC, while Asian/Pacific Islanders (AP) patients from SEER database had more poorly differentiated/undifferentiated tumors than white and black patients. Moreover, tumor size of Chinese patients was significantly larger than that in other groups of patients. Upon diagnosis, distant metastasis was less common in Chinese patients than that in white and black patients from the SEER database. This situation was also found in AP patients with GEJ‐NENs from the SEER database. Therefore, different genetic and epigenetic changes may partly explain the diversity among different races.
It is acknowledged that this study has limitations. The primary limitation is that the SEER database does not include information of Ki‐67 index and data on recurrence or disease‐free survival. The chemotherapy and specific treatment regimens were not included. The second limitation is the retrospective nature of our study. The pathological data from achieves were according to the 2010 WHO classification, although a new term of mixed neuroendocrine‐non‐neuroendocrine neoplasm(MiNEN) was proposed, considering the morphological and biological heterogeneity of gastrointestinal mixed tumors.[26](#cam41702-bib-0026){ref-type="ref"} However, all the MANECs in our entity are consistently poorly differentiated neuroendocrine carcinoma combined with adenocarcinoma. Moreover, we include 298 Chinese patients with GEJ‐NEN and a large sample of GEJ‐NEN patients from the SEER database. The cohort in our study represents the largest dataset of GEJ‐NENs to date and offers valuable information on the epidemiology and prognosis. This study provides a uniquely detailed assessment of the GEJ‐NENs to improve our understanding of the disease and guide future research.
5. CONCLUSIONS {#cam41702-sec-0014}
==============
GEJ‐NENs were highly invasive with frequent distant metastases, with predominantly poorly differentiated and undifferentiated tumors, thus showed worse survival than other gastric NENs. The clinicopathological characteristics of GEJ‐NENs among different races were distinct. Chinese patients with GEJ‐NENs are all NEC or MANEC, and the latter show distinct metastatic patterns and better survival. Surgical resection improved the survival, and there was no additional survival benefit to adjuvant chemotherapy, and prospective studies using defined diagnostic criteria are necessary to determine optimal management.
CONFLICT OF INTEREST {#cam41702-sec-0016}
====================
None declared.
The authors declare that they have no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
**Mild traumatic brain injuries (mTBI)** are among the most common neurologic conditions, representing a substantial burden in adults and children ([@B1]--[@B3]). A subset of mTBI patients suffers from acute **post-concussion symptoms** that may manifest as somatic symptoms (e.g., nausea, dizziness, headache, blurred vision, auditory disturbance, and fatigue), cognitive complaints (memory and executive function), emotional, and/or behavioral problems (e.g., disinhibition and emotional lability) ([@B4]--[@B6]).
In 10--25% of mTBI patients, post-concussion symptoms persist over time ([@B7]--[@B10]), which is often referred to as **post-concussion *syndrome* (PCS)**. PCS is usually diagnosed according to the International Classification of Diseases (ICD)-10 ([@B5]), or following Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria ([@B6]). However, over the last 15 years the concept of PCS as a reliably identifiable, unique syndrome has been questioned ([@B11], [@B12]). Therefore, we will use the term post-concussion symptoms to describe symptoms following mTBI and will refer to *persistent* post-concussion symptoms when these persist for at least 3 months after TBI.
This focused review (based on a systematic literature search until March 1st 2018, see Appendix [A](#SM1){ref-type="supplementary-material"}) summarizes current knowledge on epidemiology, controversies, etiology, assessment and treatment of post-concussion symptoms in adults and children. Understanding the various factors leading to post-concussion symptoms, and the complex interactions between temporal onset, biological, psychological and social factors, as well as the relative influence of injury-related and non-injury related factors, may contribute toward a better understanding, diagnosis and classification of post-concussion symptoms. Figure [1](#F1){ref-type="fig"} shows current topics in research on post-concussion symptoms. In addition, an insight into the wide range of assessment methods and possible treatments may provide guidance for both clinicians (e.g., physician, psychologist, neuropsychologist, neurosurgeon, nurse, physical therapist, and occupational therapist), social worker and policy-makers.
![A model for the study of post-concussion symptoms after mTBI. Permission has been obtained to model our figure based on Yeates ([@B13]), © The International Neuropsychological Society 2010, published by Cambridge University Press.](fneur-09-01113-g0001){#F1}
Materials and Methods {#s2}
=====================
Search Strategy and Selection Criteria
--------------------------------------
We searched EMBASE and MEDLINE from Jan 1th, 2010 to March 1st 2018, where landmark papers with earlier dates were also integrated. We included papers published in peer-reviewed English language journals, identifying observational, experimental, and intervention studies and reviews in civilian mTBI patients with short- and long-term (3--36 m) post-concussion symptoms or syndrome. See Appendix [A](#SM1){ref-type="supplementary-material"} for the EMBASE search strategy. Additional papers were identified by screening reference lists and citation indices and from authors\' own files.After removal of duplicates, articles were excluded on the basis of title and abstract by two reviewers (MC and DV).
To warrant a minimum level of quality, papers were selected for current review if they were either (systematic) reviews or prospective cohort studies that included ≥100 patients. Exceptions were imaging studies, where lower numbers of patients were allowed and studies about treatment, where we prioritized (randomized) controlled trials. If these studies were not available, we used either retrospective or case-control studies, studies with lower number of patients or papers published before 2010. In cases where included papers did not meet our quality threshold \[review, prospective cohort ≥100 patients or randomized controlled trials (RCT)\], this was explicitly mentioned.
Definitions and Epidemiology {#s3}
============================
Mild Traumatic Brain Injury (mTBI)
----------------------------------
The American Congress of Rehabilitation Medicine (ACRM) ([@B4]) defines mTBI as an "acute brain injury resulting from mechanical energy to the head from external physical forces," with any of the following symptoms: loss of consciousness (LOC) not exceeding 30 min, post-traumatic amnesia (PTA) of no more than 24 h, a score of no \<13 on the Glasgow Coma Scale (GCS) after 30 min post injury (or upon presentation) ([@B14]), and an (unspecified) period of confusion (feeling dazed, disoriented, and confused), or other transient neurologic abnormalities such as focal signs or seizures.
Most mTBI patients do not show trauma-related abnormalities on computed tomography (CT) scans. However, the literature on mTBI frequently distinguishes between *complicated* and *uncomplicated* mTBI and the term *complicated mTBI* is often used to refer to e.g., 5--10% of emergency department (ED) patients ([@B15]) who show abnormalities, such as subarachnoid hemorrhage, intracranial contusions, or small extra-axial hematomas. The prevalence of pediatric mTBI based on emergency department visits are likely underestimated in childhood as studies have demonstrated that most children initially seek care with their primary care doctor for these mild injuries ([@B16]). In children, findings on CT are even more rare ([@B17]) and multiple effective clinical prediction rules have been developed to reduce unnecessary CT use in children ([@B18]). Special consideration should be given for children \<2 years of age with regard to decision-making about the use of CT scans in the setting of head trauma.
Diagnosis of Post-concussion Syndrome
-------------------------------------
PCS is usually defined according to DSM-IV or ICD-10 criteria, which both focus on symptom presentation ([@B19]). These manuals agree on the prerequisite history of brain trauma for the diagnosis of post-concussional disorder \[DSM-IV ([@B6])\] or PCS \[ICD-10 ([@B5])\]. Differences between diagnostic systems are presented in Table [1](#T1){ref-type="table"}. An important difference is that DSM-IV requires immediate symptom onset and persistence for at least 3 months whereas ICD-10 does not. In addition, DSM-IV requires objective evidence of memory or attention deficits (criterion B), but ICD-10 explicitly precludes such evidence (criterion C-3). The variability in terminology and associated criteria of the DSM-IV and ICD-10 hampers accurate identification and diagnosis of patients with PCS ([@B13]). Different classification methods may result in overestimation or underestimation of symptoms, particularly when relying on subjective endorsement of symptoms by patients. This was shown in a cross-sectional study in which 61 patients were referred to a concussion clinic following mTBI ([@B20]).
######
Comparison of three definitions of post-concussion symptoms.
**ICD-10** **DSM-IV** **DSM-5**
------------------------------------------ ----------------------------------- ----------------------------------- -----------------------------------
Headache √ √ --
Dizziness √ √ --
Fatigue √ √ --
Noise intolerance √ √ --
Irritability/lability/anxiety/depression √ √ --
Sleep problems √ √ --
Concentration problems √[^A^](#TN1){ref-type="table-fn"} √[^B^](#TN2){ref-type="table-fn"} √[^B^](#TN2){ref-type="table-fn"}
Memory deficit √[^A^](#TN1){ref-type="table-fn"} √[^B^](#TN2){ref-type="table-fn"} √[^B^](#TN2){ref-type="table-fn"}
Intolerance of alcohol √ -- --
Preoccupation with symptoms √ -- --
Personality change -- √ --
Apathy -- √ --
Perceptual-motor -- -- √[^B^](#TN2){ref-type="table-fn"}
Social cognition -- -- √[^B^](#TN2){ref-type="table-fn"}
*Table shows symptoms presented in the International Classification of Diseases (ICD)-10 definition of PCS (diagnosis code F07.02), the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV definition of postconcussional disorder and the DSM-V definition of neurocognitive disorder*.
*Subjective report*.
*Objective test*.
Post-concussional disorder was not included in the last DSM-5 edition ([@B21]). Instead, DSM-5 contains "mild neurocognitive disorder due to TBI," a neurocognitive disorder, which strongly suggests---but does not formally require---performance-based, quantifiable evidence of acquired cognitive deficits after mTBI (Table [1](#T1){ref-type="table"}). Importantly, DSM-5 denotes the status of the most frequently reported post-concussion symptoms to the level of "associated features." Finally, DSM-5 emphasizes a broad range of differential diagnoses, especially when symptom severity "appears to be inconsistent with the severity of the TBI" ([@B22]).
Prevalence of Post-concussion Symptoms
--------------------------------------
Prevalence of post-concussion symptoms varies and depends on pre-injury factors ([@B10], [@B23]), patient population ([@B24]), assessment ([@B24]), and analytic strategies, diagnostic criteria ([@B24], [@B25]), and classification methods ([@B26]). Overall, single symptoms (e.g., fatigue, headache, and cognitive symptoms) are very common ([@B27]) (Figure [2](#F2){ref-type="fig"}), whereas multiple concurrent symptoms are less frequent ([@B24]).
![The prevalence of post-concussion symptoms over time. *Permission has been obtained to base our figure on* data presented in Theadom ([@B27]).](fneur-09-01113-g0002){#F2}
Neuropsychological testing consistently shows minor cognitive deficits within the first 2 weeks after injury, with some exploratory evidence suggesting deficits lasting up to 6 months ([@B28]). It has been suggested that self-reported somatic symptoms (headaches, dizziness) are more prevalent immediately after the injury (1--2 weeks) ([@B29]), whereas cognitive and emotional symptoms resolve more slowly and may still be above baseline levels long-term post-injury ([@B30], [@B31]). However, these cross-sectional analyses did not track the evolution of symptoms in single patient groups. Therefore, evidence supporting a differential trajectory between self-reported somatic and cognitive/emotional subacute symptoms is limited.
ICD-10 prevalence rates of PCS at 3 months post-injury vary between 6% ([@B32]), 22% ([@B33]), and 64% ([@B25]). DSM-IV diagnostic criteria appear to be stricter than ICD-10 criteria leading to lower estimates ([@B34]): a cohort study of patients after mTBI found a prevalence of PCS at 3 months of 64% based on ICD-10 criteria, but only a prevalence of 11% when using DSM-IV ([@B25]).
Few pediatric studies report on the prevalence of post-concussion symptoms according to ICD-10 or DSM-IV diagnostic criteria; 1-month prevalence for children recruited from ED based on ICD-10 reach 52% ([@B35]) and 3-month prevalence based on DSM-IV constitutes 29.3% ([@B36]). Some studies define symptomatic children as having an increase in at least one symptom and arrive at estimates between 24.5 and 52.5% at 1 month post injury ([@B35], [@B37]), 11--39% after 3 months, and 2.3% at 12 months ([@B35]), which makes comparison of symptom development trends between children and adults challenging. An additional complication in capturing prevalence rates in children is that younger children may not be able to describe their symptoms reliably. Therefore, such prevalence estimates should be treated with caution.
Controversies {#s4}
=============
Post-concussion symptoms are highly controversial and a major topic of debate among clinicians, methodologists, and health outcome experts. One problem is that post-concussion symptoms do not always cluster in a consistent and predictable manner ([@B12], [@B19]). Therefore, it is controversial whether they truly represent a specific, cohesive, and predictable syndrome (i.e., PCS) ([@B12], [@B19]). In addition, although the term post-concussion symptoms might suggest otherwise, these symptoms are not specific to TBI but are also frequently reported in non-brain injured trauma patients ([@B10]), including patients with whiplash injuries ([@B38]) and in healthy adults and children ([@B35], [@B39], [@B40]).
The literature on mTBI frequently uses the term "symptom" to refer to all changes experienced after a concussion. However, when focusing solely on the patient\'s self-report, the use of the term "complaint" might be more appropriate.
Similarly, the etiology of post-concussion symptoms is also debatable. Although the biopsychosocial model is often applied to explain the onset and persistence of post-concussion symptoms ([@B41]), post-concussion symptoms have also been associated with malingering, exaggeration, misattribution, and recall bias, thereby prompting concern regarding the clinical reality of post-concussion symptoms.
Acute and Persistent Post-concussion Symptoms
---------------------------------------------
Acute post-injury symptoms, such as headache, dizziness, sensitivity to light or noise, double vision or tinnitus, are associated with the development of persistent symptoms ([@B19], [@B42], [@B43]). A clinical risk score in children has identified headache, sensitivity to noise, fatigue and answering questions slowly as predictive of post-concussion symptoms at 28 days post-injury ([@B44]). In addition, the experience of post-concussion symptoms early post-injury (1 week−1 month) is consistently associated with higher odds of persistent post-concussion symptoms ([@B10], [@B45]). A study from 2015 found that 82% out of 103 patients who were experiencing post-concussion symptoms 1 year after mTBI had already reported these 1 month post injury ([@B46]).
Biological Factors and Persistent Post-concussion Symptoms
----------------------------------------------------------
Several, predominantly biological factors, such as diffuse axonal injury, neuro-inflammation, and altered cerebral blood flow have been implicated in the genesis of post-concussion symptoms after mTBI ([@B41], [@B47], [@B48]). However, these factors have not yet been analyzed in high-quality prognostic studies. The role of biological factors is supported by findings that repetitive mTBI is associated with increased symptom prevalence ([@B49], [@B50]), longer time to symptom resolution ([@B50], [@B51]), and a minimal effect of neurocognitive deficits ([@B52]). Similarly, repetitive sub-concussive impacts, e.g., in contact sports, have also been associated with minor long-term neuropsychological sequelae ([@B53]), abnormalities in both neuroimaging and in neuropsychological testing ([@B54]), and with the development of severe neurodegenerative conditions such as chronic traumatic encephalopathy (CTE) ([@B54]). Although many symptoms of CTE overlap with post-concussion symptoms (e.g., irritability, impulsivity, depression, (short-term) memory loss), current evidence on the association of repetitive sub-concussive impacts with CTE is limited and should be considered preliminary ([@B55]).
A major controversy in attempting to identify the role of biological factors in the development of post-concussion symptoms is their weak relationship with injury severity and the high prevalence of PCS-like symptoms in non-brain injured patients, as well as in healthy participants ([@B10], [@B35], [@B37]--[@B40], [@B45], [@B56]).
Even though most studies report that the rate of post-concussion symptoms is higher among brain-injured patients as compared to non-brain injured trauma controls ([@B32], [@B46], [@B57]--[@B59]), the high rate of false-positives needs to be taken into account when examining biological factors. It should be acknowledged that biological factors do not exist in isolation but need to be interpreted in the context of potentially confounding factors, e.g., pre- and post-injury physical and mental health, trauma, and psychosocial factors ([@B10], [@B58]--[@B60]).
Psychiatric, Psychological, (Psycho)-Social Factors and Post-concussion Symptoms
--------------------------------------------------------------------------------
### Psychiatric Factors
Many post-concussion symptoms (e.g., sleep difficulties, irritability and concentration problems) are similar to symptoms of the hyperarousal dimension of posttraumatic stress disorder (PTSD) ([@B59]), which may occur following exposure to severe, often life-threatening events. PTSD following mild, moderate, or severe TBI has a pooled prevalence rate of 13.8% (10.2--17.4%) ([@B61]) and appears to follow TBI more frequently than any other traumatic injuries not involving the brain ([@B47], [@B62]). Given the overlap between post-concussion and PTSD symptoms ([@B59], [@B62], [@B63]), careful differential diagnosis is required. Nevertheless, a prospective study including 534 brain-injured patients and 827 controls found that mTBI was a significant predictor for PTSD but not for post-concussion symptoms ([@B59]). It is not yet clear, whether these results also hold true for pediatric samples. A smaller prospective study comparing parent-reported post-concussion symptoms and PTSD symptoms in 186 children after mTBI and 99 children with non-head orthopedic injuries reported higher rates of post-concussion symptoms after mTBI but comparable rates of PTSD symptoms ([@B63]).
Almost half of patients with persistent post-concussion symptoms suffer from premorbid depression and anxiety ([@B47], [@B64]). Pre-injury mental health status has repeatedly been shown to predict persistent post-concussion symptoms in adult ([@B41], [@B45], [@B47], [@B65]) and pediatric populations ([@B13], [@B35]). However, the question of causality remains unclear, as psychiatric symptoms might be a reaction to experiencing persistent post-concussion symptoms, and/or mental health problems might increase the risk of reporting persisting symptoms.
### Psychological Factors
Recall biases have been shown to influence reports of post-concussion symptoms after mTBI. Patients after mTBI expecting to experience post-concussion symptoms show higher symptom rates than patients not expecting to experience post-concussion symptoms ([@B66]). Similarly, in some patients the "good-old-days" bias may lead them to underestimate pre-injury symptoms ([@B41], [@B57]). If gross overrepresentation of symptoms is suspected (malingering), performance in selected neuropsychological tests can indicate whether the patient is exerting optimal effort ([@B11], [@B67]).
Finally, symptoms commonly occurring in everyday life, such as headache, irritability, sleep disturbance and forgetfulness may be misattributed to brain trauma ([@B11], [@B33]). Extensive assessments for putative somatic origins of such common symptoms may further make one believe that these symptoms are indicative of serious brain damage, leading to hypervigilance and catastrophic attributions, comparable to behaviors seen in patients with somatoform disorders or hypochondriasis ([@B11], [@B33], [@B62], [@B67], [@B68]).
### Socio-Demographic, Social, and Personality Factors
Female sex is consistently associated with greater reporting of persistent post-concussion symptoms ([@B45]). Gender effects appear to be smaller in children ([@B35], [@B37], [@B56]). Some studies found that post-concussion symptoms are associated with lower education in adults ([@B45]) and pre-injury learning difficulties in children ([@B37]). Community integration, social support, lifestyle, and family dynamics may contribute to the development and persistence of post-concussion symptoms in adults ([@B41], [@B69]), and children ([@B13], [@B70]). However, conclusive evidence has not yet been established.
The five-factor model of Widiger and colleagues is a model of basic personality traits, consisting of five domains: neuroticism, extraversion, openness, agreeableness, and conscientiousness ([@B71]). Basic personality traits as captured in the five-factor model do not appear to be associated with persistent post-concussion symptoms ([@B10]). However, more specific traits such as high anxiety sensitivity ([@B72]), low resilience ([@B73]), coping styles ([@B33], [@B74]) or alexithymia ([@B72]) may be associated with persistence of symptoms. However, the cross-sectional design and small sample sizes in these studies hamper the establishment of firm conclusions in the area.
### Predicting Persistent Post-concussion Symptoms
The identification of risk factors might be especially useful for clinical practice when combined into a prognostic model predicting patients at risk of poor outcome. However, current models are often based on small samples ([@B9], [@B75]) and lack internal and external validation ([@B10], [@B45], [@B75], [@B76]). In addition, no model is able to reliably predict outcomes at the individual patient level ([@B45]). Therefore, identification of high-risk patients might best be accomplished by careful and dense follow-up data collection. Advances in study and modeling methodology and, possibly, the incorporation of advanced imaging, and biochemical biomarkers (see Panel [1](#Box1){ref-type="boxed-text"} for recommendations) may improve the ability to identify at-risk patients in the first week post-injury in the future.
###### Methodological recommendations for studies on post-concussion symptoms after mTBI.
Well-designed confirmatory studies with the following characteristics have been called for to better understand post-concussion symptoms and its consequences:
1. \- **Study design**: Prospective inception cohort studies with appropriate control group (e.g. non-brain injured patients, general population) and appropriate follow-up period to differentiate persistent deficits and symptoms due to post-concussion symptoms from the effects of pre-injury (neuro)psychiatric disorders and other non-mTBI factors. Longitudinal analyses strategies to monitor evolution of post-concussion symptoms in single patients.
2. \- **Instruments**: use crosswalk analysis to compare incidence rates between studies using different post-concussion symptom assessment procedures. At a minimum, aim to include at least some comparable items, i.e., items whose functioning is comparable between patient samples, and evaluate other items relative to these anchor items. **Studies on predictors/prediction models** (based on Mushkudiani et al. ([@B77]) and Steyerberg ([@B78]):
1. ° Sample size: N \> 500
2. ° Predictors should be based on theory, clinical knowledge or previous research
3. ° For every predictor considered there should be at least ten cases (i.e., patients classified as having PCS)
4. ° A liberal *p*-value (e.g., *p* \< 0·157) ([@B79]) should be used when applying selection procedures
5. ° Results should be internally validated (e.g., bootstrap validation)
6. ° Both discrimination and calibration statistics should be mentioned; a score chart is warranted for implementation in clinical practice
7. ° External validation: external validation in an independent dataset is a prerequisite before implementation in clinical practice. External validation and updating of an existing model should be prioritized against the development of a new model.
Clinical Assessment of Post-concussion Symptoms {#s5}
===============================================
Providing optimal care depends on early and reliable identification of patients at risk of developing persistent post-concussion symptoms ([@B12], [@B80]) by a multidisciplinary team. Medical examination should include a history of previous TBIs, head and neck injuries, and a detailed description of the number and extent of acute concussion symptoms, preferably using standardized instruments (see Table [2](#T2){ref-type="table"}). Special emphasis should also be placed on the assessment of co-morbid injuries and disorders, such as chronic headache, and other pain, cervical-disorders, visual or vestibular disorders, chronic fatigue, sleep, and somatoform disorders ([@B35], [@B65], [@B80], [@B81]). However, checklists alone are not sufficient to provide a *diagnosis* of persistent post-concussion symptoms as a disorder in the absence of a comprehensive multidimensional medical, neurological, and psychiatric and (neuro)psychological evaluation ([@B64], [@B82]).
######
Selection of Post-concussion symptoms assessments (adults and children) based on CDE recommendations and frequent clinical use.
**Assessments** **Examinations and instruments** **Population**
---------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------
Clinical Examination and History Standardized medical history and history of injury event, neurological and physical examination including orientation, speech fluency, memory, concentration, dyslexia, dizziness, vertigo, sleep, cranial nerves, motor, sensory and gait assessment; balance and vestibular testing; respiratory and heart rate, blood pressure; Cervical spine range of motion and tenderness; comprehensive headache assessment; neuroimaging (if mandated by neurological deficits) A/P
Standardized pre- and post-injury anamnesis of depression, anxiety, stress, dissociation, behavior, and other mental health problems retro- and prospective assessment: e. g. Structured Clinical Interview-DSM, Mini International Neuropsychiatric Interview (v 5.5),
Diagnostic Interview Schedule for Children-IV, Neuropsychiatric Rating Schedule (NPRS), Clinician-administered PTSD Scale (CAPS)
Self-reported post-concussion symptoms Health and behavior inventory[^\*^](#TN4){ref-type="table-fn"} P
Neurobehavioral symptom inventory[^\*\*^](#TN3){ref-type="table-fn"} A
Post-concussion symptom inventory[^\*\*^](#TN3){ref-type="table-fn"} P
Rivermead post-concussion symptom questionnaire[^\*^](#TN4){ref-type="table-fn"} A
Neuropsychological Impairments Behavior rating inventory of executive function[^\*\*^](#TN3){ref-type="table-fn"} P
Rey auditory verbal learning test[^\*^](#TN4){ref-type="table-fn"} A/P
California verbal learning test for children[^\*^](#TN4){ref-type="table-fn"} P
Delis-kaplan executive function system---verbal fluency[^\*^](#TN4){ref-type="table-fn"} P
Immediate post-concussion assessment and cognitive testing[^\*\*^](#TN3){ref-type="table-fn"} A/P
Trail making test (TMT)[^\*^](#TN4){ref-type="table-fn"} A
TRAILS-PRESCHOOL[^\*\*^](#TN3){ref-type="table-fn"} P
Cognitive battery-NIH toolbox[^\*\*^](#TN3){ref-type="table-fn"} A/P
Wechsler abbreviated scale of intelligence[^\*^](#TN4){ref-type="table-fn"} P
Wechsler adult intelligence scale[^\*^](#TN4){ref-type="table-fn"} A
Wechsler intelligence scale for children-iv[^\*^](#TN4){ref-type="table-fn"}/wechsler preschool and primary scale of intelligence -III P
Psychological and psychiatric status Brief-symptom-inventory-18[^\*^](#TN4){ref-type="table-fn"} A
Beck-depression inventory II[^\*\*^](#TN3){ref-type="table-fn"} A/P
Child behavior checklist[^\*\*^](#TN3){ref-type="table-fn"} P
Patient health questionnaire-9[^\*\*^](#TN3){ref-type="table-fn"} A/P
Screen for Child Anxiety Related Emotional Disorders (SCARED)[^\*\*^](#TN3){ref-type="table-fn"} P
Minnesota Multiphasic Personality Inventory (MMPI)[^\*\*^](#TN3){ref-type="table-fn"} A
Posttraumatic Stress Disorder Checklist (PCL)[^\*\*^](#TN3){ref-type="table-fn"} A
Short Mood and Feelings Questionnaire (SMFQ)[^\*\*^](#TN3){ref-type="table-fn"} A/P
Alcohol Use disorders identification test: self-report version (AUDIT)[^\*\*^](#TN3){ref-type="table-fn"} A
Symptom validity Test of memory malingering (TOMM)[^\*\*^](#TN3){ref-type="table-fn"} A/P
Medical symptom validity test[^\*\*^](#TN3){ref-type="table-fn"} A/P
Family and environment Family Assessment Device (FAD)[^\*\*^](#TN3){ref-type="table-fn"} A/P
Child and Adolescent Scale of Environment (CASE)[^\*\*^](#TN3){ref-type="table-fn"} P
Family Burden of Injury Interview (FBII)[^\*\*^](#TN3){ref-type="table-fn"} P
Common Data Elements (CDEs) recommended as basic measure;
*CDEs recommended as supplemental measure; A, Adult TBI; P, Pediatric TBI*.
Since persistence of post-concussion symptoms has been associated with pre-, peri-, and post-injury psychological distress and risk of psychiatric disorders (PTSD, depression, anxiety, substance abuse, somatoform disorders), anamnesis should also include an assessment of pre-injury and current mental health difficulties (see Table [2](#T2){ref-type="table"}) ([@B10], [@B28], [@B61], [@B64]). Finally, information on social and legal factors, such as availability of social support, life stressors, and involvement in legal proceedings needs to be collected ([@B81]).
A variety of symptom checklists exist to assess somatic, emotional, and cognitive post-concussion symptomatology, and require patients to indicate presence, absence, frequency, or intensity/severity of symptoms. Neuropsychological performance based outcomes include measures of attention, memory, concentration, orientation and executive function, and can corroborate subjective complaints of impaired cognition. However, cognitive deficits after mTBI are usually transient ([@B28]) and appear to be only weakly related to subjective complaints ([@B23]). Standard neuropsychological procedures should be followed to ensure that test results are not influenced by comorbid disorders \[e.g., attention deficit hyperactivity disorder, and dyslexia ([@B83], [@B84])\], or inadequate understanding of test and questionnaire requirements, or low effort ([@B85]). Currently, only the field of sport concussion utilizes short reliable and sensitive screening instruments (7--10 min) to identify possible symptoms ([@B86]). A comprehensive overview of instruments suitable for clinical assessment is presented in Table [2](#T2){ref-type="table"}. This overview is based on common data elements (CDE) recommendations ([@B87]--[@B89]) and frequent clinical use.
Neuroimaging and Persistent Post-concussion Symptoms
----------------------------------------------------
No consensus has been reached on the relevance of imaging indicators of brain abnormalities for prognosis and outcome after mTBI. Figure [3](#F3){ref-type="fig"} presents magnetic resonance imaging (MRI) images of patients with post-concussion symptoms. Several studies have shown that measures derived from MRI ([@B80], [@B90]--[@B92]) or magnetic resonance spectroscopy (MRS) can reveal structural or functional abnormalities in adults and children with an otherwise normal CT ([@B35]). Thus, for some patients, persistence of post-concussion symptoms may be explained by yet unknown brain abnormalities. However, current evidence is equivocal and the few large-scale, prognostic studies available suggest only small effects ([@B93]), if at all.
{#F3}
Post-concussion Symptoms and Outcome: Health Related Quality of Life, Return to Work, and Societal Costs
--------------------------------------------------------------------------------------------------------
Health outcome can be classified along three dimensions: health-related quality of life (HRQoL), functional, and economic outcome. Available studies suggested that post-concussion symptoms correlate with lower levels of life-satisfaction ([@B69], [@B94]) and HRQoL ([@B95]). HRQoL measures supplement functional and mental health outcomes with information on how health conditions influence patients\' self-reports of their subjective well-being. HRQoL represents an important outcome after TBI, as it provides well-standardized information on the recovery patterns and frequency, nature, severity, and duration of the functional consequences ([@B96]). Post-concussion symptoms have been linked to lower levels of satisfaction with life ([@B69]) and HRQoL in adults ([@B94]) and children ([@B97]). However, given the association of pre-injury physical and mental health status with persistent post-concussion symptoms, the specificity of these findings is still unclear. Further research is needed to isolate the specific effects of persistent symptoms on HRQoL ([@B14]).
Furthermore, post-concussion symptoms are associated with reduced return to work ([@B69], [@B98], [@B99]). There is a need to focus on the management of persistent post-concussion symptoms to facilitate return to work ([@B100]).
The societal costs of TBI include direct medical costs and indirect expenses related to the illness and the value of lost production due to reduced working time or impaired work performance. A large part of the total lifetime costs in the field of TBI are associated with mTBI. The high incidence of mTBI, combined with a large group of patients with long-term post-concussion symptoms, results in a substantial societal and economic burden ([@B101]).
Carefully designed longitudinal research on HRQoL, functional recovery, costs and return to work is needed to differentiate persistent deficits and symptoms due to post-concussion symptoms from the effects of pre-injury neuropsychiatric disorders and other factors not associated with mTBI ([@B14]).
Management of Patients With Post-concussion Symptoms {#s6}
====================================================
Pharmacological Interventions
-----------------------------
The evidence for pharmacological treatment of depression, anxiety, and mood lability after mTBI is limited and conflicting. A meta-analysis evaluating the effectiveness of depression treatment after mTBI found that studies using a pre-post design suggested treatment benefits from selective serotonin reuptake inhibitors ([@B102]). In contrast, the overall effects of controlled trials included in this meta-analysis did not reveal significant differences between treatment and control groups, with some evidence favoring the control condition ([@B102]). However, a recently published RCT found sertraline to be effective in preventing depression following TBI when administered early after injury ([@B103]). These findings may have considerable therapeutic implications for patients after TBI, but future studies are needed to replicate results before a change in the treatment guidelines could be recommended.
Non-pharmacological Interventions
---------------------------------
Evidence concerning the benefits of non-pharmacological interventions targeting post-concussion symptoms is limited. Early educational interventions in ED patients after mTBI may be promising in reducing the incidence and severity of post-concussion symptoms since a single-center RCT focusing on symptom management delivered via telephone counseling demonstrated reduced chronification of post-concussion symptoms during the first 3 months post-injury ([@B104]). This finding could not be replicated in a multi-center study; however the investigated patients showed mixed severity of TBI ([@B105]).
A recent study suggests that cognitive behavioral therapy (CBT) can improve HRQoL in patients with persistent post-concussion symptoms in the context of outpatient rehabilitation services ([@B106]). However, the effect of CBT on post-concussion symptoms was only marginal ([@B106]). Problem orientation and problem-solving skills seem to improve by neuropsychological rehabilitation addressing self-regulation of cognitive and emotional processes ([@B107]), but evidence is limited.
Evidence for beneficial effects of neuropsychological rehabilitation concerning post-concussion symptoms is limited. A systematic review found evidence that, when applied early, such approaches may be efficient in reducing self-reported post-concussion symptoms, anxiety and depression, but do not result in a clear reduction of cognitive impairment ([@B108]).
Intervention studies in children and adolescents are highly variable, of limited methodological quality, and evidence to support any particular intervention for post-concussion symptoms in pediatric samples is absent ([@B109], [@B110]). In adults, as in pediatric populations, well-designed prospective studies focusing on non-pharmacological multidimensional intervention that show improvement on variables such as HRQoL and return to play and work are still lacking.
Rest and Post-concussion Symptoms
---------------------------------
Historically, "rest" has been a foundation in the treatment of acute mTBI ([@B70]). Concerns have been raised regarding the expert-based consensus recommendation for rest after acute concussion, as studies in adults ([@B111], [@B112]) and children ([@B113]) indicate that prolonged rest, longer than 3 days to a week may contribute to prolonged symptomatology ([@B114]), and no reduction in post-concussion symptoms was found in a study on rest interventions ([@B115]).
Vestibular and Vision Rehabilitation Therapy
--------------------------------------------
The traumatic event resulting in mTBI may also cause concomitant cervical soft tissue damage, resulting in "whiplash-related" symptoms such as headache, dizziness, and balance dysfunction as well as cognitive, vestibular and visual dysfunction ([@B38]). A RCT comparing cervical spine physiotherapy and vestibular rehabilitation therapy (VRT) with a control condition in athletes found that among the intervention group, a significantly higher proportion of individuals were medically cleared after 8 weeks of treatment ([@B116]). However, a recent systematic review concluded that current evidence for optimal prescription and efficacy of VRT in patients after mTBI is still limited ([@B117]). In addition, large retrospective cohorts including both adults and children examining vision rehabilitation for vision disorders associated with mTBI have demonstrated clinical improvement in conditions such as convergence and accommodative insufficiency ([@B118]--[@B120]). Thus, high-level studies evaluating the effects and optimal intervention window for VRT and vision rehabilitation are required.
Headaches
---------
Headaches are among the most disabling symptoms after mTBI. Most post-traumatic headaches show clinical features of a recognized primary headache, such as migraine headaches or tension headaches. Post-traumatic migraines may respond to the same abortive and prophylactic treatments as sporadic migraines ([@B121]). In addition, non-pharmacological approaches such as biofeedback, physical therapy, CBT, either as primary or adjunctive treatments, have also been successfully applied to persistent post-concussion headaches ([@B65], [@B122]).
Conclusions and Future Directions {#s7}
=================================
Despite a sharp increase in studies investigating post-concussion symptoms, controversies and debates still exists with regard to etiology, diagnosis, classification systems, pathophysiology, natural history, prevalence, and terminology. The subjective nature of post-concussion symptoms, their low specificity, and the significant overlap with other physical, neurological, and psychiatric conditions add additional challenges to these discussions ([@B10], [@B12], [@B19], [@B39], [@B44], [@B45], [@B56], [@B59], [@B82]). The frequent overlap and idiosyncratic interplay of post-concussion symptoms with pre- and post-injury psychiatric, psychological and social factors are still under-investigated and necessitate a standardized comprehensive differential diagnosis of comorbid mental conditions, in particular depression, anxiety disorders and PTSD.
In this review, we described possible factors contributing to post-concussion symptoms from a bio-psychosocial perspective. Insights into the complex nature of post-concussion symptoms may support the risk estimate of persistent symptoms in individual patients. In addition, it may provide targets for predictive modeling which combine the different factors contributing to post-concussion symptoms. Currently, no valid model is available to predict post-concussion symptoms in adults and children ([@B45], [@B75]). Future predictive modeling studies could be improved by using solid methodology (see Panel [1](#Box1){ref-type="boxed-text"}). However, the feasibility of predictive modeling may be debated given the complex, controversial, and multifactorial nature of post-concussion symptoms. Therefore, investing in routine and economic follow-up methods (e.g., smartphone-based experience sampling approaches which have demonstrated feasibility and utility in the post-injury setting ([@B123], [@B124]) might be prioritized over predictive models.
The frequent reliance on simple symptom questionnaires for diagnosis ignores possible biases ([@B10]) and the fact that the major classification systems require several other criteria to be fulfilled, such as performance-based evidence of cognitive impairment ([@B21]). Most questionnaires were developed in and for patients with more severe deficits, thus their sensitivity and specificity in mTBI may be limited. More refined neuropsychological tests, especially those sensitive enough to assess cognition after mTBI, may support the diagnosis of post-concussion symptoms. Moreover, short screening batteries (computerized and paper and pencil) are needed for use in EDs and in general practice. This is aligned with international attempts at developing and implementing standards for clinical research (e.g., CDEs) ([@B87]), terminology and diagnosis criteria for post-concussion symptoms.
The heterogeneous nature of mTBI and post-concussion symptoms and the lack of reliable biological predictors and clinically useful gold-standard biomarkers ([@B34]) hamper the development of disease-modifying therapies. A first step may be the identification of specific biochemical ([@B125]) and imaging biomarkers that can complement clinical diagnosis, inform prognosis by identifying patients at risk for post-concussion symptom persistence, and predict treatment response ([@B90], [@B101]). Portable, lower-cost imaging modalities such as functional near infrared spectroscopy warrant further investigation to determine their clinical utility in diagnosis and management of mTBI ([@B126]).
Large-scale multidimensional, prospective longitudinal studies with several measurement points are strongly required to tackle current challenges in studying post-concussion symptoms. Such designs would allow stratified subgroup analyses to identify patients at risk for developing persistent symptoms, and might help to advance early and personalized treatment. Depending on the research question, improved designs should include control groups to provide insight into the spontaneous recovery, progression, injury severity, frequency, intensity, and fluctuation (trauma controls and healthy participants) of post-concussion symptoms.
Due to normal variation in developmental trajectories, outcomes in children after mTBI may be particularly variable. Longitudinal large sample studies (\>100) that investigate predictors of post-concussion symptoms in pediatric populations with multiple endpoints, adequate controls are especially important since high neurologic and cognitive plasticity is present here.
Although evidence for effective treatments is limited, a multi-disciplinary approach corresponding to the complex etiology of post-concussion symptoms may be the most promising. Such an approach would combine in-depth comprehensive medical and neurological diagnosis with an emphasis on psychiatric differential diagnostics and psychosocial und neuropsychological outcome assessment. Future treatment directions (repetitive transcranial magnetic stimulation, vestibular and vision rehabilitation therapy, and aerobic exercise) may offer a solution for the basic pathological processes associated with post-concussion symptoms ([@B65]).
Standardization of treatment and interventions, outcome measures ([@B87]), and follow-up assessment time-points would enhance reliability and validity of research comparisons and individualized treatment. One might speculate as to whether post-concussion symptoms represent the most valid endpoint for treatment/study after mTBI. Given their low specificity, it may well be that other outcomes (e.g., functional outcome and HRQoL) prove to be more useful.
In this focused review, we only included prospective cohort studies with at least 100 participants, and reviews, with some exceptions (Appendix [A](#SM1){ref-type="supplementary-material"}). Ten included studies did not meet these criteria ([@B20], [@B29], [@B30], [@B39], [@B40], [@B57], [@B72], [@B83], [@B84], [@B127]). For these topics, there was no prospective study with at least 100 participants available. Therefore, prospective, multicenter research with larger patient samples is needed. In addition, it should be noted that studies fulfilling our quality criteria might still be at risk of bias. Attrition is a recurrent problem ([@B45], [@B75]), that may have influenced the reported prevalence rates, the relevance of etiological factors and treatment effectiveness. In addition, some studies of etiological factors were based only on univariable analyses, while multivariable assessment is highly recommended because of the multifactorial nature of post-concussion symptoms.
To summarize, standardization of the multidimensional comprehensive diagnostics, treatment interventions, and follow-up assessment time-points may enhance reliability and validity of research comparisons and refine personalized treatment and care. This review documents the need for future studies to target the identification of potential mechanisms, new imaging techniques, comprehensive multidisciplinary assessment and treatment options. Longitudinal, well controlled studies applying standardized diagnostic assessment strategies and evidence-based interventions are needed in adult and pediatric mTBI populations to optimize recovery and reduce burden of post-concussion symptoms.
Author Contributions {#s8}
====================
SP and NvS wrote and revised the manuscript, finalized the paper based on authors, and reviewer feedback. MC conducted the search strategy, screened papers, extracted data of eligible papers, wrote, and revised the manuscript. RD-A, RR, JH, AC, CM, AG, and DV wrote and revised the manuscript. All authors critically reviewed and approved the final version of the paper.
Conflict of Interest Statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The paper has been written in the context of the CENTER-TBI and TRACK-TBI projects, large scale collaborative projects embedded within the framework of the International initiative on TBI research. CENTER-TBI has received funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement n° 602150. TRACK-TBI has received funding from the National Institutes of Neurologic Disorders and Stroke, grant n° U01 NS086090. We would like to thank Professor Andrew Maas for critically reviewing the manuscript.
Supplementary Material {#s9}
======================
The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fneur.2018.01113/full#supplementary-material>
######
Click here for additional data file.
[^1]: Edited by: Nada Andelic, University of Oslo, Norway
[^2]: Reviewed by: Solrun Sigurdardottir, Sunnaas Sykehus, Norway; Torgeir Hellstrøm, Oslo University Hospital, Norway
[^3]: This article was submitted to Neurotrauma, a section of the journal Frontiers in Neurology
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Leptin acts centrally via leptin receptor (LepRb) expressing neurons that are found in the hypothalamus and many extra-hypothalamic sites. Recent research has focused on the identification of distinct LepRb populations to understand their contribution to diverse physiological leptin effects, including the ability of leptin to modulate food reward [@bib16; @bib23].
A key component of reward behavior is the mesolimbic dopamine system, which consists of dopaminergic (DA) neurons in the ventral tegmental area (VTA) and their projections to the nucleus accumbens (NAc) as well as other cortico-limbic areas [@bib32; @bib35]. The rewarding value of nutrients, e.g. sucrose or fat, requires activation of midbrain DA neurons and is enhanced by fasting and diminished by leptin [@bib16; @bib18; @bib23], but the precise neuronal mechanisms employed by leptin to reduce nutrient reward are controversial. LepRb is expressed on some midbrain DA neurons, and leptin inhibits these LepRb-DA neurons [@bib30]. Nutrient reward is encoded by NAc dopamine release, so that leptin-inhibited VTA DA neurons could explain a decreased nutrient reward and anorexia. Conversely, NAc DA levels are decreased in severely hyperphagic, leptin deficient *ob/ob* mice compared to control animals and leptin injections increase NAc DA levels [@bib23]. Thus, NAc DA deficiency may increase motivated behavior, such as food intake, in an attempt to induce DA release and normalize NAc DA levels. This hypothesis is in line with the decreased NAc activity of obese compared to lean humans and shows striking resemblance to the low NAc activity observed in drug addicts [@bib63].
Leptin action in the lateral hypothalamus (LHA) is sufficient to increase NAc DA levels [@bib41] and involves indirect leptin action via orexin neurons [@bib43]. LHA LepRb neurons are distinct from orexin/hypocretin neurons, but they directly innervate orexin/hypocretin neurons [@bib40; @bib43]. Orexin modulates the rewarding value and consumption of sucrose: Central orexin injections promote sucrose intake [@bib24], while genetic or pharmacological blockade of orexin signaling decreases the rewarding value and intake of sucrose [@bib44]. Orexin\'s effect on reward is mediated via the VTA [@bib27; @bib70], where orexin stimulates DA neurons [@bib47]. Leptin generally inhibits orexin neurons [@bib66], so that leptin inhibition of orexin neurons would be consistent with an inhibitory effect on midbrain DA neurons.
We recently reported that a population of LepRb neurons in the LHA co-expresses the inhibitory acting neuropeptide galanin (GAL-LepRb neurons) and that LHA galanin mRNA (*Gal*) expression is stimulated by leptin [@bib40]. Central galanin peptide (GAL) injections selectively increase fat intake [@bib33; @bib38], and deficiency in *Gal* or galanin-receptor-1 (GalR1) results in decreased dietary fat intake [@bib1; @bib34; @bib71]. GAL also modulates reward circuits by inhibition of mesolimbic DA neurotransmission [@bib60], inhibition of noradrenergic LC neurons [@bib54] and counteracts opiate withdrawal behavior [@bib29; @bib52].
We thus hypothesized that GAL-LepRb neurons play a role in nutrient reward and selection, which could lead to changes in body weight. To test this, we studied mice with conditional deletion of LepRb from GAL neurons (GAL-LepRb^KO^ mice). Our data strongly suggest that GAL via GalR1 mediates inhibitory leptin action onto orexin neurons, which could mediate the differential reward modulation of sweet and fatty stimuli.
2. Results {#sec2}
==========
2.1. Generation and validation of GAL-LepRb^KO^ mice {#sec2.1}
----------------------------------------------------
We studied the physiological importance of leptin action in GAL neurons by generating mice with conditional LepRb deletion from GAL neurons (GAL-LepRb^KO^ or KO mice, [Figure 1](#fig1){ref-type="fig"}A). Correct *cre*-expression in *Gal*^Cre^ mice was validated in *Gal*^YFP^ reporter mice and compared with galanin mRNA expression from the Allen Brain Atlas ([Figure S1](#appsec2){ref-type="sec"}) and immunohistochemical staining of galanin peptide ([Figure S2](#appsec2){ref-type="sec"}). In earlier studies we used transgenic *Gal*^*tg*GFP^ mice to identify and characterize LHA GAL-LepRb neurons [@bib40]. Thus, we further verified correct reporter gene expression (GFP or YFP) and distribution of GAL-LepRb neurons in *Gal*^YFP^ mice and *Gal*^(-neo)YFP^ mice compared to *Gal*^*tg*GFP^ mice. Hypothalamic *Gal*^GFP^ neurons were similarly distributed in all mouse models and as described earlier GAL-LepRb neurons (neurons with *Gal*^GFP^ and leptin-induced phosphorylation of signal-transducer-and-activator-of-transcription-3 (pSTAT3)) were found in the LHA and the nucleus of the solitary tract (NTS; [Figure S3A--C](#appsec2){ref-type="sec"}). The presence of the *neo* cassette had no effect on correct GAL-LepRb expression and all following experiments were conducted in *Gal*^YFP^ mice.
In KO mice leptin-induced pSTAT3 was significantly decreased in the LHA and NTS compared to control wildtype mice (WT mice) ([Figure 1](#fig1){ref-type="fig"}B--D, [Figure S4](#appsec2){ref-type="sec"}, n = 4--8, p^LHA^ \< 0.01, p^NTS^ \< 0.05) and confirms the successful deletion of LepRb.
2.2. Increased body weight gain in GAL-LepRb^KO^ mice {#sec2.2}
-----------------------------------------------------
The metabolic phenotype of KO mice was assessed from 4 to 12 weeks of age (n = 8--11). At 4 weeks of age, KO mice showed a significantly lower body weight compared to WT mice ([Figure 1](#fig1){ref-type="fig"}E, p^t-test^ \< 0.05), but at 12 weeks of age body weights were undistinguishable between groups ([Figure 1](#fig1){ref-type="fig"}E). However, body weight gain over 8 weeks was significantly increased in KO mice compared to WT mice ([Figure 1](#fig1){ref-type="fig"}F, p^t-test^ \< 0.02). Furthermore, we found that body weight of KO mice was significantly increased compared to WT mice in a separate cohort of older (7--9 month old) mice ([Figure 1](#fig1){ref-type="fig"}G, n = 7, p^t-test^ \< 0.05), consistent with an increase in body weight gain. Food intake ([Figure 1](#fig1){ref-type="fig"}H), energy expenditure ([Figure 1](#fig1){ref-type="fig"}I) or locomotor activity ([Figure 1](#fig1){ref-type="fig"}J), measured between 4 and 12 weeks of age, showed no significant difference between groups. At 12 weeks of age we also evaluated fed and fasted blood glucose levels, rectal body temperature and body composition in WT and KO mice with no significant differences between genotypes ([Figure S5](#appsec2){ref-type="sec"}).
2.3. Decreased LHA galanin signaling is associated with decreased fat intake {#sec2.3}
----------------------------------------------------------------------------
Earlier data indicated that leptin induced LHA *Gal* gene expression [@bib40]. Consistent with this, KO mice showed a 60% decrease in LHA *Gal* gene expression ([Figure 2](#fig2){ref-type="fig"}A, p^t-test^ \< 0.01). Furthermore, GalR1 expression, was significantly decreased by 30% ([Figure 2](#fig2){ref-type="fig"}B, p^t-test^ \< 0.05), while GalR2 and GalR3 expression remained unchanged within the LHA ([Figure 2](#fig2){ref-type="fig"}B).
GAL mainly acts as an inhibitory neuropeptide via GalR1 and selectively modulates the ingestion of fat [@bib59], suggesting that decreased *Gal* and GalR1 mRNA can affect fat intake. To test this, naïve KO and WT mice had access to a 10% Intralipid solution (1 kcal/ml) for 1 h per day over 10 days. Intralipid solution is highly palatable for mice and the amount ingested correlates with the rewarding value of the solution [@bib18; @bib53]. As expected, both WT and KO mice increased their Intralipid consumption over 10 consecutive 1 h sessions. However, over 10 days KO mice consumed significantly less Intralipid solution compared to WT mice ([Figure 2](#fig2){ref-type="fig"}C; n = 5; p^ANOVA^ \< 0.001; \*p^Holm-Sidak^ \< 0.05--0.001). These data are consistent with the fat intake inducing effect of GAL and indicate a reduced reward value for Intralipid solution in KO mice.
Next we tested whether the altered reward behavior was unique to fat or if other palatable nutrients like sucrose were also less rewarding. In another cohort of naïve mice (n = 5--7) we offered a choice of isocaloric (1 kcal/ml) 25% sucrose and 10% Intralipid solutions over 10 consecutive days. Again, KO mice consumed less Intralipid than WT mice, but strikingly KO mice consumed more sucrose solution compared to WT mice ([Figure 2](#fig2){ref-type="fig"}D; n = 5--7; p^ANOVA^ \< 0.0001; \*p^Holm-Sidak^ \< 0.05--0.001). In fact, while WT mice ingested equal amounts of Intralipid and sucrose solutions and had no preference for either solution, KO showed a robust and significant preference for sucrose over Intralipid ([Figure 2](#fig2){ref-type="fig"}E, n = 5--7, p^ANOVA^ \< 0.001; \*p^Holm-Sidak^ \< 0.01). Therefore, lack of LepRb in GAL neurons causes a differential modulation of sucrose and fat preference. The sucrose preference in KO mice was present from the first day of exposure to the solutions ([Figure 2](#fig2){ref-type="fig"}F, p^ANOVA^ \< 0.05; \*p^Holm-Sidak^ \< 0.02), suggesting that this nutrient selection is not due to a learned behavior.
2.4. GAL-LepRb^KO^ mice work more for sugar rewards {#sec2.4}
---------------------------------------------------
We further tested whether or not the increased sucrose preference affects their motivation to work for food rewards using the incentive runway [@bib51; @bib55]. Mice have to run a straight path with increasing length (up to 75 cm) to obtain a visible food reward.
Indeed, KO mice showed an increased completion speed to obtain the sweet (fat free) treat ([Figure 3](#fig3){ref-type="fig"}A, n = 4; p^ANOVA^ \< 0.05; \*p^Holm-Sidak^ \< 0.05--0.01). Further analysis revealed that KO had more incidences of direct, undistracted runs, while WT mice showed significantly more runs with distractions such as pauses, falters and reversals ([Figure 3](#fig3){ref-type="fig"}B, C and videos S1, S2; n = 4; p^ANOVA^ \< 0.05; p^Holm-Sidak^ \< \*0.03, ^\#^ = 0.06; p^t-test^ \< 0.03, respectively). In addition, KO also ran faster to obtain the treat compared to WT mice ([Figure 3](#fig3){ref-type="fig"}D, E and videos S1, S2; n = 4; p^ANOVA^ \< 0.03; p^Holm-Sidak^ \< \*0.03, \*\* \< 0.02, ^\#^ = 0.08; p^t-test^ \< 0.03, respectively).
The following are the supplementary data related to this article:Video S1WT mouse in an incentive runway trial at session 9.Video S2KO mouse in an incentive runway trial at session 9.
2.5. GAL-LepRb neurons innervate orexin neurons {#sec2.5}
-----------------------------------------------
We further aimed to identify the anatomical sites and neurons that were innervated by both LHA GAL and LepRb neurons in order to understand how GAL-LepRb neurons may regulate food reward.
Projections of LepRb and GAL neurons were identified by injecting the adenoviral construct Ad-iZ/EGFPf [@bib41] into the LHA of LepRb^Cre^ (n = 4) or *Gal*^Cre^ mice (n = 3), which resulted in a cre-specific expression of a farnesylated enhanced green fluorescent protein (EGFPf) and a non-cre dependent β-galactosidase expression from all adenovirus infected cells ([Figure 4](#fig4){ref-type="fig"}A--F). The farnesylation anchors EGFPF to the cell membrane and enhances the visualization of thin axonal processes; the β-galactosidase expression is used to track the spread of overall virally infected cells. Correctly targeted Ad-iZ/EGFPf injection into the LHA of LepRb^Cre^ ([Figure 4](#fig4){ref-type="fig"}A) or *Gal*^Cre^ mice ([Figure 4](#fig4){ref-type="fig"}D) was verified by confined EGFPf (green) and β-galactosidase (red) expression to LHA. EGFPf positive processes from LHA LepRb and GAL neurons were found locally within the LHA ([Figure 4](#fig4){ref-type="fig"}A, D, respectively). We further confirmed that LHA LepRb neurons, strongly innervate the VTA ([Figure 4](#fig4){ref-type="fig"}B, H) [@bib41; @bib42], but LHA GAL neurons rarely projected to the VTA ([Figure 4](#fig4){ref-type="fig"}E, K), suggesting that GAL-LepRb neurons do not innervate the VTA. Instead, both -- LHA LepRb and GAL neurons -- strongly innervated the locus coeruleus (LC) ([Figure 4](#fig4){ref-type="fig"}C, F, respectively), a site that had not yet been associated with leptin function.
In a separate cohort of LepRb^Cre^ (n = 7) or *Gal*^Cre^ (n = 4) mice, we injected another adenoviral construct, Ad-iN/WED [@bib43], that resulted in cre-dependent expression of wheat germ agglutinin (WGA) and the fluorescent protein DsRed. WGA can be transported anterogradely and transsynaptically into 2nd order neurons, while DsRed remains in 1st order neurons (here LepRb or GAL neurons) ([Figure 4](#fig4){ref-type="fig"}G--L).
LHA Ad-iN/WED injections in LepRb^Cre^ or *Gal*^Cre^ mice, resulted in WGA labeling within the LHA, where 1st order neurons are identified by WGA/DsRed co-expression (= yellow neurons) and 2nd order neurons by single WGA labeling (= green neurons) ([Figure 4](#fig4){ref-type="fig"}G, J). Several 2nd order neurons were found in the LHA indicating that GAL-LepRb neurons project locally onto LHA neurons.
We further observed many 2nd order neurons in the LC labeled from LHA GAL neurons ([Figure 4](#fig4){ref-type="fig"}L), while 2nd order labeling from LHA LepRb neurons ([Figure 4](#fig4){ref-type="fig"}I) was less strong and not observed in all cases of injected animals. LC neurons do not express LepRb as shown in a LepRb^GFP^ reporter mouse with prominent GFP labeling in the hypothalamus, but complete absence of GFP labeling within the LC of the same animal ([Figure S6A, B](#appsec2){ref-type="sec"}, respectively). We further confirmed the projections of GAL-LepRb neurons to the LC with injections of the retrograde tracer fluorogold (FG) into the LC of *Gal*^YFP^ mice. GAL is densely co-expressed with noradrenergic LC neurons as indicated by co-labeling of *Gal*^GFP^ with tyrosine hydroxylase (TH) ([Figure S6C](#appsec2){ref-type="sec"}), so that the *Gal*^GFP^ signal (green label) served as an excellent visual guide for LC neurons to verify the accuracy of FG injections (red label) ([Figure S6A](#appsec2){ref-type="sec"}). We found many triple labeled FG/pSTAT3/*Gal*^GFP^ neurons in the LHA surrounding the fornix ([Figure S6B](#appsec2){ref-type="sec"}), which showed that many GAL-LepRb neurons innervate the LC. Furthermore, GAL neurons derived WGA labeled LC neurons indeed represent noradrenergic TH-positive neurons ([Figure 5](#fig5){ref-type="fig"}A).
LepRb neurons in the LHA innervate local orexin/hypocretin neurons [@bib43]. Similarly, we found that WGA labeled 2nd order neurons from LHA GAL and LepRb neurons co-labeled with orexin/hypocretin ([Figure 5](#fig5){ref-type="fig"}B, C, respectively). Importantly, neither LepRb nor GAL neurons co-localize with orexin/hypocretin [@bib40], thus further supporting that GAL-LepRb neurons innervate orexin/hypocretin neurons.
2.6. Galanin mediated inhibition of orexin neurons {#sec2.6}
--------------------------------------------------
LHA *Gal* and *GalR1* expression was decreased in KO mice ([Figure 2](#fig2){ref-type="fig"}A, B) and GAL acts via GalR1 to potently inhibit neuronal excitation in many neuronal systems, including the hypothalamus [@bib4; @bib19; @bib20]. We hypothesized that GAL may mediate the inhibitory actions of leptin on orexin neurons. Therefore, we predicted an increased activation of orexin neurons in KO mice. We analyzed KO and WT brains for basal levels of cFos (as a surrogate for neuronal activation) in orexin neurons and indeed found increased cFos/orexin co-localization, which was most prominent in the rostral portion of orexin/hypocretin neurons ([Figure 6](#fig6){ref-type="fig"}A--E, n = 5, p^ANOVA^ \< 0.003,\*p^Holm-Sidak^ \< 0.05). This was due to an increased number of cFos/ox neurons ([Figure 6](#fig6){ref-type="fig"}D), while the total number of orexin neurons was unchanged between groups ([Figure 6](#fig6){ref-type="fig"}E). Enhanced cFos expression was restricted to the LHA, while other adjacent sites, e.g. the DMH, showed similar cFos expression ([Figure S8](#appsec2){ref-type="sec"}). Further, orexin gene expression was similar in KO and WT mice ([Figure 6](#fig6){ref-type="fig"}F). However, the gene expression of LHA orexin receptor Ox2, but not Ox1, was significantly decreased ([Figure 6](#fig6){ref-type="fig"}F, \*p^t-test^ \< 0.02), indicating that within the LHA Ox1 signaling would be preferentially enhanced. We further determined that orexin neurons were co-labeled with GalR1 ([Figure 6](#fig6){ref-type="fig"}G), demonstrating that orexin neurons have the molecular capability to respond to GAL ([Figure 6](#fig6){ref-type="fig"}H).
3. Discussion {#sec3}
=============
Our data demonstrate that leptin action via GAL neurons regulates nutrient reward. Dysregulation of this system by deleting LepRb from GAL neurons caused increased reward value and consumption for sucrose, while fat consumption was decreased.
Our data uncover a novel neuronal circuit where LHA GAL-LepRb neurons directly innervate orexin neurons as well as noradrenergic locus coeruleus neurons. In contrast to LHA LepRb neurons, LHA galanin neurons and therefore GAL-LepRb neurons do not innervate the VTA directly.
Leptin stimulates LHA *Gal* gene expression, while lack of LepRb in GAL neurons decreased LHA *Gal* gene expression. GAL is an inhibitory neuropeptide and we further speculate that leptin inhibits orexin neurons via GAL→GalR1 signaling to modulate nutrient reward and body weight.
3.1. Leptin and regulation of VTA DA neurons {#sec3.1}
--------------------------------------------
Central leptin controls nutrient reward via interactions with VTA dopaminergic neurons [@bib18; @bib22; @bib23], but the exact circuits involved are unclear. There is an ongoing controversy regarding the stimulatory or inhibitory effect of leptin on DA neurons. Some VTA dopamine neurons express LepRb and leptin inhibits these DA-LepRb neurons [@bib30], suggesting that leptin decreases food reward to mediate its anorexigenic effects.
In contrast, the lack of leptin in *ob/ob* mice results in decreased DA content within the NAc. Peripheral, central and intra-LHA leptin injections increase NAc DA content and increases tyrosine-hydroxylase expression in DA neurons, indicating a stimulatory leptin effect on DA neurons [@bib23; @bib41]. This is consistent with the DA deficiency theory, which argues that a low DA content would enhance behaviors like feeding, aimed at increasing NAc DA content and avoiding potentially unpleasant effects of low NAc DA levels [@bib8]. Thus, inhibitory and stimulatory leptin effects on DA neurons are plausible and may both contribute to the modulation of food reward.
The presented data demonstrate that sucrose preference further translates into enhanced work for a sweet treat in KO mice. KO mice demonstrate increased reward value for sucrose and sugar, indicated by increased sucrose consumption and incentive runway performance. Orexin action is well known to modulate the rewarding value of food, by inducing NAc DA release via activation of VTA DA neurons [@bib61; @bib62]. This enhances the motivation to work for sucrose reward, which is blocked by orexin receptor inhibitors [@bib5; @bib12; @bib27; @bib44]. Thus, the increased sucrose reward value in KO mice can be explained by the observed increase in baseline activation of orexin neurons.
Leptin inhibits orexin neurons, possibly via inhibitory GABAergic LHA LepRb neurons [@bib43]. In addition, our data strongly support a role of GAL to mediate inhibitory leptin effects onto orexin neurons: 1) LHA GAL and LepRb neurons innervate local orexin neurons 2) Leptin regulates LHA *Gal* gene expression, with leptin being sufficient to increase LHA *Gal* mRNA [@bib40] and LepRb expression on GAL neurons being necessary to maintain normal LHA *Gal* mRNA expression. 3) GalR1 mRNA, the predominant receptor responsible for inhibitory GAL actions [@bib4; @bib20], is decreased in the LHA of KO mice. 4) GalR1 is co-expressed with orexin neurons. 5) KO mice show an increased percentage of activated orexin neurons. Indeed, a recent study confirmed in slice preparations that GAL inhibits orexin neurons [@bib26]. Thus, our study is the first to highlight a direct interaction of LHA galanin neurons with orexin neurons and we speculate that the inhibitory acting neuropeptide GAL could modulate food reward by inhibiting orexin neurons.
LHA LepRb neurons also express the inhibitory neurotransmitter GABA and innervate the VTA and local orexin neurons [@bib41; @bib43]. Our study confirms LHA LepRb → VTA projections, but LHA GAL neurons do not innervate the VTA. Therefore, we conclude that LHA GAL-LepRb neurons are distinct from LHA LepRb → VTA projecting neurons.
A subset of LHA LepRb neurons and LHA GAL neurons co-express the neuropeptide neurotensin (Nts) [@bib40]. LHA neurotensin neurons, but not LHA GAL neurons, project directly to the VTA, thus we conclude that LHA Nts-LepRb → VTA projecting neurons are distinct from GAL-LepRb neurons.
LHA Nts-LepRb neurons [@bib42] and GAL-LepRb neurons both innervate orexin neurons, further suggesting that LHA LepRb→orexin projecting neurons may indeed co-express GAL and neurotensin. However, orexin neurons do not express neurotensin receptors [@bib49], while we found GalR1co-expressed with orexin neurons and GalR1 mRNA down-regulation in KO mice. Thus, we further suggest that GAL, not neurotensin, regulates orexin neurons and that this involves inhibitory GAL actions via GalR1.
We found a strong projection of GAL-LepRb neurons to the LC. LHA Nts neurons do not project to the LC [@bib42]; and personal communication with Dr. MG Myers); thus, we further conclude that GAL-LepRb → LC projecting neurons are distinct from LHA Nts neurons. We have further clarified this classification of LHA LepRb populations in [Figure 6](#fig6){ref-type="fig"}H.
3.2. Fat intake and galanin {#sec3.2}
---------------------------
Selective deletion of LepRb in GAL neurons caused a decrease in fat intake, which cannot be explained by anorexigenic leptin action. The concept that GAL controls macronutrient selection was first introduced by Leibowitz and colleagues, who found that central GAL injections selectively increased fat intake, while carbohydrate and protein intake remained unchanged [@bib59]. The reported effects of GAL on macronutrient selection were small and varied dependent on the initial nutrient preferences [@bib56; @bib57]. More recently, GAL\'s effect on fat intake was further supported in GAL deficient mice, which ate less of a high fat diet compared to WT mice [@bib1; @bib34]. Similarly, GalR1 null mice consume less high fat diet [@bib71], therefore low *Gal* and GalR1 mRNA expression in the LHA of GAL-LepRb^KO^ mice could sufficiently explain the observed decrease in fat consumption. Furthermore, in a macronutrient choice paradigm *Gal*-KO mice preferred carbohydrates over fat [@bib1]. This effect is very similar to the observed sucrose preference over Intralipid in GAL-LepRb^KO^ mice and strongly suggests that GAL indeed mediates the differential effects on sucrose and fat intake in GAL-LepRb^KO^ mice.
Similar to sucrose, fat rich solutions like Intralipid are highly rewarding for rodents and similarly involve reward circuits that are associated with DA release [@bib36; @bib58]. Thus, a differential regulation of sucrose and fat reward value cannot be explained exclusively by a regulation of DA neurons and suggests that other reward circuits are modulated in KO mice.
Nutrient reward is also modulated by the opioid system, a pathway that interacts with the dopaminergic system as well as other central sites. Importantly, activation or inhibition of μ-opioid receptors (MOR) in the NAc of rats robustly modulates fat intake, while chow intake remains unchanged and sucrose intake is only minimally affected [@bib64; @bib65; @bib67; @bib68]. Indeed, several studies found that fat intake stimulates the endogenous opioid enkephalin and *Gal* expression simultaneously [@bib6], and GAL induced fat intake is prevented by selective blockade of MOR [@bib7]. Thus, it may be possible that sucrose intake can be regulated by the enhanced orexin release in the VTA, while fat intake remains suppressed due to the blunted GAL action on MOR. Further studies will need to address these distinct aspects of nutrient reward.
3.3. Leptin and galanin interaction with the locus coeruleus {#sec3.3}
------------------------------------------------------------
GAL-LepRb neurons strongly innervate noradrenergic neurons in the LC. The LC is the major projection sites of orexin neurons and orexin and LC neurons both control arousal and reward [@bib9; @bib10; @bib11]. The LC is the sole NE source for many central sites (e.g. prefrontal cortex, PFC), while other sites (e.g. hypothalamus) receive additional NE input from the brainstem (NTS). Noradrenergic LC neurons do not express LepRb, and whether leptin regulates LC function has not been investigated to our knowledge. However, leptin injections decrease and leptin deficiency increases hypothalamic NE levels (even though the source of NE was unclear in these studies), while central blockade of NE signaling substantially decreases hyperphagia in leptin deficient mice [@bib13; @bib14; @bib15; @bib37].
In line with this, chemical deletion of noradrenergic LC fibers, but not noradrenergic NTS fibers, results in decreased food and sucrose consumption [@bib2] as well as impaired incentive runway behavior [@bib3], further suggesting that LC neurons could at least contribute to the observed sucrose preference and rewarding value of sweet treats in KO mice. The functional connectivity of orexin and LC neurons has been convincingly shown for arousal behavior and may be reflected in the improved runway behavior, where KO mice are less distracted during trials.
The LC is clearly an important mediator of opioid signaling and best known for its role in physical dependence and opiate withdrawal behavior (recently reviewed in [@bib45]). Importantly, orexin and GAL have reciprocal effects on opiate withdrawal, which further supports an inhibitory role of GAL for orexin neurons. GAL decreases opiate withdrawal behavior via GalR1 [@bib28; @bib29], while orexin increases opiate withdrawal behavior [@bib25]. Thus, future experiments will have to further depict if the LC indeed plays a role in GAL-LepRb mediated nutrient reward.
In summary, our data highlight an important role of GAL-LepRb neurons in nutrient reward. We show evidence that galanin mediates the inhibitory actions of leptin on orexin neurons via GalR1 and suggest that this contributes to the differential regulation in the rewarding value of sucrose and Intralipid.
4. Material and methods {#sec4}
=======================
4.1. Animals {#sec4.1}
------------
Mice were bred and housed at 22 °C on a 12-hour light/dark cycle. Food and water were available *ad libitum* unless otherwise specified. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Pennington Biomedical Research Center. Hemizygous BAC transgenic *Gal*^TgGFP^ mice with green fluorescent protein (GFP) expression under the control of the *Gal* promoter (*Gal*^TgGFP^, Stock Tg(*Gal*-EGFP)109Gsat, \#0163420UCD) were obtained from the Mutant Mouse Regional Resource Center (MMRRC, <http://www.mmrrc.org>), a NCRR-NIH funded strain repository, and was donated to the MMRRC by the NINDS funded GENSAT BAC transgenic project (<http://www.gensat.org>).
4.2. Generation of Gal^Cre^ mice {#sec4.2}
--------------------------------
An IRES-cre sequence was inserted into the murine *Gal* gene between the Stop codon and polyadenylation site; an *frt*-flanked *neo* cassette was inserted downstream of the polyadenylation sites. The construct was electroporated into mouse ES cells (albino C57/B6 background), screened for construct insertion and injection into blastocysts to generate chimeras. Chimeras were bred to C57/B6 and germ line transmission was determined by coat color. Correct genotypes and 5'-recombination was confirmed by PCR with transgene spanning amplicons (fwd: 5′-TTG AAA CCT GCC CTG ACT CTC AGC A, reverse: 5′-AGG GAA ACC GTT GTG GTC TGA CTA) and 3′-recombianation (fwd: 5′- CCA TCA GAA GCT GAC TCT AGC GAA, rev: 5′-CTT GCT AGC TCT TCC CCA ACT CTA).
4.3. Experimental mice {#sec4.3}
----------------------
*Gal*^Cre^ mice were crossed with LepRb^fl/fl^ mice ([@bib46] and kindly provided by Dr. Streamson Chua) to generate Lepr^fl/fl^ mice (referred to as WT mice) and *Gal*^cre/+^; Lepr^fl/fl^ mice (referred to as GAL-LepRb^KO^ or KO mice). *Gal*^YFP^ reporter mice were generated by breeding with B6.129X1-*Gt(ROSA)26Sor*^*tm1(EYFP)Cos*^/J (stock\#006148) and compared to transgenic *Gal*^*tg*GFP^ mice, which we used earlier to identify and characterize GAL-LepRb neurons in the LHA [@bib40]. In some mice the *frt*-flanked *neo* cassette was removed from the genome by crossing *Gal*^YFP^ mice with 129S4/SvJaeSor-*Gt(ROSA)26Sor*^*tm1(FLP1)Dym*^/J mice (stock\#003946) (*Gal*^(-neo)YFP^ reporter mice) to prove that reporter expression pattern was not compromised by the presence of the *neo* cassette.
Genotyping of experimental animals was performed by PCR: *Gal*^Cre^ (cre primers: 5′-CCT CTC CCA AGC GGC CGG AGA ACC (fwd), 5′- CCG GCT CCG TTC TTT GGT GGC CCC TTC GCG (rev); wt-cre primers: 5′-TCC TGA GAC CAT GTC CAC TG (fwd), 5′-CTG CCA CTC CTG TGA TCT GA (rev)); Lepr^fl^ (mLepr106: 5′-GTC TGA TTT GAT AGA TGG TCT T (fwd), mLepr105: 5′-ACA GGC TTG AGA ACA TGA ACA (fwd), mLepr65A: 5′-AGA ATG AAA AAG TTG TTT TGG (rev)). We used the latter primers also to screen for potential germline *Lepr* excision as described elsewhere [@bib39]; such animals were excluded from breeding and experiments.
4.4. Metabolic phenotyping {#sec4.4}
--------------------------
Male KO and WT littermates (n = 11--15) were individually housed at 4 weeks of age; food intake and body weight was measured weekly until twelve weeks. At 12 weeks of age body composition was determined by NMR (minispec-mq series Bruker Billerica), fed and over-night fasted blood glucose was evaluated with a glucometer (One Touch Ultra Mini). After that mice were acclimated to the Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments) for 3 days and energy expenditure and locomotor activity was recorded continuously over 4 days. In another cohort of 7--9 month old, male KO and WT littermates (n = 7) body weight was again evaluated. Differences between groups were statistically analyzed with a repeated measure 2-way ANOVA or a *Student\'s* t-test.
4.5. Microdissection and qPCR {#sec4.5}
-----------------------------
Microdissection was performed as described earlier [@bib21] and visual landmarks, such as the mammillothalamic tract, fornix and the third ventricle were used to strategically dissect the LHA. RNA was prepared following manufacturers recommendations (ToTALLY RNA kit, Ambion) and 350 ng RNA was converted to cDNA (RETROscript Kit, Ambion). For qPCR 10.5 ng cDNA was used in triplicates to analyze multiple TaqMan assays (Applied Biosystems): *Gapdh* (housekeeping gene; Mm99999915_g1), Galanin (Mm01236508_m1), GalR1 (Mm00433515_m1), GalR2 (Mm00726392_s1), and GalR3 (Mm00443617_m1), orexin (Mm04210469_m1), OX1 (Mm01185776_m1), OX2 (Mm01179312_m1). Reactions were pipetted by a robotic liquid handling system (Perkin Elmer Multiprobe II EX) and run on the 7500 HT Fast Real-Time PCR System (Applied Biosystems). Fold induction of gene expression was calculated using the ^ΔΔ^CT method as recommended for TaqMan assays.
4.6. Nutrient preference and reward {#sec4.6}
-----------------------------------
To test the consumption of palatable fat we provided a fat emulsion (10% Intralipid, Baxter) in a sipper bottle for 1 h per day over 10 days to naïve WT and KO mice (n = 4--5) and compare their cumulative fat intake using a repeated measure 2-way ANOVA. Similarly, we tested their preferences for isocaloric solutions (1 kcal/ml) of sucrose (25% sucrose) and Intralipid (10%) in a two-bottle-choice test in naïve WT and KO mice (n = 5--9), again with 1 h daily access over 10 days; bottle positions were alternated every other day to prevent side preferences. Statistical differences were analyzed via 3-way repeated-measure ANOVA and post-hoc analysis.
WT and KO mice were further tested in an incentive runway [@bib51]. Briefly, mice (n = 4) were food restricted (*ad lib* food restricted from 12:00--4:00 p.m.) and allowed overnight access to the sugar treat (Froot Loops^®^, Kellogg\'s) to avoid neophobic responses. Incentive runway training was performed every other day for a total of 13 training sessions. Each session consisted of 5 runway trials (between 8:00a.m. and 12:00p.m.), sessions 1--3 were strictly acclimation trials with access to the goal box + treat. Session 4 included a 15 cm runway distance from the start to the goal box. With each subsequent session the runway length was increased in 15 cm increments until reaching a maximal distance of 75 cm and mice could consume the treat for 30 s after fulfilling their task. Session were recorded and analyzed in slow motion to determine completion speed (time from leaving the start box to reaching the goal box \[sec\]/runway length \[cm\]), running speed (=completion speed of direct runs in seconds/cm), number of direct and distracted runs (distractions = pauses, falters and reversals). Differences between groups were analyzed by repeated measure 3-way ANOVA (completion speed over time) or *Student\'s* t-test for two-group comparison.
4.7. Viral tracing studies {#sec4.7}
--------------------------
Stereotaxic surgeries were performed as reported earlier [@bib40]. The LHA was targeted at coordinates: X −0.9 mm, Y −1.3 mm, Z −5.2 mm relative to Bregma according to the Paxinos Mouse Brain Atlas [@bib50]. Two adenoviral constructs (Ad-IZ/EGFPf and Ad-iN/WED; described in detail earlier [@bib41; @bib43]) were acutely injected into the LHA of *Gal*^Cre^ or LepRb^Cre^ mice. Ad-iZ/EGFPf results in cre-inducible expression of farnesylated EGFP and visualizes cell bodies and processes of cre-expressing neurons, the viral injection spread can be monitored by β-galactosidase expression in non-cre expressing neurons. Ad-iN/WED results in cre-inducible expression of DsRed and wheat germ agglutinin (WGA). WGA is anterogradely and transsynaptically transported into second order neurons, while DsRed remain in first order cre-expressing neurons. Deeply anesthetized mice received 250--350 nl adenovirus (10 nl/20 s, 2--6 × 10^12^ PFU/ml) unilaterally. After 7--10 days incubation time mice were perfused and brains extracted for histological analysis.
*Leptin-induced phosphorylation of* signal-tranducer-and-activator-of-transcription-3 *(pSTAT3)*:
The histochemical visualization of LepRb expressing neurons was done by detection of leptin-induced pSTAT3, which is a signaling target of LepRb and has been shown by us and others as an excellent marker of functional LepRb neurons in the hypothalamus [@bib17; @bib21; @bib31; @bib48]. Mice received a bolus injection of leptin (5 mg/kg i.p.) to induce phosphorylation of STAT3, a downstream signaling target of LepRb. Mice were perfused 1 h post-injection and brains were extracted for further histological analysis as described earlier [@bib69].
4.8. Immunohistochemistry {#sec4.8}
-------------------------
Free-floating immunohistochemistry was done after pretreatment (1% peroxide in ice cold methanol for 20 min, 0.3% glycine in phosphate buffered saline (PBS) for 10 min and 0.03% SDS in PBS for 10 min) and blocking (3% normal donkey serum in PBS/0.25% Triton X-100 for 1--3 h). All primary and secondary antibodies were diluted in blocking solutions. Primary antibodies were incubated at 4 °C at least over night or for 48 h and secondary antibodies were incubated at room temperature for 2 h. For rabbit anti-pSTAT3 (1:1000, Cell Signaling) and rabbit anti-cFos (1:5000, Millipore/Calbiochem) staining was developed with ABC/3,3′ diaminobenzidine tetrahydrochloride (Pierce DAB Substrate Kit, ThermoScientific) as described earlier [@bib69]. Double labeling was done with either DsRed (1:1000, rabbit anti-DsRed, Santa Cruz), WGA (1:1000, goat anti-WGA, Vector Laboratories), GFP (1:000, chicken anti-GFP; AbCAM), or orexin/hypocretin-A (1:1000, goat anti-orexin/hypocretin-A, Santa Cruz) was visualized with fluorophor-labeled secondary antibodies (1:200 Alexa 568 or 488, Invitorgen or 1:400 Dylite, Jackson ImmunoResearch). Sections were mounted onto gelatin-subbed slides and cover slipped with ProLong Anti-fade mounting medium (Invitrogen) for further microscopy analysis.
4.9. Estimate of cell counts {#sec4.9}
----------------------------
For quantification of pSTAT3 in the LHA and NTS from leptin treated WT and KO mice (n = 4--8 per genotype) 2 sections per anatomical location were imaged using the Olympus BX51 brightfield microscope and Olympus DP30BW digital camera. Signals were amplified by identical changes in brightness and contrast for all analyzed brain sections. The number of pSTAT3 positive cell nuclei was quantified and compared between groups with a *Student\'s t-test*.
WT and KO mice (n = 5 per genotype) we analyzed for baseline cFos in LHA orexin neurons. Four sections containing the entire population of LHA orexin neurons were identified for each animal, organized anatomically in a rostral to caudal manner and the total number of orexin neurons and the number of cFos expressing orexin neurons were counted (similar as described previously [@bib40]). Differences between groups were evaluated with a 2-way ANOVA and posthoc-test.
4.10. Statistic {#sec4.10}
---------------
Statistical analysis was done with SPSS Statistics (repeated measure 3-way ANOVA) (IBM, Armonk, NY) or SigmaPlot 11.2 (all other statistics) (Systat Sofware Inc, San Jose, CA). The individual tests used are noted with the according experiments. Significant differences were accepted at a p-value \< 0.05.
Conflict of interest {#appsec1}
====================
All authors declare no conflict of interest.
Appendix A. Supplementary data {#appsec2}
==============================
The following is the supplementary data related to this article:
This work was supported by AHA053298N, P/F DK020572-30, P20 RR02195, P/F NORC \#2P30-DK072476-06 (HM) and 2T32 DK064584 (EQC). This work utilized the facilities of the Cell Biology and Bioimaging Core, supported in part by COBRE (NIH P20-RR021945) and CNRU (NIH 1P30-DK072476) center grants from the National Institutes of Health. Partial support was provided through the Animal Phenotyping Core supported through NIDDK NORC Center Grant \#s2P30 DK072476 entitled 'Nutritional Programming: Environmental and Molecular Interactions' at the Pennington Biomedical Research Center, and the Islet Cell Biology Core of the DRTC at the University of Chicago (DK020595) for generation of adenoviral vectors.
Supplementary data related to this article can be found at [http://dx.doi.org/10.1016/j.molmet.2015.07.002](10.1016/j.molmet.2015.07.002){#intref0025}.
{#fig1}
{#fig2}
![*KO mice work more for sugar rewards*. Incentive runway testing of WT and KO mice (n = 4 each genotype) for completion speed (cm/sec = runway length \[cm\]/time from leaving start box till entering goal box \[sec\]) (A. p^ANOVA^ \< 0.05; \*p^Holm-Sidak^ \< 0.05--0.01), the number of direct runs (in contrast to distracted runs) (B., C.; p^ANOVA^ \< 0.05; p^Holm-Sidak^\<\*0.03, ^\#^ = 0.06; p^t-test^ \< 0.03, respectively) and running speed (mean completion speed from all direct runs) (D., E.; p^ANOVA^ \< 0.03; p^Holm-Sidak^\<\*0.03, \*\* \< 0.02, ^\#^ = 0.08; p^t-test^ \< 0.03, respectively). Each session consisted of 5 trials and sessions that were held every other day. *WT* = *wildtype*; *KO* = *knock out*.](gr3){#fig3}
{#fig4}
{#fig5}
{#fig6}
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
The incidence of lung cancer in the People's Republic of China has doubled in the past decade[@b1-ceor-8-097] likely because of aging population, poorly controlled cigarette smoking, and air pollution associated with rapid economic growth.[@b2-ceor-8-097],[@b3-ceor-8-097] Similar to the tumor histology distribution of lung cancer in industrialized countries, \>80% of diagnosed lung cancer in Chinese patients is non-small-cell lung cancer (NSCLC).[@b4-ceor-8-097] Because of the challenges associated with early detection,[@b5-ceor-8-097] NSCLC is often diagnosed at advanced stage[@b6-ceor-8-097] in Chinese patients, and chemotherapy is the main therapeutic option[@b7-ceor-8-097] that may extend survival and improve quality of life in patients with advanced NSCLC over best supportive care alone.[@b8-ceor-8-097] With substantially increased reimbursement coverage for hospital care in the People's Republic of China,[@b9-ceor-8-097] health resource utilization associated with chemotherapy for advanced NSCLC has become an important consideration for both clinical and reimbursement decision-making.
Many advances have been made for treating advanced NSCLC in the past decade. One of those advances is the role of tumor histology in predicting clinical effects of chemotherapy for advanced NSCLC.[@b10-ceor-8-097] Pemetrexed treatment was found be more effective and associated with less toxicity than gemcitabine treatment in the first-line setting[@b11-ceor-8-097] when treating advanced nonsquamous non-small-cell lung cancer (AdvNS-NSCLC). Pemetrexed treatment may be associated with lower consumption of health resources if the improved clinical effects and better safety profile translate into fewer treating disease-related symptoms and adverse events (AEs). Thus, we conducted this real-world cohort study to test this hypothesis by comparing the allocation of hospital costs per treatment cycle (HCTC) associated with pemetrexed--platinum and other platinum-based doublets commonly used in the first-line setting for AdvNS-NSCLC in a retrospective cohort of Chinese patients.
Methods
=======
This study was designed as a retrospective cohort study including Chinese patients identified from Hunan Province Tumor Hospital (HNPTH) and Xiangya Hospital (XYH), the two major tertiary hospitals providing cancer care to patients living in Hunan province, People's Republic of China. The observation time set for patient identification was from January 1, 2010, to December 31, 2012. The study protocol was reviewed and approved by the ethics review boards of HNPTH and XYH.
Patient identification
----------------------
The electronic hospital admission registry databases in the two hospitals were used to search for patients who were hospitalized for lung cancer between January 1, 2010, and December 31, 2012. The identified patients with NS-NSCLC or histologically unclassified lung cancer were linked with their hospital records to confirm their tumor histology and tumor stage according to the definitions made by the International Staging Committee of the International Association for the Study of Lung Cancer in 2009.[@b12-ceor-8-097] The medical records of patients with biopsy or cytology-confirmed NS-NSCLC, mainly including adenocarcinoma or large-cell carcinoma, were further reviewed for any records of platinum-based doublet treatment in the first-line setting after the diagnosis of stage IIIb or IV cancer. To have a sufficient sample size for data analysis, our study only included patients receiving cisplatin- or carboplatin-based doublets with pemetrexed (given with supplementation of folic acid and vitamin B12 and approved to treat advanced NSCLC with cisplatin in the first-line setting), docetaxel, paclitaxel, gemcitabine, or vinorelbine. Patients receiving tyrosine kinase inhibitor, epidermal growth factor receptor monoclonal antibody, and/or anti-angiogenic therapy in the first-line setting were excluded in order to control their confounding effects on tumor response and clinical toxicity associated with the studied platinum-based doublets. This study also excluded patients who initialized first-line chemotherapy out of HNPTH or XYH or who had missing information on hospital costs during follow-up.
Data extraction
---------------
The follow-up time defined for data extraction was set from the hospitalization with the first administration of platinum-based doublets to the hospitalization with the last administration of platinum-based doublets. We reviewed the hospital records before the first administration of the studied doublets to extract baseline characteristics of patients that included demographic information, type of health insurance plan, smoking status, physical function assessed by the Eastern Cooperative Oncology Group's performance status, baseline marrow function, and disease information on tumor stage, tumor histology, and metastasis status. We also reviewed hospital prescription records to extract treatment information on administration doses and schedule of the studied platinum-based doublets. Additionally, the prescription records for granulocyte colony-stimulating factor (G-CSF), erythropoietin, thrombopoietin, interleukin 11, and blood products (blood transfusion and/or platelet infusion) were extracted for the patterns of treating hematological AEs[@b13-ceor-8-097] associated with the studied platinum-based doublets. The extracted information also included tumor response, which was assessed every two treatment cycles using Response Evaluation Criteria in Solid Tumors (Version 1.0)[@b14-ceor-8-097] and clinical toxicity, which was assessed by the Common Terminology Criteria for Adverse Events (Version 3.0) with modified criteria for anemia[@b15-ceor-8-097] in the two study hospital settings. The laboratory blood testing results for hemoglobin, white blood cell count, neutrophilic granulocyte count, and platelet count during follow-up were also extracted as supplemental information to confirm hematological AEs. Finally, hospital discharge billing records associated with each hospitalization during follow-up were reviewed to extract hospital costs associated with billable medications and services. Because the billing records only provided the cost sum by category, we tracked the prescriptions of chemotherapy drugs (platinum agent and cytotoxic agent) to estimate the chemotherapy drug costs. The hospital costs were classified into three categories in this study: chemotherapy drug costs, nonchemotherapy drug costs, and nondrug care costs. The perspective of the hospital costs was the People's Republic of China's health system, and any hospital costs were taken into account irrespective of their reimbursement status.
Outcome measures
----------------
The primary outcome measure in our study was HCTC. The extracted hospital costs from the two study hospitals were categorized by platinum agents, nonplatinum cytotoxic agents, nonchemotherapy drugs, and nondrug care for the allocation of hospital costs. The secondary outcome measures in our study included tumor response, which was classified as complete response, partial response, stable disease, and progressive disease (PD), defined by Response Evaluation Criteria in Solid Tumors (Version 1.0). The secondary outcome measures also included clinical toxicity measured by hematological and nonhematological AEs. Because early treatment discontinuation often occurred within two treatment cycles because of PD, there is often lack of tumor response assessment in these patients. To include the patients with early treatment discontinuation in the data analysis, we further classified tumor response as tumor control (defined as complete response, partial response, or stable disease) and treatment failure (defined as PD or no tumor response assessment associated with early treatment discontinuation) for data analysis. To control the bias associated with missing information on hematological toxicity assessment associated with platinum-based doublets, we used both recorded hematological AE information from hospital medical notes and homological toxicity assessment based on laboratory blood testing results to measure occurrence and severity of hematological AEs associated with studied platinum-based doublets.
Data analysis
-------------
One-way analysis of variance and Fisher's exact test were used to describe the differences in patients' baseline characteristics and treatments used to prevent and/or treat hematological AEs in patients receiving the five studied doublets. Propensity score methods were used to create matched pairs for pemetrexed--platinum versus the other four studied platinum-based doublets, respectively, after balancing baseline characteristics of patients and treatments for hematological AEs. The matching condition was set as propensity score difference between matched pairs \<0.001 when using the greedy approach.[@b16-ceor-8-097] McNemar's test was used for head-to-head comparisons of tumor response and occurrences of AEs between propensity score-matched treatment groups. Wilcoxon signed rank test was used to compare the allocation of HCTC and HCTC for nonchemotherapy drugs and nondrug care between matched treatment groups. We further used multiple logistic or linear regression analyses with generalized estimating equation to adjust imbalanced baseline variables (*P*\<0.5 after propensity score matching) in propensity score-matched patients to confirm the observed differences in tumor response, clinical toxicity, and the allocation of HCTC between the matched treatment groups for pemetrexed--platinum versus the other four studied platinum-based doublets.[@b17-ceor-8-097] Finally, we used vinorelbine--platinum as reference to rank the association between five studied platinum-based doublets and the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care in all patients, which included patients stratified by their tumor response and hematological AEs using multiple linear regression analyses with adjustment of baseline characteristics and treatments for hematological AEs. Statistical significance was defined as two-sided *P*-value \>0.05 in this study, and SAS 9.2 was used to perform the data analyses described earlier.
Results
=======
The initial search of electronic hospital admission registry databases identified 4,558 patients who were hospitalized for lung cancer. We first excluded 3,054 patients without chemotherapy treatment in hospital and 698 patients with ineligible histology or lack of tumor histology information (333 with squamous histology, 207 with mixed squamous and nonsquamous histology, 91 with small-cell histology, and 67 without biopsy or cytology-confirmed tumor histology). We further excluded 140 patients with tumor stage less than IIIb, 179 patients due to treatment received (166 patients receiving first-line chemotherapy other than the studied five platinum-based doublets and 13 patients receiving tyrosine kinase inhibitor or epidermal growth factor receptor monoclonal antibody in the first-line setting), and 40 patients due to missing hospital billing information. The final data analyses were based on 447 patients who met all eligibility criteria, including 259 patients receiving five studied doublets in HNPTH and 188 patients treated by pemetrexed-, docetaxel-, or gemcitabine-contained doublet in XYH. The patient identification processes in the two hospitals are illustrated in [Figure 1](#f1-ceor-8-097){ref-type="fig"}.
Patient baseline characteristics and patterns of care
-----------------------------------------------------
Of the 447 eligible patients, 34.9% received pemetrexed--platinum (n=156), 15% received docetaxel--platinum (n=67), 8.7% received paclitaxel--platinum (n=39), 24.6% received gemcitabine--platinum (n=110), and 16.8% received vinorelbine--platinum (n=75). The comparisons of baseline characteristics of patients across the five treatment groups ([Table 1](#t1-ceor-8-097){ref-type="table"}) observed significant differences in the distributions of public health insurance plan for urban residents (35.9%--58.3%, *P*=0.015), Eastern Cooperative Oncology Group performance status of 0 (4.5%--25.6%, *P*\<0.001) and 1 (69.2%--91.0%, *P*=0.004), adenocarcinoma histology (95.5%--100%, *P*=0.014), and pleural metastasis (11.9%--32.1%, *P*=0.001). Further comparisons of treatment patterns observed highly uneven distribution of the studied platinum- based doublets by hospital setting, hospital admission year, platinum agents, and treatments for treating hematological AEs. For example, pemetrexed was used more frequently in XYH than in HNPTH (58.5% versus 17.8%, *P*\<0.001). The most frequently used doublets in the three hospital admission years were vinorelbine--platinum in 2010 (77.3%, *P*\<0.001), gemcitabine--platinum in 2011 (36.4%, *P*=0.002), and pemetrexed--platinum in 2012 (43%, *P*\<0.001). Cisplatin was used more frequently in the combination treatment with vinorelbine (93.3%, *P*\<0.001), and carboplatin was used more frequently in the combination treatment with paclitaxel (43.6%, *P*\<0.001). G-CSF was used most frequently in patients receiving paclitaxel treatment (76.9%, *P*=0.061), and interleukin 11 was used most frequently in patients receiving vinorelbine treatment (16%, *P*\<0.001). The five studied platinum-based doublets were each administered every 3 weeks. Pemetrexed, docetaxel, and paclitaxel were each administered once at day 1 per treatment cycle, while gemcitabine and vinorelbine were administered at both day 1 and day 8 per three-week treatment cycle. The administered doses of the five studied doublets were highly consistent with the recommended doses. The completed treatment cycles associated with the five studied doublets significantly differed even though the average completed treatment cycles had a small range (two to three cycles, *P*\<0.001). The comparisons of treatment patterns across the five treatment groups are summarized in [Table 1](#t1-ceor-8-097){ref-type="table"}.
Comparisons of tumor response, clinical toxicity, and allocation of HCTC between propensity score-matched treatment groups
--------------------------------------------------------------------------------------------------------------------------
Propensity score methods created 61 matched pairs for pemetrexed--platinum versus docetaxel, 39 matched pairs for pemetrexed--platinum versus paclitaxel--platinum, 93 matched pairs for pemetrexed--platinum versus gemcitabine--platinum, and 73 matched pairs for pemetrexed--platinum versus vinorelbine--platinum for head-to-head comparisons.
Tumor response
--------------
The head-to-head comparisons of tumor response indicated that pemetrexed--platinum was associated with significantly higher tumor control rates as compared to the docetaxel (62.3% versus 24.6%, relative risk \[RR\] 2.533, *P*\<0.001), gemcitabine (61.3% versus 40.9%, RR 1.500, *P*=0.009), or vinorelbine doublets (63% versus 30.1%, RR 2.091, *P*\<0.001). After adjusting imbalanced baseline variables between matched treatment groups, treatment with pemetrexed--platinum was associated with a significantly lower risk of treatment failure as compared to the other four doublets (odds ratio \[OR\] ranged from 0.081, *P*\<0.001, for the comparison with vinorelbine--platinum to 0.276, *P*=0.001, for the comparison with gemcitabine--platinum). The head-to-head comparisons of tumor response are summarized in [Table 2](#t2-ceor-8-097){ref-type="table"}.
Clinical toxicity
-----------------
Head-to-head comparisons of occurrences of AEs indicated that pemetrexed--platinum was highly comparable to docetaxel--platinum regarding clinical toxicity but was associated with fewer hematological AEs than the other three studied doublets. After further adjusting imbalanced baseline variables between the matched treatment groups, pemetrexed--platinum had significantly lower risk of anemia (OR 0.023, *P*=0.007) than paclitaxel--platinum; significantly lower risks of leukopenia (OR 0.248, *P*=0.003), anemia (OR 0.092, *P*\<0.001), thrombocytopenia (OR 0.172, *P*\<0.001), and any hematological AE (OR 0.099, *P*\<0.001) than gemcitabine--platinum; and significantly lower risks of anemia (OR 0.063, *P*\<0.001) and any hematological AE (OR 0.153, *P*=0.002) than vinorelbine--platinum. For nonhematological AEs, pemetrexed--platinum had significantly lower rates of nausea than paclitaxel--platinum (46.2% versus 71.8%, RR 0.643, *P*=0.025); significantly lower rates of arthralgia (4.3% versus 18.3%, RR 0.235, *P*=0.002), cough (2.2% versus 24.7%, RR 0.087, *P*\<0.001), and fatigue (16.1% versus 58.1%, RR 0.278, *P*\<0.001) than gemcitabine--platinum; and significantly lower rates of fatigue (6.8% versus 42.5%, RR 0.161, *P*\<0.001), nausea (42.5% versus 82.2%, RR 0.517, *P*\<0.001), and vomiting (26% versus 45.2%, RR 0.576, *P*=0.013) than vinorelbine--platinum. The head-to-head comparisons of the occurrence rates of hematological and nonhematological AEs between the propensity score-matched treatment groups for pemetrexed--platinum versus the other four studied doublets are summarized in [Table 2](#t2-ceor-8-097){ref-type="table"}.
Allocation of HCTC
------------------
Comparisons of the allocation of HCTC demonstrated that pemetrexed--platinum was associated with significantly higher nonplatinum cytotoxic drug costs (median differences ranged from RMB 4,636 to RMB 7,332 \[1 RMB= US\$0.16\]) but significantly less HCTC for nonchemotherapy drugs and nondrug care (median difference ranged from --RMB 3,251 to --RMB 1,478) than the other four studied doublets. Pemetrexed--platinum was comparable to the other four studied platinum-based doublets regarding the HCTC for platinum agent and nondrug care. Because the saved costs for nonchemotherapy drugs and nondrug care associated with pemetrexed--platinum treatment did not completely offset the high acquisition costs of pemetrexed, the total HCTC associated with pemetrexed--platinum remained significantly higher than the other four studied platinum-based doublets (median increase ranged from RMB 1,692 to RMB 7,400). Further adjusting imbalanced baseline variables after propensity score matching observed that the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care associated with pemetrexed--platinum treatment was significantly less than that for docetaxel (coefficient −0.246, *P*=0.003), paclitaxel (coefficient −0.351, *P*\<0.001), or gemcitabine doublet (coefficient −0.194, *P*=0.001). The head-to-head comparisons of the allocation of HCTC between propensity score-matched treatment groups for pemetrexed--platinum versus the other four studied doublets are summarized in [Table 3](#t3-ceor-8-097){ref-type="table"}.
Ranking the studied doublets by their impact on HCTC for nonchemotherapy drugs and nondrug care in patients stratified by tumor response and hematological toxicity
-------------------------------------------------------------------------------------------------------------------------------------------------------------------
The multiple linear regression analysis ranked pemetrexed--platinum to have the lowest coefficient (−0.174, *P*=0.015) for the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care among the five studied platinum-based doublets in 409 patients irrespective of their status of tumor control and hematological AEs ([Figure 2A](#f2-ceor-8-097){ref-type="fig"}). The coefficient associated with pemetrexed--platinum was also ranked the lowest for the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care in 272 patients experiencing any hematological AE (coefficient −0.199, *P*=0.013; [Figure 2B](#f2-ceor-8-097){ref-type="fig"}), in 73 patients experiencing neutropenia (coefficient −0.426, *P*=0.021; [Figure 2C](#f2-ceor-8-097){ref-type="fig"}), and in 119 patients experiencing leukopenia (coefficient −0.406, *P*=0.001; [Figure 2D](#f2-ceor-8-097){ref-type="fig"}). However, the coefficient associated with both docetaxel- (coefficient 0.261, *P*=0.006) and gemcitabine-contained doublets (coefficient 0.252, *P*=0.021) for the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care was significantly higher than vinorelbine--platinum in 272 patients experiencing any hematological AE. No other significant differences were observed regarding the coefficients associated with the five studied platinum-based doublets for the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care in patients experiencing tumor control ([Figure 2E](#f2-ceor-8-097){ref-type="fig"}), treatment failure ([Figure 2F](#f2-ceor-8-097){ref-type="fig"}), no hematological AE ([Figure 2G](#f2-ceor-8-097){ref-type="fig"}), anemia ([Figure 2H](#f2-ceor-8-097){ref-type="fig"}), or thrombocytopenia ([Figure 2I](#f2-ceor-8-097){ref-type="fig"}).
Discussion
==========
To our knowledge, this study is the first to assess real-world data to demonstrate that superior tumor response and better safety profile associated with chemotherapy could save hospital costs for nonchemotherapy drugs and nondrug care in the first-line setting for AdvNS-NSCLC in Chinese patients. Among the five platinum-based doublets frequently used to treat AdvNS-NSCLC in Chinese patients, pemetrexed was the most expensive nonplatinum cytotoxic agent. However, our study observed that pemetrexed treatment was associated with superior tumor response and less clinical toxicity, which could reduce the utilization of nonchemotherapy drugs and nondrug care and offset the drug acquisition costs of pemetrexed--platinum. Additionally, our study also observed that pemetrexed--platinum was associated with the lowest hospital costs for nonchemotherapy drugs and nondrug care in patients experiencing neutropenia or leukopenia, the two common hematological AEs associated with chemotherapy. This may suggest that pemetrexed treatment could further save hospital costs for nonchemotherapy drugs and nondrug care by causing less severe hematological toxicity. Thus, our study is a great example to demonstrate economic benefits associated with tumor responses and clinical toxicity of chemotherapy when treating cancer patients.
The superior tumor response and better safety profile associated with pemetrexed treatments in the real-world first-line setting for AdvNS-NSCLC were consistent with what were observed in Chinese patients receiving pemetrexed treatment in the second-line setting.[@b18-ceor-8-097] Our study observed superior tumor response but highly comparable clinical toxicity associated with pemetrexed treatment when compared with docetaxel--platinum doublet. Because the highly comparable clinical toxicity between the two doublets should consume similar hospital resources for AE management, the observed superior tumor response associated with pemetrexed treatment was the only known factor contributing to the saved hospital costs for nonchemotherapy drugs and nondrug care associated with pemetrexed treatment. A recent Phase III trial reported that the increased utilization of health resources associated with maintenance therapy in tumor-controlled patients was mainly related to hematological AE management, including blood transfusion, G-CSF, and anti-infection medications.[@b19-ceor-8-097] Thus, we believe that the better safety profile associated with pemetrexed treatment could further save hospital costs for nonchemotherapy drugs and nondrug care when compared with the paclitaxel-, gemcitabine-, or vinorelbine-based doublet, the three cytotoxic agents usually associated with significant hematological toxicity. Additionally, the better safety profile of pemetrexed treatment was mainly related to hematological toxicity. The saved hospital costs for nonchemotherapy drugs and nondrug care associated with pemetrexed treatment could be the result of less use of health resources for hematological AEs. With the rare use of blood transfusions and platelet infusions in our study cohort, the reduced hospital costs for nonchemotherapy drugs associated with pemetrexed treatment may suggest reduced utilization of nonchemotherapy drugs managing hematological toxicity. Future studies are needed to further confirm our hypothesis on the saved drug costs for hematological toxicity in patients receiving pemetrexed treatment.
Our study ranked the five studied doublets for their impact on hospital costs for nonchemotherapy drugs and nondrug care in patients stratified by tumor response and hematological AE to further explore any other factors that could affect hospital costs for nonchemotherapy drugs and nondrug care. Pemetrexed--platinum doublet was associated with significantly lower hospital costs for nonchemotherapy drugs and nondrug care in patients who experienced neutropenia or leukopenia, the two conditions usually treated with G-CSF and antibiotics.[@b20-ceor-8-097] This finding suggests that the hematological toxicity associated with pemetrexed treatment could be less severe. Because the small sample size does not allow us to adjust possible confounding effects associated with tumor response and nonhematological AEs in these patients, future studies are needed to confirm our hypothesis regarding the impact of AE severity on hospital costs for nonchemotherapy drugs and nondrug care. Another important factor contributing to lower hospital costs for nonchemotherapy drugs and nondrug care associated with pemetrexed treatment is the treatment administration schedule. Both gemcitabine and vinorelbine were administered twice at days 1 and 8 per treatment cycle, and the length of hospital stay per treatment cycle was increased by 4--5 days when compared to pemetrexed treatment, which was administered only once at day 1 per treatment cycle. Thus, the shorter hospital stay associated with pemetrexed treatment undoubtedly reduced hospital costs for nonchemotherapy drugs and nondrug care.
This study has several significant implications on clinical practice, research, and health policy-making. First, the generated clinical and economic evidences in this study could further reduce uncertainty associated with tumor response, clinical toxicity, and medical costs of platinum-based doublets for AdvNS-NSCLC in the first-line setting. Second, our study design and study methods can be used in other settings to explore the economic impact of clinical effectiveness and toxicity associated with chemotherapy. Third, the real-world tumor response, clinical toxicity, and hospital costs associated with the five studied doublets in our study can be applied to future cost-effectiveness analyses and budget impact analysis, which have been increasingly used to support reimbursement decision-making in the People's Republic of China. Finally, the tumor response associated with pemetrexed treatment in our study is much stronger than previous studies that mainly included Caucasian patients. We had a hypothesis that Chinese ethnicity could be more sensitive to pemetrexed than other cytotoxic agents. Thus, future studies are needed to confirm our hypothesis regarding the impact of ethnicity on clinical and economic benefits of pemetrexed treatment for AdvNS-NSCLC in the first-line setting.
There are several limitations associated with the retrospective nature of this study. First, about half of the eligible patients had no tumor response assessment because of early treatment discontinuation. Because our study was unable to identify the cause of treatment discontinuation, missing information on tumor response in patients with early treatment discontinuation could bias our comparisons on tumor response. Second, our study was unable to capture clinical toxicity associated with the studied platinum-based doublets outside of the two participating hospitals. The longer hospital stay associated with gemcitabine and vinorelbine doublets increased observation time for treatment toxicity and might overestimate the clinical toxicity associated with the two treatments. Third, the hospital settings were not adjusted in our analysis because paclitaxel- and vinorelbine-contained doublets were not used in XYH. Fourth, this study was unable to make full adjustment of potential confounding effects associated with social economic status on hospital costs for nonchemotherapy drugs and nondrug care due to the lack of information. Because the price of vinorelbine was much lower than other nonplatinum cytotoxic agents, it may be used more often in patients with lower socioeconomic status. Therefore, the confounding effects associated with possible lower socioeconomic status in patients receiving vinorelbine treatment could have overestimated the cost saving associated with pemetrexed treatment for nonchemotherapy drugs and nondrug care. Fifth, selection bias could also be introduced by the propensity score methods that only selected matched patients for comparisons of measured outcomes. The *P*-values of multiple comparisons between propensity score-matched treatment groups were not further adjusted to reduce the risk of type I error. Finally, the significant economic gaps and demographic differences across Chinese cities[@b11-ceor-8-097] might affect the generalizability of our study results based on two hospitals in a provincial capital.
Conclusion
==========
In summary, this retrospective cohort study demonstrated that the superior tumor response and better toxicity profile associated with pemetrexed--platinum doublet was also related to lower hospital costs for nonchemotherapy drugs and nondrug care, mainly for nonchemotherapy drugs, when compared with other platinum-based doublets frequently used in the first-line setting for AdvNS-NSCLC in Chinese patients. However, the saved hospital costs for nonchemotherapy drugs and nondrug care associated with pemetrexed treatment were not higher enough to completely offset the increased drug acquisition cost of pemetrexed relative to other cytotoxic agents.
The authors thank Lisa M Hess, the principal research scientist of Eli Lilly and Co, for her valuable comments and editorial support. They also thank the staff of Normin Health Changsha Representative Office for their logistics and administration support to this study. This research project was supported and monitored by an unrestricted health outcome research grant from Eli Lilly and Co, People's Republic of China.
**Disclosure**
Yicheng Yang, Narayan Rajan, Yun Chen are employees of Eli Lilly and Co. Canjuan Yang and Jianfeng Li are employees of Normin Health Changsha Representative Office. Dr Wendong Chen is the founder of Normin Health and receives consulting fee and research funds from Eli Lilly and Co. The other authors report no conflicts of interest in this work.
{#f1-ceor-8-097}
######
Impact of the studied five platinum-based doublets on the log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care in patients stratified by their tumor control status and hematological AEs.
**Notes:** Patients (**A**) irrespective of their status of tumor control and hematological AEs, (**B**) experiencing any hematological AE, (**C**) experiencing neutropenia, (**D**) experiencing leukopenia, (**E**) with tumor control, (**F**) without tumor control, (**G**) without any hematological AE, (**H**) experiencing anemia, and (**I**) experiencing thrombocytopenia. The graphs only included baseline variables with *P*-value \<0.5. Bold values represent statistically significant results, *P*\<0.05.
**Abbreviations:** HCTC, hospital costs per treatment cycle; AEs, adverse events; ECOG, Eastern Cooperative Oncology Group; G-CSF, granulocyte colony-stimulating factor; EPO, erythropoietin; IL-11, interleukin 11.
######
A summary of baseline characteristics of patients and treatment pattern associated with the five studied platinum-based doublets in the first-line setting for AdvNS-NSCLC
Studied platinum-based doublet Pemetrexed/Platinum Docetaxel/Platinum Paclitaxel/Platinum Gemcitabine/Platinum Vinorelbine/Platinum *P*-value
-------------------------------------------------- --------------------- -------------------- --------------------- ---------------------- ---------------------- ----------- ---- ------- ------ ----- --------- ------- ---- ------- ------ -------------
**Demography**
Age (years) -- 57.1 10.6 -- 55.6 9.4 -- 52.8 9.2 -- 55.6 9.0 -- 57.5 8.8 0.053
BMI (kg/m^2^) -- 21.9 2.8 -- 22.4 3.1 -- 22.2 3.2 -- 21.9 2.8 -- 21.3 2.9 0.687
BSA (m^2^) -- 1.6 0.2 -- 1.6 0.2 -- 1.6 0.2 -- 1.6 0.1 -- 1.6 0.2 0.724
Male (%) 94 60.3 -- 45 67.2 -- 28 71.8 -- 73 66.4 -- 52 69.3 -- 0.551
Nonsmoking (%) 92 59.0 -- 28 41.8 -- 20 51.3 -- 52 47.3 -- 35 46.7 -- 0.115
**Public health insurance type (%)**
Urban residents 91 58.3 -- 28 41.8 -- 14 35.9 -- 46 41.8 -- 32 42.7 -- **0.015**
Rural residents 54 34.6 -- 30 44.8 -- 17 43.6 -- 55 50.0 -- 35 46.7 -- 0.123
**ECOG performance status (%)**
0 7 4.5 -- 3 4.5 -- 10 25.6 -- 5 4.6 -- 11 14.7 -- \<**0.001**
1 142 91.0 -- 58 86.6 -- 27 69.2 -- 99 90.0 -- 60 80.0 -- **0.004**
2 7 4.5 -- 5 7.5 -- 2 5.1 -- 4 3.6 -- 4 5.3 -- 0.804
**Baseline marrow function**
Hemoglobin (g/L) -- 127.0 16.2 -- 125.0 16.4 -- 131.0 12.9 -- 126.0 16.0 -- 128.0 17.9 0.757
Neutrophilic -- 4.4 2.3 -- 4.5 2.3 -- 4.3 1.8 -- 4.7 2.2 -- 5.0 1.9 0.662
granulocyte count
(10^9^/L)
White cell count (10^9^/L) -- 6.7 2.7 -- 6.9 3.3 -- 6.6 2.6 -- 7.0 2.6 -- 7.4 2.4 0.489
Platelet count (10^10^/L) -- 21.9 8.6 -- 23.6 8.5 -- 22.9 7.2 -- 22.8 8.5 -- 25.0 8.1 0.440
**Disease stage and histology (%)**
Stage 4 142 91.0 -- 58 86.6 -- 30 76.9 -- 92 83.6 -- 61 81.3 -- 0.087
Adenocarcinoma type 156 100.0 -- 64 95.5 -- 38 97.4 -- 110 100.0 -- 74 98.7 -- **0.014**
**Number of metastasis sites (%)**
1 90 57.7 -- 25 37.3 -- 19 48.7 -- 55 50.0 -- 36 48.0 -- 0.086
2 30 19.2 -- 17 25.4 -- 7 18.0 -- 25 22.7 -- 21 28.0 -- 0.552
≥3 20 12.8 -- 6 9.0 -- 4 10.3 -- 9 8.2 -- 3 4.0 -- 0.284
**Site of metastasis (%)**
Brain 24 15.4 -- 18 26.9 -- 5 12.8 -- 12 10.9 -- 11 14.7 -- 0.096
Bone 69 44.2 -- 24 35.8 -- 12 30.8 -- 46 41.8 -- 31 41.3 -- 0.547
Liver 13 8.3 -- 11 16.4 -- 4 10.3 -- 12 10.9 -- 5 6.7 -- 0.356
Pleural 50 32.1 -- 8 11.9 -- 5 12.8 -- 29 26.4 -- 11 14.7 -- **0.001**
**Hospital setting**
HNPTH 46 29.5 -- 47 70.2 -- 39 100.0 -- 52 47.3 -- 75 100.0 -- \<**0.001**
XYH 110 70.5 -- 20 29.9 -- 0 0.0 -- 58 52.7 -- 0 0.0 -- \<**0.001**
**Admission year**
2010 38 24.4 -- 45 67.2 -- 22 56.4 -- 48 43.6 -- 58 77.3 -- \<**0.001**
2011 51 32.7 -- 19 28.4 -- 5 12.8 -- 40 36.4 -- 11 14.7 -- **0.002**
2012 67 43.0 -- 3 4.5 -- 12 30.8 -- 22 20.0 -- 6 8.0 -- \<**0.001**
**Dosage of chemotherapeutical agent (mg/m^2^)**
Cisplatin -- 75.0 4.9 -- 72.6 9.6 -- 71.4 6.8 -- 74.1 17.3 -- 73.7 8.3 \<**0.001**
Carboplatin -- 248.4 76.1 -- 261.5 48.6 -- 306.3 50.3 -- 286.0 56.6 -- 280.9 40.5 \<**0.001**
Cytotoxic agent -- 528.6 60.6 -- 73.2 6.8 -- 148.4 10.9 -- 2,343.8 390.1 -- 50.0 7.3 ***--***
**Treatment pattern**
Distribution of cisplatin 132 84.6 -- 50 74.6 -- 22 56.4 -- 98 89.1 -- 70 93.3 -- \<**0.001**
Treatment cycles -- 3.0 1.3 -- 2.0 1.1 1 2.0 1.1 -- 2.0 1.4 -- 2.0 1.2 \<**0.001**
Hospital episodes per -- 1.0 0.2 -- 1.0 0.0 0 1.0 0.2 -- 1.0 0.2 -- 1.0 0.2 0.236
cycle
Hospital stay length -- 9.5 5.6 -- 9.5 5.1 4 10.5 5.5 -- 14.1 6.0 -- 14.5 5.9 \<**0.001**
per cycle
**Treatment for hematological AE**
G-CSF 82 52.6 -- 39 58.2 -- 30 76.9 -- 62 56.4 -- 48 64.0 -- 0.061
EPO 4 2.6 -- 0 0.0 -- 1 2.6 -- 4 3.6 -- 2 2.7 -- 0.649
IL-11 7 4.5 -- 0 0.0 -- 3 7.7 -- 13 11.8 -- 12 16.0 -- \<**0.001**
TPO 1 0.6 -- 0 0.0 -- 0 0.0 -- 0 0.0 -- 1 1.3 -- 0.707
Blood transfusion 1 0.6 -- 0 0.0 -- 0 0.0 -- 1 0.9 -- 3 4.0 -- 0.240
Platelet infusion 1 0.6 -- 0 0.0 -- 1 2.6 -- 2 1.8 -- 0 0.0 -- 0.433
**Note:** Bold values represent statistically significant results, *P*\<0.05.
**Abbreviations:** AdvNS-NSCLC, advanced nonsquamous non-small-cell lung cancer; BMI, body mass index; BSA, body surface area; ECOG, Eastern Cooperative Oncology Group; HNPTH, Hunan Province Tumor Hospital; XYH, Xiangya Hospital; G-CSF, granulocyte colony-stimulating factor; EPO, erythropoietin; IL-11, interleukin 11; TPO, thrombopoietin.
######
Adjusted comparisons on tumor response and AEs between propensity score-matched treatment groups for pemetrexed/platinum versus the other four studied doublets in the first-line setting for AdvNS-NSCLC
Comparison Pemetrexed/Platinum Docetaxel/Platinum Pemetrexed/Platinum versus docetaxel/platinum Pemetrexed/Platinum Paclitaxel/Platinum Pemetrexed/Platinum versus paclitaxel/platinum Pemetrexed/Platinum Gemcitabine/Platinum Pemetrexed/Platinum versus gemcitabine/platinum Pemetrexed/Platinum Vinorelbine/Platinum Pemetrexed/Platinum versus vinorelbine/platinum
------------------------------------------------------------ --------------------- -------------------- ----------------------------------------------- --------------------- --------------------- ------------------------------------------------ --------------------------- ---------------------- ------------------------------------------------- --------------------- ---------------------- ------------------------------------------------- --------------------- ------ ---- ------ ------- ------------- --------------------- ------ ---- ------ ------- -------------
**Tumor response**
PR 9 14.8 4 6.6 2.250 0.166 4 10.3 5 12.8 0.800 0.739 10 10.8 2 2.2 5.000 **0.021** 8 11.0 4 5.5 2.000 0.248
SD 29 47.5 11 18.0 2.636 0.002 18 46.2 8 20.5 2.250 0.025 47 50.5 36 38.7 1.306 0.086 38 52.1 18 24.7 2.111 \<**0.001**
PR + SD 38 62.3 15 24.6 2.533 \<**0.001** 22 56.4 13 33.3 1.692 0.061 57 61.3 38 40.9 1.500 0.009 46 63.0 22 30.1 2.091 \<**0.001**
PD 12 19.7 19 31.1 0.632 0.127 8 20.5 10 25.6 0.800 0.593 18 19.4 13 14.0 1.385 0.251 12 16.4 16 21.9 0.750 0.414
Unknown 11 18.0 27 44.3 0.407 0.002 9 23.1 16 41.0 0.563 0.108 18 19.4 42 45.2 0.429 \<**0.001** 15 20.5 35 47.9 0.429 0.001
Adjusted risk of PD or unknown tumor response (OR, 95% CI) 0.170, 0.060--0.484 0.001 0.093, 0.014--0.621 0.014 0.276, 0.126--0.603 **0.001** 0.081, 0.025--0.257 \<**0.001**
**Hematological AE**
Neutropenia 17 27.9 14 23.0 1.214 0.532 6 15.4 10 25.6 0.600 0.248 20 21.5 16 17.2 1.250 0.465 13 17.8 16 21.9 0.813 0.532
Leukopenia 13 21.3 15 24.6 0.867 0.670 3 7.7 13 33.3 0.231 0.012 17 18.3 33 35.5 0.515 0.006 10 13.7 25 34.2 0.400 0.003
Anemia 16 26.2 18 29.5 0.889 0.695 9 23.1 16 41.0 0.563 0.071 27 29.0 66 71.0 0.409 \<**0.001** 19 26.0 49 67.1 0.388 \<**0.001**
Thrombocytopenia 8 13.1 14 23.0 0.571 0.134 7 17.9 12 30.8 0.583 0.132 18 19.4 43 46.2 0.419 \<**0.001** 13 17.8 28 38.4 0.464 0.011
Any hematological AE 30 49.2 34 55.7 0.882 0.465 18 46.2 23 59.0 0.783 0.251 45 48.4 81 87.1 0.556 \<**0.001** 32 43.8 57 78.1 0.561 \<**0.001**
**Nonhematological AE**
Alopecia 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- --
Arthralgia 3 4.9 5 8.2 0.600 0.480 1 2.6 0 0.0 -- 1.000 4 4.3 17 18.3 0.235 0.002 2 2.7 0 0.0 -- 0.500
Cough 1 1.6 1 1.6 1.000 1.000 0 0.0 3 7.7 0.000 0.250 2 2.2 23 24.7 0.087 \<**0.001** 1 1.4 3 4.1 0.333 0.317
Dermatitis 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- --
Diarrhea 0 0.0 1 1.6 0.000 1.000 0 0.0 0 0.0 -- -- 1 1.1 0 0.0 -- 1.000 0 0.0 1 1.4 0.000 1.000
Dyspnea 0 0.0 1 1.6 0.000 1.000 1 2.6 2 5.1 0.500 0.564 2 2.2 8 8.6 0.250 0.058 2 2.7 0 0.0 -- 0.500
Edema 1 1.6 0 0.0 -- 1.000 0 0.0 0 0.0 -- -- 0 0.0 1 1.1 0.000 1.000 1 1.4 0 0.0 -- 1.000
Fatigue 9 14.8 5 8.2 1.800 0.248 5 12.8 4 10.3 1.250 0.706 15 16.1 54 58.1 0.278 \<**0.001** 5 6.8 31 42.5 0.161 \<**0.001**
Nausea 28 45.9 31 50.8 0.903 0.564 18 46.2 28 71.8 0.643 0.025 45 48.4 58 62.4 0.776 0.053 31 42.5 60 82.2 0.517 \<**0.001**
Peripheral neuropathy 0 0.0 1 1.6 0.000 1.000 0 0.0 0 0.0 -- -- 0 0.0 2 2.2 0.000 0.500 0 0.0 3 4.1 0.000 0.250
Rash 2 3.3 0 0.0 -- 0.500 0 0.0 0 0.0 -- -- 2 2.2 4 4.3 0.500 0.414 2 2.7 0 0.0 -- 0.500
Stomatitis 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- -- 0 0.0 1 1.1 0.000 1.000 0 0.0 0 0.0 -- --
Vomiting 15 24.6 14 23.0 1.071 0.835 10 25.6 16 41.0 0.625 0.180 24 25.8 35 37.6 0.686 0.071 19 26.0 33 45.2 0.576 0.013
Weight loss 0 0.0 1 1.6 0.000 1.000 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- -- 0 0.0 0 0.0 -- --
**Adjusted risk of hematological AE (OR, 95% CI)**
Neutropenia 1.691, 0.511--5.601 0.390 0.000, \<0.001--\>999.999 1.000 1.679, 0.593--4.751 0.329 1.566, 0.399--6.138 0.520
Leukopenia 0.750, 0.173--3.248 0.701 0.000, \<0.001--\>999.999 1.000 0.248, 0.100--0.616 **0.003** 0.352, 0.100--1.243 0.105
Anemia 0.943, 0.263--3.382 0.928 0.023, 0.001--0.356 **0.007** 0.092, 0.039--0.217 \<**0.001** 0.063, 0.018--0.220 \<**0.001**
Thrombocytopenia 0.491, 0.106--2.268 0.362 0.001, 0.000--35.315 0.190 0.172, 0.070--0.422 \<**0.001** 0.242, 0.056--1.036 0.056
Any hematological AE 1.143, 0.365--3.582 0.819 0.203, 0.037--1.119 0.067 0.099, 0.039--0.247 \<**0.001** 0.153, 0.048--0.491 **0.002**
**Note:** Bold values represent statistically significant results, *P*\<0.05.
**Abbreviations:** AEs, adverse events; AdvNS-NSCLC, advanced nonsquamous non-small-cell lung cancer; RR, relative risk; PR, partial response; SD, stable disease; PD, progressive disease; OR, odds ratio; CI, confidence interval.
######
Adjusted comparisons on the allocation of HCTC between propensity score-matched treatment groups for pemetrexed/platinum versus the other four studied doublets in the first-line setting for AdvNS-NSCLC
Comparison Pemetrexed/Platinum Docetaxel/Platinum Pemetrexed/Platinum versus docetaxel/platinum *P*-value Pemetrexed/Platinum Paclitaxel/Platinum Pemetrexed/Platinum versus paclitaxel/platinum *P*-value Pemetrexed/Platinum Gemcitabine/Platinum Pemetrexed/Platinum versus gemcitabine/platinum *P*-value Pemetrexed/Platinum Vinorelbine/Platinum Pemetrexed/Platinum versus vinorelbine/platinum *P*-value
---------------------------------------------------------------------------------------------------------------------------------- -------------------------- -------------------- ----------------------------------------------- ----------- --------------------- --------------------- ------------------------------------------------ ------------------------- --------------------- ---------------------- ------------------------------------------------- ----------- --------------------- ---------------------- ------------------------------------------------- ----------- --------- --------- --------- --------- ------------- ----------------------- --------- --------- --------- --------- --------- -------------
**Allocation of HCTC (RMB)[a](#tfn5-ceor-8-097){ref-type="table-fn"}**
Platinum agent ¥235 ¥229 ¥250 ¥229 −¥15 ¥0 0.160 ¥254 ¥229 ¥294 ¥267 −¥40 −¥38 0.060 ¥233 ¥229 ¥238 ¥229 −¥5 ¥0 0.675 ¥234 ¥229 ¥237 ¥229 −¥3 ¥0 0.330
Nonplatinum cytotoxic agent ¥11,638 ¥8,704 ¥2,734 ¥2,116 ¥8,904 ¥6,588 \<**0.001** ¥11,107 ¥8,704 ¥1,667 ¥1,637 ¥9,439 ¥7,067 \<**0.001** ¥11,284 ¥8,704 ¥5,718 ¥4,068 ¥5,567 ¥4,636 \<**0.001** ¥11,275 ¥8,704 ¥1,366 ¥1,372 ¥9,909 ¥7,332 \<**0.001**
Nonchemotherapy drugs ¥4,885 ¥4,193 ¥8,023 ¥6,695 −¥3,138 −¥2,502 \<**0.001** ¥5,407 ¥4,203 ¥7,670 ¥6,570 −¥2,262 −¥2,367 **0.006** ¥4,830 ¥4,061 ¥6,627 ¥5,612 −¥1,797 −¥1,551 \<**0.001** ¥5,115 ¥4,203 ¥6,163 ¥5,888 −¥1,048 −¥1,684 **0.012**
Nondrug care ¥5,330 ¥4,158 ¥5,757 ¥4,776 −¥427 −¥618 0.360 ¥5,741 ¥4,038 ¥6,148 ¥5,253 −¥408 −¥1,215 0.357 ¥5,561 ¥4,490 ¥6,094 ¥5,533 −¥533 −¥1,043 0.096 ¥5,545 ¥4,660 ¥5,731 ¥4,819 −¥186 −¥159 0.985
Total ¥22,192 ¥19,264 ¥16,763 ¥14,584 ¥5,428 ¥4,680 \<**0.001** ¥22,509 ¥19,470 ¥15,780 ¥14,367 ¥6,729 ¥5,103 \<**0.001** ¥21,976 ¥19,470 ¥18,677 ¥17,778 ¥3,300 ¥1,692 **0.001** ¥22,170 ¥19,773 ¥13,498 ¥12,374 ¥8,672 ¥7,400 \<**0.001**
HCTC for supportive care (RMB)a ¥10,319 ¥9,053 ¥13,780 ¥11,479 −¥3,461 −¥2,426 **0.003** ¥11,148 ¥9,280 ¥13,818 ¥12,532 −¥2,670 −¥3,251 **0.009** ¥10,459 ¥9,053 ¥12,721 ¥11,581 −¥2,262 −¥2,528 **0.001** ¥10,660 ¥9,202 ¥11,894 ¥10,679 −¥1,234 −¥1,478 0.058
Log~10~ scale of HCTC for nonchemotherapy drugs and nondrug care (coefficient, 95% CI)[b](#tfn6-ceor-8-097){ref-type="table-fn"} −0.246, −0.406 to −0.085 **0.0027** −0.351, −.547 to −0.156 0 \<**0.001** −0.194, −0.309 to −0.078 **0.001** −0.128, −0.265--0.009 0.066
**Notes:**
Head-to-head comparison between propensity score-matched treatment groups.
Multiple logistic regression analysis with further adjustment of imbalanced baseline variables in propensity score-matched patients. 1 RMB = US\$0.16 in 2012. Bold values represent statistically significant results, *P*\<0.05.
**Abbreviations:** AdvNS-NSCLC, advanced nonsquamous non-small-cell lung cancer; HCTC, hospital costs per treatment cycle; RMB, Chinese currency yuan; CI, confidence interval.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Many neurodegenerative diseases such as Alzheimer\'s disease (AD), synucleinopathies (including Parkinson\'s disease, PD), Huntington\'s disease (HD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia can be included under the definition of "prion-like disease" as they share some neuropathological features with the prion diseases (transmissible spongiform encephalopathies, TSEs). Overall, the prion and prion-like diseases have high social impact and cost as they include the most common age-related disorders. The proteins directly involved in prion-like diseases \[Amyloid β (Aβ) for AD, tau for tauopathies, and α-synuclein (α-syn) for synucleinopathies\] show similarities to prion protein in terms of the mechanism of seeding (Yamamoto et al., [@B253]; Nonaka et al., [@B159]) and spreading (Frost et al., [@B71]; Hansen et al., [@B94]) and of cell interaction modalities (Yamamoto et al., [@B253]; Nonaka et al., [@B159]). In the central nervous system (CNS), prion-like proteins can aggregate in an ordered "cross-β" assembly following a "nucleated growth" process and they can produce fibers that precipitate forming intracellular amyloid-like inclusions or extracellular amyloid deposits associated to neurodegeneration. Recently, Prusiner and collaborators demonstrated the ability of α-syn to serially propagate the infection both *in vitro* and *in vivo* systems defining it as a new prion protein (Prusiner et al., [@B176]; see Section "α-syn as New Human Prion" for more details). Prion proteins, α-syn and prion-like proteins (Aβ and tau) can directly interact with several metal ions affecting the conformation of the protein and its tendency to aggregate in oligomers and fibrils.
Epidemiological studies show that the occupational exposure to environmental metals such as manganese, copper, lead, iron, mercury, zinc, and aluminum is a risk factor for the development of neurodegenerative and prion-like diseases (Zayed et al., [@B256]; Rybicki et al., [@B189]; Gorell et al., [@B84]; Benedetto et al., [@B12]; Fukushima et al., [@B73]; Cannon and Greenamyre, [@B33]). On the other hand, metal ions and especially transition metals are key elements in organisms\' life. They act like cofactors of many enzymes and are essential in cell respiration and metabolism due to their ability to accept or donate electrons passing from a reduced to an oxidized state (Barnham and Bush, [@B9]). However, transition metal ions can also be potentially dangerous for the cell as they can participate in redox reactions leading to the formation of reactive oxygen species (ROS) that can oxidize intracellular proteins, lipids, and nucleic acids. In particular, the radical-mediated oxidation of a protein can strongly affect its molecular structure by the generation of protein-protein linkages, the oxidation of amino acid side chains and even by the fragmentation of the polypeptide chain and the level of oxidized proteins increases during aging in many animals (for detail refer to Stadtman, [@B210]; Valensin et al., [@B230]).
For this reason, in the CNS any factor which induces a metal dyshomeostasis and the consequent production of ROS and cell injury may contribute to the onset of these neurodegenerative diseases. On the contrary, any factor able to reduce metal dyshomeostasis and to limit the production of ROS and free radicals can play a neuroprotective action. In addition to these general aspects, metal ions can have a more direct involvement in prion and prion-like neurodegenerative diseases for their ability to directly bind to prion and prion-like proteins and to affect the amount of amyloid aggregates.
The diet, source of metal ions and antioxidant agents such as polyphenols for the organism, is an aspect to take into account in addressing the issue of neurodegeneration. In recent decades, the food consumption in Western countries has undergone a rapid change that quickly led to the shift from a seasonal diet based on the consumption of fresh vegetable food and some meat derived from extensive farming to a massive consumption of packaged food and meat resulting from factory farms where animals are usually fed with a non-natural diet and subjected to drug treatments. The so-called mad cow disease is a striking example of how the change in farming method can contribute to the spread of prion diseases. In addition, the use of pesticides and herbicides in intensive agriculture determines the inclusion in the food chain of chemical substances that potentially can have harmful effects in the CNS even reacting with prion and prion-like proteins. Such changes in human diet lead to a variation in the nutrient intake that may affect the antioxidant and metal bioavailability in the CNS. Epidemiological data suggest that the Mediterranean diet (MeDi), based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases.
In this review, metal role in the onset of prion diseases (TSEs and synucleinopathies) and in AD, as an example of prion-like disease, is dealt with from a nutritional, cellular and molecular point of view.
Prion protein as a model protein for prion diseases {#s2}
===================================================
The cellular prion protein (PrP^C^) is a highly conserved cell surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed in all mammalian tissues, particularly in the CNS (Harris, [@B96]). A conformationally-modified isoform of PrP^C^ called "Scrapie" (PrP^Sc^) is the major component of prions, the infectious particles at the basis of rare and inexorably fatal neurodegenerative disorders, called TSEs or prion diseases. TSEs develop on genetic, sporadic, or infectious grounds (Prusiner, [@B175]), and include bovine spongiform encephalophathy (BSE) in cattle, scrapie in sheep and goat, chronic wasting disease (CWD) in cervids and Creutzfeldt-Jacob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome in humans. TSEs are characterized by spongiform modifications in the brain, amyloid deposits with neuronal loss, and synaptic dysfunction. PrP^C^ and PrP^Sc^ though sharing the same primary structure and post-translational modifications (Stahl et al., [@B211]), are characterized by a different secondary structure. The β sheet-rich overall structure confers to PrP^Sc^ different physico-chemical and biologic features, including increased tendency to aggregate and resistance to proteolysis, the ability to self-propagate in a host organism and the acquisition of novel neurotoxic properties (Prusiner, [@B175]). The peculiarity of PrP^Sc^ is its ability to self-propagate in exponential fashion in cells, by acting as a model for the protein misfolding (Soto, [@B207]). PrP^C^ is tethered to the outside of the plasma membrane through its GPI anchor in plasma membrane subdomains called lipids rafts (Stahl et al., [@B212]) and it can shift to caveolae mediating signal transduction events (Toni et al., [@B223]). PrP^C^ NMR structure shows a flexible and disordered N-terminal domain (NT) (23--124 residues) and a C-terminal globular domain (CT) (125--228) that contains three α-helices, two short anti-parallel β-strands, and a short C-terminal tail (Zahn et al., [@B254]). This structure is stabilized by a single disulfide bond between Cys-179 and Cys-214 (human sequence). The NT contains five highly conserved proline- and glycine-rich octapeptide repeats (OR) (Harris, [@B96]). In spite of PrP^C^\'s intimate involvement in TSE prion propagation, its function in cell physiology remains enigmatic, also because PrP-KO mice, in which the gene coding for PrPC was ablated (PrP-KO) by different gene-targeting strategies, showed no defects in embryonic and postnatal development and no behavioral alterations (Büeler et al., [@B28]; Manson et al., [@B136]; Mallucci et al., [@B133]) and only marginal phenotypes under normal conditions were observed in these mice (Criado et al., [@B42]; Nazor et al., [@B155]). These phenotypes could be due to the activation of compensatory mechanisms that hide the PrP-KO phenotype under physiological conditions and makes it detectable only under specific stress conditions or during aging (for a review see Ref. Peggion et al., [@B168]). However, several functions have been attributed to PrP^C^ (Peggion et al., [@B168]), some of which related to the ability of this protein to bind transition metal ions.
Prion protein binds transition metals {#s3}
=====================================
Several metal ions are able to bind with different affinities to the PrP^C^, affecting its conformation and tendency to aggregate and influencing PrP^C^/PrP^Sc^ conversion. The OR region of PrP^C^ is the main metal binding site that can interact with Cu^2+^, Zn^2+^, and Mn^2+^ (Jackson et al., [@B100]; Walter et al., [@B241]; Brown, [@B23]) and it confers high stability to the protein (Benetti et al., [@B13]).
At physiological pH, PrP^C^ is able to bind four Cu^2+^ ions with high affinity in specific sites of OR region (Brown et al., [@B25]; Thompsett et al., [@B218]) and two additional Cu^2+^ at residues 96 and 111 with lower affinity (Jones et al., [@B106]). Many studies suggest the involvement of PrP^C^ in copper homeostasis (Brown et al., [@B25]; Wong et al., [@B251]) as extracellular Cu^2+^ ions stimulate PrP^C^ endocytosis (Pauly and Harris, [@B166]; Brown and Harris, [@B27]), particularly at the pre-synaptic membrane (Vassallo and Herms, [@B234]) and regulate PrP^C^ expression (Toni et al., [@B222]).
PrP^C^ has been associated with metal-dependent enzymatic functions (Brown et al., [@B26]; Rachidi et al., [@B181]) and, consequently, with the enhancement of cell anti-oxidant potentials (White et al., [@B248]; Mitteregger et al., [@B146]; Peggion et al., [@B168]). Recently, a Cu^2+^-dependent neuroprotective role of PrP^C^ by mediating N-methyl-d-aspartate (NMDA) receptor-nitrosylation (Gasperini et al., [@B77]) has been demonstrated.
Experimental evidence supports the involvement of copper not only in PrP physiology but also in its pathology. In fact, the binding of copper to PrP would make the protein a relatively easy target of metal-catalyzed oxidation and it would lead to structural modifications that favor the PrP^C^--PrP^Sc^ conversion and the following protein aggregation. Experimental evidence shows that both histidine and methionine residues can be oxydated and involved in these events (Requena et al., [@B184]; Colombo et al., [@B39]). Currently available data are sometimes conflicting and does not allow a clear interpretation of Cu^2+^ role in prion disease. *In vitro* and *in vivo* experiments show that the addiction of Cu^2+^ induces the conversion of PrP^C^ into PrP^Sc^, increases the protease resistance and the infectivity of the protein, and overall accelerates prion disease (Pauly and Harris, [@B166]; Qin et al., [@B179]; Quaglio et al., [@B180]; Kuczius et al., [@B116]; Kim et al., [@B110]; Canello et al., [@B32]), while Cu^2+^ chelation delays the beginning of the disease (Sigurdsson et al., [@B199]; Siggs et al., [@B198]). On the other hand, similar experiments indicate that the presence of Cu^2+^ inhibits conversion of PrP^C^ into PrP^Sc^ and consequently its accumulation, delaying the onset of the disease in infected cells (Hijazi et al., [@B97]; Bocharova et al., [@B17]; Orem et al., [@B161]; Mitteregger et al., [@B145]). One possible explanation of these contrasting results comes from *in vitro* studies suggesting that Cu^2+^ ions can exert different effects on PrP^C^/PrP^Sc^ conversion depending on whether the template is constituted by soluble recombinant/purified PrP proteins or by preformed fibrils (Liu et al., [@B125]). Moreover, it has been reported that point mutations in PrP^C^ gene may cause coordinational changes at the copper site, favoring the conversion of PrP^C^ into PrP^Sc^ (D\'Angelo et al., [@B46]; Giachin et al., [@B78]). Other possible explanations come from analyses of the ecosystems that support defined clusters of sporadic TSE. These studies suggest that Cu ions, such as Zn ions, can exert different effects on PrP^C^/PrP^Sc^ conversion depending on whether these transition metals are free or bound to scavenger co-factors in the CNS (Purdey, [@B178]).
TSEs are characterized by dys-metal homeostasis and by increased oxidative stress. Elevated levels of reactive oxygen intermediate species, nitric oxide and lipid peroxidation markers were detected both in the brains of mice infected with scrapie strains (Wong et al., [@B250]) and in sporadic CJD frontal cortex homogenates (Freixes et al., [@B70]). The continuous conversion of PrP^C^ into PrP^Sc^ deprives neurons from the control of metal balance. However, whether the imbalance of metals is the cause or the consequence of TSEs is not yet known. PrP^C^ protein is able to bind Mn^2+^ at His96 and in the C-terminal region between residues 91 and 230 (Treiber et al., [@B224]; Brazier et al., [@B21]), protecting cells against Mn^2+^-induced oxidative stress (Choi et al., [@B37]). Manganese induces spontaneous PrP^Sc^ conversion (Brown et al., [@B24]) and increases the infectivity of PrP^Sc^ in cultured cells (Davies and Brown, [@B49]), while Mn^2+^ chelation decreases the amount of PrP^Sc^ present in infected mice brains (Brazier et al., [@B22]). Mn^2+^ levels are increased in blood and brain samples from subjects affect by BSE, scrapie, CJD, and from experimentally infected mice. Moreover, the increased manganese levels were inversely correlated to copper concentrations (Wong et al., [@B252]; Thackray et al., [@B217]).
The influence of the environment on prion diseases is supported by studies indicating a geographical increase of CJD prevalence in people who have lived in a region of Slovakia where manganese is a major pollutant. High Mn^2+^ levels and increased Mn^2+^/Cu^2+^ ratios were observed in these CJD brains (Slivarichová et al., [@B204]). The link between Mn^2+^ and prion infection was also evidenced at the cellular level (Pass et al., [@B165]).
PrP^C^ binds also Zn^2+^ regulating its homeostasis, although with lower affinity and at low concentrations than Cu^2+^. Zinc, as copper, increases endocytosis of the prion protein causing the metal internalization and its elimination from the synaptic cleft. It has been also speculated that PrP^C^ acts as a sensor to monitor Zn^2+^ extracellular levels that may trigger a PrP^C^-induced signaling (Rana et al., [@B182]). Moreover, PrP^C^ may affect Zn^2+^ uptake via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Watt et al., [@B245]). Studies conducted on several PrP^C^-peptides demonstrate that Zn^2+^ induces the increase of PrP^Sc^ aggregation while in the presence of full length PrP^C^, Zn^2+^caused a reduction of PrP^Sc^ deposition (Jobling et al., [@B102]; Kenward et al., [@B108]).
PrP^C^ contributes to maintaining iron homeostasis in the brain by regulating the iron uptake through the interaction with transferrin receptor pathway (Waheed et al., [@B238]) and by working as a ferric reductase in the transport of Fe^3+^ from endosomes to the cytosolic ferritin (Singh et al., [@B203]). Iron dyshomeostasis involves an incorrect nerve myelinizzation, altered neurotransmission, and affect bioenergetic processes resulting in neuronal death.
In sporadic-CJD brains, iron is sequestered in heat- and SDS-stable ferritin-PrP^Sc^ protein complexes, inducing an iron deficiency phenotype (Singh et al., [@B200]). So, despite the increased redox-activity of iron, its sequestration in complex aggregate proteins leads to a functional iron deficit. Moreover, Basu et al. ([@B10]) demonstrated that the exposure to a source of reduced iron, such as inorganic ferrous chloride (FeCl~2~) induced the conversion of PrP^C^ to PrP^Sc^ implicating a role for this metal in the generation and propagation of PrP^Sc^.
Synucleinopathies as prion diseases {#s4}
===================================
The term "synucleinopathies" defines a group of human neurodegenerative disorders characterized by the presence of amyloidogenic α-synuclein (α-syn) inclusions that can occur in neurons and glia cells of the central and peripheral nervous system. These high social impact diseases include PD, Parkinson\'s disease dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and a number of less well-characterized neuroaxonal dystrophies (Goedert, [@B80]).
Patients affected by synucleinopathies can show both motor and non-motor symptoms. PD is a progressive neurological disorder characterized by depression, anosmia, sleep disturbances, bradykinesia, resting tremor, rigidity, and postural instability (Fahn, [@B67]). Dementia is a frequent complication of PD characterized by deficits in attention, recognition memory and visual perception, by sleep disturbances, hallucinations and paranoid ideas (Perry et al., [@B172]; McKeith and Mosimann, [@B142]; Vann Jones and O\'Brien, [@B233]; Garcia-Ptacek and Kramberger, [@B75]). MSA (also previously referred as Shy--Drager syndrome, olivopontocerebellar atrophy, and striatonigral degeneration) is characterized by muscle rigidity, tremor, cerebellar dysfunctions that include ataxia, and impairment of the autonomic nervous system (Goedert, [@B80]).
The amyloidogenic α-syn inclusions are intracellular proteinaceous bodies containing aggregates of hyperphosphorylated α-syn rich in β-sheets (Baba et al., [@B7]; Wakabayashi et al., [@B239]; Spillantini, [@B208]; Fujiwara et al., [@B72]) that are called Lewy bodies (LBs) and Lewy neurites (LNs) in PD, PDD, and LDB (Spillantini et al., [@B209]), glial cytoplasmic inclusions in MSA (Gai et al., [@B74]), and axonal spheroids in neuroaxonal dystrophies (Newell et al., [@B157]). A causative role for α-syn in the development of synucleinopathies has been established in PD via the discovery of mutations in the α-syn gene *SNCA* (A30P, E46K, H50Q, G51D, and A53T) resulting in autosomal-dominant PD (Kruger et al., [@B115]; Zarranz et al., [@B255]; Appel-Cresswell et al., [@B4]; Lesage et al., [@B121]; Proukakis et al., [@B174]; Pasanen et al., [@B164]).
α-syn belongs to synuclein family together with β- and γ-syns. These proteins are particularly expressed at the level of the central (α- and β-syn) and peripheral (γ-syn) nervous system. In the CNS, α-syn is expressed in both neurons and glial cells and in neurons it is mainly localized in the cytoplasm and in presynaptic terminals, but a nuclear localization has been also reported (Vivacqua et al., [@B235]). The syn family members were sequenced in species representative of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved and fulfill important physiological functions (Toni and Cioni, [@B220]; Toni et al., [@B221]; Figure [1](#F1){ref-type="fig"}). However, the exact physiological roles of these proteins have not been fully clarified yet.
), amphibians (***Xenopus laevis***, [NP_001080623](NP_001080623)), reptiles (***Anolis carolinensis***, [XP_003221349](XP_003221349)), birds (***Taeniopygia guttata***, [NP_001041718](NP_001041718)), and mammals (***Homo sapiens***, NP_001139526)**. Sequences were aligned with Clustal Omega (<http://www.ebi.ac.uk/Tools/msa/clustalo/>). Asterisks indicate identity of amino acids; double dots indicate amino acids with the same polarity or size; dots indicate semiconserved substitutions. Cu^2+^ binding sites are indicated by arrowheads, Cu^+^ binding regions are underlined, manganese, and iron binding sites are highlighted in gray and Ca^2+^ binding regions are double underlined. Negatively charged residues are indicated in bold characters. Circled P letter indicates residues whose phosphorylation increases the α-syn binding affinity for Cu^2+^, Pb^2+^, and Fe^2+^.](fnins-11-00003-g0001){#F1}
Human α-syn is a naturally unfolded protein composed of 24 negatively charged residues (Asp and Glu) that belongs to the class of "intrinsically disordered proteins" (Uversky et al., [@B229]; Figure [1](#F1){ref-type="fig"}). Its primary structure can be subdivided in the N-terminal region (NT) (1--60), the NAC (non-amyloid component) segment (61--95), and C-terminal region (96--140) (CT) (Figure [1](#F1){ref-type="fig"}). As in the case of the prion protein PrP^C^/PrP^Sc^, α-syn can exist in different conformations in the cell, passing from an unstructured conformation in solution to a high α helix percentages (63--71%) when linked to the phospholipid vesicles (Davidson et al., [@B47]) or to a major anti-parallel β-sheet conformation when aggregated into fibrils (Weinreb et al., [@B247]; El-Agnaf et al., [@B61]; Narhi et al., [@B153]). The NT can assume α-helix conformation binding to lipid membranes (Davidson et al., [@B47]) and from these features probably depend the involvement of α-syn in vesicle trafficking (Burré et al., [@B29]), neurotransmitter release and synaptic plasticity (Abeliovich et al., [@B1]; Murphy et al., [@B152]). The NAC segment is a highly hydrophobic region containing the amino acid sequence GVTAVAQKTVE capable of acquire antiparallel β-sheet conformation responsible for the aggregation and precipitation of α-syn proteins in insoluble fibrils (Narhi et al., [@B153]). The CT is an unstructured negatively charged region able to bind metal ions and proteins that probably acts as a scaffold for the recruitment of additional protein to the membrane (Eliezer et al., [@B64]).
α-syn as a new human prion {#s5}
==========================
A series of experimental evidence collected in the last 15 years supports the existence of a "prion like" mechanism in synucleinopathies according to which the α-syn pathology can spread between cells following neuroanatomical traits and the amyloidogenic β-syn can act as a template to guide the conversion of soluble, natively, unfolded α-syn to a conformationally-altered, aggregated form able to transmits from cell-to-cell. Recently, Prusiner and co-workers have defined the α-syn as a new human prion (Prusiner et al., [@B176]). Main experimental results are summarized in Figure [2](#F2){ref-type="fig"} (for further details refer to the figure caption).
![**Schematic overview of the main evidence in support of the prion nature of human α-syn. (A)** *In vitro* experiments, recombinant α-syn monomer can generate amylodogenic fibrils able to infect cultured cells. **(B)** In the idiopathic PD the formation of proteinaceous inclusion bodies begins in the dorsal motor nucleus of the vagus nerve and advances from there essentially upwards through susceptible regions of the medulla oblongata, pontine tegmentum, midbrain, and basal forebrain until it reaches the cerebral cortex (circled numbers refer to the synuclein progression in the CNS). **(C)** 12--22 years after transplantation into the striatum of individuals with PD, grafted nigral neurons developed α-syn positive LBs that stained positively also for ubiquitin providing evidence that the disease can propagate from host to graft cells. **(D)** Dopaminergic neurons extracted from the ventral mesencephalon of E12.5 C57BL/6 mice were grafted to the striatum of 6-week old transgenic mice overexpressing h α-syn. After 6 months, the grafted dopaminergic neurons showed putative intracellular h α-syn positive punctae demonstrating *in vivo* transfer of α-syn between host cells and grafted dopaminergic neurons. **(E)** Cell-produced α-syn is secreted via an exosomal calcium-dependent mechanism. The extracellular α-syn is taken up by cells through endocytosis and inside the cell interacts with intracellular α-syn forming dimers. **(F)** (1) Homozygous TgM83^+/+^ mice expressing A53T h α-syn in CNS neurons developed intracytoplasmic neuronal α-syn inclusions and a severe and complex motor impairment leading to paralysis and death starting from the 8th month; (2) hemizygous TgM83^+/−^ mice developed the same symptoms between 22 and 28 months of age; (3) Tg mice that express wild type h α-syn developed no motor impairment and revealed normal neuropil staining pattern expected for the protein up to the age of 28 months; (4) TgM83^+/+^ mice inoculated with brain homogenates from sick 12 or 18 month-old TgM83^+/+^ mice showed the characteristic motor clinical signs of illness after 97 days post-inoculation (dpi); (5) bigenic Tg mice (M83^+/−^; *Gfap*-luc) inoculated with brain homogenates from spontaneously ill 10 month-old TgM83^+/+^ sample showed symptoms of synucleinopathies 160 dpi; (6) C57B1/6S Δα-syn mice (presenting a deletion of the α-syn locus) inoculated with brain homogenates from sick, 12 or 18 month-old TgM83^+/+^ mice, show no signs of disease and were still alive and healthy 14 months post-inoculation; (7) brain homogenate from healthy 2 month-old TgM83^+/+^ inoculated in TgM83^+/+^ mice did not induce an acceleration in the onset of α-syn pathology in TgM83^+/+^ mice; (8) Tg mice (M83^+/−^; *Gfap*-luc) inoculated with brain homogenate from two independent confirmed cases of MSA began to exhibit signs of neurologic illness, most commonly ataxia and circling behavior, at about 90 dpi; (9) WT C57BL6/C3H mice inoculated with synthetic pre-formed α-syn fibrils (PFF) obtained *in vitro* from recombinant mouse or h α-syn progressively developed α-syn cytoplasmatic accumulation at 30 dpi that evolved in dense perinuclear LB-like inclusions by 90 and 180 dpi; (10) α-syn^−/−^ mice inoculated with PFF did not develop α-syn deposits; (11) no phosphorylated α-syn, ubiquitin or p62-positive pathology was observed in the brain of WT C57BL/6J mice inoculated with human or mouse α-syn monomer; (12) WT C57BL/6 mice inoculated with nigral LB-enriched fractions from post mortem PD brain resulted in progressive nigrostriatal neurodegeneration and diffusely α-syn accumulation within nigral neurons and anatomically interconnected regions by 4 up to 17 months post-inoculation; (13) WT C57BL/6 mice inoculated with nigral non-LB fractions from post mortem PD brain showed no nigrostriatal degeneration; (14) WT C57BL/6 mice inoculated with α-syn-immunodepleted nigral LB fractions from post mortem PD brain showed no phospho α-syn-positive cells and any evidence of pathology; (15) α-syn^−/−^ C57Bl6Sv129 mice inoculated with nigral LB fractions from post mortem PD brain did not produce any α-syn pathology or evidence of nigrostriatal lesions; (16) WT C57BL/6J mice brain injected with DLB insoluble fraction from post mortem PD brain showed in the 50% of cases immunopositive structures for anti-phosphorylated α-syn at 15 months post injection (17--21) recombinant full length mouse fibrillar α-syn (mfib) or fibrillar human 21-140 α-syn (hfib) were injected in the hind limb muscle of 2 month-old TgM83^+/+^ (17), of TgM83^+/−^ (18--19), of WT C3H/C57BL6 (20), or of α-*syn*^−/−^ (21) mice. The injected mice died or had to be killed due to paralysis within 57--88 (TgM83^+/+^) or 121 (TgM83^+/−^) dpi. Mice also developed α-syn inclusion pathology that was nearly indistinguishable morphologically in anatomic distribution from that seen in aged (\>8 month-old) untreated TgM83^+/+^ mice. Four of the seven mice that had sciatic nerve transection showed no motor deficits 200 dpi (19). On the contrary, WT and α-syn^−/−^ mice developed no motor phenotype or α-syn pathology at 12 months post injection (20--21); (22) The injection of human Δ71--82 α-syn in TgM83^+/+^ mice resulted in delayed onset of disease (120 dpi) and incomplete penetrance of the pathology. **(G)** Inoculation with MSA brain homogenate in TgM83^+/−^ mice caused CNS dysfunction and the accumulation of large aggregates of phosphorylated α-syn and widespread astrocytic gliosis with mean incubation periods of 106--143 dpi (primary transmission). The inoculations in TgM83^+/−^ mice with brain homogenates from ill TgM83^+/−^ mice previously inoculated with MSA showed a shorter incubation period (92--113) (secondary transmission). The brain homogenate from MSA patients and from both serially infected TgM83^+/−^ mice were able to infect cultured cells. Figure drawings refers to the following references: (**A**; Desplats et al., [@B52]; Luk et al., [@B132]; Emmanouilidou et al., [@B65]; Nonaka et al., [@B159]; Volpicelli-Daley et al., [@B237]; Narkiewicz et al., [@B154]); (**B**; Braak et al., [@B20]); (**C**; Kordower et al., [@B112]; Li et al., [@B122], [@B123]; Kurowska et al., [@B117]); (**D**; Hansen et al., [@B94]); (**E**; Emmanouilidou et al., [@B65]; Hansen et al., [@B94]); \[**F**: 1--3 (Giasson et al., [@B79]); 4, 6, 7 (Mougenot et al., [@B149]); 5, 8 (Watts et al., [@B246]); 9, 10 (Luk et al., [@B131]); 11, 16 (Masuda-Suzukake et al., [@B141]); 12--15 (Recasens and Dehay, [@B183]); 17--21 (Sacino et al., [@B190])\]; (**G**; Prusiner et al., [@B176]).](fnins-11-00003-g0002){#F2}
Pre-formed α-syn fibrils (PFF) rich in β-sheets with morphologies and structural characteristics similar to those extracted from LBs can be obtained *in vitro* from the polymerization of α-syn unstructured monomers. The α-syn fibrillation is a nucleated polymerization process in which the unfolded monomer undergoes self-assembly to form oligomeric intermediates (nuclei) at the onset of aggregation, followed by monomer accretion and fibril growth. A similar mechanism has been described in PrP aggregation process. PFF addiction to the culture medium induced the formation of protein aggregates in the cytoplasm of neuron cells (Figure [2A](#F2){ref-type="fig"}).
Studies of Braak et al. ([@B20]) performed on numerous post-mortem cases of idiopathic PD support the capacity of pathological α-syn to spread in the human CNS suggesting the existence of a path of diffusion of the α-syn pathology from the dorsal motor nucleus of the vagus and in the anterior olfactory structure to the other brain regions (Figure [2B](#F2){ref-type="fig"}).
In order that a "prion-like" disease can be spread in the CNS, the causative agent must be transferred from one cell to another to induce the formation of amyloidogenic inclusions.
Monomeric and oligomeric forms of α-syn have been detected in human body fluids (plasma, saliva and cerebrospinal fluid; Borghi et al., [@B18]; El-Agnaf et al., [@B62], [@B63]; Tokuda et al., [@B219]; Vivacqua et al., [@B236]) and *in vitro* and *in vivo* experiments showed that α-syn can be released from a donor cell, be taken up by a recipient cell, and then seed aggregation of endogenous α-syn within the recipient cell (Figures [2C--E](#F2){ref-type="fig"}).
Transgenic (Tg) mice expressing wild type (WT) human α-syn (h α-syn), or mutant A53T h α-syn M83 mice have been employed to provide compelling evidence for the detrimental role of α-syn inclusion formation in CNS neurons (Figure [2F](#F2){ref-type="fig"}). These studies highlight how the expression of the mutant A53T h α-syn in mice leads to neurodegeneration by promoting the formation of filamentous inclusions (Figures [2F1--3](#F2){ref-type="fig"}).
In order to further investigate whether α-syn can be spread with a "prion like" mechanism, several studies have been conducted based on the experimental approach used in the study of prion disease transmission: solutions containing α-syn protein of various origins have been administered to cell cultures or injected in WT or Tg mice to evaluate whether such administration may induce the formation of LB-like inclusion (Figures [2A,F4--21,G](#F2){ref-type="fig"}). Both recombinant α-syn (in the form of monomers, oligomers or fibrils; Figures [2F9,10,16--21](#F2){ref-type="fig"}) and brain homogenate from murine (Figures [2F4--7](#F2){ref-type="fig"}) or human subjects affected by α-syn pathologies (Figures [2F8,11--15](#F2){ref-type="fig"}) have been used.
On the whole these studies suggest that the injection of a lysate containing pathological α-syn in the brain of WT mice or Tg A53T h α-syn mice, or even in the hind limb peripheral nerve of Tg A53T h α-syn mice induces/accelerates the onset of the disease depending on the amount of pathological α-syn administrated and that α-syn expression in the host animal is necessary for the development of the pathology. Finally, the serial propagation of the infectivity in α-syn pathology, a prerequisite for a prion agent, was demonstrated by primary and secondary transmission in TgM83^+/−^ mice using pathological α-syn from MSA brain homogenates (Figure [2G](#F2){ref-type="fig"}). Collectively, these studies strongly support the prion nature of the pathological h α-syn.
Role of metals in synucleinopathies {#s6}
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The involvement of metals in synucleinopathies is suggested by epidemiological studies showing a positive correlation between the occupational exposure to specific metals and the onset of PD and manganism, a disorder that shares many phenomenological features with PD, such as cognitive decline, psychiatric alteration and movement abnormalities (Zayed et al., [@B256]; Rybicki et al., [@B189]; Gorell et al., [@B84]; Benedetto et al., [@B12]; Fukushima et al., [@B73]). Elevated levels of several metal ions have been reported in the substantia nigra of people affected by PD (Gorell et al., [@B83]; Ayton and Lei, [@B5]). Furthermore, iron and aluminum have been detected in the LBs demonstrating the accumulation of metal ions in α-syn amyloidogenic aggregation (Dexter et al., [@B53]; Hirsch et al., [@B98]). In contrast, a lower concentration of copper was detected in brain regions of PD patients characterized by an accumulation of iron, if compared with controls (Dexter et al., [@B53]; Davies et al., [@B48]). Several lines of evidence support the possible involvement of iron in the pathogenesis of PD and specific reviews have been published on the subject (Götz et al., [@B86]; Mounsey and Teismann, [@B150]).
The risk of developing synucleinopathies increases with age. Interestingly, also the concentration of iron in the brain increases with age (Acosta-Cabronero et al., [@B2]; Pirpamer et al., [@B173]). A recent study has demonstrated an age-induced increase in the expression of both divalent metal transporter (DMT1) and α-syn in mice cells (Lu et al., [@B128]). This result describes a cellular scenario in which the cytoplasmic amount of both divalent metal ions and α-syn increase with age.
Many metal ions (mono, di and trivalent) can bind to α-syn by means of its negative charged residues, reducing their repulsion and affecting the protein tendency to aggregate in fibrils. This feature, also observed in PrP prion protein, can be closely related to the onset and development of synucleinopathies. Metal ions show different binding affinities for α-syn but the binding sites are similar for the majority of metal ions. Generally, divalent cations (such as Fe^2+^, Mn^2+^, Co^2+^, Ni^2+^, Ca^2+^, and Cu^2+^) bind to the CT of α-syn (Nielsen et al., [@B158]; Lowe et al., [@B127]; Binolfi et al., [@B16]) but binding sites for copper have been described also in NT and NAC segment (Camponeschi et al., [@B31]; Moriarty et al., [@B148]). Among metal ions, copper shows the highest binding affinity estimated in the μM range and it can bind to residues Met-1, Asp-2, His-50, Asp-119, Asp-121, and Glu-123 (Binolfi et al., [@B16], [@B15]; Rodríguez et al., [@B186]; Figure [1](#F1){ref-type="fig"}). Manganese and iron can bind to residues Asp-121, Asn-122, and Glu-123 with an affinity in the range 1--50 mM (Golts et al., [@B81]; Binolfi et al., [@B16]) whereas the Fe^3+^ shows an affinity of 1.2 × 10 ^13^ M^−1^ (Peng et al., [@B170]).
The binding between metal ions and α-syn can be affected by pH-values (Drew et al., [@B56]) and protein modification such as phosphorylation (Lu et al., [@B129]), acetylation (Moriarty et al., [@B148]), and puntiform mutation (Drew et al., [@B57]). At acidic pH-values, Cu^2+^ binding sites shift toward the CT (Drew et al., [@B56]). The phosphorylation of residues Ser-129 and Tyr-125 increases the binding affinity for Cu^2+^, Pb^2+^, and Fe^2+^ (but not Fe^3+^) and determine a shift of the binding sites from the NT to CT (Lu et al., [@B129]). Among PD linked mutations, the A30P mutation favors the binding of Cu^2+^ to His50 whereas E46K, A53T did not seem to affect copper binding (Drew et al., [@B57]).
The rate of α-syn fibril formation *in vitro* was significantly accelerated by several divalent and trivalent metal ions such as Cu^2+^, Fe^3+^, Co^3+^, and Mn^2+^ (Uversky et al., [@B228]). The addition of Cu^2+^ to α-syn leads to a dynamically stable β-sheet conformation that serves as a nucleation point for a second- β strand (Rose et al., [@B187]) producing compact conformers toxic for the cell (Curtain et al., [@B44]). Moreover, μM concentrations of Fe^3+^ induce the formation of large SDS-resistant cytotoxic oligomers capable of forming pores in the lipid bilayer when added to the cells (Kostka et al., [@B114]). Interestingly, different metal ions induce the formation of α-syn oligomer**s** with different characteristics: Co^3+^ and Ca^2+^ produced annular oligomers (Lowe et al., [@B127]) whereas other metal ions (Ni^2+^, Fe^3+^, Cu^2+^, Mg^2+^, Cd^2+^, Zn^2+^) induced spherical oligomers (Nielsen et al., [@B158]; Figure [1D](#F1){ref-type="fig"}).
The potential involvement of α-syn in oxidative stress is an important issue as synucleinopathies, and in general neurodegenerative diseases, are associated with high levels of oxidative stress in the brain (Eskici and Axelsen, [@B66]; Schildknecht et al., [@B195]; Dixon and Stockwell, [@B54]). However, conflicting evidences on the causative or protective role of α-syn aggregation in ROS generation are available. On the one hand, it was shown that the oxidation of copper bound to α-syn can lead to the formation of hydrogen peroxide that exhibits a cytotoxic behavior in the cell (Lucas et al., [@B130]; Wang et al., [@B243]) promoting events proposed to be strongly related to the etiology of PD (Dell\'Acqua et al., [@B51]) such as dityrosine crosslink (Lucas et al., [@B130]), dopamine oxidation (Meloni and Vasak, [@B143]), and methionine sulfoxidation (Ayton et al., [@B6]). On the other hand, a recent study showed that the copper/α-syn binding can exert a protective role against ROS. Pedersen et al. ([@B167]) found that the levels of ROS and the rate at which they are generated, are significantly reduced when copper is bound to α-syn (and Aβ) particularly when the protein is in oligomeric and fibrillar form. These observations suggest that copper bound to the protein is less accessible to the solvent and therefore less capable of reacting with ascorbate, resulting in a reduced ROS formation, but further studies are necessary to elucidate this issue.
Recently, new data has been obtained on the interaction between iron and α-syn. In cell cultures the over expression of α-syn induced increased levels of intracellular iron and resulted in partial redistribution of iron from the cytoplasm to perinuclear inclusions (Ortega et al., [@B162]). Moreover, the expression of A53T mutant h α-syn aggravates Fe^2+^ mediated toxicity which results in an increased oxidative stress and DNA damage (Chew et al., [@B36]). Interestingly, intranasal treatment with desferrioxamine (DFO), a chelator widely used in clinical settings for the treatment of iron overload, down-regulated the expression of both α-syn and divalent metal transporter 1(DMT1; Guo et al., [@B91]).
Which exactly is the physiological function of the binding between metal ions and α-syn and whether it has an evolutionary significance is not yet completely clarified. The comparative analysis of α-syn amino acid sequences of representative species of teleost fish (*Xiphophorus maculatus*), amphibians (*Xenopus laevis*), reptiles (*Anolis carolinensis*), birds (*Taeniopygia guttata*), and mammals (*Homo sapiens*) shows that most of the negatively charged amino acids and sites able to bind copper, iron, and manganese ions in mammals are well-conserved among vertebrate species (Figure [1](#F1){ref-type="fig"}): Met-1, Asp-2 are conserved in all the five sequences analyzed, whereas, Asn-122, and Glu-123 are conserved in all tetrapoda species considered. Asp-119 is not conserved only in *X. laevis* in which it is substituted by Asn, His-50 lacks in *X. maculatus* and *A. carolinensis*, whereas Asp-121 is conserved only in *X. maculatus*. Interestingly, Tyr-125 and Ser-129, residues whose phosphorylation increases the affinity for Cu^2+^, Pb^2+^, and Fe^2+^, are perfectly conserved among the tetrapoda species analyzed.
Although there is variability in the number of syn isoforms in non-mammalian vertebrates (Toni and Cioni, [@B220]) and more data and detailed studies are necessary for a thorough analysis, the data here reviewed suggest that the binding of metal ions to α-syn may be involved in evolutionary conserved, as not yet clear, physiological functions and that the concomitant presence in the cytoplasm of high amount of α-syn protein and high metal ions concentrations makes the cell more prone to the formation of amyloid aggregates and the organism more susceptible to disease development.
The amount of metal ions in the brain undergo changes during the course of life (Acosta-Cabronero et al., [@B2]; Pirpamer et al., [@B173]), and the metal ions concentration may vary in the different brain regions (Ayton and Lei, [@B5]). Given the broad spectrum of effects of metal ions on the cell physiology, this means that in the course of human life the change in metal ions concentration can make the nervous system more prone and susceptible to the onset of neurodegenerative prion diseases. In this sense, the metal intake through the diet could influence the onset of prion and prion-like neurodegenerative diseases.
Alzheimer\'s disease as a prion-like disease {#s7}
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Alzheimer\'s disease (AD) is a neurodegenerative disorder involving progressive cortical and hippocampal neuron loss, clinically characterized by progressive and irreversible cognitive deficits and behavioral alterations that affect memory, learning ability, and the quality of life. There are two distinct clinical manifestations of AD, familial and sporadic, both characterized by the aggregation of misfolded proteins, inflammation, and metabolic failure. Although AD is a genetically complex autosomal dominant disease, caused by mutations in the amyloid precursor protein (APP) gene, presenilin 1 (PSEN1), or presenilin 2 (PSEN2; Bertram et al., [@B14]), the majority of AD cases are sporadic. Pathologically, AD is characterized by amyloid deposits that consist of extracellular aggregated Aβ, neurofibrillary tangles (NFTs) composed by the hyperphosphorylated tau protein, and by neuronal losses (Hardy and Higgins, [@B95]). The Aβ peptides are produced by the cleavage of the APP through the so called "amyloidogenic pathway." In this way, the sequential activity of β-secretase (BACE1; Cole and Vassar, [@B38]) and γ-secretase, a multiprotein complex that contains both PSEN1 or PSEN2 (Steiner, [@B213]), produces APP fragments of different length (39--42 amino acids) among which Aβ42 is considered particularly neurotoxic and more prone to self-aggregation than shorter peptides.
Research conducted over the past two decades have shown many similarities between Aβ and prion properties and for this reason Aβ is generally included among the prion-like proteins. Recently, comprehensive reviews on this fascinating subject have been published to which refer for in-depth details and specific references (Tatarnikova et al., [@B216]; Ugalde et al., [@B227]; Walker et al., [@B240]). Aβ, like PrP, may undergo conformational changes assuming a tertiary structure rich in β sheets that promotes the self-assembly of the protein in oligomeric and fibrillar aggregates with neurotoxic properties (Haass and Selkoe, [@B92]; Klein, [@B111]). Interestingly, insoluble Aβ seeds are relatively resistant to proteinase K (PK) whereas Aβ soluble fraction is largely PK sensitive and it loses its seeding activity after PK digestion (Langer et al., [@B120]).
Several experiments based on the intracerebral injection of AD brain homogenate in non-human primates and in transgenic mice and rats (Baker et al., [@B8]; Meyer-Luehmann et al., [@B144]; Rosen et al., [@B188]) showed an increase in the amount of Aβ deposits in the receiving brain that augmented with longer incubation time and with higher concentration of the injected homogenates. On the contrary, the use of both control brain homogenate and AD brain homogenate in which Aβ had been depleted by antibodies did not induce the Aβ accumulation (Meyer-Luehmann et al., [@B144]; Duran-Aniotz et al., [@B60]), suggesting that the pathological Aβ contained in the AD brain can be the promoting agent. In support of this hypothesis, the deposition of Aβ can be observed also after the injection of high concentrations of multimeric synthetic Aβ accompanied by long periods of incubation (Stöhr et al., [@B214]).
The deposits of seed-induced Aβ, initially found only in tissues near the site of injection, gradually propagate along axons to different brain regions with stereotypic temporal-spatial spreading patterns which are partly reminiscent of the spreading characteristics of prions. From this point of view, the deposition of the pathological protein in AD can be explained by the initial formation of protein seeds early in pathogenesis, followed by a prion-like spread of misfolding and aggregation events along the neuroanatomical pathways.
Role of metals in AD {#s8}
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Although genetic, biochemical, and neuropathological data indicate that amyloid formation plays a central role in AD pathogenesis (Selkoe, [@B196]), the aetiopathology of this disease remains unclear. Compelling evidence suggest the existence of a correlation between the imbalance of metal homeostasis in the brain and the pathogenesis of AD. Transition metals could participate in AD pathogenesis by interacting with Aβ peptide, promoting its aggregation and facilitating ROS production and oxidative stress. Focal accumulation of Zn, Cu, and Fe might also deprive other brain tissues of these essential metals, leading to aberrant neuronal function. The total amount of copper (390 μM), zinc (1055 μM), and iron (940 μM) have been reported to be increased in AD brain as compared to normal age-matched samples (copper, 70 μM; zinc, 350 μM; iron, 340 μM; Lovell et al., [@B126]). At a cellular level, the analysis of the amount of metal ions in samples from AD patients revealed increased zinc and iron (Deibel et al., [@B50]; Bouras et al., [@B19]; Duce et al., [@B59]), decreased levels of copper (Bouras et al., [@B19]; Smith et al., [@B205]; Duce et al., [@B59]) and imbalances in aluminum, silicium, and mercury.
Concerning Zinc, Bush et al. ([@B30]) provided the first biochemical evidence that this metal ion is able to bind Aβ and to cause its aggregation and precipitation initiating the plaque formation. Zinc also interferes with Aβ processing, by inhibiting γ-secretase activity and increasing PS1 expression (Lammich et al., [@B118]). Through the activation of tau-kinases, Zn can also impact on tau-related neurotoxicity (Mo et al., [@B147]). The dyshomeostasis of Zn in the brain can lead to an enhanced susceptibility to the excitotoxicity of glutamate and to oxidative stress (Oteiza et al., [@B163]; Takeda et al., [@B215]), being this metal an inhibitory neuromodulator of glutamate release in the hippocampus and antagonizing the catalytic properties of the redox-active transition metals.
Copper, like Zn, is synaptically released and acts as a potent mediator of Aβ aggregation (Mantyh et al., [@B137]). It binds with high affinity to an amino terminal tyrosine residue in Aβ inducing its oligomerization and neurotoxicity. Moreover, the elevated copper amount was detected in amyloid plaques. The cytotoxicity induced by the Cu-Aβ complex involves also oxidative stress events, as this complex catalytically generates hydrogen peroxide (Huang et al., [@B99]), through the reduction of Cu^2+^ to Cu^+^, accompanied with the oxidation of endogenous molecules such as thiols, cytochrome c oxidase, ascorbate and lipids (Turnbull et al., [@B226]; Puglielli et al., [@B177]). The resulting lipid peroxidation leads to the formation of peroxyl radicals, which once formed, can then be rearranged into compounds like malondialdehyde (MDA; Jomova and Valko, [@B105]) and 4-hydroxy-2-nonenal (HNE), detected at higher levels in AD brain and AD transgenic mouse models (Haeffner et al., [@B93]; Nelson and Alkon, [@B156]). However, although Cu^2+^ is increased in amyloid plaques, it is decreased in AD neuronal tissue which in turn could deprive Cu-binding proteins such as superoxide dismutase and ceruloplasmin and impairs their function. Copper also influences the fate of APP processing, given its participation in APP processing into non-amyloidogenic derivatives while its deficiency reduces Aβ degradation (Cater et al., [@B34]).
Concerning Fe, its deposition in neurons causes oxidative stress via the Fenton reaction, producing abnormalities in RNA, yielding a great reduction in protein synthesis and initiating several Fe-induced apoptotic signaling pathways (Salvador and Oteiza, [@B191]). Fe indirectly damages proteins such as Ca^2+^-ATPase, glutamate transporter, Na^+^/K^+^-ATPase as well as N-methyl-D-aspartate (NMDA) receptor, and lipids such as cholesterol ceramides and unsaturated fatty acids through the formation of hydrogen peroxide and the hydroxyl radical (Kaplán et al., [@B107]; Mark et al., [@B138]; Muñoz et al., [@B151]; Shinkyo et al., [@B197]). Elevated cellular Fe levels can cause cell death independently of ROS toxicity by the phenomenon known as ferroptosis, a type of Ras-related cell death pathway (Dixon et al., [@B55]), while its increased concentration at synapses may also leads to increased Aβ production by altering its processing. In fact iron can act by decreasing the protein furin, which modulates γ--secretase activity, and increased iron concentration favor γ--secretase activity and enhances the amyloidogenic pathway. Although metal dyshomeostasis in AD is primarily due to metal sequestration by amyloid plaques and NFTs, the disruption of the vesicular trafficking has also to be considered since it prevents metal ion containing vesicles to arrive at the axon terminal. The disruption of axonal transport affects also the mitochondrial transport preventing the replacement of the old organelles with the new ones and the production of the energy required for the reuptake of Cu/Zn from the postsynaptic neuron. The acquisition of knowledge of the role of metals in AD neurodegeneration has prompted the discovery of metal-chelating compounds (such as desferrioxamine and deferiprone) conjugated to nanoparticles favoring their passing through the blood-brain barrier that *in vivo* prevent the toxic actions of transition metals (Li et al., [@B124]). Presently, only one family of metal-binding agents, PBT2 \[5,7-dichloro-2-((dimethylamino)-methyl)-8-hydroxyquinoline\], is testing in clinical trials for the treatment of AD. PBT2 binds excesses of copper, zinc, and possibly iron in the brain, thereby diminishing the amount of amyloid plaque formation and relocating these metal ions to depleted cellular and neuronal compartments (Crouch et al., [@B43]).
Mediterranean diet, prion and prion-like diseases, and neurodegeneration {#s9}
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The traditional Mediterranean diet (MeDi) is characterized by an abundant consumption of plant foods, a moderate intake of fish and wine, and low intakes of meats and dairy products, with extra-virgin olive oil as the main fat source. Adherence to the MeDi has also been associated with a lower risks of AD, PD, dementia, and cognitive decline (Scarmeas et al., [@B194], [@B193]; Féart et al., [@B68]; Singh et al., [@B201]) while no epidemiological data are available for the rare sporadic CJD. Human epidemiological study, such as the Northern Manhattan Study have confirmed that MeDi is associated with a lower white matter hyperintensities (VMH) burden evaluated by magnetic resonance imaging, a marker of small vessel damage and neurodegeneration (Gardener et al., [@B76]). Many studies have demonstrated that MeDi is associated with a significant reduction in AD risk. Results based on observational studies (Valls-Pedret and Ros, [@B231]) and long-term randomized clinical trials have clearly shown that a correlation between MeDi and AD exists with strong level of scientific evidence (Valls-Pedret et al., [@B232]).
Higher MeDi adherence was also associated with reduced odds for PD, after adjustment for all covariates while on the contrary, lower MeDi adherence was associated with earlier PD age-at-onset (Alcalay et al., [@B3]).
In PD, there is considerable evidence for ROS-mediated damage in post mortem brain samples as well as in other tissues, even outside of the central nervous system. Oxidative damage to nucleic acids, lipids and proteins in both the brain and peripheral tissues in human PD has been clearly evidenced (Sanders and Greenamyre, [@B192]).
Even if the neurobiological basis of the relationship between the MeDi to brain health has not been elucidated yet, various specific nutrient components of this diet have been examined in relation to cognitive performance including dietary fatty acids, antioxidants, amount of fruits and vegetables consumption, vitamins (particularly B6, B12, and folate; Smith and Blumenthal, [@B206]). The MeDi is a very complex eating pattern, with a multitude of single components that could cause beneficial neuroprotective effects (Jacobs et al., [@B101]; Gotsis et al., [@B85]). In the midst of this multitude, plant polyphenols, which occur mainly in fruit, vegetables, and wine (Manach et al., [@B134]) have recently shown to possess beneficial effects with regard to overall health, as well as cognitive functions (Baur and Sinclair, [@B11]; Witte et al., [@B249]). Plant polyphenols represent an abundant class of plant secondary metabolites found in herbal-rich food and beverages, with no \<8000 phenolic structures having been identified in plants (Tsao, [@B225]).
For example, oleuropein aglycone (OLE), the main polyphenol in the extra-virgin olive oil has clearly showed to ameliorate memory dysfunction, neuronal loss and neurodegenerative damages in AD animal models (Grossi et al., [@B89]). Dietary supplementation of OLE (50 mg/kg of diet), strongly improved the cognitive performance of young/middle-aged TgCRND8 mice, a model of Aβ deposition. In these mice, a reduced β-amyloid levels and plaque deposits was associated with a strong increase of autophagic markers expression and of lysosomal activity (Grossi et al., [@B90]). In human population, serum concentration of some phytochemicals, characteristic of the Mediterranean diet (such as lutein, zeaxantin, and β-carotene), were consistently related to better cognition functions in the Georgia centenarian study (Johnson et al., [@B104]).
Much effort has been undertaken in the way of understanding the neuroprotective effects of polyphenols, using both *in vitro* and *in vivo* models (Johnson et al., [@B104]). The molecular mechanisms of their neuroprotective actions can be classified as anti-inflammatory, antioxidant (free radical scavenging and metal chelation), anti-amyloid action, or through direct modulation of cell signaling pathways, such as metalloproteinases inhibition or their activity on transcription factors such as NF-kB (Gomes et al., [@B82]; Crascì et al., [@B41]). Their ability to simultaneously and synergistically modulate multiple molecular targets, suggests a greater potential also for therapeutic intervention in AD and PD (Grodstein et al., [@B88]; Johnson et al., [@B103]). Since transition metal and particularly iron have been clearly involved in neurodegenerative disorders and aging (Ward et al., [@B244]), in the next section, we focus on the free radical scavenging and metal chelation effects of some polyphenols.
Polyphenols as metal chelators and free radical scavengers {#s10}
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Polyphenols are well-established metal chelators and some of them possess the ability to bind and chelate many different bivalent metals, such as Cu^2+^, Zn^2+^, and Fe^2+^ (Singh et al., [@B202]; Mandel et al., [@B135]). In this way, the rate of Fenton reaction directly diminishes and the oxidation caused by reactive hydroxyls radicals can be prevented (Perron and Brumaghim, [@B171]). Moreover, polyphenols decrease metal absorption by exerting their chelating activities also into the small intestine (Landete, [@B119]). In humans participating in the PAQUID cohort study, higher levels of polyphenols (flavonoids) were associated with \~50% reduction of the risk to develop dementia (Commenges et al., [@B40]).
In animal models, it has been clearly demonstrated that various polyphenols, capable to chelate divalent metals and with antioxidants activities, possess neuroprotective effects. Dairam et al. ([@B45]) have observed that curcumin modulates iron in rat-brain homogenates. In a rat model of Parkinson\'s disease, curcumin has shown neuroprotective effects by decreasing neuron degeneration (Du et al., [@B58]). Epigallocatechin Gallate (EGCG) is the most effective antioxidant polyphenol in green tea and there is clear evidence that EGCG is very effective in metal chelation of Fe^2+^, Zn^2+^ and Cu^2+^ (Chan et al., [@B35]). In SH-SY5Y neuroblastoma cells, EGCG has exhibited stronger iron chelation compared to desferrioxamine (Reznichenko et al., [@B185]).
At the molecular level, different polyphenols have shown to inhibit Aβ42 fibril formation by directly interacting both with Aβ42 and transition metals. For example, glycosylated polyphenols (such as verbascoside present in the olive tree) and their esterified derivatives regulate metal-free and metal-induced Aβ42 aggregation and disaggregation at 50 μM concentrations (Korshavn et al., [@B113]).
EGCG, at 100 μM completely inhibited the formation of Aβ42 fibril and was capable to reduce the amount of fibrils present when EGCG was added demonstrating its ability of remodeling preformed fibrils (Chan et al., [@B35]). Curcumin, one of the principal polyphenols in turmeric (*Curcuma longa*) exhibited moderate metal chelation and antioxidant activity, and it is a known inhibitor of Aβ fibril formation *in vitro* (Ono et al., [@B160]).
Red wine is famous for its polyphenol content. The major polyphenols found in red wine extracts include resveratrol, quercetin, catechin, epicatechin, tyrosol, gallic acid, and procyanidins. Many different *in vitro* studies have described the potent free radical scavenging effects of red wine polyphenols, including direct scavenging of reactive oxygen and nitrogen species, such as peroxides, superoxide, the hydroxyl radical, and the peroxynitrite anion, as well as sequestering of highly redox-active metal ions (Wang and Brumaghim, [@B242]). Treatment of neuronal and astrocytic cell lines with these polyphenolic compounds suppressed ROS production and significantly improved cell viability (Martin et al., [@B139], [@B140]). Resveratrol, mostly present in berries, grapes and wine, was protective in human neuroblastoma cells exposed to Aβ or to Aβ-metal complexes through its scavenging properties (Granzotto and Zatta, [@B87]). Several polyphenols present in wine have been demonstrated to protect mitochondria from ROS damages in *in vitro* studies. These include resveratrol, quercetin, anthocyanidins, and proanthocyanidins (Fernández-Moriano et al., [@B69]). Moreover, resveratrol administration, as a dietary supplement, significantly attenuates 6-hydroxydopamine-induced oxidative damage, and dopamine depletion in a rat model of Parkinson\'s disease (Khan et al., [@B109]).
Since resveratrol is able to penetrate the blood--brain barrier and exert strong neuroprotective effects, even at low concentrations, it has been used in a clinical trial that has currently completed phase II in 120 patients with possible AD diagnosis (NCT01504854). Ongoing trials are investigating the efficacy of a mixture of grape polyphenols in mild cognitive impairments (MCI) and moderate AD (i.e., NCT02502253).
To date, although some polyphenols can efficiently cross the blood-brain barrier, the literature suggests that single polyphenol may not attain concentrations within the brain that are sufficient to exert an effective metal chelation and free radical scavenger activities. Nevertheless, MeDi is characterized by a high consumption of many different polyphenols-rich foods and beverages that may exert their neuroprotective effects in a time-dependent manner. Epidemiological studies suggest that the activities of these complex mixtures of multitarget compounds could be protective and really effective at preventing or delaying prion-like diseases with a high prevalence in the population, such as AD and PD (Alcalay et al., [@B3]; Pelletier et al., [@B169]).
Conclusions {#s11}
===========
Neurodegenerative diseases such as TSEs, synucleinopathies, AD, Huntington\'s diseases, and ALS belong to the prion and prion-like diseases, disorders in which specific proteins precipitate in amyloid aggregates in the nervous system as a result of conformational change. Among these diseases, AD and PD are disorders of concern for their high social impact and cost as they are the most widespread neurodegenerative diseases in the populations of Western European countries and the USA. Epidemiological studies and experimental evidence at the cellular and biochemical level show the influence of the dyshomeostasis of metals in the onset of such neurodegenerative diseases. It is noteworthy that different risks of developing AD and PD have been associated with different nutritional habitus. Among them, the Mediterranean diet was found to be the most protective, with significant reduced odds for PD and AD. This diet is characterized by a very high content of different multifunctional polyphenols capable to bind metals (transition metals in particular) and amyloid aggregates, acting by specific regulators of aggregation and cytotoxicity of metal-free and metal-associated prion and prion-like proteins. Many of these polyphenols can cross the blood-brain barrier and serve as specific and multitarget compounds capable to regulate ROS damages, inflammation and aggregation of misfolded proteins in neuronal tissues.
Having classified the major neurodegenerative diseases within the classes of prion and prion-like diseases implies that we must accept the idea that nutritional factors are capable to modify the mechanisms of neuronal damage induced by prion and prion-like proteins and that the Mediterranean diet can exert a protective role in these diseases.
Author contributions {#s12}
====================
MT, MM, and ES drew the manuscript. MT, MM, AD, EA, and ES wrote the manuscript.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors ES and states that the process nevertheless met the standards of a fair and objective review.
This study was supported by the Sapienza University of Rome (Progetti di ricerca 2015 to MT) and by the University of Bologna (RFO2015 to ES).
[^1]: Edited by: Federico Benetti, International School for Advanced Studies, Italy
[^2]: Reviewed by: Jesus R. Requena, University of Santiago de Compostela, Spain; Chaoyang Li, Wuhan Institute of Virology, China
[^3]: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection^[@CR1]^. The World Health Organization has recently acknowledged sepsis as a major health care issue^[@CR2]^. Indeed, incidence of septic syndromes is continuously increasing, affecting more than 35% of patients hospitalized in intensive care units (ICU) in Europe^[@CR3]^. Moreover, septic shock, *i.e*. subset of sepsis with acute circulatory failure, is associated with mortality up to 45% in the ICU despite improvements in early care of organ dysfunctions^[@CR4]^.
Thus, research effort focuses on finding specific treatment for sepsis. In the last few years, there has been renewed interest for the role and underlying mechanisms of sepsis-induced immunosuppression. Indeed, it has been demonstrated that immune alterations observed after sepsis were associated with increased risk of secondary infections and mortality^[@CR5]^. Recently, new therapeutic approaches targeting these alterations in patients with persistent immunosuppression have emerged. Relevance of this strategy has been lately illustrated by encouraging results from a phase II trial using interleukin 7 as an immunoadjuvant therapy targeting adaptive immunity defects in septic shock patients^[@CR6]^. Nevertheless, pathophysiology of immune dysfunctions developed after sepsis remains incompletely understood. Yet, exhaustive characterisation of these immune alterations is fundamental to highlight potential new targets and biomarkers, which would allow development of immune-based therapy of this hitherto deadly disease.
Such comprehensive studies of sepsis-induced host response and immune alterations were long limited by technology issues. Emergence of innovative single cell technologies widens possibilities to improve our understanding of sepsis pathophysiology in humans^[@CR7],[@CR8]^. Particularly, mass cytometry (CyTOF) is a promising alternative to multicolour flow cytometry allowing to measure above 40 parameters simultaneously^[@CR9]^. Indeed, CyTOF uses monoclonal antibodies conjugated with metal isotopes, avoiding common autofluorescence and spectral overlap issues occurring with conventional flow cytometry. These properties allow broad phenotypic exploration of several immune populations at once.
In this study, we used for the first time CyTOF technology to analyse immune alterations in a cohort of patients with septic shock. As a preliminary proof of concept study, the main objective was to evaluate feasibility and opportunities provided by mass cytometry in this clinical context. We designed a mass cytometry panel comprising 25 antibodies to explore mononuclear cell features from septic shock patients and healthy donors. Unsupervised analysis strategies such as SPADE^[@CR10]^ and viSNE^[@CR11]^ were used to highlight novel aspects of immune alterations developed after septic shock.
Results {#Sec2}
=======
Set up of blood processing and mass cytometry analysis {#Sec3}
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First, we tested 4 freezing protocols which differed in freezing and thawing media and in freezing/thawing steps (see Supplementary Table [S1](#MOESM1){ref-type="media"}). We observed similar yield in number of cells available after thawing between the 4 protocols. However, protocol \#4 was more convenient and easy to use, and included the use of DNAse during the thawing step, which allowed to avoid cell aggregates, especially in septic shock patients' samples. Protocol \#4 was thus selected to build the biobank.
Next, we collected 10 samples from healthy volunteers and 11 samples from septic shock patients included at day 3 after the onset of septic shock. As significant loss in number of cells is commonly observed during freezing/thawing process^[@CR7]^, we first assessed the recovery of proportion of lymphocyte subpopulations measured by flow cytometry before freezing and after thawing steps. Using the analytical conditions described above to process our samples, no loss of any particular lymphocyte population was noted, allowing further data analysis (Fig. [1](#Fig1){ref-type="fig"}). The number of viable cells available after the whole process was adequate for a relevant mass cytometry analysis.Figure 1No deletion of a particular lymphocyte sub-populations is noted during the whole mass cytometry analysis process. Before freezing (white boxes) and after thawing (grey boxes) processes, proportions of lymphocyte sub-populations in mononuclear cells were analysed by flow cytometry in 10 samples from healthy volunteers and 11 samples from septic shock patients.
CyTOF analysis in septic shock patients {#Sec4}
---------------------------------------
Due to limited access to CyTOF platform, we restrained the number of samples analysed by mass cytometry to 5 healthy volunteers and 5 septic patients. As our mass cytometry panel was designed for studying mononuclear cells, we selected the 5 septic shock samples presenting with the highest proportion and number of mononuclear cells in peripheral blood mononuclear cells (PBMC) layer after Ficoll. Clinical and biological data for the 5 septic shock patients are presented in Supplementary Table [S2](#MOESM1){ref-type="media"}. Median \[IQR\] age of the healthy volunteers (HV) was 61 \[53--63\] years old and 40% were male.
The analysis pipeline used throughout the study is illustrated in Fig. [2](#Fig2){ref-type="fig"}. To characterize and visualize mononuclear cells' subpopulations in the observational cohort, we ran a SPADE clustering analysis. SPADE algorithm first downsamples data to capture rare populations. Then it hierarchically clusters phenotypically similar cells into "nodes" and represents these nodes and their relationships in a minimum spanning tree format (*i.e*. SPADE tree). The main issue with SPADE analysis is the definition of the optimal number of cell clusters. This is difficult to determine empirically. Firstly, to address this problem, we determined with X-shift algorithm the optimized cluster number for our dataset, as described elsewhere^[@CR12]^. X-shift algorithm uses weighted *k-*nearest-neighbour density estimation to define the optimal number of cell clusters in high dimensional data. For this, X-shift evaluates the impact of different numbers of nearest neighbours on the number of cell clusters, with the optimal number of cell clusters defined at the "switch point" between under-clustering and over-fragmenting data. In our dataset, optimal cluster number was found at 78. Secondly, from this result we ran a SPADE analysis seeking 78 nodes. For further analysis, only nodes with an average proportion ≥0.5% of total cells were selected, in order to exclude sparse clusters where low cell numbers could potentially increase the error of reported medians. This threshold resulted in exclusion of approximately 6% of total cells, thus retaining 51 nodes for further analysis (see Supplementary Fig. [S1A](#MOESM1){ref-type="media"}). The individual SPADE trees for control and septic samples are shown in Supplementary Fig. [S1B](#MOESM1){ref-type="media"}. We thus finally identified cell types for each of the 51 cell nodes drawing a heatmap depicting the mean expression of 19 lineage or cellular differentiation markers (Fig. [3](#Fig3){ref-type="fig"}).Figure 2Analysis pipeline used throughout the study. The study was built through several steps: set up of mass cytometry analysis, process of samples on mass cytometer and data analysis. In this last step, several multidimensional data analysis tools were used, such as SPADE and viSNE, to highlight differences between septic shock patients and healthy donors in terms of cellular abundance and expression levels of exhaustion, activation and differentiation markers.Figure 3Expression of phenotypic markers used for node characterisation. Median expression for each marker was averaged from all 10 samples for each of the 51 nodes. Results are shown on heatmap after hierarchical clustering. Colour intensity specifies the expression level. Combined expression of several markers allowed to identify characteristic leukocyte populations, but also rare cell subsets. For instance, two populations of CD3^+^ cells but double negative CD4^−^CD8^−^ were noted, both expressing TCRγδ, but showing different features, such as the expression level of CD8, CD16 and CD56.
Node distribution shows different proportions in septic shock patients compared to healthy donors {#Sec5}
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Next, we compared node proportions between septic patients and healthy volunteers. We drew a new heatmap representing proportions of each node in each individual (Fig. [4A](#Fig4){ref-type="fig"}). Clustering highlighted two groups of nodes, including respectively nodes with increased and decreased proportions in septic patients compared to healthy donors. Intriguingly, one septic shock patient showed a pattern of node's proportions very similar to healthy volunteers'. When analysing clinical data for this patient, result of HLA-DR expression on monocyte (mHLA-DR) was 20,445 antibodies/cell (AB/C) whereas the four other septic patients had a mHLA-DR \< 10,000 AB/C. As we focused on sepsis-induced immunosuppression, we decided to exclude this patient in further analyses, to avoid a risk of masking potential differences between controls and septic patients in this small cohort.Figure 4Significant variations in node proportions are observed after septic shock. (**A**) For the 5 healthy donors and 5 septic shock patients, individual proportions of each SPADE node were plotted onto a heatmap. The red and blue colours indicate increased and decreased proportions respectively. Order of nodes was determined by non-supervised hierarchical clustering. On the right side of the heatmap were clustered nodes that seemed decreased in septic patients compared to controls. On the left side were clustered nodes that looked increased in septic shock patients. Septic patient presenting mHLA-DR \> 20,000 AB/C and excluded from further analyses is indicated by a star. (**B**) To highlight SPADE nodes with important variations in cell proportions between healthy donors and septic patients, nodes were ranked by descending order according to their average proportions in controls (n = 5) and in septic shock patients (n = 4). The rank difference between healthy volunteers and septic patients was calculated and plotted for each node. A difference (controls - septic patients ranks) under 0 reflected a decrease of cell number within the node in septic patients, whereas a positive difference suggested an increased node proportion in septic patients compared to healthy donors. Arbitrary, proportion variation was considered non-negligible when a node's rank differed more than 1/3 of total node number, reflecting a gain or loss of more than 17 positions (red lines). (**C**) Potentially increased (left plot) or decreased (right plot) nodes' frequencies were plotted for septic shock patients (n = 4; grey boxes) and healthy donors (n = 5; white boxes) samples as boxplots with individual values. Node proportions between controls and septic shock patients were compared using Mann-Whitney tests. \*p \< 0.05.
To identify nodes with the most important variations in cell proportion between HV and septic shock patients, we ranked nodes according to their average proportions in HV and in patients respectively. For each node, the rank difference between HV and septic patients reflected increased or decreased proportion after septic shock. For further analysis, we selected only the nodes with a rank difference of more than 1/3 of total node number (Fig. [4B](#Fig4){ref-type="fig"}). Thus, we identified 9 increased nodes and 8 decreased nodes after septic shock. Interestingly, all 8 decreased nodes were CD4 or CD8 T cells while the 9 increased nodes were mainly non-T cells. Especially, 4 nodes were B cell clusters, highlighting a potential enrichment after septic shock in this usually sparsely represented leukocyte subpopulation.
So as to confirm these changes between groups, we analysed node frequencies across individuals in the 17 selected nodes, especially to get rid of average variations that could hide high inter-individual variations. Figure [4C](#Fig4){ref-type="fig"} shows significant proportion increases in nodes \#6, 43 and 49 in septic shock patients (p \< 0.05; Mann-Whitney). These 3 nodes appeared to be conventional monocytes CD14^+^HLA-DR^+^, particular monocytes CD14^+^HLA-DR^low^ and a population of CD56^−^CD16^+^ cells respectively. A significant decrease in node \#18 representing specific effector memory CD8 T cells was also observed in patients (p \< 0.05; Mann-Whitney). Finally, we noted a trend towards increased proportions of nodes \#25 and 45 (p = 0.065; Mann-Whitney), which represented naïve B cells and plasmablasts respectively.
Specific phenotype changes are observed on leukocytes after septic shock {#Sec6}
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In order to highlight the broad potential of mass cytometry data analysis, we next investigated phenotypic changes in activation and exhaustion markers' expression, using three different approaches: unsupervised heatmap, coexpression study and viSNE algorithm. These approaches were applied on monocytes, CD4 T cells and B cells respectively, selecting SPADE nodes according to the expression of lineage markers CD14, CD3/CD4, and CD19 (Fig. [2](#Fig2){ref-type="fig"}).
### Uniform modifications in monocyte expression of 4 specific markers after septic shock {#Sec7}
First, we were interested in monocyte activation and immunoregulatory profile after septic shock. We drew a heatmap of individual expression of activation markers (CD4, CD14, CD16, CD38, CCR6, CCR7, CD45RA) and immune checkpoint inhibitors (PD-L1 and Tim-3) on each of the 4 monocytes nodes from SPADE (*i.e*. nodes \#3, \#6, \#20 and \#43) (Fig. [5](#Fig5){ref-type="fig"}). PD1 and Lag-3 were not expressed at all on monocytes. Unsupervised clustering revealed three groups of markers depending on their modulation of expression on monocytes after septic shock. Interestingly, markers with increased (purple rectangle) or decreased (green rectangle) expression in septic patients were uniformly modulated on all monocyte nodes. Indeed, septic patients presented with an increased expression of CD38 and PD-L1, as well as a decreased expression of HLA-DR and CD4 on all septic monocyte nodes compared to healthy donors, except on node \#3 for the latter parameter.Figure 5Expression of 4 monocyte markers is regulated after septic shock. After normalisation, median expression of phenotypic markers on each SPADE node of monocytes was shown on heatmap after hierarchical clustering for 4 septic shock and 5 healthy donors. The red and blue colours indicate over- and under-expressed markers respectively and the colour intensity specifies the expression level. Rectangles in green and purple indicate respectively markers down-regulated and up-regulated on particular nodes in septic shock patients compared to controls.
### High heterogeneity in PD1+ CD4 T cells after septic shock, without coexpression of other immune checkpoint inhibitors {#Sec8}
Considering the expanding importance of immunostimulating therapies targeting immune checkpoint inhibitors, we studied expression of PD1, PD-L1 and Tim-3 on the 18 CD4 T cell nodes from SPADE (Fig. [6A](#Fig6){ref-type="fig"}). This was performed by representing individual values and box-plots of MFI of each marker on all CD4 T cell nodes in HV and patients. In addition, showing background signal measured on a mass-minus-one (MMO) sample for each marker was helpful to compare co-expressions. No difference of Tim-3 or PD-L1 expressions was observed between HV and septic patients. In HV, PD1 was systematically expressed on a unique node (\#63). However, in septic shock patients PD1 exhibited increased expression on node \#63 and appeared on several other nodes, *i.e*. nodes \#27, \#29, \#38, \#54 and \#57. Co-expression of PD1 and PD-L1 was rarely observed. Interestingly, Tim-3 was expressed only on 2 nodes, and was not co-expressed with neither PD1 nor PD-L1. Next, we studied expression of HLA-DR, CD38, CD25 and CD127 on these five PD1^+^ nodes to investigate whether the increased expression of PD1 was associated with expression of activation markers (Fig. [6B](#Fig6){ref-type="fig"}). There was almost no expression of these markers on node \#38, which was the only node of double positive CD4^+^ CD8^+^ T cells. Nodes \#27 and \#63 did not express HLA-DR nor CD38, but were CD25^+^ and showed high expression of CD127, suggesting potential resting memory cells. Node \#54 had a profile of activated effector cells, HLA-DR^+^ CD38^+^ CD25^low^ and CD127^−^. Finally, high expressions of HLA-DR, CD38 and CD25 on nodes \#29 and \#57 were observed, in association with a loss of CD127 expression. This latter phenotype could be a feature of either activated effector CD4 T cells or regulatory T cells. None of these nodes had significant increased proportion in septic shock patients compared to healthy volunteers.Figure 6PD1 expressing CD4 T cells show high heterogeneity after septic shock and do not co-express other immune checkpoint inhibitors. Expressions of several exhaustion (**A**) and activation (**B**) markers on CD4 T cell nodes from SPADE were compared in septic shock patients (n = 4, dark grey boxes) and healthy volunteers (n = 5, white boxes). The light grey areas represent global median expression on CD4 T cells from mass-minus-one control, characterising the threshold of positive expression for each marker. PD-1 expressing nodes are highlighted underlined.
### B cell commitment to plasma cell differentiation in septic shock patients {#Sec9}
Finally, given the enrichment in B cell populations in septic shock patients, we went further in B cell study. We ran a viSNE analysis on all CD19^+^ cells from SPADE tree (Fig. [7](#Fig7){ref-type="fig"}). ViSNE is a well-established CyTOF analysis tool that displays high dimensional data on a two-dimensional map. Cells are plotted on a continuum of expression with phenotypically related cells clustered together, often manifesting as "islands", which can be identified by colouring cells according to their expression of specific marker. A gap in island distribution between two conditions can indicate an altered cell type or phenotype. Here, viSNE maps from patients and controls did not show the same pattern, suggesting a reorganisation of B cell subpopulations after septic shock. To further investigate this observation, cells were coloured on viSNE plots according to cell surface expression of CD38 or CCR6, two markers of B cell differentiation^[@CR13]^. We observed that, in both healthy donors and septic shock patients, CD38^+^ and CCR6^+^ cells did not present same location on the viSNE map, indicating that they belonged to different cell subtypes. Moreover, we could highlight an increase of CD38^+^ cells in septic patients, as well as a decrease of CCR6^+^ cells compared to healthy donors. These observations suggested a shift from B cells to plasma cell differentiation in septic shock patients.Figure 7B cells shift towards plasma differentiation after septic shock. Cells from the 10 CD19^+^ nodes from SPADE tree (red nodes) were selected before clustering with the viSNE algorithm. Individual viSNE plots are shown for septic patients (n = 4) and healthy donors (n = 5). ViSNE is a visualization tool based on the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm. ViSNE establishes the two dimensional representation of single-cell data sets that best preserves their respective local and global geometry. ViSNE plots represent individual cells in a visual similar to a scatter plot, while using all pairwise distances in high dimension to determine each cell's location in the plot. tSNE algorithm provided each cell with a unique x- and y-coordinate (tSNE1 and tSNE2) according to each cell's expression of 19 parameters of the CyTOF panel. The relative positions of cells on the two-dimensional plot thus reflect their similarity in terms of expression pattern for the 19 parameters included in the panel. In the current analysis, we used color as a third dimension to visualize specific markers expression levels on each cluster. B cell expressions of CD38 and CCR6 are shown on the figure. Red color intensity represents high expression level of the indicated marker. Blue color intensity represents low expression level of the indicated marker. Plots were visually compared between septic patients and controls to highlight differences in marker intensity and cellular distribution.
Discussion {#Sec10}
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Exhaustive characterisation of sepsis-immune alterations has not been completed yet, limiting the identification of potential new specific targets of immunotherapies. In this study, we hypothesized that high dimensionality afforded by CyTOF could be a useful tool to detect novel and unexpected immune features in septic shock patients. To date, very few studies have used CyTOF technology to explore infectious diseases in clinical cohorts^[@CR14]--[@CR16]^ and none specifically in septic shock. So far, CyTOF has been more often employed as an exploration tool of *in vitro* models of infections^[@CR17]--[@CR19]^. Here, we set up a proof of concept study with a mass cytometry panel of 25 markers, in order to compare immune phenotype of mononuclear cells from septic shock patients and healthy volunteers.
CyTOF represents a powerful high dimensional single-cell analysis technique that overcomes major limitations of flow cytometry. In particular, through the use of antibodies coupled to rare elemental metal isotopes, mass cytometry can detect discrete isotope peaks without significant overlap. This significantly ameliorates the need for compensation, a major limiting factor of flow cytometry. In addition, mass cytometry has no "autofluorescence" equivalent. This limits the loss of resolution that could be observed in flow cytometry because of high background level, thus making data analysis easier. Finally, signal intensity is more homogeneous with isotopes than fluorochromes, which sometimes show important brightness differences between the highest and dimmest, complicating panel design. This leads to higher multiplexing capability and easier panel design in CyTOF than in flow cytometry, enabling simultaneous measurement of several tens of markers. Thus, CyTOF appears more appropriate than conventional flow cytometry to take over high dimensional data analysis.
However, cell loss during freezing steps and CyTOF acquisition, high amount of cells needed, low throughput and complex high dimensional data analysis are many hurdles that might prevent the use of CyTOF in clinical studies of patients with septic shock^[@CR7]^. Here, we show that pre-analytical process did not lead to any specific cell subset depletion among mononuclear cells and that final cell number was adequate for a relevant mass cytometry analysis. Moreover, reliability of our data was validated by non-supervised approaches used throughout the pipeline analysis. First, expression of lineage and differentiation markers clustered SPADE nodes in coherent distinct leukocyte subsets leading to identification of cell types corresponding to each node. Then, non-supervised approaches were able to highlight some established hallmarks of septic shock immune alterations. Particularly, in septic shock patients we observed a decreased expression of HLA-DR on monocytes^[@CR20]^, an increase of CD14^+^HLA-DR^low^ monocyte proportions^[@CR21]^ and an overexpression of PD1 and PD-L1 on CD4 T cells and monocytes respectively^[@CR22]^.
As hypothesised, CyTOF single cell data analysis allowed discrimination of many cell subsets, enabling to study specific rare populations and to highlight new features of sepsis-induced immunosuppression. First, a global approach on all SPADE nodes led to the identification of particular leukocyte subpopulations with modifications of their proportion after septic shock. These changes would have probably been missed with bulk analysis considering low abundance of the cell populations described. For example, we noted an increased proportion of CD3^−^CD19^−^CD14^−^CD16^+^CD56^−^ cells in septic shock patients. This population could represent an interesting subtype of CD56 negative NK cells, described as an aberrant functionally altered NK subset, as they present with low capacity of proliferation, cytokine secretion, degranulation and expression of activation receptors. They were observed with elevated levels in individuals chronically infected with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV)^[@CR23]^ but, to our knowledge, never described in septic shock. While this NK population could be of interest in sepsis-induced immunosuppression physiopathology, we cannot exclude the possibility that these cells represent some immature granulocytes frequently observed in septic shock patients' samples. As our mass cytometry panel did not contain markers dedicated to granulocyte characterization, we were not able to discriminate the two hypotheses, which now deserve to be further studied in septic shock patients. We also noted a trend toward B cell enrichment among mononuclear cells in septic shock patients, which had been very recently described by standard flow cytometry^[@CR24]^.
Then, we studied changes in cellular phenotypes after septic shock through more specific approaches. First, we focused on monocyte SPADE nodes. Remarkably, among the 9 markers used in the heatmap analysis, unsupervised clustering highlighted an immune signature of 4 markers (HLA-DR, CD4, CD38 and PD-L1) differentially expressed in healthy volunteers and septic shock patients. This uniform response might be used as potential biomarker of sepsis outcomes and then should be confronted to clinical recovery in larger cohorts, e.g. development of hospital acquired infection or mortality. Next, we were interested in the characterisation of CD4 T cell nodes expressing PD1, as many novel immunostimulating therapies target this molecule. Increase of PD1 expression did not occur on all CD4 T cell subsets after septic shock. Interestingly, CD4 T cells exhibiting PD1 upregulation did not coexpress other immune checkpoint inhibitors Tim-3 and PD-L1. Furthermore, increased expression of PD1 was observed on CD4 T cells presenting with different activation status, as some PD1^+^ cells clearly exhibited activated phenotypes whereas some corresponded to resting memory T cells. Here, CyTOF revealed to be a powerful tool to highlight and study cellular heterogeneity, as demonstrated recently for exhausted CD8 T cells in other clinical contexts^[@CR16]^. In particular, our results imply that exhaustion phenomenon might not occur in all cell subsets after septic shock, or at least might not be systematically associated with PD1 overexpression. These observations should be kept in mind and further explored when considering anti-PD1 strategy to treat septic patients. They strengthen the need of developing biomarkers of patients' immune status before using immunotherapies. Finally, when focusing on CD19^+^ cells, we observed a shift of B cell subsets towards plasma cell differentiation after septic shock. Thus we confirmed a feature of sepsis described recently for the first time by our group^[@CR24]^. It was striking to be able to detect this phenomenon in such a small cohort.
The aim of the present study was to assess feasibility and opportunities provided by CyTOF use in sepsis clinical cohorts. Thus, by nature, as a proof of concept study, our work has some limitations. In particular, we included small numbers of HV and septic shock patients. However, even in such a small cohort, non-supervised analyses of high dimensional data strikingly succeeded in highlighting both well-established (*e.g*., low mHLA-DR) and novel characteristics (*e.g*., increased plasma cells) of sepsis-induced immunosuppression. This illustrates the power of CyTOF technology. Furthermore, CyTOF was able to identify one septic patient with a singular immune pattern compared to others. This was confirmed by unusually high mHLA-DR expression measured by traditional flow cytometry. Again, this exemplifies CyTOF capacity to detect heterogeneity within septic shock patients' immune patterns. Thus, upon analysis of larger cohorts, CyTOF could be relevant to reveal new features of sepsis pathophysiology on the one hand, and to stratify patients according to their immune status and trajectories over time on the other hand. This latter point is an absolute requirement in future studies evaluating immunomodulating therapies in clinical cohorts. Analysing data from more patients would also open the possibility to use new interesting algorithms and software tools, as Citrus, which are able to automatically identify immune signatures in cellular populations^[@CR12],[@CR25]^.
Beyond the present study, there are several limiting factors for mass cytometry implementation in clinical studies. Indeed, CyTOF is a time consuming method, requiring 3 days of experiment from freezing to data acquisition. High dimensional data leads to days of data analysis, involving innovative bioinformatics tools such as SPADE and viSNE and representing analytical challenge for non-experts. However, mass cytometry represents a powerful tool to characterize immune alterations and observe the individual variability that must be targeted when developing immunomodulating therapies in context of personalised medicine. This has been illustrated in trauma context, where mass cytometry was used to link single-cell profiling of immunologic states to post-traumatic outcomes^[@CR26]^, or in cancer context, revealing specific markers associated with melanoma immunotherapy response^[@CR27]^. CyTOF could be relevant to highlight such immune signatures in sepsis.
In conclusion, in this study, we paved the way to the use of mass cytometry in sepsis clinical studies by demonstrating its reliability and feasibility in this context. We must now considered the use of standardized protocols on whole blood samples^[@CR28]--[@CR30]^ that would allow the use of this technology in multicentre studies. In addition, these preliminary results now deserve to be completed by larger studies combining extra and intracellular markers to explore signalling pathways and transcription factors, in order to progress in exhaustive characterisation of sepsis-induced immunosuppression.
Patients and Methods {#Sec11}
====================
Study population {#Sec12}
----------------
This clinical study was conducted on septic shock patients admitted to the ICU of Edouard Herriot Hospital (Hospices Civils de Lyon, Lyon, France). This project is part of a global study on ICU-induced immune dysfunctions. It has been approved by our Institutional Review Board for ethics ("Comité de Protection des Personnes Sud-Est II"), which waived the need for written informed consent since this study was observational with low risk for the patients and no specific blood sampling procedure beside routine blood sampling was required (\#IRB 11236). This study is also registered at the French Ministry of Research and Teaching (\#DC-2008-509) and recorded at the Commission Nationale de l'Informatique et des Libertés. Oral information and non-opposition to inclusion in the study were mandatory and recorded in patients' clinical files. This study is registered at NCT02803346.
Septic shock patients were identified according to the diagnostic criteria of the 1992 consensus definitions of the American College of Chest Physicians/Society of Critical Care Medicine^[@CR31]^. Patients were excluded if under 18 years of age or presented with aplasia or pre-existent immunosuppression. Septic shock diagnosis was based on the combination of an identifiable site of infection with persisting hypotension --- despite fluid resuscitation --- and evidence of a systemic inflammatory response manifested by at least two of the following criteria: a) temperature \>38 °C or \<36 °C; (b) heart rate \>90 beats/min; (c) respiratory rate \>20 breaths/min; (d) white blood cell count \>12,000/mm3 or \<4,000/mm3. The onset of septic shock was defined by the beginning of vasopressor therapy.
Peripheral blood was collected in patients 3 days after the onset of septic shock during routine blood sampling procedure, since previous studies showed that this time-point corresponds to the nadir of sepsis-induced immunosuppression^[@CR5]^. Clinical and biological parameters were collected, including demographic characteristics, date and cause of ICU admission, status at day 28 after inclusion, type of infection, comorbidities allowing to calculate Charlson comorbidity index^[@CR32]^. Severity scores were recorded: the Simplified Acute Physiology Score II (SAPSII; range 0--194)^[@CR33]^ and the Sepsis-related Organ Failure Assessment (SOFA; range 0--24)^[@CR34]^.
Concomitantly, healthy volunteers were obtained from the blood bank Etablissement Français du Sang (EFS) in Lyon, France. According to EFS standardized procedures for blood donation, informed consent was obtained from healthy donors and personal data were anonymized at the time of blood donation and before the transfer of blood to the research lab.
Sample preparation {#Sec13}
------------------
PBMC from septic patients and healthy volunteers were isolated from 7 mL of blood freshly drawn on anticoagulated tubes with Lympholyte®-H Cell Separation Media (Cedarlane, Burlington, Canada) centrifugation. PBMC were immediately cryoconserved after isolation in Roswell Park Memorial Institute medium 1640 (RPMI) complemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and glutamine (Eurobio Abcys, Courtaboeuf, France) containing 40% AB human serum (SAB) and 10% dimethylsulfoxyde (DMSO) (Sigma-Aldrich, Saint-Louis, MO).
Mass cytometry {#Sec14}
--------------
Depending on the sample, between 0.5 and 2.5 million thawed PBMC were incubated during 5 min with a viability reagent (5 μmol/L Cell-ID™ Cisplatin; Fluidigm, South San Francisco, CA) and then washed twice in Dulbecco's Phosphate-Buffered Saline (DPBS) containing 0.1% Bovine Serum Albumin (BSA). For staining, cells were incubated for 40 min at 4 °C in 100 μL DPBS/0.1% BSA containing antibody cocktail prepared with 25 antibodies listed in Supplementary Table [S3](#MOESM1){ref-type="media"}. Pre-metal tagged antibodies were purchased from Fluidigm. Cells were washed twice in DPBS/0.1% BSA and then fixed overnight at 4 °C with 1 mL of MaxPar® Fix&Perm Buffer (Fluidigm) containing 1 µL of Cell-ID™ Intercalator-Ir 125 µM (Fluidigm). The following day, cells were washed twice with DPBS and finally once with deionized MaxPar® Water (Fluidigm). Cells were resuspended in 1×EQ Four Element Calibration Beads (Fluidigm Sciences) according to the manufacturer's instructions. Samples were collected on a Helios mass cytometer (Fluidigm). Events were normalized prior to analysis as described elsewhere^[@CR35]^. Normalized data were imported into Cytobank (Santa Clara, CA) and manually gated: first, singulets were identified as ^191^Ir^+\ 193^Ir^+^ events and then viable cells as ^195^Pt negative events. Eventually, normalization beads were excluded by gating on 140Ce negative events (see Supplementary Fig. [S2A](#MOESM1){ref-type="media"}). To analyse proportions of main PBMCs' populations, cells were first gated according to their expression of CD3. CD4 and CD8 T cells were identified within CD3^+^ cells, while CD19^+^, CD14^+^ and CD16^+^/CD56^+^ non-T cells were used to classify B cells, monocytes and NK cells respectively (see Supplementary Fig. [S2B](#MOESM1){ref-type="media"}). When studying individual expression of phenotypic markers, we used a mass-minus-one control to set positive threshold. It was constituted of thawed PBMC stained only with lineage markers CD3, CD4, CD8, CD14, CD19, CD16 and CD56.
Flow cytometry {#Sec15}
--------------
Proportions of CD4 and CD8 T cells, as well as monocytes in PBMC were determined by flow cytometry using a four-color monoclonal antibody panel consisting of CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5 (Beckman Coulter, Hialeah, FL). A second antibody panel was used to measure B cells and NK cells' proportions, using CD19-PB/CD3-FITC/CD16-CD56-PE/CD45-APC-AF750 (Beckman Coulter). Samples were run on a Navios flow cytometer (Beckman coulter). Listmodes were subsequently analysed using Kaluza software (Version 1.2). Briefly, in both panels, lymphocytes were first gated out of total events on a biparametric CD45/SSC dot-plot, as CD45^hi^ SSC^lo^ events. Then, T cell subpopulations were defined as CD4^+^ or CD8^+^ CD3^+^ lymphocytes while B and NK lymphocytes were characterized as CD3 negative cells expressing respectively CD19 and CD56/CD16.
Data processing and analysis pipeline {#Sec16}
-------------------------------------
The whole analysis pipeline is illustrated in Fig. [2](#Fig2){ref-type="fig"}.
### X-shift {#Sec17}
The 06/29/2017 version of VorteX was downloaded and manually gated singulet viable events from the 5 healthy donors and 5 septic shock samples were uploaded into the VorteX clustering environment^[@CR36]^. The imported settings described previously were used^[@CR12]^: minimal Euclidean length of the profile: 1.0, import maximum: 10,000 events, and merge all files into one dataset. All 24 parameters were selected for clustering. After the data were imported, the following clustering settings were used: numerical transformation: arcsinh (x/f), f = 5.0, noise threshold: 1.0, feature rescaling: none, normalization: none, distance measure: angular distance, clustering algorithm: X-shift (gradient assignment), density estimate: N nearest neighbors (fast), number of neighbors for density estimate (K): from 150 to 5, with 30 steps, and number of neighbors for mode finding (N): determine automatically. Following clustering, the elbow point for cluster number was calculated as K = 35, which corresponded to 78X-shift--defined clusters within the dataset.
### Spanning-tree Progression Analysis of Density-normalized Events {#Sec18}
SPADE analysis was performed on manually gated singulet viable events from the 5 healthy volunteers and 5 septic shock samples on Cytobank. Raw median intensity values were transformed to a hyperbolic arcsine (arcsinh) scale with a cofactor of 5. The target number of nodes was adjusted to 78 as defined with the X-shift clustering tool. Clustering channels included all 25 parameters except for CD138, because of elemental contamination in 2 samples. Percentage downsampling was 10%. SPADE trees for each individual across all 24 markers were downloaded from Cytobank. Intensity and cellular abundance of each node from each individual were exported for further analysis.
### Visual Stochastic Network Embedding {#Sec19}
All CD19^+^ nodes from SPADE tree were selected and subjected to viSNE analysis. Non-B lineage markers as well as CD138 were excluded from viSNE clustering. Equal event sampling was selected to allow comparison between individuals, using the lowest common denominator across samples, which was 3055 events. Other viSNE settings were applied: number of iterations 2000, perplexity 30, theta 0.5. Cellular phenotype was studied visualizing characteristic markers' distribution and expression on viSNE plots and through generation of overlays on Cytobank.
### Determination of node frequency variations between patients and controls {#Sec20}
To select nodes with important variations in cell proportions between healthy donors and septic patients, nodes were ranked in each group by descending order according to their average proportions. A rank difference (control - septic patient ranks) under 0 reflected a decrease of cell proportion in septic patients, whereas a positive difference suggested an increased proportion in septic patients compared to healthy donors. Nodes with proportion variations differing of more than one tertile of total node ranks were selected and individual frequencies were compared between septic shock patients and healthy donors with appropriate statistical test.
Statistical analysis {#Sec21}
--------------------
Statistical analyses, heatmaps and graphics exploring cellular abundance and expression were performed on RStudio software. Non-parametric Mann-Whitney tests were used to compare data between septic shock patients and controls. Non-parametric Wilcoxon paired test was used when comparison within the same sample was performed. Values from continuous variables are presented as medians with Q1-Q3 interquartile ranges \[IQR\] between brackets. p value \< 0.05 was considered as significant.
Electronic supplementary material
=================================
{#Sec22}
Supplementary Information
**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
=================================
**Supplementary information** accompanies this paper at 10.1038/s41598-018-35932-0.
The authors would like to thank Fayrouz Mehennaoui and Valérie Cerro from E. Herriot Hospital, for their valuable help in patient inclusion process of the Immunosepsis study. The authors also thank équipex PHENOCAN (ANR -11-EQPX-0035 PHNEOCAN).
F.V., G.M. and M.G. conceived the study. T.R., S.D. and F.B. included patients in the cohort. M.G. and T.A. performed the experiments. M.G., C.M. and F.P.L. analysed the data. M.G. wrote the manuscript. F.V., G.M., F.P.L., C.M., T.R., S.D., F.B. and T.A. reviewed the manuscript.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
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"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
============
HIV infection via the penis accounts for a majority of infections among the 17 million men estimated to be living with HIV globally, yet remains a relatively understudied route of virus transmission. It is the primary route of HIV acquisition among heterosexual men and is of relevance to MSM who practice insertive anal intercourse^[@CR1]^. Current strategies to reduce one's risk of acquiring HIV infection via the penis include condom usage, male circumcision and taking antiretroviral drugs for preexposure prophylaxis (PrEP)^[@CR2]--[@CR5]^. Regrettably, each of these prevention strategies has limitations that curb effectiveness. For example, even when used consistently, condoms are \<80% effective in reducing HIV incidence among uninfected partners of HIV-discordant heterosexual couples^[@CR2]^. Similarly, male circumcision, in which all or part of the penile foreskin is removed surgically, confers incomplete protection against HIV--reducing infection risk by 50--60%^[@CR5]^. With regard to PrEP, once-daily oral tenofovir disoproxil fumarate (TDF) or TDF-emtricitabine (FTC) combination reduced HIV infection risk by 63% or 84%, respectively, among heterosexual men with a known HIV-infected partner^[@CR3]^. Among MSM, clinical trials have shown daily oral TDF--FTC to reduce overall HIV incidence either by 44% (with significantly greater reductions (92--95%) among trial participants who had detectable study drugs in plasma)^[@CR6]^, or by 86% (PROUD Study)^[@CR7]^. In addition, on-demand dosing with TDF--FTC, rather than daily dosing, demonstrated an 86% reduction in HIV incidence as compared with placebo controls (IPERGAY Study)^[@CR8]^. While oral TDF--FTC can be highly efficacious in preventing HIV infection, users' lack of adherence to drug regimens is a major behavioral impediment that limits PrEP effectiveness^[@CR9]^.
As a result, development of long-acting HIV intervention products that require relatively less frequent dosing remains a top priority in the HIV prevention and treatment fields. Passive immunization using recombinant monoclonal antibodies capable of neutralizing diverse HIV isolates (broadly neutralizing antibodies (bNAbs)) is a promising approach to achieve long-acting HIV prevention. The Antibody-Mediated Prevention (AMP) Study currently is evaluating bNAb VRC01, when administered via intravenous infusion once every 8 weeks, for safety and efficacy to prevent HIV infection among high-risk women (HVTN 703/HPTN 081; NCT02568215) and among men and transgender persons who have sex with men (HVTN 704/HPTN 085; NCT02716675)^[@CR10]^. However, because HIV transmission among MSM occurs more commonly via the rectal than the penile route^[@CR1],[@CR11]^, the AMP Study is not poised specifically to evaluate antibody-mediated protection against penile HIV acquisition.
Many other bNAbs, which exhibit greater potency or neutralization breadth than VRC01 are being developed and are in clinical evaluation^[@CR12]--[@CR14]^. Some of the most clinically advanced among these are 10--1074, which targets the base of the third variable loop and surrounding glycans on the HIV envelope protein (Env), and 3BNC117, which targets the CD4 binding site^[@CR15],[@CR16]^. Both have been shown to be safe and well tolerated in HIV-infected and uninfected individuals, and capable of suppressing HIV viremia in viremic subjects^[@CR12],[@CR17]--[@CR20]^.
During preclinical development, HIV prevention products or vaccines often are evaluated for protective efficacy in nonhuman primate models. These infection models are predicated upon exposing macaques to a single high-dose, or repeated low-doses of simian-human immunodeficiency virus (SHIV), most commonly via rectal or vaginal routes^[@CR21],[@CR22]^. bNAbs 10--1074 and 3BNC117 have been shown to protect macaques against rectal infection with SHIV~AD8-EO~^[@CR23],[@CR24]^. However, their efficacy in preventing infection via the other major mucosal routes of HIV infection---namely penile or vaginal, has not been described. Nor is it known how these bNAbs' correlates of protection compare among all mucosal infection routes.
In the absence of penile animal model data, it is difficult to determine how efficacious PrEP regimens are in preventing HIV infection across the penile mucosa during insertive intercourse (vaginal or rectal) with an infected individual. Myriad differences exist between penile, vaginal, and rectal mucosa that may modulate HIV susceptibility or PrEP pharmacokinetics. These include differences in epithelial thickness or composition, presence or absence of mucus, mechanical trauma incurred via sexual intercourse, or prevalent microbiota^[@CR25]--[@CR32]^. Despite the public health relevance of penile HIV infection, preclinical studies of the efficacy of PrEP or other biomedical preventions against this route are limited, due largely to a lack of relevant animal models. This represents a knowledge gap with unknown impact on current and future translational efforts to develop new candidate HIV interventions for men.
Recently, we developed a repeat-exposure penile SHIV infection model in rhesus macaques to evaluate the protective efficacy of biomedical preventions against HIV^[@CR33]^. The model is predicated upon repeatedly exposing multiple penile tissues that are relevant for HIV acquisition in humans, including the inner foreskin, glans, and distal urethra, to limiting doses of SHIV~SF162P3~ (i.e., doses that do not result in systemic infection among all animals following a single challenge) using a nontraumatic technique that precludes inadvertent perturbation of the penile epithelium^[@CR33]^. Here, we utilized this penile infection model to assess the protection conferred by passive bNAb immunization. We chose to evaluate 10--1074, which exhibits potent neutralization of SHIV~SF162P3~, and show that a single subcutaneous dose of 10--1074 durably protected macaques against repeated penile SHIV~SF162P3~ challenges.
Because the efficacy of 10--1074 against vaginal challenge had not been evaluated, we also tested its protective efficacy in a repeat low dose challenge model. However, the greatest burden of HIV infection of women is in sub-Saharan Africa, where multiple HIV-1 subtypes predominate^[@CR34]^. In vivo, in vitro, and in silico modeling predicts that bNAbs used in combination will achieve greater neutralization coverage^[@CR35]--[@CR40]^. To this end, several phase-I studies evaluating bNAb combinations are ongoing^[@CR14]^, and one that evaluated administering 10--1074 in combination with 3BNC117 recently has been reported^[@CR20]^. Therefore, we elected to evaluate 10--1074 in combination with 3BNC117. Because SHIV~SF162P3~ is not sensitive to neutralization by 3BNC117, we chose to challenge vaginally with SHIV~AD8-EO~, which is sensitive to both 3BNC117 and 10--1074. As such, our penile and vaginal challenge models used SHIVs that are similarly susceptible to neutralization by 10--1074, with IC~80~ values of 0.20 or 0.25 μg ml^−1^ against the replication competent challenge virus stock of SHIV~SF162P3~ or SHIV~AD8-EO~, respectively. We show that subcutaneously delivered 10--1074 and 3BNC117 exhibited different pharmacokinetics including a longer plasma persistence of 10--1074 than 3BNC117, providing an opportunity to measure efficacy against vaginal challenge of the bNAb combination and of 10--1074 alone. We used probit regression analyses to compare plasma bNAb concentrations that reduced penile or vaginal infection.
Results {#Sec2}
=======
Protection by 10--1074 against repeated penile SHIV challenges {#Sec3}
--------------------------------------------------------------
We sought to determine the protective efficacy that passive immunization with 10--1074 conferred against SHIV acquisition by the penile route in rhesus macaques. Six macaques in the treatment group (Group-1) received a single subcutaneous injection of 10--1074 (human IgG~1~ isotype; does not contain the LS or any other half-life modifying mutations) at 10 mg per kg body weight at 1 week prior to the start of SHIV challenges (Fig. [1a](#Fig1){ref-type="fig"}). We performed nontraumatic penile challenges with SHIV using a 'no contact' technique that eliminated inadvertent perturbation of the penile mucosa during the inoculation procedure itself, while simultaneously exposing multiple susceptible penile tissues to the challenge virus^[@CR33]^. For each virus challenge, macaques received 200 TCID~50~ SHIV~SF162P3~ into the prepuce pouch and 16 TCID~50~ SHIV~SF162P3~ into the distal urethra. Ten historical control animals (Group-2) had received no antibodies and had been challenged identically with the same stock of SHIV~SF162P3~ (Fig. [1a](#Fig1){ref-type="fig"}). Penile SHIV~SF162P3~ challenges were repeated once weekly until systemic infections were confirmed via positive RT-qPCR plasma viral load assay for macaques in the treatment group (Fig. [1b](#Fig1){ref-type="fig"}) or control group (Fig. [1c](#Fig1){ref-type="fig"}). Macaques that received a single injection of 10--1074 became infected following a median of 15.5 weekly challenges (range = 5--19) and were protected significantly longer than untreated control animals, which became infected following a median of 2.5 challenges (range = 1--12) (*P* = 0.0007, Log-Rank test) (Fig. [1d](#Fig1){ref-type="fig"}). Of note, the earliest infection among macaques administered 10--1074 occurred in animal 10--139 following five SHIV~SF162P3~ challenges and corresponded with an abrupt decline in the neutralization activity of 10--1074 in plasma between 3 and 5 weeks post injection, due to the development of an antidrug antibody (ADA) response (Fig. [1b](#Fig1){ref-type="fig"}, Supplementary Fig. [1](#MOESM1){ref-type="media"})^[@CR41],[@CR42]^. Protection among 10--1074-treated macaques that did not exhibit such ADA responses was relatively greater, showing median protection against 17 weekly challenges (range = 13--19). No differences in levels of peak viremia, or area under the curve (AUC) through 7 weeks post infection were observed between 10--1074-treated and control groups (Supplementary Fig. [1](#MOESM1){ref-type="media"}).Fig. 1Passive immunization of macaques with bNAb 10--1074 delays SHIV acquisition following repeated penile SHIV challenges.**a** Study design to assess the protective efficacy of 10--1074 against repeated penile SHIV challenges. Rhesus macaques (*N* = 6; Indian origin) were injected subcutaneously once with 10--1074 (10 mg kg^−1^). Beginning 1 week later, macaques were challenged repeatedly, once per week, via the penis with SHIV~SF162P3~ (200 TCID~50~ into the prepuce pouch and 16 TCID~50~ into the distal urethra) until systemic SHIV infection was confirmed via plasma viral load assay. Control macaques (*N* = 10) received no antibody, but were challenged identically. Plasma viral load (vRNA copies ml^−1^) determined in 10--1074-treated macaques (**b**) or untreated controls (**c**). Symbols denote individual animals. **d** Percentages of macaques remaining uninfected at 7 days following administration of the indicated cumulative number of SHIV challenges. Statistical difference between groups was measured using a two-sided log-rank test (*P* = 0.0007). **e** Plasma levels of 10--1074 (μg ml^−1^) were determined via TZM-bl neutralization assays using 10--1074-sensitive pseudovirus X2088.c9. **f** Plasma concentrations of 10--1074 in individual macaques (*N* = 6) at the time of SHIV breakthrough (values from **e**).The solid line denotes the group median; the dotted line denotes the limit of detection for 10--1074. Source data are provided in [Supplementary Tables](#MOESM1){ref-type="media"} or a [Source data](#MOESM3){ref-type="media"} file. SHIV infection data for animals in the control group have been reported previously in a paper describing the development of our penile SHIV infection model^[@CR33]^ and are reproduced here to facilitate comparison between the treatment and control groups.
Plasma levels of 10--1074 exhibited a mean maximum concentration (*C*~max~) of 36.0 ± 13.3 μg ml^−1^ at 7 days following antibody injection, which was the earliest postinjection timepoint assayed (Fig. [1e](#Fig1){ref-type="fig"}). Plasma levels of 10--1074 exhibited an average half-life of 15.5 ± 4.0 days and remained above the level of detection (0.10 μg ml^−1^) for 15--20 weeks following injection in 5 of 6 treated animals (Fig. [1e](#Fig1){ref-type="fig"}; (Supplementary Table [1](#MOESM1){ref-type="media"})). At the time of SHIV breakthrough (i.e., first detectable plasma viremia), the median plasma concentration of 10--1074 among all treated macaques was 0.50 μg ml^−1^ (range: \<0.10--0.70 μg ml^−1^) (Fig. [1f](#Fig1){ref-type="fig"}). Excluding the animal that developed an early ADA response against 10--1074 and had an undetectable level of 10--1074 at the time of SHIV breakthrough, the median plasma concentration of 10--1074 at the time of SHIV breakthrough was 0.60 μg ml^−1^ (range of 0.20--0.70 μg ml^−1^).
To evaluate antibody-mediated protection against penile infection, we used probit regression to estimate the per-challenge probability of SHIV infection as a function of imputed plasma 10--1074 levels (Fig. [2](#Fig2){ref-type="fig"}), excluding animal 10--139 who exhibited an early ADA response. Among no-antibody controls, each SHIV challenge presented a 0.25 chance of infection (Fig. [2](#Fig2){ref-type="fig"}; 95% CI 0.13--0.41). The probit model estimated \>100-, 9.5-, and 1.6-fold reductions of this infection probability to \<0.001, 0.002, or 0.093 for plasma 10--1074 concentrations of 5.0, 3.0, or 1.0 μg ml^−1^, respectively. Bootstrap fitting of the probit regression model estimated a 0.01 probability of infection following a single penile SHIV challenge at a plasma 10--1074 concentration of 2.28 μg ml^−1^ (95% CI: 1.27, 3.47 μg ml^−1^).Fig. 210--1074 antibody concentration predicts the probability of infection following penile SHIV~SF162P3~ challenge.**a** Probit regression analysis was used to estimate the per-challenge probability of penile SHIV acquisition as a function of imputed 10--1074 concentration in plasma. The per-challenge probability of infection among untreated control animals was 0.25 (95% CI 0.13--0.41) and is denoted in blue. Tick marks represent SHIV challenge events that either did (red) or did not (green) result in systemic infection. The solid black line is the model-based prediction; dotted black lines depict 90% pointwise confidence intervals. **b** Model-based estimates of SHIV infection probabilities following a single penile challenge and reduction of infection probability as compared with controls, at the indicated plasma concentrations of 10--1074.
Protection by bNAbs against repeated vaginal SHIV challenges {#Sec4}
------------------------------------------------------------
bNAbs 10--1074 and 3BNC117 are among the most clinically advanced second generation bNAbs and have not been evaluated previously in nonhuman primate models for protection against vaginal SHIV challenge. Because bNAbs targeting nonoverlapping epitopes on HIV Env could be used in combination to achieve additive neutralization activity or increased breadth of neutralization^[@CR35]--[@CR40]^, we evaluated protection against repeated vaginal SHIV challenges among macaques that received a combination of 10--1074 and 3BNC117, or 3BNC117 singly.
Macaques in the treatment groups (Group-1, -2) received a one-time subcutaneous injection of both 10--1074 and 3BNC117 (10 mg each kg^−1^, Group-1) or 3BNC117 singly (10 mg kg^−1^, Group-2) 1 week prior to the start of vaginal SHIV~AD8-EO~ challenges. Both bNAbs were of human IgG~1~ isotype and did not contain the LS or any other half-life modifying mutations. Macaques in the control group (Group-3) were not administered any antibodies and were challenged identically with SHIV~AD8-EO~ (Fig. [3a](#Fig3){ref-type="fig"}). Macaques in all groups received intramuscular injections of depot medroxyprogesterone acetate (DMPA; 30 mg) 2 weeks prior to the first SHIV challenge (corresponding to 1 week prior to bNAb administration in the treatment groups), and every 4 weeks thereafter, to normalize vaginal SHIV susceptibility via progestin-mediated thinning of the vaginal mucosa^[@CR43]--[@CR45]^ (Fig. [3a](#Fig3){ref-type="fig"}). Vaginal SHIV~AD8-EO~ challenges (300 TCID~50~) were repeated once weekly until systemic infections were confirmed via positive RT-qPCR viral load assay for macaques in the combination treatment group (Fig. [3b](#Fig3){ref-type="fig"}), 3BNC117-only group (Fig. [3c](#Fig3){ref-type="fig"}), or untreated control group (Fig. [3d](#Fig3){ref-type="fig"}).Fig. 3Passive immunization with bNAbs 10--1074 and 3BNC117 in combination, or 3BNC117 singly, delays SHIV acquisition following repeated vaginal challenges in DMPA-treated macaques.**a** Rhesus macaques (Chinese origin) were injected subcutaneously once with a combination of bNAbs 10--1074 and 3BNC117 (10 mg each kg^−1^; *N* = 5) or 3BNC117 singly (10 mg kg^−1^; *N* = 6). Commencing 1 week later, macaques were challenged repeatedly, once per week, intravaginally with SHIV~AD8-EO~ (300 TCID~50~) until systemic SHIV infection was confirmed via plasma viral load assay. Control macaques (*N* = 3) received no antibody but were challenged identically. Animals in both groups received DMPA (30 mg) intramuscularly at 2 weeks before the first SHIV challenge and every 4 weeks thereafter to normalize SHIV susceptibility. Plasma viral loads (vRNA copies ml^−1^) for macaques in the combination bNAb group (**b**), single bNAb group (**c**) or untreated control group (**d**). Symbols denote individual animals. **e** Percentages of macaques remaining uninfected at 7 days following administration of the indicated cumulative number of SHIV challenges. Statistical differences between groups were analyzed using a two-sided log-rank test. Plasma levels of 10--1074 (μg ml^−1^) or 3BNC117 (μg ml^−1^) were determined via TZM-bl neutralization assays using 10--1074-sensitive pseudovirus X2088.c9 or 3BNC117-sensitive pseudovirus Q769.d22 for macaques that received both 10--1074 and 3BNC117 (**f**) or 3BNC117 alone (**g**). **h** Plasma concentrations of 10--1074 or 3BNC117 in individual macaques (*N* = 5 in Group-1, *N* = 6 in Group-2) at the time of SHIV breakthrough (values from **f** (Group-1) or **g** (Group-2)). Solid lines denote group medians; dotted lines indicate the limits of detection for 10--1074 (black) or 3BNC117 (gray). Source data are provided in [Supplementary Tables](#MOESM1){ref-type="media"} or a [Source data](#MOESM3){ref-type="media"} file.
Macaques that received a single injection of only 3BNC117 showed breakthrough SHIV infections following a median of five challenges (range = 4--7) and were protected significantly longer than were untreated controls, which became infected following a median of two SHIV challenges (range = 1--3) (*P* = 0.0018, Log-Rank test; Fig. [3e](#Fig3){ref-type="fig"}). More importantly, macaques in the 10--1074 + 3BNC117 combination group were protected against a median of 11 weekly challenges (range = 10--12), which was significantly greater than that conferred following administration of 3BNC117 alone (*P* = 0.0012, Log-rank test) or observed among untreated controls (*P* = 0.0042, Log-Rank test) (Fig. [3e](#Fig3){ref-type="fig"}). Levels of peak viremia and early SHIV replication, measured as vRNA AUC through 7 weeks post infection, were similar among all groups (Supplementary Fig. [3](#MOESM1){ref-type="media"}).
Beginning 1 week following antibody injection, plasma levels of 10--1074 and 3BNC117 were measured among macaques that had received these two antibodies in combination. The maximum concentration of 10--1074 (mean *C*~max~ 10--1074 = 35.7 ± 9.6 μg ml^−1^) was 6 times as high as 3BNC117 (mean *C*~max~ 3BNC117 = 5.9 ± 2.6 μg ml^−1^) (*P* = 0.0022, paired *t*-test; Fig. [3f](#Fig3){ref-type="fig"}), and both maxima were observed at the earliest timepoint (7 days) that was sampled following antibody injection. Among macaques that received only 3BNC117, the mean maximum plasma level of 3BNC117 (mean *C*~max~^3BNC117^ = 7.0 ± 3.2 μg ml^−1^; Fig. [3g](#Fig3){ref-type="fig"}) also was observed at the earliest timepoint sampled following antibody injection (7 days post injection) and was similar in magnitude to that observed for 3BNC117 among animals in the combination treatment group (Fig. [3f](#Fig3){ref-type="fig"}; Supplementary Tables [2](#MOESM1){ref-type="media"}, [3](#MOESM1){ref-type="media"}). Thus, no adverse interaction by 10--1074 on 3BNC117 pharmacokinetics was observed in vivo.
In macaques that were administered 3BNC117 singly, the median plasma 3BNC117 concentration at the time of SHIV breakthrough was 0.50 μg ml^−1^ (range = 0.24--0.80 μg ml^−1^) (Fig. [3h](#Fig3){ref-type="fig"}). In contrast, at the time of SHIV breakthrough, macaques that had been administered both bNAbs exhibited a median plasma concentration for 10--1074 of 0.10 μg ml^−1^ (range = 0.10--0.30 μg ml^−1^), but undetectable levels of 3BNC117 (Fig. [3h](#Fig3){ref-type="fig"}). Moreover, among all macaques in the combination bNAb group, 3BNC117 levels were at, or had been below, the level of detection (0.24 μg ml^−1^) for a median of 4 weeks (range = 3--8 weeks) prior to SHIV breakthrough. This argues that the more durable protection observed in the combination antibody group was due to 10--1074, which persisted relatively longer than 3BNC117. As such, these macaques were protected through a period of effective 10--1074 monotherapy. This allowed us to estimate levels of 10--1074 that were protective against vaginal SHIV~AD8-EO~ infection, and enabled a comparison of correlates of protection for 10--1074 between the penile and vaginal challenge models.
We first used probit regression to estimate the per-challenge probability of SHIV infection as a function of imputed bNAb levels in plasma among animals in the vaginal challenge cohorts (Fig. [4](#Fig4){ref-type="fig"}). Among macaques that received 3BNC117 singly, this model estimated infection probabilities of \<0.001, 0.016, or 0.36 at 3BNC117 concentrations of 5.0, 3.0, or 1.0 μg ml^−1^, respectively. These estimates represent \>100-, 31-, and 1.4-fold reductions in infection probability, respectively, from that of no-antibody controls whose per-challenge infection probability was 0.50 (95% CI: 0.11--0.88). Bootstrap fitting of this model estimated a 0.01 probability of infection following a single vaginal SHIV challenge at a plasma 3BNC117 concentration of 3.22 μg ml^−1^ (95% CI: 1.62, 4.69 μg ml^−1^).Fig. 4Antibody concentration predicts the probability of infection following vaginal SHIV~AD8-EO~ challenge.Probit regression analysis was used to estimate the per-challenge probability of vaginal SHIV acquisition as a function of imputed 3BNC117 concentrations in plasma among macaques that were administered 3BNC117 singly (**a**) or 10--1074 concentrations in plasma among animals that received 10--1074 in combination with 3BNC117 (**b**). The per-challenge probability of infection among untreated control animals was 0.50 and is denoted in blue. Tick marks represent SHIV challenge events that either did (red) or did not (green) result in systemic infection. The solid black or gray line is the model-based prediction; dashed black lines depict 90% pointwise confidence intervals. Model-based estimates of SHIV infection probabilities following a single vaginal SHIV challenge at the indicated plasma concentrations of 3BNC117 (**c**) or 10--1074 (**d**).
Among macaques in the combination treatment group, probit regression estimated per-challenge infection probabilities of \<0.001, \<0.001, or 0.014 at 10--1074 concentrations of 5.0, 3.0, or 1.0 μg ml^−1^, respectively. These estimates represent \>100-, \>100-, and 36-fold reductions as compared with untreated controls. Bootstrap fitting of the model showed a plasma 10--1074 concentration of 1.13 μg ml^−1^ (95% CI: 0.51, 1.53 μg ml^−1^) predicted a 0.01 probability of infection following a single vaginal SHIV~AD8-EO~ challenge. Thus, among vaginally challenged macaques, the estimated plasma bNAb concentration corresponding to a 0.01 probability of infection was three times as high for 3BNC117 \[3.22 μg ml^−1^ (95% CI: 1.62, 4.69 μg ml^−1^)\] than 10--1074 \[1.13 μg ml^−1^ (95% CI: 0.51, 1.53 μg ml^−1^)\], which reflects the relatively lower neutralization potency of 3BNC117, as compared with 10--1074, against the challenge virus.
In contrast, comparison of the correlates of 10--1074-mediated protection between the penile and vaginal SHIV challenge models showed no significant difference---the estimated plasma 10--1074 concentration that corresponded to a 0.01 probability of SHIV~SF162P3~ infection via the penis \[2.28 μg ml^−1^ (95% CI: 1.27, 3.47 μg ml^−1^)\] was not different from that against SHIV~AD8-EO~ infection vaginally \[1.13 μg ml^−1^ (95% CI: 0.51, 1.53 μg ml^−1^)\].
Discussion {#Sec5}
==========
HIV infection of the penis has contributed substantially to the global HIV epidemic, as a majority of the \~18 million men living with HIV worldwide became infected through heterosexual transmission^[@CR46]^. However, preclinical animal modeling to assess the efficacy of candidate HIV interventions against penile infection has lagged that targeting rectal or vaginal infection routes. Here, using a recently developed macaque model^[@CR33]^, we provide a preclinical evaluation of bNAb-mediated protection against penile SHIV infection.
Our results demonstrated durable protection (up to 19 weeks) against penile SHIV~SF162P3~ infection of macaques following a single subcutaneous injection of 10--1074. Probit regression modeling estimated that 10--1074 reduced the per-challenge penile infection risk by \>2-logs, from 0.25 among untreated controls to \<0.001 among macaques with a plasma 10--1074 concentration of 5.0 μg ml^−1^. In phase-I studies among HIV uninfected people, 10--1074 concentrations \> 5.0 μg ml^−1^ were observed for at least 8 weeks following either a single infusion of 10--1074 at 3, 10, or 30 mg kg^−1^ or repeated infusions, once every 8 weeks, of 10--1074 in combination with 3BNC117 at 10 mg kg^−1 [@CR17],[@CR20]^. If directly translatable, our results suggest that relatively infrequent dosing with 10--1074 might provide a high level of protection against penile HIV infection among men.
Our vaginal challenge study also provided information on correlates of bNAb-mediated protection. Among macaques administered 10--1074 and 3BNC117 in combination, protection was attributed to 10--1074 due to the observed difference in antibody pharmacokinetics following subcutaneous administration that resulted in a period of effective 10--1074 monotherapy preceding SHIV~AD8-EO~ infection. Probit regression modeling estimated that per-challenge vaginal infection risk was reduced by \>2-logs, from 0.50 among untreated controls to \<0.001 among macaques with a plasma 10--1074 concentration of 3.0 μg ml^−1^ or greater.
A limitation in directly comparing the correlates of 10--1074-mediated protection against penile or vaginal infection in this study is that the underlying animal models utilized different challenge virus strains and doses. Here, our penile and vaginal challenge models used SHIVs that are similarly susceptible to neutralization by 10--1074, with IC~80~ values of 0.20 or 0.25 μg ml^−1^ against the replication competent challenge virus stock of SHIV~SF162P3~ or SHIV~AD8-EO~, respectively. Regarding challenge virus dose selection, model feasibility necessitates that each SHIV exposure presents a relatively higher risk of infection to macaques, than does exposure of men or women to an HIV-infected source, for which the per-exposure infection rates are low (insertive vaginal---0.0004, insertive rectal---0.0011, receptive vaginal---0.0008)^[@CR1]^. For these macaque studies, challenge virus doses were selected empirically to normalize the number of challenges required to infect animals via penile or vaginal routes and resulted in a median of 2 or 2.5 challenges, respectively. Importantly, the bootstrapped estimate of the per-challenge probability of infection among untreated control animals in the penile SHIV~SF162P3~ model \[0.25 (95% CI: 0.13--0.41)\] was not significantly different from that among vaginal SHIV~AD8-EO~ controls \[0.50 (95% CI: 0.11--0.88)\]. Thus, macaques were challenged with SHIVs, via a penile or vaginal route, under model conditions of comparable stringency. These model-specific differences notwithstanding, we found no significant difference between the plasma 10--1074 concentration that reduced per-exposure infection risk to 0.01 against penile SHIV~SF162P3~ or vaginal SHIV~AD8-EO~ challenges, and risk reduction was greater than 100-fold for both groups when plasma 10--1074 concentrations were 5.0 μg ml^−1^ or higher.
Likewise, these correlates of protection against penile or vaginal infection compare favorably with those reported for 10--1074-mediated protection against repeated rectal SHIV~AD8-EO~ challenges among macaques^[@CR23]^. For example, at a plasma 10--1074 concentration of 1.0 μg ml^−1^, 63 or 84% reductions in per-challenge infection risk were observed among macaques challenged repeatedly with SHIV~SF162P3~ via the penis or SHIV~AD8-EO~ rectally^[@CR23]^. These findings are consistent with an earlier study showing passively administered bNAb PGT126 protected similarly against a single high-dose challenge with SHIV~SF162P3~ via rectal or vaginal routes^[@CR47]^.
The site(s) and mechanism(s) by which passively administered bNAbs confer protection against penile or other mucosal routes of infection are not known fully. Transudation or active (FcRn-mediated) transport of bNAbs from circulation to mucosal tissues or secretions would position these effector molecules at the point of virus entry, where their neutralization or Fc-related antiviral functions may be expected to have greatest impact to prevent either initial infection or local amplification and systemic spread of a nascent infection^[@CR48]--[@CR51]^. Observation that the plasma correlates of 10--1074-mediated protection were not different against penile or vaginal infection may reflect similar bNAb pharmacokinetics among these anatomic compartments or may reflect common antiviral effects occurring elsewhere than the mucosal portal of virus entry. Thus, a second limitation of this study was the lack of mucosal sampling to determine bNAb pharmacokinetics in penile tissues or secretions. However, because our primary study objective was protective efficacy, we chose not to collect penile samples from animals being challenged, out of concern that the collection procedures themselves would alter susceptibility to SHIV acquisition. Future studies to characterize the pharmacokinetics of bNAbs in penile tissues or secretions, following passive administration, should be informative in this regard.
We note that our penile infection model is a model of HIV infection among uncircumcised men. Because 60--70% of adult men globally are not circumcised^[@CR52],[@CR53]^, this model has broad public health relevance. However, penile tissues other than the foreskin are relevant for HIV infection, as evidenced by the incomplete protection that circumcision confers against penile infection^[@CR54]--[@CR56]^, as well as the wide distribution of HIV target cells among other penile tissues, including the urethra and glans^[@CR25],[@CR57],[@CR58]^. Although our current challenge model simultaneously exposes multiple penile tissues to SHIV, it is of interest to determine whether removal of the foreskin via circumcision would alter the capacity of bNAbs to protect remaining penile tissues from infection. A final model limitation is that the nontraumatic technique used for penile challenges with SHIV does not account for any trauma that might occur to the penis during sexual intercourse.
In summary, we have utilized a recently developed macaque model of penile HIV infection to demonstrate protective efficacy of a passively administered bNAb against this route of infection. We determined plasma 10--1074 concentrations as correlates of protection against penile or vaginal SHIV acquisition and compared these to values reported to protect against rectal SHIV infection. Despite model-specific differences, the findings from these macaque studies show that large reductions in infection probability for all major routes of HIV acquisition could be demonstrated at clinically relevant levels of circulating bNAbs. Such an overall indication from macaque modeling should facilitate dose selection of bNAbs for clinical advancement of immunoprophylaxis against HIV.
Methods {#Sec6}
=======
Macaques {#Sec7}
--------
Thirty adult rhesus macaques (*Macaca mulatta*) were used to perform the penile or vaginal SHIV challenge studies, respectively. All animals were housed at the Centers for Disease Control and Prevention (CDC; Atlanta, GA) in accordance with the *Guide for the Care and Use of Laboratory Animals* (8th edition) in an AAALAC-accredited facility, according to institutional standard operating procedures. For housing, macaques were maintained in cages that met or exceeded the minimum size requirements as stipulated in the Guide. Animals were provided enrichments that included objects to manipulate, assortments of fresh fruits and vegetables, suitable feeding methods (foraging and task-oriented), and humane interactions with caregivers and research staff. Prior to the initiation of virus challenges, compatible macaques were pair-housed to the extent possible. Animal studies were approved by the CDC Institutional Animal Care and Use Committee (IACUC, protocol 2804GARMONC). To minimize animal discomfort or suffering, all biomedical procedures were performed on animals under ketamine (10 mg kg^−1^) or Telazol (2--6 mg kg^−1^) anesthesia.
Challenge virus stocks {#Sec8}
----------------------
Preparation and characterization of the cell-free SHIV~SF162P3~ stock used to perform penile challenges has been described^[@CR33]^---the undiluted stock had a titer of 2430 TCID~50~ ml^−1^, determined on whole, unstimulated primary rhesus PBMCs. A cell-free stock of SHIV~AD8-EO~, which was used to perform vaginal challenges, was prepared as follows. Infectious virus was obtained in supernatants of 293T cell cultures at 48 h following FuGene 6 (Promega)-mediated transfection of plasmid pSHIV AD8-EO (kindly provided by Malcolm Martin, NIAID). Virus in 293T culture supernatants was amplified in rhesus macaque PBMCs following in vitro depletion of CD8^+^ cells (Dynabeads CD8, ThermoFisher) and stimulation with Concanavalin-A (Sigma-Aldrich). Supernatants were clarified via centrifugation, aliquoted and stored in the vapor phase of liquid nitrogen. The undiluted SHIV~AD8-EO~ stock had a titer of 3600 TCID~50~ ml^−1^, determined on whole, unstimulated primary rhesus PBMCs.
Antibodies and passive immunization {#Sec9}
-----------------------------------
Monoclonal antibodies 10--1074 and 3BNC117 were produced in the laboratory of M.C.N. and formulated individually for injection at concentrations ranging between 49.5 and 53 mg ml^−1^ in 5 mM acetate, 280 mM trehalose, 0.05% Tween20 (pH 5.2) or 10 mM [l]{.smallcaps}-histidine, 280 mM trehalose, 0.05% Tween20 (pH 5.5), for 10--1074 and 3BNC117, respectively^[@CR17],[@CR18]^. Antibodies were administered via subcutaneous injection in macaques at 10 mg kg^−1^ in the upper back (3BNC117 on the left side, 10--1074 on the right side) via 22G1 needle; injection volumes were \<2 ml per injection site.
Virus challenges {#Sec10}
----------------
SHIV challenges were performed once weekly, via penile or vaginal routes, until systemic infection was confirmed by detection of vRNA in plasma. Penile SHIV challenges were performed by administering 200 TCID~50~ into the prepuce (foreskin) pouch and 16 TCID~50~ virus into the distal urethra. Urethral inoculations were performed using a 'no-contact' technique in which the lobes of the glans were manually flared to expose the navicular fossa and a 20 μl inoculum volume was expelled from a micropipettor tip positioned 1--5 mm above the center of the exposed urethral opening^[@CR33]^. This technique avoids all contact of the pipet tip with urethral tissue and precludes inadvertent abrasion of the urethral epithelium. SHIV~SF162P3~ was used for penile challenges as it is sensitive to neutralization by bNAb 10--1074, and the penile model had been optimized with SHIV~SF162P3~. For vaginal SHIV challenge, macaques were nontraumatically administered 300 TCID~50~ virus, in a 1 ml volume, via an inserted pediatric nasogastric feeding tube of adjusted length. SHIV~AD8-EO~ was selected for vaginal studies because we wanted to evaluate two bNAbs---3BNC117 and 10--1074, both of which neutralize this virus.
Viral load assay {#Sec11}
----------------
Viral RNA in plasma was quantified using a real-time reverse transcription PCR assay to detect the SIV *gag* gene^[@CR59]^. SHIV virions were concentrated from 1 ml plasma samples via ultracentrifugation (100,000 g for 30 min at 4 °C) and subjected to RNA extraction using NucliSens reagents (Biomerieux). One-step RT-PCR was performed using the SuperScript III Platinum One-Step qRT-PCR Kit with ROX (ThermoFisher) with forward primer (SIVp1f1) 5′-GCCAACAGGCTCAGAAAATTTAA-3′, reverse primer (SIVp1r1) 5′-TCCTCAGTGTGTTTCACTTTCTCTTC-3′ and probe 5′-HEX-AGCCTTTATAATACTGTCTGCGTCATCTGGTGC-BHQ1-3′. The limit of detection was 60 viral RNA copies per ml.
Determination of 10--1074 and 3BNC117 concentrations {#Sec12}
----------------------------------------------------
Concentrations of 10--1074 and 3BNC117 in plasma were determined using TZM-bl neutralization assays using pseudovirus X2088.c9, which is sensitive to neutralization by 10--1074 but resistant to 3BNC117, or Q769.d22, which is sensitive to 3BNC117, but resistant to 10--1074^[@CR20]^. Inhibitory dilution 50% (ID~50~) titers of pseudovirus neutralization by plasma samples were determined via five-parameter curve fitting. Plasma antibody concentration was calculated by multiplying a sample's ID~50~ titer by the inhibitory concentration 50% value from 10--1074 or 3BNC117 reference lots, which were assayed in parallel. Plasma samples were measured against murine leukemia virus to detect any nonspecific activity. All samples were assayed in duplicate. Induction of endogenous (rhesus) antibody responses against SHIV Env following repeated SHIV challenges, but prior to systemic infection, could confound use of the TZM-bl assay to determine plasma bNAb concentrations. SHIV~SF162P3~ Env-specific antibody responses were not detected prior to SHIV breakthrough among macaques that received 10--1074 and were challenged repeatedly with SHIV~SF162P3~ via the penis (Supplementary Fig. [4](#MOESM1){ref-type="media"}).
Detection of antidrug antibody (ADA) responses {#Sec13}
----------------------------------------------
ADA responses were evaluated in macaque plasma samples via ELISA. ELISA plates (96-well, high binding plates (Corning)) were coated overnight with 1 μg 10--1074 per well, then blocked with phosphate buffered saline containing 1% bovine serum albumin (PBS-1%BSA). Plasma samples were diluted 1:50 in PBS-1%BSA, added to the plates (100 μl per well) and incubated for 1.5--2.0 h at 37 °C. Plates were washed and incubated with mouse antirhesus IgG heavy chain-horseradish peroxidase (HRP) preadsorbed (Abcam) for 30 min at 37 °C. Plates were washed and assayed for HRP activity by the addition of tetramethylbenzidine (1-Step Ultra TMB, ThermoFisher). Color development was arrested via the addition of sulfuric acid to a final concentration of 1 N and absorbance at 450 nm was measured using a BioTek ELx808 plate reader.
Statistical analysis {#Sec14}
--------------------
Statistical comparisons between groups using *t*-, Mann--Whitney, or log-rank tests were conducted using Prism 7.0 (GraphPad Software, Inc., San Diego, CA). Probit modeling and bootstrap analyses were conducted in R version 3.4.4.
Reporting summary {#Sec15}
-----------------
Further information on research design is available in the [Nature Research Reporting Summary](#MOESM2){ref-type="media"} linked to this article.
Supplementary information
=========================
{#Sec16}
Supplementary Information Reporting Summary
Source data {#Sec17}
===========
Source Data
**Peer review information** *Nature Communications* thanks Ian McGowan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
**Supplementary information** is available for this paper at 10.1038/s41467-020-16928-9.
Funding for this study was from CDC intramural funds. The findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or National Institutes of Health.
D.A.G., J.McN, M.C.N, and W.H. developed the concept and designed the experiments; D.R.A., P.G., J.M., and K.K. performed experiments or animal procedures; A.G. generated bNAbs used in this study; M.S.S. and T.S. determined bNAb concentrations; M.N. performed probit modeling and bootsrap analyses; D.A.G wrote the paper; All authors edited or reviewed the paper.
All relevant data are available from the authors. The source data underlying Figs. [1](#MOESM3){ref-type="media"}e, [3f](#MOESM3){ref-type="media"}, and [3g](#MOESM3){ref-type="media"} are included in Supplementary Tables [1](#MOESM1){ref-type="media"}, [2](#MOESM1){ref-type="media"}, and [3](#MOESM1){ref-type="media"}, respectively. The source data underlying Figs. [1b, c, f](#Fig1){ref-type="fig"}, [3b, c, d, h](#Fig3){ref-type="fig"} are provided as a Source Data File.
There are patents on 3BNC117 (US Provisional Application No. 61/715,642) and 10--1074 (US Provisional Application No. 61/486,960) on which MCN is an inventor. 10--1074 and 3BNC117 are included in licensing agreements between The Rockefeller University and Gilead Sciences. The authors declare no further competing interests.
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"pile_set_name": "PubMed Central"
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INTRODUCTION
============
Due to progress in cerebrovascular imaging methods, such as magnetic resonance (MR) angiography and three-dimensional CT angiography, there is increased interest in cerebrovascular disease in ageing society. Many cerebrovascular diseases are worked up through health screening, which prevent cerebrovascular events. However, cerebral catheter angiography remains the gold standard method for examining the vasculature of patients with cerebrovascular disease. In most institutions, the right femoral artery is usually selected as the main vascular access for performing cerebral angiography, because of the clinicians\' familiarity and its convenience. Transfemoral angiography has some limitations. Many patients suffer from hematomas or bruising in the groin. Additionally, pseudoaneurysms or arteriovenous fistulae of the puncture site are rare but do occur. It cannot be used when patients have extensive atherosclerotic disease or atypical anatomy in their aortic arch or brachiocephalic vessel, femoral artery occlusion or abnormality. To avoid these problems and limitations, we performed transradial access for the preceding year.
The purpose of this study is to describe the efficacy of transradial angiography by a single operator\'s experience for one year, especially in elderly patients.
MATERIALS AND METHODS
=====================
Patient selection
-----------------
Between March 2011 and February 2012, 244 patients underwent selective cerebral angiography with digital subtraction angiography in our institution. For 126 patients (51.64%), the procedure was performed via a transradial approach for the evaluation of cerebrovascular disease. These patients ranged in age from 17 to 78 years (mean age, 56.2 years) and included 73 women and 53 men. There were 47 patients over the age of 60; 25 women and 22 men. Additionally, 91 of the total 244 patients receiving a cerebral angiography were over the sixty, 47 underwent transradial access and 44 underwent transfemoral access. Patients requiring interventional procedures were excluded. After a one-year neurointerventional learning period with 322 cerebral angiographic cases in another institution, we shifted the initial access from the right femoral artery to the right radial artery in all patients requiring cerebral angiography. The indications for cerebral angiography are listed in [Table 1](#T1){ref-type="table"} for patients over the age of 60. We did not attempt radial access in 40 of these patients for various reasons ([Table 2](#T2){ref-type="table"}). Our institutional review board approved these informed consents.
Instruments and techniques
--------------------------
Patients were brought to the angiographic suite. All procedures were performed for diagnostic purposes. We performed all the transradial approaches via the right side. If the right side was not available, we did not proceed with the transradial approach because performing this procedure via the left radial artery was very inconvenient. In these cases, we performed a right transfemoral approach. All patients underwent a modified Allen\'s test of the right wrist to evaluate satisfactory collateral perfusion prior to the procedure. If the test was abnormal, we did not proceed with radial artery access. After ulnar artery patency was proven via modified Allen\'s test, patients were prepared and draped with the right radial artery site exposure at the wrist and the right arm abducted at a 70-degree angle. The right inguinal area was also draped as well, for conversion of the access route from the wrist to the groin if necessary. A small amount of 2% lidocaine was infiltrated into the subcutaneous tissue on the radial artery around the styloid process of the radius, and the artery was punctured at the point of maximum pulsation using a 20-gauge needle with a modified Seldinger technique. A 5-French introducer sheath (Terumo Radifocus, Tokyo, Japan) was then placed within the artery. To prevent vasospasm of the artery, a diluted solution of nitroglycerine (200 µg/mL; 0.25 mL), verapamil (2.0 mg), heparin (5000 IU/mL) was injected intra-arterially through the side port of the introducer, which was then continuously flushed with pressurized (300 mm Hg), heparinized saline (5000 IU per 1000 mL of normal saline) during the procedure. A 5-French Simmons type 2 catheter (Terumo Radifocus, Tokyo, Japan) was inserted and forearm angiography performed through the catheter to confirm there was satisfactory collateral perfusion. Under fluoroscopy monitoring, a 5-French Simmons 2 catheter was advanced over a 0.035-inch hydrophilic guidewire (Terumo Radifocus, Tokyo, Japan) through the radial artery to the brachial artery, then through the axillary artery into the subclavian artery in all cases. Guidewire was subsequently advanced to the ascending aorta and turned back at the aortic valve for the reconstitution of the natural Simmons configuration of the catheter. After the catheter was reconstituted, the catheter tip was controlled to select the orifice of the target artery \[the innominate artery, left common carotid artery (CCA) or the left subclavian artery\], and then advanced to the distal selection \[the internal carotid artery (ICA) or vertebral artery\]. After the procedure, the catheter and sheath were removed, and a superficial pressure dressing with a radial artery hemostatic device (Hangshou Alicon Pharm Co., LTD, Zhejian, China) was applied to the radial artery puncture site. Patients were observed for three hours before discharge. During this observation period, their activity was not limited to bed rest.
RESULTS
=======
In patients over the age of 60, radial access was successful in 47 of 51 cases, a success rate of 92.2%. Transfemoral approach was performed in the four patients with failed radial artery access. This was attributed to failure of the radial puncture, loop formation or tortuosity at the proximal end of the radial artery in two patients, and severe vasospasm of the radial artery following multiple puncture trials in two patients ([Fig. 1](#F1){ref-type="fig"}). All supra-aortic vessels were successfully catheterized. However, selective catheterization rates of the left-sided distal vessels were lower, with success rates of 89.7% (26/29) for the right ICA and 75% (27/36) for the left ICA. Patients were excluded when it was deemed impossible or dangerous to catheterize the ICA because of severe stenosis or atheromatous plaques. We did not attempt to select the vertebral artery routinely, if there was not found the legions at previous CT angiography or MR angiography. Two procedures using the same radial artery were performed in two patients (4.3%). Among these patients, radial artery occlusion or stenosis was not demonstrated on follow-up cerebral angiography. In all patients in whom the transradial angiography was successful, the diagnostic adequacy and quality of images were satisfactory.
Pain in the forearm or arm developed in three patients during the procedures, but it was usually mild and transient. Vasospasm of the radial artery was noted in two cases. Spasms were effectively resolved with the combined solution of nitroglycerin, verapamil, and heparin. Procedure-related vascular complications, such as puncture site hematoma, hand ischemia, pseudoaneurysm, arteriovenous fistula and arterial dissection, were not seen in our series. No patients had functional disability of the hand, and no complaints of discomfort were reported on the questionnaire. However, intraprocedural thrombosis developed in one patient and aggravation of a pre-existing neurologic deficit was noted ([Fig. 2](#F2){ref-type="fig"}). Chemical thrombolysis was performed through the transradial route and the thrombus was removed completely. The patient\'s symptom was improved after one day without further sequelae.
DISCUSSION
==========
The transradial approach was introduced for the first time in 1998 in coronary angiography[@B2]. It was contributed to reduce puncture site complication and the discomfort of the patients. Transradial approach has become popular among a majority of interventional cardiologists as a diagnostic and therapeutic tool in a number of centers worldwide because of its low puncture site complication rate, as compared with the transfemoral approach, for patients undergoing anticoagulation therapy[@B8]-[@B11]. The transradial approach in cerebral angiography and neurointervention was introduced in 2000[@B14]. Some authors expected that the transradial approach could be a gold standard for cerebral angiography[@B17], although the transfemoral approach is usually favored in almost centers because of familiarity and convenience.
The femoral artery is the most common puncture site used for cerebral angiography, because most angiographers and neurointerventionalists are comfortable using this access for the entire cerebral vascular system. However, there are some limitations. Transfemoral approach cannot be used when patients have extensive atherosclerotic disease in their aortic arch, atypical anatomy of their aortic or brachiocephalic vessels, dissection of the thoracic artery, iliofemoral occlusive disease, or groin infection[@B1],[@B3],[@B7]. Additionally, this procedure can lead to several complications, such as retroperitoneal hematoma, pseudoaneurysm formation, arteriovenous fistula formation, femoral nerve injury, lower limb ischemia, and pulmonary embolism[@B7],[@B12],[@B13],[@B15],[@B18]. One of the major limitations of the transfemoral approach is the requirement for 4 to 6 hours of leg immobilization and bed rest to prevent bleeding complications at the femoral access site[@B13]. At times, duration of the leg immobilization was greatly reduced by application of percutaneous closure devices. However, these devices were expensive.
Transradial approach has the most important advantage of easy hemostasis. Compared with a groin hematoma formation rate with transradial approach reported as high as 10%[@B16]. Transfemoral approach may cause more suffering for the patients, particularly for those with advanced age, degenerative spine or spinal lesions, or benign prostate hypertrophy, because these conditions require complete bed rest for at least several hours following femoral artery puncture[@B19]. Furthermore, after the transfemoral approach, patients are usually kept in the hospital overnight for observation. In contrast, transradial approach does not require bed rest and allows the patient to be ambulatory immediately without the restriction of arm movement after procedure. In our institution, after transfemoral approach, we perform manual compression for 15 minutes without the use of additional percutaneous groin closure devices in the angiographic suite. However, transradial approach does not take the time for the compression. It is not necessary to compress the puncture site manually, as a simple compression device is sufficient to achieve hemostasis without special precautions. We generally observe patients for six hours with leg immobilization after transfemoral angiography. It is quite uncomfortable for patients and may lead to anxiety toward future angiographic trials. However, transradial angiography does not require prolonged observation. It takes 3 hours for observing patients and allows cerebral angiography to be easily performed on out-patient basis, useful for the image follow-up of patients after neurointerventional procedures.
The transradial approach is especially useful for patients with obesity or severe atherosclerotic change of bilateral femoral arteries, as the radial artery is an easier and safer access route[@B15]. In these cases, femoral artery puncture may often fail due to its deep location and weak pulsation. However, radial artery is superficially located on the underlying firm tendon, relatively fixed, easily compressible, and, most important, not an end artery. Since no major nerves or veins run in the vicinity of the radial artery over the radial styloid, neuropathies or arteriovenous fistulas are less likely to occur[@B15].
Transradial cerebral angiography has some advantages after the administration of anticoagulants or with effective antiplatelet therapy[@B13],[@B15],[@B17]. In the elderly, as the rate of atherosclerotic cerebrovascular and coronary diseases increase, anticoagulation or antiplatelet therapies are more common. These conditions are a contraindication for femoral approach, secondary to the potential for neurologic compromise caused by a large hematoma[@B17]. It is usually necessary to discontinue the anticoagulant or antithrombotic agents before the procedure to reduce the risk of serious hemorrhagic complications. Additionally, it may be difficult and time consuming to restart and readjust the level of anticoagulation after angiography, if this has been stopped for the angiogram. However, transradial approach does not require these agents to be discontinued. 17 (36.2%) of the 47 patients in our series received anticoagulant and/or antiplatelet therapy perioperatively in our series, no major hemorrhagic complications experienced, such as significant puncture site hematoma, pseudoaneurysm, or arteriovenous fistula formation. Therefore, it is reasonable to expect that anticoagulant and antiplatelet therapies need not be discontinued for cerebral angiography via transradial approach. In addition, it may actually reduce the intraprocedural thromboembolic complications if these agents are continued or added. We also recommend the use of pressurized, heparinized saline for the continuous flushing of the introduce sheath in order to prevent thrombosis.
With advancing patient age, vascular tortuosity and atheromatous change are increasing. Transradial approach is very useful in patients with significant tortuosity or ectatic changes of the aortic arch, and in patients with acute right vertebral artery course from the subclavian artery[@B12]. In some patients with very tortuous supra-aortic branches or bovine type left CCA, it is necessary to exchange Simmons or other specialized catheters for transfemoral access. However, transradial approach allow easy catheterization without exchanging the catheter. Additionally, it is more suitable in the right vertebral and right carotid systems. At times, the right vertebral artery is extremely difficult to catheterize from a transfemoral approach. In this condition, the right vertebral artery is easily catheterized from the transradial approach. Stability of guiding catheters in the right vertebral and carotid systems is outstanding from a transradial approach[@B17]. Catheter stability is excellent because the catheter system is confined to vessels with relatively small diameters, whereas guiding-catheter instability from a transfemoral approach often occurs because of transverse ectatic and large-diameter aortic arches[@B17].
Transradial cerebral angiography has some limitations. The success rate of selective catheterization is relatively low, especially for the left vertebral and internal carotid artery[@B12]. However, it is not a severe problem in vascular evaluation, because in most cases the target vessels were successfully visualized with the left common carotid artery injection and the left subclavian artery and/or the right vertebral artery injection. In our institution, to avoid vessel wall damage in the vertebral artery, we placed the catheter tip just to the orifice or the proximal portion of the vertebral artery. Otherwise, we prefer the transfemoral approach if the left vertebral artery is absolutely necessary to evaluate.
As a transradial approach is technically somewhat difficult and more challenging, it has a steep and significant learning curve to increase the success rate. It is difficult to negotiate the wire into the aortic arch, because the vessels originating from the arch become tortuous with advancing age. Additionally, potential trauma to the radial artery is more likely to occur in elderly patients, in whom the radial artery may be quite tortuous[@B13]. The success rate of transradial cerebral angiography is reported to be 92.7-99%[@B6],[@B10],[@B13],[@B15],[@B19]. Hildick-Smith et al.[@B5] have reported that the success rate of a transradial approach ranged from 88% for beginners to 94% for experienced specialists. In our series, the success rate was 92.2% in elderly patients (\>60 years old). We failed in 4 patients; two had loop formations of the radial artery, and two experienced severe vasospasm due to multiple puncture failures ([Fig. 1](#F1){ref-type="fig"}). Some authors worry about procedure-related thromboembolism with a reversed-angle catheter such as Simmons[@B12]. They suggest that the whirling motion of the bulky catheter within a tortuous atheromatous aorta is dangerous. We had one patient who experienced intraprocedural thrombosis. However, this complication rate is comparable with that of transfemoral access[@B4],[@B12]. It is important that there is gentle catheter manipulation in the aortic arch to reduce the chance of disturbing atheromatous plaques.
CONCLUSION
==========
Transradial cerebral angiography is a useful routine or alternative method for patients in whom transfemoral cerebral angiography is less favorable. With advancing patient age, transradial angiography is helpful to overcome vessel tortuosity and patient discomfort.
I thank our members of the cerebral angiographic suite, Young-Chool Min and Young-Tak Jung for the procedural assistant, and Yoon-Kyung Ha for data record and collection. This study was supported by a grant from Kosin University College of Medicine in 2012.
{#F1}
{#F2}
######
Indications for transradial cerebral angiography in patients with age \>60 years
######
The reasons for initial transfemoral access in patients with age \>60 years
There were four cases excluded due to failure of transradial access; two loop formations of radial artery and two multiple puncture failures
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Protein is an essential nutrient for the survival of humans and animals \[[@CR1], [@CR2]\]. Protein in mature cereal grains, in particular, provides a substantial portion of the world's plant protein, and its concentration determines the nutritional quality and end use properties of the grain \[[@CR3], [@CR4]\]. Barley (*Hordeum vulgare* L.) is one of the earliest domesticated crops in the world. Approximately 25% of its production has relatively lower GPC and is suitable for malting and brewing, while the remaining 75% with relatively higher GPC is used for feed and food (<http://faostat.fao.org>/). Hence, there is increasing need for breeding barley cultivars with high GPC, but this has been hindered by the relatively low heritability of GPC due to the significant interaction between environmental and genetic factors \[[@CR5], [@CR6]\]. Based on a statistical methodology, the genetic factors (quantitative trait loci, QTLs) that involved in determination of GPC can be elucidated \[[@CR7]\]. Thus, identification and utilization of environmentally stable QTLs associated with GPC will provide an alternative but promising strategy for high GPC barley breeding.
To date, numerous studies have been conducted on dissecting the genetic basis of GPC, and QTLs have been mapped on all seven barley chromosomes. In particular, several consensus QTLs mapped on chromosomes 2H, 4H, 5H, 6H, and 7H have been repeatedly detected by multiple studies \[[@CR8]--[@CR15]\]. For example, two QTLs on chromosomes 5HS and 6HS located in the *Bmac0096*-*Bmag0323* and *ABG458*-*HVM74* intervals, respectively, have been repeatedly detected \[[@CR10]--[@CR12], [@CR16]\]. Moreover, these two loci have also been identified by genome-wide association studies (GWAS) \[[@CR17]--[@CR19]\]. In addition, two genes (*HvNAM1* and *HvNAM2*) on chromosomes 6H and 2H in barley, which were suggested to be orthologous to *TtNAM-B1*, contributed a substantial effect on GPC \[[@CR17], [@CR20]\]. Notably, a recent study revealed that a single nucleotide polymorphism (SNP) within the second intron of *HvNAM2* was associated with GPC, which is useful in developing high quality barley cultivars \[[@CR17]\]. Although these identified QTLs/genes for GPC that could be expressed under multiple environments might be valuable for GPC improvement in barley, most of the genetics studies focused on breeding and selection for low-protein barley \[[@CR21], [@CR22]\].
A saturated genetic linkage map will improve the precision of QTL localization and estimation of phenotypic variance, especially for some small and medium-sized QTLs \[[@CR23]\]. Due to the abundance of SNPs in plant genome, SNP markers have been widely used in genetic linkage map construction \[[@CR24]--[@CR26]\]. High-density SNP linkage maps have been largely used in QTL detection for yield and quality in barley \[[@CR27]--[@CR29]\]. However, QTL mapping for GPC based on a high-density SNP map has rarely been reported. Here, to identify QTLs for GPC, a RIL population including 190 lines derived from a cross between the Chinese landrace ZGMLEL with high GPC (\> 20%) and the Australian cultivar Schooner was used for linkage and QTL analyses. Furthermore, near-isogenic line (NIL) populations were developed to validate the environmentally stable QTLs.
Methods {#Sec2}
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Plant materials {#Sec3}
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A RIL population (generations F~9~ to F~11~) containing 190 RILs derived from two spring barley varieties, ZGMLEL and Schooner, was employed to identify QTLs controlling for GPC. ZGMLEL is a hull-less landrace with high GPC, while Schooner is a hulled cultivar with low GPC. All the RILs and their parental lines were kindly provided by Dr. Yawen Zeng (Yunnan Academy of Agricultural Sciences, China).
For NIL development, one RIL line (RIL7) was crossed with the recurrent parent (Schooner). Because the QTLs on chromosomes 6H (i.e. *QGpc.ZiSc-6H.2*, *QGpc.ZiSc-6H.3* and *QGpc.ZiSc-6H.4*) and 7H (i.e. *QGpc.ZiSc-7H.1*, *QGpc.ZiSc-7H.2* and *QGpc.ZiSc-7H.3*) were linked in coupling phase, the QTL clusters were introgressed into the Schooner background as a unity, respectively. After three backcross generations (BC~3~), individuals that solely exhibited heterozygosity at one QTL region were self-pollinated to produce its corresponding BC~3~F~2~ populations. Finally, three NIL populations, that is BC~3~F~2~-I (region 2H), BC~3~F~2~-II (region 6H) and BC~3~F~2~-III (region 7H), were developed for the validation their corresponding QTLs. The number of progenies in BC~3~F~2~-I, BC~3~F~2~-II and BC~3~F~2~-III populations were 249, 205 and 213, respectively.
Field experiments {#Sec4}
-----------------
Field experiments were carried out in three locations, including Shangzhuang Experiment Station of CAU (China Agricultural University) in Beijing, Wangtaibao Experiment Station of NAAFS (Ningxia Academy of Agriculture and Forestry Sciences) in Ningxia Hui Autonomous Region, and Dishang Experiment Station of HAAFS (Hebei Academy of Agriculture and Forestry Science) in Hebei Province. The RIL population and the two parents were grown during three growing seasons from 2013 to 2015, providing data for six environments. Location-year information and corresponding weather data are presented in Additional file [1](#MOESM1){ref-type="media"}: Table S1. In field trials, each plot consisted of 2 rows that were 2 m long with approximately 20 plants per row. The middle ten plants in each line were bulk-harvested at maturity and measured for grain protein concentration (GPC).
Three BC~3~F~2~ populations for QTL validation were planted in Beijing (2016). Individuals were grown in 2-m-long rows with a 0.25-m row spacing. Within each row, 15 plants were evenly sown. At maturity, all the panicles were harvested from single-plant and sun-dried. Grain protein concentration (GPC), grain yield (GY) and thousand grain weight (TKW) were scored on a single-plant basis.
The field experiments were in accordance with local practice. All the trails were conducted under optimum irrigation. Nitrogen (N) was supplied at a rate of 220 kg/ha, including 70 kg/ha of N as diammonium phosphate and 80 kg/ha of N as urea applied before sowing. In addition, 70 kg/ha of N as urea was applied at booting stage.
Phenotypic evaluation and statistical analysis {#Sec5}
----------------------------------------------
Mature grains of RIL population and BC~3~F~2~ populations were ground to a powder using a Cyclotec 1093 sample mill (Hoganas City, Sweden). Then, the ground powder was dried to a constant mass in an 80 °C oven. The total nitrogen content was determined using the Kjeldahl method with a FOSS Kjeltec ™ 2300 and then the GPC was calculated using a factor of 5.83 \[[@CR30]\]. GY and TKW of the NIL populations were measured on a single-plant basis. TKW was determined using a camera-assisted phenotyping system, which was provided by Hangzhou Wanshen Detection Technology Co., Ltd. (Hangzhou, China).
The basic statistical analysis was performed using SPSS version 20.0 (SPSS, Chicago, IL, USA). The Shapiro-Wilk test was conducted using R software (V. 3.2.2) for the normality test. The best linear unbiased prediction (BLUP) for GPC across the six environments was calculated using SAS^®^ V.8 (SAS Institute Inc. 2000) with the PROC MIXED procedure. Under the random-effect model, environments were treated as fixed, and genotype and genotype-environments interactions were considered as random factors. The broad sense heritability (*h* ~*B*~ ^2^) on a family basis was calculated using SAS^®^ V.8 (SAS Institute Inc. 2000) with the PROC GLM procedure, which was calculated according to the following formula: *h* ~*B*~ ^2^ = *ó* ~*g*~ ^2^ */ (ó* ~*g*~ ^2^ *+ ó* ~*ge*~ ^2^ */n + ó* ^2^ */nr)* where *ó* ~*g*~ ^2^ = genotypic variance, *ó* ~*ge*~ ^2^ = genotype by environmental variance, *ó* ^2^ = the residual error variance, *n* = the number of environments, and *r* = number of replicates.
Linkage and QTL analyses {#Sec6}
------------------------
The RIL population was genotyped using the barley 9 K SNP chip developed from the RNA-seq data of barley varieties \[[@CR31]\]. Additionally, a total of 21 polymorphic SSR markers were employed to genotype the RIL population, and most of the SSR sequences were obtained from <http://wheat.pw.usda.gov/GG3/>. Only markers with less than 5% missing data were selected for map construction. The genetic linkage map was constructed using RECORD 2.0 \[[@CR32]\] and JoinMap 4.0 \[[@CR33]\]. Markers with identical segregation were first removed using RECORD 2.0. After removing the redundancy, the unique markers were grouped using JoinMap 4.0 with a LOD value of 10. Finally, the marker order was established using the maximum likelihood mapping algorithm and the map distance was calculated using the Kosambi mapping function. The probe sequences of the SNP assigned to barley chromosomes were queried using the BLAST algorithm against barley reference genome sequence to locate chromosomal positions with a cutoff criterion of E-value ≤1e-10. The quality of the genetic map was validated using the alignments between SNP map and barley reference genome. Only the best hit of the SNP against the reference genome was selected for the collinearity analysis when the SNP was located to multiple paralogous positions in the genome.
The average GPC data in each environment and BLUP values across six environments were collected for QTL analysis. WinQTLCart2.5 software with the composite interval mapping (CIM) method was used to identify QTLs for GPC. The walking speed was set to 1 cM. Model 6 was chosen for QTL analysis, with 5 control markers and 10 cM window size defaults. The LOD threshold was set via 1000 permutations at *P* ≤ 0.05, and these QTLs were considered "identified QTLs". A 2-LOD support with a 99% confidence level was chosen for each identified QTL. The identified QTLs detected in different environments with overlapping confidence intervals were regarded as the same in this study. The QTLs were named following the rules of Blake and Blake \[[@CR34]\].
Marker development {#Sec7}
------------------
The flanking markers of the QTLs were employed to define the target region, which could be used to compare to the barley Genome Zipper developed by Mayer et al. \[[@CR35]\]. Gene sequences of three grasses (rice, sorghum, and *Brachypodium*) were used as queries to blast against the database "assembly_WGSMorex" at IPK Barley BLAST Server (<http://webblast.ipk-gatersleben.de/barley_ibsc/>). The Morex contigs with best hit were employed to search for simple sequence repeat using the SSR Hunter software. Finally, the selected sequences were used to design SSR markers using the Primer3 software (<http://frodo.wi.mit.edu/primer3/>).
Results {#Sec8}
=======
Analysis of GPC {#Sec9}
---------------
The basic statistics of minimum, maximum, mean standard deviation and coefficient of variation for GPC in six environments are listed in Table [1](#Tab1){ref-type="table"}. The GPC of parent ZGMLEL ranged from 20.52% to 22.88% in the six environments evaluated, which was significantly higher (*P* \< 0.01) than that of Schooner (16.35--17.20%) (Table [1](#Tab1){ref-type="table"}; Additional file [2](#MOESM2){ref-type="media"}: Table S2). Moreover, the 190 RILs exhibited a wide range of variation in GPC, with coefficients of variation (CVs) ranging from 6.94% to 7.80% in the six environments. The Shapiro-Wilk for testing normality was performed for GPC based on the mean value collected from six environments (Fig. [1](#Fig1){ref-type="fig"}). In all of the six environments, GPC showed normal distribution, suggesting a quantitative nature of GPC in barley. Remarkably, the broad sense heritability (*h* ~*B*~ ^2^) for the GPC of the RILs was 80.67%, indicating that the GPC variance was mostly determined by genetic factors.Table 1Parental and population minimums, maximums, means, standard deviations and coefficient of variations for grain protein concentration (GPC)Environ. ^a^Parental linesRIL populationZGMLELSchoonerMin.Max.MeanSD ^b^CV(%) ^c^*h* ~*B*~ ^*2*^(%) ^d^E122.02 ± 0.5316.45 ± 0.3716.0823.3619.231.346.9580.67E221.54 ± 0.2016.80 ± 0.6216.0123.0519.251.367.07E322.41 ± 0.2316.56 ± 0.1914.0823.7519.871.557.80E420.52 ± 0.2116.35 ± 0.2515.7222.9319.251.387.18E521.70 ± 0.3316.70 ± 0.2614.7822.2818.831.316.94E622.88 ± 0.1017.20 ± 0.0916.3524.8320.801.507.22^a^E1, 2013-Beijing; E2, 2014-Beijing; E3, 2014-Hebei; E4, 2014-Ningxia; E5, 2015-Ningxia; E6, 2015-Hebei^b^SD is the standard deviation^c^CV is the coefficient of variation^d^ *h* ~*B*~ ^*2*^ is the broad sense heritability estimated across all six environments Fig. 1Histogram for grain protein concentration (GPC) in the ZGMLEL × Schooner population based on the mean value collected from six environments. The *Y*-axis represents the density of each group. The unit of the *X*-axis is GPC data
Construction of a high-density genetic linkage map {#Sec10}
--------------------------------------------------
Of the 7864 SNP markers on the chip, 1526 (19.40%) were polymorphic between ZGMLEL and Schooner. After removing 53 SNPs with over 5% missing data, we used 1473 SNP markers and 21 polymorphic SSR markers to construct the linkage map. The resultant linkage map comprised nine linkage groups that contained 1011 unique loci and spanned 2353.48 cM. These linkage maps had an average locus interval of 2.33 cM (Table [2](#Tab2){ref-type="table"}, Additional file [3](#MOESM3){ref-type="media"}: Table S3, Additional file [4](#MOESM4){ref-type="media"}: Figure S1). The identity and polarity of linkage groups were determined by BLAST against the barley reference sequence databases \[[@CR36]\].Table 2Summary of the genetic linkage map constructed with the ZGMLEL × Schooner populationChr.No. of linkage groupsNo. of markersNo. of lociLength (cM)Average locus interval (cM)No. of loci assigned to barley genomeCovered physical length (Mb)Total length of barley genome (Mb)Coverage ratio (%)1H2154/4476/31157.98/40.062.08/1.29153/44463.81 (0.25--464.06)464.1299.932H1302203468.202.31291411.63 (1.82--161.43, 372.38--624.40)628.3465.513H1203112204.371.82196472.31 (20.53--558.95)564.4395.404H1169121260.492.15156540.69 (0.03--540.72)544.1799.365H2125/6597/51314.67/146.143.24/2.87116/57354.43 (2.25--84.96, 285.64--557.36)561.4163.136H1252184322.401.75237342.26 (3.59--175.79, 363.74--533.80)538.7663.527H1180136421.173.10161599.46 (1.10--600.56)601.6099.64Total9149410112353.482.3314113184.593902.8381.60
To further validate the quality of the map, SNP flanking sequences were employed to align with the barley reference sequence. Of 1473 SNP markers, 1411 (95.79%) were successfully assigned to the barley genome (Table [2](#Tab2){ref-type="table"}; Additional file [5](#MOESM5){ref-type="media"}: Table S4). A good collinearity of the genetic map with the barley reference genome sequence was observed ([Fig. 2](#Fig2){ref-type="fig"}), indicating a high quality of the genetic linkage map. However, several chromosome intervals were inconsistent with the reference genome sequence, i.e., chromosomes 2H at 76.43--204.46 cM, 4H at 125.91--127.33 cM, and 5H-1 at 100.55--146.27 cM.Fig. 2Alignments between the genetic linkage map in the ZGMLEL × Schooner population and barley physical map. The *X*-axis represents the genetic distance in centiMorgan (cM). The *Y*-axis represents the physical distance in million base pairs (Mb)
Of the 5.1 Gb size of barley genome, 3.1 Gb has been successfully anchored to the physical map through population sequencing (POPSEQ) \[[@CR36]\]. We calculated the coverage ratio for each barley chromosome. Chromosomes 1H, 3H, 4H and 7H displayed similar coverage ratios at 99.93, 95.40, 99.36, and 99.64%, respectively, and chromosomes 2H (65.51%), 5H (63.13%) and 6H (63.52%) exhibited lower ratios (Table [2](#Tab2){ref-type="table"}).
QTL mapping of GPC {#Sec11}
------------------
A total of 17 QTLs were detected for GPC, which are randomly distributed among chromosomes 2H (3 QTLs), 4H (3 QTLs), 5H (3 QTLs), 6H (4 QTLs), 7H (4 QTLs) ([Fig. 3](#Fig3){ref-type="fig"}; Table [3](#Tab3){ref-type="table"}). With an exception of *QGpc.ZgSc-6H.1*, ZGMLEL contributed effects for increased GPC at other 16 QTLs. These 16 significant QTL had LOD values ranging from 2.51 to 15.51 and explained the GPC variation from 2.4% to 19.86%. Schooner contributed effects for increased GPC at the locus of *QGpc.ZgSc-6H.1*, which had a LOD value of 3.37 and accounted for 5.70% of GPC variation. This indicated that the favorable alleles for increased GPC were mainly inherited from the feed barley ZGMLEL. In the present study, significant QTLs that could be detected in no less than three environments as well as in the combined analysis were defined as environmentally stable QTLs. According to this criterion, 6 of 17 significant QTLs were environmentally stable QTLs, which were identified on chromosomes 2HL (1), 4HS (1), 6HL (1), and 7HS (3).Fig. 3QTL locations for grain protein concentration (GPC) in the ZGMLEL × Schooner population. A centiMorgan (cM) scale is shown on the left. Vertical bar represents a 2-LOD interval for each QTL. Black ellipses represent the approximate locations of the centromeres. Black triangles indicate the environmentally stable with increasing allele from ZGMLEL. Red and pink triangles represent the putative QTLs that were detected only in less than three environments with increasing allele from ZGMLEL and Schooner, respectively. Blue and red shadows on the physical map represent the approximate positions of the QTL identified in previous studies and the present study, respectively. References from previous studies are presented under the physical map. The known positions of the *vrs1* and *int-c* loci are shown with black arrows (Ramsay et al. \[[@CR72]\]) Table 3QTLs detected for grain protein concentration (GPC) in the ZGMLEL × Schooner populationQTLPos. (cM)Nearest markerLOD*R* ^*2*^(%) ^a^Additive ^b^LOD2_intervalEnviron.*QGpc.ZiSc-2H.1*253.8*SCRI_RS_171032*3.875.700.32243.5--261.0E1263.1*SCRI_RS_126439*3.283.700.26252.2--270.4E5263.1*SCRI_RS_126439*2.512.400.24256.0--264.0E6264.1*SCRI_RS_126439*3.784.470.23259.9--271.7C ^c^*QGpc.ZiSc-2H.2*294.3*BOPA1_7236--1384*3.775.690.34291.2--298.7E4*QGpc.ZiSc-2H.3*309.3*SCRI_RS_170162*5.148.580.41305.3--314.4E4*QGpc.ZiSc-4H.1*13.4*BOPA1_7385--763*3.655.800.382.5--23.9E314.4*BOPA1_7385--763*2.573.140.255.0--23.9E412.4*BOPA1_7385--763*3.335.260.352.6--14.9E612.4*BOPA1_7385--763*3.554.200.221.0--15.9C*QGpc.ZiSc-4H.2*216.5*BOPA2_12_31139*4.585.680.33212.3--217.6E5*QGpc.ZiSc-4H.3*223.6*SCRI_RS_160461*5.596.100.34219.1--228.6E5*QGpc.ZiSc-5H.1*200.4*BOPA1_9745--628*4.717.080.41188.6--206.7E4*QGpc.ZiSc-5H.2*212.4*BOPA1_3928--513*4.136.560.39206.7--217.6E4*QGpc.ZiSc-5H.3*280.6*BOPA1_10318--572*4.124.860.30272.9--285.9E5282.6*BOPA1_10318--572*2.784.520.33278.9--285.2E6283.9*SCRI_RS_218201*3.313.370.20278.9--285.2C*QGpc.ZiSc-6H.1*146.4*BOPA1_3048--1349*3.375.70−0.37129.7--159.6E2*QGpc.ZiSc-6H.2*275.7*Bmac0040*8.9616.480.58275.6--278.2E2274.7*Bmac0040*10.0916.440.58269.7--278.2E4275.7*Bmac0040*13.8418.780.48271.2--278.2C*QGpc.ZiSc-6H.3*278.2*Bmag0612*9.1613.420.51278.0--283.3E1280.2*Bmag0612*8.9315.520.56278.2--285.6E2278.2*Bmag0612*4.677.010.43278.2--283.9E3282.2*Bmag0612*10.8516.450.59278.2--286.6E4282.2*Bmag0612*15.5119.860.61277.6--286.6E5278.2*Bmag0612*6.178.560.46278.2--284.3E6280.2*Bmag0612*13.8217.900.46278.2--283.8C*QGpc.ZiSc-6H.4*290.7*BOPA1_1852--509*5.779.990.44287.7--292.7E1286.3*SCRI_RS_124549*5.648.450.46287.3--292.3E6*QGpc.ZiSc-7H.1*98.1*GBM1464*7.259.970.4393.8--99.5E198.2*GBM1464*6.6611.220.4698.1--101.7E296.4*SCRI_RS_152122*3.194.440.3095.0--100.2E498.2*GBM1464*5.836.380.3498.1--100.2E596.4*SCRI_RS_152122*4.817.150.4494.6--101.7E698.2*GBM1464*10.5712.370.3898.1--99.2C*QGpc.ZiSc-7H.2*108.9*SCRI_RS_138111*9.4715.430.56107.1--111.9E2106.8*SCRI_RS_178619*3.724.840.33104.2--111.8E4107.8*SCRI_RS_178619*6.587.510.38104.7--111.5E5109.9*SCRI_RS_138111*4.376.880.47106.8--112.5E6107.8*SCRI_RS_178619*10.7113.290.41107.0--112.2C*QGpc.ZiSc-7H.3*116.0*SCRI_RS_208698*8.5515.070.55112.5--117.6E2119.6*BOPA1_2669--1012*6.297.330.37113.7--120.9E5116.0*SCRI_RS_208698*3.355.670.42113.0--117.0E6116.0*SCRI_RS_208698*9.4712.530.39113.0--117.6C*QGpc.ZiSc-7H.4*151.4*EBmac0401*5.117.480.47143.8--155.6E6^a^ *R* ^*2*^ is the phenotypic variation explained by the identified QTL^b^Positive value represents the increasing allele from ZGMLEL, while a negative value is from Schooner^c^C is the combined QTL analysis based on the BLUP across six environments
Three QTLs associated with GPC were detected on chromosome 2HL. Only the one flanked by *SCRI_RS_171032* and *BOPA2_12_30901*, *QGpc.ZgSc-2H.1*, was considered being environmentally stable, which could be detected under three environments. *QGpc.ZgSc-2H.1* had a LOD value of 3.78 and explained 4.47% of the GPC variation for the combined analysis. The other two putative QTLs, *QGpc.ZgSc-2H.2* and *QGpc.ZgSc-2H.3*, were observed in one environment and explained 5.69--8.58% of GPC variation.
Among three significant QTLs for GPC detected on chromosome 4H, one stable QTL was identified on chromosome 4HS and designated *QGpc.ZgSc-4H.1*. This QTL had a minor effect, with a LOD value of 3.55, and explained 4.20% of the GPC variation for the combined analysis. *QGpc.ZgSc-4H.2* and *QGpc.ZgSc-4H.3* could be detected only in one environment, which had LOD and *R* ^*2*^ values ranging from 4.58 to 5.59 and 5.68% to 6.10%, respectively.
Chromosome 5HL carried three putative QTLs significantly associated with GPC, which were designated *QGpc.ZgSc-5H.3*, *QGpc.ZgSc-5H.1*, and *QGpc.ZgSc-5H.2*. *QGpc.ZgSc-5H.3* had a LOD value of 3.31 and explained 3.37% of the GPC variation for the combined analysis. The other two QTLs had the LOD values ranging from 4.13 to 4.71, and explained the GPC variation from 6.56% to 7.08%.
Four significant QTLs were identified on chromosome 6HL and were designated *QGpc.ZgSc-6H.1*, *QGpc.ZgSc-6H.2*, *QGpc.ZgSc-6H.3*, and *QGpc.ZgSc-6H.4*. Schooner contributed the effects for increasing GPC at *QGpc.ZgSc-6H.1*, and ZGMLEL contributed increased GPC at the other three loci. *QGpc.ZgSc-6H.3* was the most stable QTL for GPC, which could be detected in all six environments and explained as much as 17.90% of the GPC variation for the combined analysis. Two putative QTLs, *QGpc.ZgSc-6H.2* and *QGpc.ZgSc-6H.4*, were identified in two environments and explained 8.45--18.78% of GPC variation.
Four QTLs were found to be significantly associated with GPC on chromosome 7HS. Among these significant QTLs, three were environmentally stable QTLs, and they were designated *QGpc.ZgSc-7H.1*, *QGpc.ZgSc-7H.2*, and *QGpc.ZgSc-7H.3*. These three stable QTLs were detected in three to five environments, explaining 12.37--13.29% of GPC variation for the combined analysis. The last putative QTL, *QGpc.ZgSc-7H.4*, was detected at E6 and accounted for 7.48% of GPC variation.
QTL validation {#Sec12}
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To study the three genomic regions on chromosomes 2H, 6H and 7H that possessing environmentally stable QTLs for GPC in more depth, three BC~3~F~2~ populations were developed and named BC~3~F~2~-I, BC~3~F~2~-II and BC~3~F~2~-III, respectively. Accordingly, three sets of SSR markers were used for foreground selection, i.e., *2L10*, *2L11* and *2L12* for BC~3~F~2~-I, *6L89*, *6L155* and *6L147* for BC~3~F~2~-II, and *7S40*, *7S69*, *7S87* and *7S89* for BC~3~F~2~-III (Additional file [6](#MOESM6){ref-type="media"}: Figure S2, Additional file [7](#MOESM7){ref-type="media"}: Table S5). A total of 76 SSRs were used for background selection of the BC~3~F~1~ individuals. Finally, three BC~3~F~1~ individuals that exhibit heterozygosity solely at genomic regions 2H, 6H or 7H were selfed to produce their corresponding BC~3~F~2~ populations. These three BC~3~F~1~ individuals shared 93.42, 92.10 and 94.74% similarities in genetic background with the recurrent parent, respectively.
To determine whether the stable QTLs affect the GPC in NIL populations, we compared the GPC between two homozygous groups. Based on the genotype of flanking markers (*2L10* and *2L11* on 2H, *6L89* and *6L147* on 6H, *7S87* and *7S40* on 7H), two homozygous groups, namely, ZGMLEL homozygous (ZZ) and Schooner homozygous (SS) were classified in each NIL population. The evaluation results for GPC showed that plants with ZZ genotype in BC~3~F~2~-I, BC~3~F~2~-II and BC~3~F~2~-III had an average GPC of 13.82, 14.18 and 14.20%, respectively. In contrast, plants with SS genotype in BC~3~F~2~-I, BC~3~F~2~-II and BC~3~F~2~-III had an average GPC of 13.15, 13.19 and 13.48%, respectively, which is similar to the recurrent parent, Schooner (13.32%) (Additional file [8](#MOESM8){ref-type="media"}: Table S6). Based on the GPC value, highly significant difference was found between two homozygous genotypes in each NIL population (*P* \< 0.01) (Table [4](#Tab4){ref-type="table"}, Additional file [8](#MOESM8){ref-type="media"}: Table S6). The allelic effects of the three populations were in the same direction as the original allele, with alleles from ZGMLEL increasing GPC. These results suggested that the stable QTLs on chromosomes 2H, 6H and 7H had significant effect on GPC, which was in agreement with the detection in RIL population.Table 4Variation between two homozygous genotypic groups of three NIL populations for grain protein concentration (GPC)PopulationGPC (mean ± SE ^c^) (%)*P*-valueTKW (mean ± SE) (g)*P*-valueGY (mean ± SE) (g)*P*-valueZZ ^a^SS ^b^ZZSSZZSSBC~3~F~2~-I13.82 ± 0.0913.15 ± 0.052.40E-1050.40 ± 0.1950.55 ± 0.180.727.06 ± 0.126.74 ± 0.110.06BC~3~F~2~-II14.20 ± 0.1113.47 ± 0.062.85E-0850.44 ± 0.3351.02 ± 0.250.226.79 ± 0.136.71 ± 0.100.58BC~3~F~2~-III14.18 ± 0.1713.19 ± 0.125.33E-0644.19 ± 0.4045.82 ± 0.670.086.73 ± 0.236.40 ± 0.220.28^a^ZZ represents ZGMLEL homozygote; ^b^ SS represents Schooner homozygote; ^c^ SE represents standard error
Previous studies reported that there was negative relationship between GPC and grain yield \[[@CR37]\]. Thus, we measured thousand kernel weight (TKW) and grain yield per plant (GY) for the three BC~3~F~2~ populations but found no significant difference for TKW and GY (Table [4](#Tab4){ref-type="table"}; Additional files [8](#MOESM8){ref-type="media"}: Table S6).
Discussion {#Sec13}
==========
The advantages and disadvantages of the present genetic linkage map {#Sec14}
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QTL mapping is a reliable way to resolve the genetic basis of GPC, and a high-density map will increase the accuracy of QTL detection \[[@CR23]\]. In the present study, a high-density map comprised of 1473 SNP and 21 SSR, and spanned 2354.48 cM in length. Notably, our genetic map has a good collinearity with the barley reference genome, which is suitable for the identification of QTLs \[[@CR38]\]. However, several chromosome intervals were inconsistent with the reference genome sequence. This could be partially explained by the following reasons: 1) a suppressed recombination frequency at the centromere region, 2) the presence of partially homologous sequences or duplication, and 3) the deficiency of polymorphic markers. In addition, four chromosomes (1H, 3H, 4H and 7H) had high genome coverage (95.40--99.93%), while three (2H, 5H and 6H) showed low genome coverage (63.13--65.51%), which might be caused by the lack of polymorphic markers within the centromeric region. Due to the low recombination frequency in the centromeric region, we speculated that it would not influence the identification of the QTLs.
Compared with two SNP maps reported by Close et al. \[[@CR24]\] and Muñoz-Amatriaín et al. \[[@CR25]\], the whole genome of our map expanded in genetic distance by 41.75 and 111.51%, respectively, with individual chromosome extended by 26.44% to 83.89% and 24.30% to 160.97%, respectively (Additional file [9](#MOESM9){ref-type="media"}: Table S7). Missing genotype data of each line and large number of heterozygotes could lead to expanded genetic distance \[[@CR39], [@CR40]\]. Consistent with this, similar phenomenon was observed in our SNP genotype data, which could partially contribute to the large whole genetic distance. The casual reason will be an interestingly area to further investigation.
Extensive variation for GPC in barley {#Sec15}
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Determining the phenotypic variation of GPC in a segregating population is a prerequisite for elucidating its genetic foundation and for breeding barley cultivars with desirable GPC. Extensive variation in GPC in different barley genotypes has been reported previously. For example, analysis of 59 cultivated and 99 Tibetan wild barley accessions showed that the GPC ranged from 8.02% to 13.50% and Tibetan wild barley had much higher GPC than cultivated barley \[[@CR17]\]. QTL analysis provides an efficient way to look for associations between the phenotypic variance and the markers segregating in a bi-parental population \[[@CR41], [@CR42]\] and has been widely used in dissecting GPC variation in barley populations. However, the lack of parental lines with high GPC in most previous studies may have hindered the detection of possible major QTLs for GPC \[[@CR10], [@CR43]\]. In the current study, the rare accession ZGMLEL, with consistently high GPC (20.52--22.88%), and an Australian cultivar, Schooner, with relatively low GPC (16.35--17.20%), were used to construct the mapping population. A relatively high broad sense heritability (80.67%) was found, suggesting that QTLs/genes controlling GPC are less environmentally influenced in the ZGMLEL × Schooner population. Thus, the ZGMLEL × Schooner population is a perfect material for identifying QTLs for GPC. Environmentally stable QTLs detected in this way might be suitable for marker-assisted selection (MAS) in barley breeding, which is anticipated to increase efficiency of the genetic improvement for GPC.
Novel QTLs controlling GPC on chromosomes 4H and 7H {#Sec16}
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To enhance the GPC of barley, novel genes or QTLs with increased effects are of interest for breeding purposes. In our study, two genomic regions harboring four stable QTLs (*QGpc.ZgSc-4H.1*, *QGpc.ZgSc-7H.1*, *QGpc.ZgSc-7H.2*, and *QGpc.ZgSc-7H.3*) appeared to be novel ([Fig. 3](#Fig3){ref-type="fig"}, Additional file [10](#MOESM10){ref-type="media"}: Table S8).
A significant QTL *QGpc.ZgSc-4H.1* for GPC was identified in the telomeric region of chromosome 4HS and was steadily expressed in three environments. QTLs affecting GPC have been identified on 4HS \[[@CR8], [@CR43], [@CR44]\] and 4HL \[[@CR15], [@CR45], [@CR46]\]. For example, Marquez-Cedillo et al. identified a QTL for GPC at the region of the *intermedium-c* (*int-c*) locus, which is obviously different from *QGpc.ZgSc-4H.1* ([Fig. 3](#Fig3){ref-type="fig"}) \[[@CR8]\]. Therefore, *QGpc.ZgSc-4H.1* likely represents a new locus for GPC, although its contribution to the variation of GPC was relatively small.
Remarkably, three neighboring QTLs (*QGpc.ZgSc-7H.1*, *QGpc.ZgSc-7H.2*, and *QGpc.ZgSc-7H.3*) were detected on chromosome 7HS. QTLs for GPC on chromosome 7H have been extensively reported \[[@CR8], [@CR12]--[@CR14]\]. For example, Emebiri et al. \[[@CR12]\] reported mapping of two QTLs for GPC in the telomeric and centromeric regions that are probably within the physical intervals of 15.8--40.0 and 261.8--277.6 Mb, respectively ([Fig. 3](#Fig3){ref-type="fig"}) \[[@CR12]\]. Marquez-Cedillo et al. \[[@CR8]\] and Abdel-Haleem et al. \[[@CR14]\] identified a consensus QTL near the *nud* locus on chromosome 7HL \[[@CR8], [@CR14]\]. However, the location of these QTLs was different from that detected in the present study. Therefore, these three QTLs in adjacent intervals are likely to be new QTLs, which might be due to the utilization of specific genetic materials in the present study.
Consensus QTL regions for GPC on chromosomes 2H and 6H {#Sec17}
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An efficient method to introgress favorable alleles into elite germplasm is to select consensus QTLs that steadily affect GPC in different genetic backgrounds and environments \[[@CR47]\]. For example, Emebiri reported that pyramiding two consensus QTLs on chromosomes 6HS and 5HS could significantly decrease GPC levels by 4% compared to the commercial check \[[@CR22]\]. In this study, two genomic regions on chromosomes 2HL and 6HL for GPC were coincident with QTLs reported in previous studies ([Fig. 3](#Fig3){ref-type="fig"}; Additional file [10](#MOESM10){ref-type="media"}: Table S8). For example, a stable QTL *QGpc.ZgSc-2H.1* on chromosome 2HL was coincident with the locus reported by Marquez-Cedillo et al. \[[@CR8]\]. Another major QTL, *QGpc.ZgSc-6H.3*, explaining the highest GPC variance was mapped at a similar locus to *Qpro6a* detected in the Morex/Steptoe DH population \[[@CR15]\]. However, the additive effect of our loci (0.43--0.59%), however, is higher than *Qpro6a* (0.14%), which might be caused by the special materials used in our study.
To date, two homologous genes, *HvNAM1* and *HvNAM2* associating with GPC on the short arm of chromosomes 6H and 2H, respectively, have been widely studied \[[@CR17], [@CR48], [@CR49]\]. For example, Cai et al. \[[@CR17]\] performed a multi-platform candidate gene-based association analysis using 59 cultivated and 99 Tibetan wild barley genotypes and found that the haplotypes of *HvNAM1* and *HvNAM2* markers were associated with GPC in barley. In the present study, two identified QTLs, *QGpc.ZgSc-6H.3* and *QGpc.ZgSc-2H.1* associated with GPC were also detected on chromosomes 6H and 2H, respectively, while they were both located on the long arms, demonstrating that *HvNAM1* and *HvNAM2* were obviously different from the QTLs detected in this study.
QTLs for GPC linked in coupling phase on chromosomes 6H and 7H {#Sec18}
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Neighboring QTLs associated with many important traits, such as yield and quality, that are linked in coupling phase are commonly observed in primary QTL analysis \[[@CR50], [@CR51]\]. Previous studies have tried to dissect QTLs in coupling phase using nearly isogenic lines (NILs) or residual heterozygous lines (RHLs), and found that coupling QTLs were partially attributed to tightly linked independent QTLs \[[@CR52]--[@CR55]\]. For example, Han et al. identified two QTLs each for malt extract and for α-amylase and two to three for diastatic power in a complex QTL region using advanced segregation populations \[[@CR53]\]. In this study, we detected two genomic regions on chromosomes 6HL and 7HS, each of which harbored linked QTLs for GPC. Region 6H contains three neighboring QTLs, i.e., one environmentally stable QTL (*QGpc.ZgSc-6H.3*) and two putative QTLs (*QGpc.ZgSc-6H.2* and *QGpc.ZgSc-6H.4*). These three QTLs with favorable alleles from one parent (ZGMLEL) were in coupling phase. A shadow QTL, significant but false, is caused by a real QTL in an adjacent marker interval \[[@CR55]\]. Since *QGpc.ZgSc-6H.2* and *QGpc.ZgSc-6H.4* were located close to the stable QTL *QGpc.ZgSc-6H.3*, it is difficult to determine whether these two loci were shadow or genuine QTLs. Similarly, region 7H also contains three linked QTLs that were in coupling phase. Unlike the region 6H, region 7H harbored three environmentally stable QTLs and showed similar effects on GPC. Further studies are needed to dissect these two complex regions using advanced population.
The contribution of stable QTLs to GPC {#Sec19}
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QTL effect was generally not precisely estimated in primary QTL analysis due to the genetic noise in mapping populations \[[@CR56]--[@CR62]\]. In view of this point, NILs were proposed and developed as an ideal population for QTL validation, especially for the QTL with a minor effect \[[@CR63], [@CR64]\]. In the present study, three genetic intervals harboring five stable QTLs, *QGpc.ZgSc-2H.1*, *QGpc.ZgSc-6H.3*, *QGpc.ZgSc-7H.1*, *QGpc.ZgSc-7H.2*, and *QGpc.ZgSc-7H.3* were identified and had additive effects of 0.23, 0.46, 0.38, 0.41, and 0.39% in the RIL population, respectively. Of these loci, three located on chromosome 7HS were linked together and resided in one genomic region. Three NIL populations, BC~3~F~2~-I, BC~3~F~2~-II and BC~3~F~2~-III, were developed according to a standard process of consecutive backcross, which targeted the genomic regions of chromosomes 2H, 6H and 7H, respectively. These stable QTLs were validated in the corresponding NIL population and their contribution to GPC could be directly compared between two homozygous groups in a similar genetic background. In populations I, II and III, the average GPC of plants carrying homozygous ZGMLEL were 0.66, 0.99 and 0.71% higher than that of plants carrying homozygous Schooner, respectively, providing further evidence for the reliability of these stable QTLs. Interestingly, the locus on chromosome 2HL (*QGpc.ZgSc-2H.1*) exhibited a strong potential increased in GPC in the Schooner background, which illustrated the conclusion that near-isogenic lines could be used to identify a quantitative locus even though it showed a relatively small effect on the phenotype \[[@CR65]\]. Thus, the minor QTL, *QGpc.ZgSc-2H.1*, is feasible for cloning using NILs with least genetic noise. The estimation of the combination of three loci (*QGpc.ZgSc-7H.1*, *QGpc.ZgSc-7H.2*, and *QGpc.ZgSc-7H.3*) in BC~3~F~2~-III, however, was somewhat lower than expected by the sum of the individual effects of three loci, which might be caused by QTL × QTL interactions, QTL × environment interactions or QTL × new genetic background interactions.
A relatively lower GPC of BC~3~F~2~ populations (11.66--16.82%) grown in Beijing during year 2016 was observed as compared with the RIL population grown in Beijing during year 2013 (16.08--23.36%) or 2014 (14.08--23.75%). This provided further evidence that GPC is largely modified by environmental conditions, which may be due to the alteration of weather condition in 2016. Previous studies indicated high temperature during grain filling stage could result in enhanced GPC \[[@CR66], [@CR67]\]. Interestingly, the number of days after flowering with a maximum temperature above 30 °C in Beijing during 2013 and 2014 were 21 and 23 days, respectively, which is obviously more than that of 2016 (17 days) (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Collectively, we speculated that the lower of GPC in 2016 may could be partially attributed to the alteration of temperature as compared to the other years.
Potential application of stable QTL for MAS in barley breeding {#Sec20}
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Since grain protein concentration is greatly influenced by environmental factors, breeding high-GPC cultivars only through phenotypic evaluation has been proved to be less effective \[[@CR68], [@CR69]\]. Hence, selection of genomic regions containing QTLs that could express steadily under multiple environments is an efficient way to cultivate barley varieties \[[@CR70]\]. Here, we report mapping of six environmentally stable QTLs for GPC that might be useful during barley breeding. Furthermore, we verified the effect of five stable QTLs located on chromosomes 2HL (*QGpc.ZgSc-2H.1*), 6HL (*QGpc.ZgSc-6H.3*) and 7HS (*QGpc.ZgSc-7H.1*, *QGpc.ZgSc-7H.2*, and *QGpc.ZgSc-7H.3*) using three NIL populations. In many cases, improvement of GPC is always accompanied by a significant reduction in grain yield \[[@CR20], [@CR71]\]. Notably, our preliminary data revealed that no significant difference was found for TKW and GY between two different homozygous groups for each QTL region. However, to further investigate the effect of QTL for GPC on yield, it is necessary to carry out experiment using lines, instead of single-plant strategy. Taken together, the identification of SSR marker intervals flanking these stable QTLs on chromosomes 2H (*2L10*-*2L12*), 6H (*6L89*-*6L147*) and 7H (*7S87*-*7S40*) may provide favorable regions for marker-assisted introgression into the elite barley germplasm.
Conclusions {#Sec21}
===========
Based on genotyping 190 RILs in a genome-wide scale and measuring of GPC collected from six environments, six environmentally stable QTLs were significant associated with GPC, among which four QTLs on chromosomes 4H and 7H were firstly identified in the present study. Furthermore, three genomic regions harboring five stable QTLs on chromosomes 2H, 6H and 7H were validated using NIL populations, suggesting the reliability of QTLs detected in primary population. The markers linked to the stable QTLs would be valuable for MAS in barley breeding.
Additional files
================
{#Sec22}
Additional file 1: Table S1.Location-year information and climate data for field trails. (DOC 39 kb) Additional file 2: Table S2.Phenotypic data of two parental lines and 190 RILs under six individual environments and the combined analysis. (XLSX 31 kb) Additional file 3: Table S3.Map locations of all markers and genotypic data of 190 RILs. (XLSX 1050 kb) Additional file 4: Figure S1.Illustration of nine linkage groups constructed using 190 RILs. Notes: A centiMorgan (cM) scale is shown on the left. Black ellipses represent the approximate position for chromosome centromeres. The marker names are not shown. Detail information of the linkage group is provided in Additional file [3](#MOESM3){ref-type="media"}. (DOC 159 kb) Additional file 5: Table S4.Alignments of SNP markers to barley reference sequence. (XLSX 76 kb) Additional file 6: Figure S2.New genetic linkage maps in the target region of the stable QTLs detected in ZGMLEL × Schooner RIL population. (A) chromosome 2HL, (B) 6HL, and (C) 7HS. New SSR markers are showed in bold and underlined. (DOC 299 kb) Additional file 7: Table S5.Markers developed to select the target regions of stable QTLs on chromosomes 2H, 6H and 7H. (DOC 38 kb) Additional file 8: Table S6.Phenotypic data of two homozygous groups in each NIL population. (XLSX 36 kb) Additional file 9: Table S7.Genome and chromosome size comparisons of this genetic map with previously reported maps. Notes: ^a^ the consensus map reported by Close et al. \[[@CR24]\]; ^b^ the consensus map reported by Muñoz-Amatriaín et al. \[[@CR25]\]. (DOC 38 kb) Additional file 10: Table S8.Genomic regions harboring environmentally stable QTLs for grain protein concentration (GPC) in the ZGMLEL × Schooner population. Notes: QTLs in bold represent the environmentally stable QTLs. (DOC 35 kb)
GPC
: Grain protein concentration
GY
: Grain yield
MAS
: Marker-assisted selection
NIL
: Near-isogenic line
QTL
: Quantitative trait locus
RIL
: Recombinant inbred line
SNP
: Single nucleotide polymorphism
SSR
: Simple sequence repeat
TGW
: Thousand grain weight
**Electronic supplementary material**
The online version of this article (doi:10.1186/s12870-017-1067-6) contains supplementary material, which is available to authorized users.
Not applicable.
Funding {#FPar1}
=======
This work was financially supported by the National Natural Science Foundation of China (Grant No. 31271710 and No. 31260326).
Availability of data and materials {#FPar2}
==================================
All data generated or analysed during this study are included in the supplementary files as well as in the manuscript.
MY and ZN conceived the project; YZ developed the ZGMLEL/Schooner RIL population; CF, HW, YY, MZ, JL, and SW carried out experiments; GG provided the facilities and assisted in measuring the grain protein concentration; CF analyzed experimental results; CF, ZN, HZ, and MY wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
Not applicable.
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no conflict of interest.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
| {
"pile_set_name": "PubMed Central"
} |
Kuo Chih-Jung,Liu Hun-Ge,Lo Yueh-Kuei,Seong Churl-Min,Lee Kee-In,Jung Young-Sik and Liang Po-Huang(2009), Individual and common inhibitors of coronavirus and picornavirus main proteases, FEBS Letters, 583, doi: 10.1016/j.febslet.2008.12.059
1. Introduction {#feb2s0014579309000167-sec-1}
===============
Picornaviruses (PV) are small nonenveloped RNA viruses with a single strand of genomic RNA of 7500--8000 nucleotides [\[1\]](#feb2s0014579309000167-bib-bib1){ref-type="ref"}. The members of PV include rhinoviruses (RV), enteroviruses (EV), coxsackieviruses (CV), polioviruses, echoviruses, encephalomyocarditis viruses, meningitis virus, foot and mouth viruses, hepatitis A virus, and so on. Among them, RV is the major cause of the common cold, whereas EV and CV infection can cause hand, foot, and mouth diseases in human and animals. In severe cases, EV can damage the central nervous systems leading to viral meningitis, encephalitis, and severe myocarditis, as well as fatal pulmonary edema \[[2](#feb2s0014579309000167-bib-bib2){ref-type="ref"}, [3](#feb2s0014579309000167-bib-bib3){ref-type="ref"}, [4](#feb2s0014579309000167-bib-bib4){ref-type="ref"}, [5](#feb2s0014579309000167-bib-bib5){ref-type="ref"}\]. CV strain B is a major human pathogen that causes meningitis and mycocarditis leading to heart failure in young adults and congestive heart failure [\[6\]](#feb2s0014579309000167-bib-bib6){ref-type="ref"}. In these PV, a chymotrypsin‐like protease (named 3C^pro^) is required to process polyproteins into mature proteins for viral replication, which represents a promising anti‐viral drug target [\[7\]](#feb2s0014579309000167-bib-bib7){ref-type="ref"}.
On the other hand, coronaviruses (CoV) are the positive‐stranded RNA viruses with larger genome of 27--32 kb, which typically cause respiratory and enteric diseases, pneumonia, exacerbation of asthma, neurological symptoms, and myocarditis in humans and domestic animals. An outbreak of severe acute respiratory syndrome (SARS), caused by a novel human CoV, was spread from China to 29 countries in 2003, infecting a total of ∼8000 people and killing ∼800 patients [\[8\]](#feb2s0014579309000167-bib-bib8){ref-type="ref"}. SARS‐CoV contains a 3C‐like protease (3CL^pro^) analogous to the 3C^pro^ of PV, responsible for processing two overlapping polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Other members of human CoV including CoV‐229E, CoV‐OC43, CoV‐HKU1, and CoV‐NL63 also require a 3CL^pro^ in the maturation of viral proteins.
Several inhibitors have been developed to inhibit the 3C^pro^ of RV and EV \[[9](#feb2s0014579309000167-bib-bib9){ref-type="ref"}, [10](#feb2s0014579309000167-bib-bib10){ref-type="ref"}, [11](#feb2s0014579309000167-bib-bib11){ref-type="ref"}, [12](#feb2s0014579309000167-bib-bib12){ref-type="ref"}\] and 3CL^pro^ of SARS‐CoV \[[13](#feb2s0014579309000167-bib-bib13){ref-type="ref"}, [14](#feb2s0014579309000167-bib-bib14){ref-type="ref"}, [15](#feb2s0014579309000167-bib-bib15){ref-type="ref"}\]. However, their inhibitors can not be mutually used without modification. For example, AG7088, a potent inhibitor of RV and other picornaviral 3C^pro^ [\[16\]](#feb2s0014579309000167-bib-bib16){ref-type="ref"}, failed to inhibit SARS‐CoV 3CL^pro^ [\[17\]](#feb2s0014579309000167-bib-bib17){ref-type="ref"}. Unlike the 3CL^pro^, which is dimeric and in which each subunit is composed of three domains, the 3C^pro^ is a monomer with only the two catalytic domains. The structure‐based sequence alignment ([Fig. 1](#feb2s0014579309000167-fig1){ref-type="fig"} ) shows some sequence differences, which may alter inhibitor specificity. In this study, we performed high throughput screening using a library of ∼6800 compounds to find five novel inhibitors of the SARS‐CoV 3CL^pro^, 4 of which also inhibited another human CoV‐229E 3CL^pro^, but did not inhibit the 3C^pro^ from RV14, CVB3, and EV71. But, one compound was found to almost equally inhibit these 3CL^pro^ and 3C^pro^. From computer modeling, we rationalized the binding discrepancy of the inhibitors against these proteases. The information is useful to further develop more potent individual or common inhibitors of 3C^pro^ and 3CL^pro^ of PV and CoV for anti‐viral drug discovery.
{#feb2s0014579309000167-fig1}
2. Methods {#feb2s0014579309000167-sec-2}
==========
2.1. Expression and purification of the proteases {#feb2s0014579309000167-sec-2.1}
-------------------------------------------------
Two types of proteases including 3CL^pro^ from SARS‐CoV and CoV‐229E and 3C^pro^ from CVB3, EV71, and RV14 were used to assay the inhibitors in this study. The SARS‐CoV 3CL^pro^ and EV71 3C^pro^ were prepared as reported previously \[[12](#feb2s0014579309000167-bib-bib12){ref-type="ref"}, [18](#feb2s0014579309000167-bib-bib18){ref-type="ref"}\]. For expressing CVB3, RV14, and CoV‐229E proteases, the genes were cloned from viral cDNAs by using polymerase chain reaction (PCR) as reported elsewhere.
2.2. Primary screening {#feb2s0014579309000167-sec-2.2}
----------------------
For screening, 0.05 μM SARS 3CL^pro^, 6 μM fluorogenic substrate Dabcyl‐KTSAVLQSGFRKME‐Edans, and 50 μM of approximately 6800 compounds provided by Korea Chemical Bank (Daejeon, Korea) were used. Enhanced fluorescence of the reactions in the buffer of 20 mM Bis‐Tris at pH 7.0 was monitored at 538 nm with excitation at 355 nm using a fluorescence plate reader. The compounds which inhibited more than 50% of the protease activity at 50 μM were selected for the next assay run at 10 μM.
2.3. IC~50~ determination {#feb2s0014579309000167-sec-2.3}
-------------------------
The five hits that inhibited SARS‐CoV 3CL^pro^ at 10 μM were also evaluated against CoV‐229E 3CL^pro^, EV71 3C^pro^, CVB3 3C^pro^, and RV14 3C^pro^. In the assay solution, the activities of these proteases (0.5 μM) with 10 μM fluorogenic substrate in the buffers of 10 mM MES at pH 6.5 and 6.0 (the optimal pH for EV71 and RV14 proteases, respectively) and 10 mM HEPES at pH 7.5 (for CoV‐229E and CVB3 proteases) were measured in the presence of various concentrations of the inhibitors to obtain the IC~50~ values.
2.4. Computer modeling of the inhibitors binding with the proteases {#feb2s0014579309000167-sec-2.4}
-------------------------------------------------------------------
For the modeling analysis, we used the crystal structure of SARS 3CL^pro^ in complex with a peptide inhibitor (PDB code 1UK4) [\[19\]](#feb2s0014579309000167-bib-bib19){ref-type="ref"}, the structures of CoV‐229E 3CL^pro^ and CVB3 3C^pro^ solved by us, and the structural model of EV71 3C^pro^ constructed from the structure of RV 3C^pro^ (PDB code 1CQQ) [\[20\]](#feb2s0014579309000167-bib-bib20){ref-type="ref"}. Docking process was performed using an automated ligand‐docking subprogram of the Discovery Studio Modeling 1.2 SBD (Accelrys Inc., San Diego, CA), with a set of parameters chosen to control the precise operation of the genetic algorithm. Docking runs were carried out using standard default settings "grid resolution" of 5 Å, "site opening" of 12 Å, and "binding site" selected for defining the active site cavity.
3. Results {#feb2s0014579309000167-sec-3}
==========
3.1. Screening of the protease inhibitors {#feb2s0014579309000167-sec-3.1}
-----------------------------------------
We first screened against a library of ∼6800 compounds for inhibiting SARS‐CoV 3CL^pro^. From the primary screening, there were 66 compounds which showed more than 50% inhibition of the enzyme activity at 50 μM. We further tested their inhibitory activities at 10 μM and five of them (21155, 22723, 27548, 43146, and 48511) showed IC~50~ values smaller than 10 μM. According to their dose--response curves as shown in [Fig. 2A--E](#feb2s0014579309000167-fig2){ref-type="fig"} , the five hits 21155, 22723, 27548, 43146, and 48511 displayed IC~50~ values of 7.2 ± 0.7, 10.6 ± 1.3, 7.0 ± 0.8, 3.3 ± 0.2, and 8.1 ± 0.9 μM, respectively, against the SARS 3CL^pro^. Similar inhibition results were observed for 3CL^pro^ of CoV‐229E (data summarized in [Table 1](#feb2s0014579309000167-tbl1){ref-type="table"} ), but not for 3C^pro^. However, 43146 inhibited both 3C^pro^ and 3CL^pro^ with IC~50~ values of 10.3 ± 1.1 μM, 5.4 ± 0.2 μM, 3.3 ± 0.3, and 5.2 ± 0.6 μM, respectively, against CoV‐229E 3CL^pro^, CVB3 3C^pro^, EV71 3C^pro^ and RV14 3C^pro^ ([Fig. 3A--D](#feb2s0014579309000167-fig3){ref-type="fig"} and summarized in [Table 1](#feb2s0014579309000167-tbl1){ref-type="table"}). This compound contains a dihydropyrazole ring with three substituents, two phenyl groups and a lengthy N‐butyl‐benzimidazolylamino‐toluene.
{ref-type="table"}.](FEB2-583-549-g002){#feb2s0014579309000167-fig2}
{#feb2s0014579309000167-fig3}
######
Summary of IC~50~ values (μM) of the five hits with SARS‐CoV 3CL^pro^, and other 3C(L) proteases
Compound ID Structure SARS 3CL 229E 3CL CVB3 3C EV71 3C RV14 3C
------------- --------------------------------------------- ------------ ------------ ----------- ----------- -----------
21155  7.2 ± 0.7 5.6 ± 1.0 \>50 \>50 \>50
22723  10.6 ± 1.3 12.4 ± 0.8 \>50 \>50 \>50
27548  7.0 ± 0.8 6.6 ± 0.3 \>50 \>50 \>50
48511  3.3 ± 0.2 1.8 ± 0.7 \>50 \>50 \>50
43146  8.1 ± 0.9 10.3 ± 1.1 5.4 ± 0.2 3.3 ± 0.3 5.2 ± 0.6
3.2. Inhibition potencies of the 43146 analogues {#feb2s0014579309000167-sec-3.2}
------------------------------------------------
Since 43146 inhibited 3CL^pro^ and 3C^pro^, its analogues including 45240, 68638, 55688, and 55585 obtained from another compound library were evaluated. As shown in [Table 2](#feb2s0014579309000167-tbl2){ref-type="table"} , all of them showed good potencies against the five proteases. The most potent compound was 45240, and its IC~50~ values in inhibiting the 3C(L) proteases were measured to be 2.5 ± 0.2 μM (SARS‐CoV 3CL^pro^), 2.6 ± 0.4 μM (CoV‐229E 3CL^pro^), 1.2 ± 0.3 μM (CVB3 3C^pro^), 0.5 ± 0.1 μM (EV71 3C^pro^), and 1.7 ± 0.1 μM (RV14 3C^pro^) ([Table 2](#feb2s0014579309000167-tbl2){ref-type="table"}). This compound contains four rings, three phenyl groups and one imidazole, surrounding a central dihydropyrazole ring, without the lengthy side chain as seen in 43146. Compound 68638 with benzylcyclohexane ring fused with the dihydropyrazole ring and acetyl and iodobenzyl groups attached to the central ring showed less inhibition against both 3C^pro^ and 3CL^pro^ ([Table 2](#feb2s0014579309000167-tbl2){ref-type="table"}). The other two compounds, 55688 and 55585, with shorter side chains attached to the benzimidazolyl group showed similar inhibitory activities as compared to 43146 ([Table 2](#feb2s0014579309000167-tbl2){ref-type="table"}).
######
IC~50~ values (μM) of compound 43146 analogs with SARS‐CoV 3CL^pro^, and other 3C(L) proteases
Compound ID Structure SARS 3CL 229E 3CL CVB3 3C EV71 3C RV14 3C
------------- --------------------------------------------- ----------- ------------ ----------- ------------ -----------
45240  2.5 ± 0.2 2.6 ± 0.4 1.2 ± 0.3 0.5 ± 0.1 1.7 ± 0.1
68638  9.8 ± 0.8 12.4 ± 0.8 7.0 ± 0.8 10.6 ± 1.3 5.3 ± 1.1
55688  8.0 ± 0.5 9.6 ± 0.3 6.1 ± 0.5 8.5 ± 0.6 7.7 ± 1.0
55585  8.4 ± 0.2 10.2 ± 0.7 6.5 ± 0.6 4.7 ± 0.2 6.4 ± 0.3
3.3. Computer modeling of 21155, 22723, 27548, and 48511 binding to the proteases {#feb2s0014579309000167-sec-3.3}
---------------------------------------------------------------------------------
These inhibitors are competitive inhibitors with respect to the substrate (data not shown), indicating they bind in the active site. To rationalize the binding discrepancy of these inhibitors against these proteases, their binding modes with SARS‐CoV 3CL^pro^ and four other proteases were modeled and some of them are shown in [Fig. 4](#feb2s0014579309000167-fig4){ref-type="fig"} . The first four inhibitors of SARS‐CoV 3CL^pro^ are more rigid because the thiazolopyridine in 21155, the dichlorobenzoquinolinone in 22723, the isoindoledione in 27548, and the oxazole in 48511 adopt planar structures and the three substituents of the oxazole ring in 48511 are fixed in a conformer, due to the 1,2‐steric interaction between the acetate group and the N‐aryl imino group as well as the biaryl interaction between the phenyl and oxazole to prohibit their free rotation. All these compounds can be considered as two rigid aromatic moieties connected by a small linker. Based on the computer modeling, each of these aromatic moieties is bound to S1 or S2 site of SARS protease by forming H‐bonds and hydrophobic interactions ([Fig. 4A--D](#feb2s0014579309000167-fig4){ref-type="fig"}). As shown in the computer modeling, Glu166 side chain of SARS 3CL^pro^ forms H‐bonds with these four inhibitors. However, the corresponding amino acid residue in 3C^pro^ is Gly164, which lacks the side chain to form H‐bond with any of these compounds (also see [Fig. 4](#feb2s0014579309000167-fig4){ref-type="fig"}F), leading to loss of inhibition. In addition, the 3C^pro^ have more open but shallow S2 site (due to its partial blockage by Leu127) than 3CL^pro^ according to the crystal structures of RV 3C^pro^ [\[20\]](#feb2s0014579309000167-bib-bib20){ref-type="ref"} and CVB3 3C^pro^ (Lee et al., unpublished results) compared to 3CL^pro^ (also see [Fig. 4](#feb2s0014579309000167-fig4){ref-type="fig"}F). Thus, 3C^pro^ can not hold these compounds tightly.
{#feb2s0014579309000167-fig4}
3.4. Binding modes of 43146 and its analogues to the proteases {#feb2s0014579309000167-sec-3.4}
--------------------------------------------------------------
In contrast, the compound 43146 is more flexible, because the dihydropyrazole is not planar, and the phenyl group is linked to the sp^3^‐hybridized carbon of the dihydropyrazole ring, so it is free for rotation. Different from the binding modes of the other 4 inhibitors, the diphenyl 4,5‐dihydro‐1H‐pyrazole moiety of 43146 fits well at the S1′ and S2 sites in the SARS 3CL^pro^ ([Fig. 4](#feb2s0014579309000167-fig4){ref-type="fig"}E) with the rest of the molecule at the S3 site and beyond. With this binding mode, the compound was predicted to also bind well in the 3C^pro^, consistent with the inhibition data. In fact, RV 3C^pro^ prefers a phenyl group at the S2 site as evidenced by its strong inhibition by AG7088 which has a P2‐fluorophenylalanine. Thus, it could be rationalized by computer modeling that only 43146 among the five hits can inhibit the three 3C^pro^ in addition to the 3CL^pro^.
The analogues of 43146, including 45240, 68638, 55688, and 55585, bind in the 3C^pro^ and 3CL^pro^ active sites with similar modes to that of 43146 (data not shown). Compared to 43146, 55688 and 55585 only have minor structural difference with shorter alkyl groups attached to the benzimidazole ring, so that they showed similar inhibition against the proteases. The fused ring system and the phenyl group in 68638 may also span from S1′ to S2 sites in both kinds of proteases, yielding similar inhibition. However, 45240 showed a significantly better inhibition against the 3C^pro^ than 43146. Apparently, the lengthy side chain attached to the phenyl group in the compound did not provide additional interaction with the protease, consistent with the binding mode shown in [Fig. 4](#feb2s0014579309000167-fig4){ref-type="fig"}E. However, the additional interaction is provided by the pyridine ring bound near the more open S1′ site in 3C^pro^.
4. Discussion {#feb2s0014579309000167-sec-4}
=============
AG7088 is the best inhibitor identified so far for 3C^pro^, which not only inhibits the 3C^pro^ from RV, but also those from CV and EV [\[16\]](#feb2s0014579309000167-bib-bib16){ref-type="ref"}. However, it did not inhibit 3CL^pro^ from SARS‐CoV [\[17\]](#feb2s0014579309000167-bib-bib17){ref-type="ref"}. This may be partially due to the blockage of its P1‐lactam ring by the relatively larger Glu166 side chain and also the S2 site of 3CL^pro^ is narrower although it is deeper. Therefore, when the P2‐phenylalanine is changed to non‐planar leucine or cyclohexane without changing the P1‐lactam, they became good inhibitors of SARS 3CL^pro^ [\[21\]](#feb2s0014579309000167-bib-bib21){ref-type="ref"}. Unlike AG7088, which is a ketomethyl isostere of a tripeptide‐conjugated ester, compound 43146 is not peptide‐like. From the random screening as shown in the study, we have found a starting point toward the development of non‐peptide multiple‐function inhibitors against CoV and PV. With further modification of these individual and common inhibitors of the viral proteases, we hope to find solution for the possible reoccurrence of SARS and other diseases caused by the viruses with the 3C^pro^ and 3CL^pro^.
This work was supported by a grant from Academia Sinica to PHL, and we thank Korea Chemical Bank for providing their chemical library with which this work was conducted.
| {
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The study was performed at the Massachusetts General Hospital in Boston, MA. Massachusetts General Hospital is a teaching affiliate of Harvard Medical School in Boston, MA.
Introduction
============
In recent years, increasing attention has been paid to the sizable and growing costs of critical care services. The percentage of the United States\' gross domestic product used for critical care services increased from the year 2000 to 2005 by 13.7%, from 0.58% to 0.66% \[[@B1]\]. Optimal use of intensive care unit (ICU) resources is an important goal for individual hospitals and healthcare systems and is an essential component of the effort to contain healthcare expenditures. At our institution, we found that many patients who met criteria for transfer out of the ICU remained in the ICU for a longer time than necessary. We investigated the incidence of delayed transfer of surgical ICU patients and sought to determine the causes for delays and to estimate the costs associated with them. We hypothesized that transfers out of our ICU are delayed by 1 or more days at least 20% of the time, and that the majority of delays are due to a lack of available floor beds.
Materials and methods
=====================
Setting
-------
After approval by the hospital\'s Institutional Review Board, the study was conducted in the Surgical Intensive Care Unit (SICU) of the Massachusetts General Hospital from January 24, 2010, to July 31, 2010. Informed consent was not necessary because of the observational nature of the study. Patient care was not affected by the study, and individual patient information was not used during the analysis of data. MGH is a 900-bed university-affiliated tertiary care center with 1.5 million outpatient visits and 47,000 inpatient admissions annually. The hospital is a level 1 trauma center. The SICU is a 20-bed unit with \>1,400 annual admissions, primarily from the complex trauma, vascular, thoracic, and general surgical populations. It is staffed 24 hours per day by attending intensivists in addition to critical care fellows and residents.
Transfer process
----------------
A patient was classified as ready for transfer to the floor by consensus among the patient\'s intensivist, surgeon, and SICU nurse, based on guidelines for discharge criteria, with emphasis on hemodynamic and respiratory stability. After a patient was deemed ready for transfer, a request to the admissions office was placed to locate an appropriate floor bed. Hospital-based nursing triage supervisors were aware of the bed availability throughout the hospital and supported the process. When a bed became available, the patient was transferred out of the SICU.
Reasons for delay
-----------------
Before the study, we surveyed intensivists, surgeons, SICU nurses, nursing triage supervisors, and administrators to determine the perceived reasons for transfer delay. Based on these responses, we predefined the following four reasons: lack of available surgical-floor bed, lack of an appropriate bed for infectious contact precautions, change of primary service (Surgery to Medicine), and lack of available patient attendant (\"sitter\" for mildly delirious patients).
Demographic and clinical factors
--------------------------------
All patients who were admitted to the SICU during the study period were included in the study, without regard for demographic or clinical factors.
Data collection and definition of delay
---------------------------------------
During the study period, a SICU intensivist (DWJ) contacted the SICU charge nurse each night to identify those patients who were delayed in transfer. Patients were classified as delayed in transfer when the intensivist, surgeon, and SICU nurse were in agreement that the patient could be transferred to the floor, yet the patient remained in the SICU past 00:01 that night. The intensivist and charge nurse discussed each case to determine which of the four reasons (or \"other\") best characterized the patient\'s reason for delay. Duration of ICU stay, duration of hospital stay, admitting surgical service, destination (type of surgical floor), date deemed ready for transfer, date/time of actual transfer, and daily hospital census were recorded throughout the study period.
Cost analysis
-------------
We used the difference between the 2005 national average cost of a day in the ICU (\$3,518) and a day in a non-ICU hospital bed (\$1,153) to estimate the extra costs associated with patients remaining in the SICU unnecessarily \[[@B1]\]. This difference (\$2,365) was multiplied by the total number of delay days (246) to generate the estimated total costs of the delays.
Statistics
----------
All data analysis was performed by using Stata 10 (Stata-Corp LP, College Station, TX, USA). Continuous variables with a normal distribution are expressed as mean and standard deviation (SD). Ordinal variables are expressed as median and range. The χ^2^test was used to compare absolute numbers and proportions. The Spearman rho was used to evaluate the association between the daily hospital census and the daily number of SICU beds occupied by patients delayed in transfer.
Results
=======
During the study period, 731 patients were transferred from the SICU to the floor. Of these patients, 160 (22%) experienced a delay in transfer of at least 1 day. The delays in transfer ranged from 1 to 6 days (mean, 1.5 days; median, 2 days; Figure [1](#F1){ref-type="fig"}). The most common reason for delay was lack of availability of a surgical-floor bed (71% (114 of 160)). The lack of an appropriate room for infectious contact precautions accounted for 18% (28 of 160) of delays. The remaining causes were change of primary service (Surgery to Medicine) at 7% (11 of 160), and lack of available patient attendant (\"sitter\" for mildly delirious patients) at 3% (five of 160).
{#F1}
The cost associated with delays in transfer was estimated to be \$581,790 for the entire study period, or \$21,547 per week.
The daily hospital census was positively correlated with the number of SICU beds occupied by delayed patients (Spearman rho = 0.27; *P*\< 0.0001). Delayed patients were significantly more likely than nondelayed patients to be transferred during night shifts, between 19:00 to 06:59, (21% (33 of 160) versus 12% (67 of 571); χ^2^= 10.6; *P*\< 0.005).
Discussion
==========
In this study, we found that delay in transfer from the SICU occurred in 22% of cases, with lack of availability of surgical-floor beds as the most common reason, accounting for 71% of delayed transfers. The ICU beds in our hospital constitute approximately 15% of the total number of beds. Although the absolute numbers and ratios of ICU beds-to-floor beds vary from institution to institution, our hospital\'s ratio of 15% ICU-to-total hospital beds is equal to the national average of 15% \[[@B1]\]. Recent studies describe similar incidence and financial impacts \[[@B3],[@B4]\]. Our results are similar to those from a 2004 study in an Australian combined medical-surgical ICU that found that 27% of ICU transfers were delayed by at least 8 hours, and that 81% of these delays were due to lack of available floor beds \[[@B5]\]. Whereas it is currently impossible to estimate the overall incidence of ICU-transfer delays in the United States and abroad, our study and these reports from other institutions suggest that the problem is widespread and contributes significantly to inefficiencies in healthcare systems.
ICU transfer delays have a large financial impact. Time spent in the ICU costs more than time spent on a regular floor, so delays in transfer naturally increase the overall cost of care. The cost difference between ICU days and floor days is driven mostly by the difference in staff-to-patient ratios in the ICU versus on the floor, including higher ratios of nurses, physicians, and therapists to patients.
The cost analysis in this study was performed by using data from the report of the United States critical care bed numbers, occupancy rates, payer mix, and costs published by Halpern and Pastores in 2010 \[[@B1]\]. By using national average costs, the results of this study are more generally applicable to the healthcare community at large.
A limitation of this method of cost analysis is that it assumes that ICU costs remain constant throughout the ICU stay. A study by Dasta and colleagues in 2005 \[[@B6]\] showed that costs are highest during the first 2 days of an ICU stay, and that higher costs are associated with mechanical ventilation. Patients who are stable and ready for discharge to the floor incur fewer costs than do critically ill patients. At our institution, the majority of daily ICU cost per patient is attributable to room and board, which includes nursing care. The daily cost of room and board in our ICU does not change after a patient is deemed ready for transfer to the floor, and the amount is not dramatically different from the estimated cost of an ICU day in the Halpern report; therefore, the cost-analysis model is valid for estimation of increased costs.
We analyzed the actual costs for delayed versus nondelayed patients (from the time each patient was deemed ready for transfer out of the ICU). Although actual patient-charge information is not publishable per our hospital\'s policies, the total additional costs of delays in transfer were greater than what would be predicted by the cost data in the Halpern report.
The increased costs associated with delayed transfer in our single-ICU study should prompt clinicians and administrators to examine closely ways to reduce or eliminate such delays. Preliminary results from a recent retrospective study showed similar increases in hospital costs associated with delayed transfer from the ICU \[[@B3]\]. Although the methods used in that study were different from ours, both studies came to the same conclusion: delays in transfer out of the ICU constitute a significant and costly problem.
A large number of delays were related to infectious contact precautions (28%). In accordance with Centers for Disease Control (CDC) guidelines, patients in our institution known to be infected or colonized with multidrug-resistant organisms or *Clostridium difficile*are required either to have single rooms or to share a room with patients with the same organism \[[@B7]\]. Providers caring for these patients are required to use contact precautions: gowns and gloves in addition to hand cleaning. A possible contribution to this element of the delay problem is that a majority of our hospital\'s non-ICU rooms are not private. Recently published studies appear to offer conflicting results regarding the efficacy of contact precautions; still, the recommendation to keep affected patients separated from unaffected patients is unlikely to change in the near future \[[@B8],[@B9]\].
Delays in transfer were associated with high hospital census. At times during the study period, our hospital\'s census was \>95% occupancy, and these times correlated with an increased number of SICU beds occupied by delayed patients. It has been shown that efficiency of acute care units is impaired when hospital occupancy rates exceed 85% \[[@B10]\]. Excessive hospital occupancy can lead to a bottleneck effect in which completely occupied floor beds prohibit transfers out of the ICU and thus prohibit the admission of critically ill surgical patients into the ICU. Our hospital is a level-1 trauma center and referral center for complex surgical operations. Prevention of SICU admissions because of excessively occupied floor beds results in trauma victims and critically ill surgical patients being admitted to nonsurgical ICUs.
Patients who were delayed in transfer were more likely than nondelayed patients to be transferred during night shifts. This is notable because of the previously published data that show that patients who are transferred at night have an increased likelihood of readmission to the ICU \[[@B11],[@B12]\] and that patients who are readmitted to the ICU have an increased risk of hospital mortality \[[@B13]\]. The reason for the observed association between ICU transfer delay and nighttime transfer is not clear and requires further study.
Investigators recently reported a marked reduction in the incidence of delayed OR-to-SICU transfer after the implementation of measures aimed at facilitating early transfer of medically suitable patients out of the SICU \[[@B4]\]. In this study, the authors noted delays in OR-to-ICU transfer related to impeded SICU throughput and demonstrated a reduction in such delays after interventions reduced time to transfer out of the SICU. Similar delays occur in our institution, and they place a tremendous burden on OR personnel, surgeons, anesthesiologists, and OR administrators. The successful interventions by Young *et al.*\[[@B4]\] demonstrate that reduction in delayed transfer from the SICU can have a measurably positive impact on the hospital as a whole.
Possible contributions to the problem include the occupation of floor beds by patients ready to be discharged from the hospital, inefficiency in discharging patients from the ICU to home, and the subspecialization of surgical-floor beds. When a surgical floor is at capacity and discharge-ready patients remain in the floor\'s beds, transfers to the floor from the SICU are potentially delayed. Interventions to reduce the number of floor beds occupied by discharge-ready patients would likely reduce SICU-transfer delays.
Physicians and nurses in our ICU have relatively little experience in discharging patients home. Some of the patients who experienced long delays in transfer from the SICU to the floor were likely ready for discharge home. Efforts to improve recognition of discharge-ready patients and to educate staff members in the process of discharging home might be beneficial in reducing the unnecessary occupation of ICU beds. Patients in our hospital are transferred from the SICU to service-specific floors (for example, aortic surgery patients are transferred to a floor dedicated solely to vascular surgery). Only when clinically essential (for example, to create an available SICU bed for a patient with severe traumatic injuries) are patients transferred to floors other than their service-specific floor. De-specialization of surgical floors might reduce the incidence of delayed transfer from the SICU. The benefit of specialty surgical floors to patients, presumed by intensivists and surgeons at many large tertiary care centers, requires study for confirmation.
The discussion regarding the United States\' need to curb its ever-increasing healthcare expenditures must include consideration of the costs associated with provision of critical care. Multiple reports have projected a shortfall of critical care services in the years to come \[[@B14],[@B15]\]. Considering these projections and the budgetary constraints that the healthcare system faces, intensivists and hospital administrators must optimize the efficiency of each dollar spent in the ICU. In a system with too few ICU beds and inadequate financial resources, floor-ready patients occupying ICU beds represent a double-edged sword.
Although planning for the projected increased need for ICU beds seems prudent, improved utilization of existing ICU resources is an essential component of the strategic planning needed to address the problem. Reduction or elimination of delays in transfer from the ICU have the potential to increase ICU-bed capacity effectively without the physical creation of new ICU beds. Previous publications have discussed the importance of ICU outflow in overall ICU resource utilization, yet few studies of this problem have been conducted \[[@B16]\]. The paucity of literature addressing the problem in our study suggests that the issue has not been adequately quantified and analyzed. Further study of ICU-transfer delays, including studies of interventions to improve the problem, will likely improve patient care and resource utilization.
This study has several limitations. The study was performed in a single surgical ICU of a major academic medical center. Similar academic centers have reported delays in SICU transfer, but it is unknown whether such delays occur commonly in nonacademic settings. We used days as the time variable. It might be beneficial to use hours in future studies, more precisely to quantify the problem. Our cost analysis was based on national average costs of ICU and floor days. Future studies could collect and analyze cost data of the hospital or hospitals where the study is being conducted and account for the reduction in actual costs that occurs as patients\' care needs lessen over time. Hospital census was used as a proxy for surgical-floor census. Future studies could analyze the actual census of destination floors for delayed ICU patients.
Conclusions
===========
Delay in transfer from the SICU is common and is associated with increased cost of hospitalization. Because the most common reason for this delay was insufficient availability of surgical-floor beds, future efforts should include emphasis on increasing the availability of floor beds.
Key messages
============
• Delay in transfer from the SICU is common and costly.
• The most common reason for delay is insufficient availability of surgical-floor beds.
• Delay in transfer is associated with high hospital census.
Abbreviations
=============
CDC: Centers for Disease Control; ICU: intensive care unit; IRB: institutional review board; OR: operating room; SICU: surgical intensive care unit.
Competing interests
===================
The authors declare that they have no competing interests, financial or nonfinancial.
Authors\' contributions
=======================
DWJ designed the study, engaged in daily communication with ICU charge nurses, collected and recorded all data, analyzed all data, and drafted the preliminary, revised, and final versions of the manuscript. US provided input on the design of the study, assisted in designing the data-collection instrument, and edited the manuscript extensively. EAB provided input on the design of the study, performed all statistical analysis, created Figure [1](#F1){ref-type="fig"}, and edited the manuscript. BC and RL provided expertise on the financial elements of the study, conducted analysis of cost differences between delayed and nondelayed patients, and edited the manuscript. RMP conceived of the study, managed the IRB application, obtained IRB approval, provided input on the design of the study, and edited the manuscript. All authors read and approved the manuscript before submission.
Acknowledgements
================
We thank the nursing staff of the SICU at Massachusetts General Hospital for their assistance with data collection during the study period. The source of all funding for the entire study was the Department of Anesthesia, Critical Care and Pain Medicine at the Massachusetts General Hospital. The study was supported by the Department of Anesthesia, Critical Care & Pain Medicine, Massachusetts General Hospital. No extramural funding was used to support the study.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Snow crabs belong to the subphylum Crustacea, Order Decapoda, Family Majidae, and Genus *Chionoecetes*. These *Chionoecetes* are found in colder water at depths less than 2000 m where it is muddy or sandy [@pone.0070887-Lim1]. Three kinds of *Chionoecetes* are prominent on the east coast of Korea, including snow crab (*Chionoecetes opilio*), red-tanner crab (*Chionoecetes japonicus*), and the hybrid Neodo-Daege (*Chionoecetes* sp.) [@pone.0070887-Kim1]. *Chionoecetes* fishing is a major industry and source of income in the area [@pone.0070887-Korean1]--[@pone.0070887-Kim3]. However, contamination of the East Sea of Korea from human activities has raised serious sanitation concerns that could potentially threaten this industry [@pone.0070887-Yong1]. Research on antibiotic resistance has primarily focused on human disease; there is limited understanding of antibiotic resistance genes in natural environments. The relationship between environmental microorganisms and human pathogens is not clear; a recent report showed that soil bacteria and human pathogens shared an antibiotic resistome [@pone.0070887-Forsberg1]. In this study, we analyzed the microbiota within parts of *Chionoecetes* using culturing methods, and the culturable microbial isolates were tested for antibiotic resistance. Here, we report the microbial populations and antibiotic resistance of isolates from the internal organs of *Chionoecetes*. These results may be used to monitor snow crab populations and to identify potentially dangerous changes in microbiota that could threaten the snow crab industry.
Materials and Methods {#s2}
=====================
Collection of snow crabs {#s2a}
------------------------
No specific permissions were required for these locations or activities because many snow crabs were sold daily at the market near the Harbor. Wild-caught, uncooked snow crabs (*Chionoecetes* spp.) were collected from a retail seafood shop at Jukbyeon Harbor and were placed into clean, resealable plastic bags. Samples were stored in a cooler during transfer from Jukbyeon Harbor to the laboratory where they were then stored at 4°C until processed. The morphological characteristics of snow crabs can be distinguished by their carapace color, the arrangement of granules on the lateral carapace, and the presence or absence of spines on the lateral carapace [@pone.0070887-Kim2]. The crabs were originally caught in the East Sea of Korea [@pone.0070887-Chun1], [@pone.0070887-Kim3]. There are no-take periods from June through November for snow crab (*Chionoecetes opilio*) and July through August for red-tanner crab (*Chionoecetes japonicus*), and no prohibition for Neodo-Daege (*Chionoecetes* sp.) for management of the snow crab industry.
Sample preparation {#s2b}
------------------
Samples were divided into the following six parts: guts (D), gills (G), heart (H), leg meat (LS), carapace meat (S), and carapace juices (J). Each sample was homogenized with 10 mL of 10 mM potassium phosphate buffer, and 100 µL of each sample was spread onto agar plates.
Enumeration of microbial populations {#s2c}
------------------------------------
The serial dilutions were spread plated on various media to determine microbial counts of the *Chionoecetes* spp. in sterile water, and the dilutions were dispensed onto agar plates. For all experiments reported herein, we cultured aerobic microbes on the following media: YPD (yeast extract, peptone, dextrose, BD Bioscience, USA) with chloramphenicol (100 mg/L) and streptomycin (100 mg/L) for yeast; PDA (potato dextrose agar, BD Bioscience) with chloramphenicol (100 mg/L) and streptomycin (100 mg/L) for fungi; R2A (BD Bioscience); NA (nutrient agar, BD Bioscience); TSA (tryptic soy agar, BD Bioscience) for general bacteria; or MA (marine agar, BD Bioscience). The plates were incubated aerobically at 28°C for 1 wk, and average CFU (colony-forming units) values were obtained from triplicate plate counts.
Isolation of bacteria {#s2d}
---------------------
All colonies from individual plates of one plate or two plates from the plates of colony counts were picked up and cultured separately. In total, 381 individual isolates were transferred to fresh plates three times and then processed for sequencing of 16S rRNA and ITS genes.
Sequencing {#s2e}
----------
The primers used to amplify the 16S rRNA and ITS genes for bacteria and other microbes were 27F and 1492R [@pone.0070887-Lane1] and ITS1 and ITS4 [@pone.0070887-White1], respectively. The PCR reaction was performed with 20 ng of genomic DNA as the template in a 30-µL reaction mixture by using EF-*Taq* DNA polymerase (Solgent, Korea). The thermocycler conditions were 95°C for 5 min, followed by 35 cycles of 95°C for 2 min, 55°C for 60 s, and 72°C for 60 s, then a final extension step for 10 min at 72°C. Thereafter, the amplification products were purified using a multiscreen filter plate (Millipore Corp., Bedford, MA, USA). Sequencing reactions were performed using a PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Bio- systems, Foster City, Calif., USA). The DNA samples containing the extension products were added to Hi-Di formamide (Applied Biosystems, Foster City, CA). The mixture was incubated at 95°C for 5 min followed by 5 min on ice, and then analyzed using an ABI Prism 3730XL DNA analyzer (Applied Biosystems, Foster City, CA). DNA sequencing of isolates was performed by Macrogen Inc. (Seoul, Korea).
Phylogenetic trees {#s2f}
------------------
The 16S rRNA and ITS sequences were aligned using the Nearest Alignment Space Termination (NAST) aligner [@pone.0070887-DeSantis1]. Aligned sequences were then compared to the Lane mask using the Greengenes website [@pone.0070887-DeSantis2]. Sequence matching to the Ribosomal Database Project [@pone.0070887-Cole1] was used to find GenBank sequences representing the most closely related type of strain for each isolate. These type strains were included as references in the phylogeny using the Greengenes Automatic Taxonomic Classification algorithm [@pone.0070887-DeSantis2]. Phylogenetic trees were constructed using neighbor-joining [@pone.0070887-Saitou1] with MEGA5 for Windows [@pone.0070887-Tamura1]. Evolutionary distances were calculated with the Kimura 2-parameter method. Bootstrap analyses of the neighbor-joining data were conducted based on 1000 samples to assess the support for inferred phylogenetic relationships.
Antibiotics {#s2g}
-----------
The antibiotics (content per disc) used in the study were ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), kanamycin (30 µg), penicillin (10 unit), rifampicin (5 µg), tetracyclin (30 µg), ticarcilin (75 µg), and vancomycin (30 µg). The antibiotic discs were purchased from BD Bioscience (San Jose, CA).
Antibiotic susceptibility test {#s2h}
------------------------------
Bacteria were considered susceptible to a particular antibiotic if the bacteria formed a clear zone around a disc on the media (disc diffusion susceptibility testing). After autoclaving the nutrient agar and cooling the agar to 50--55°C, bacterial colonies were mixed into the autoclave medium flask and poured onto petri dishes. Each antibiotic susceptibility testing disc (BD Bioscience) was placed onto a plate and incubated at 28°C for 24 or 48 h. The results indicated whether the isolates were resistant or susceptible to each antibiotic.
PCR assays for detection of resistance genes and sequencing of the PCR products {#s2i}
-------------------------------------------------------------------------------
Bacteria were tested through PCR method with the primers of antibiotic resistance genes as shown in [Table 1](#pone-0070887-t001){ref-type="table"}. For the PCR, the reaction mixtures contained 10 µl 2× DNA polymerase enzyme (PowerAmp™ 2× premix), 4 µl primer mixtures, 5 µl template DNA and sterile distilled water to bring the final volume to 20 µl. The PCR was performed with TaKaRa PCR Thermal Cycler Dice TP600 (TaKaRa, Japan). The reaction was started with a 15-min denaturation step at 95°C. In the PCRs, the temperature cycles consisted of 30 sec at 95°C, followed by 1 min at 58°C and 1 min at 72°C and each cycle was repeated 35 times. The final cycle was followed by incubation of the reaction mixture for 10 min at 72°C. Amplified PCR products were analyzed by gel electrophoresis in 2% agarose gels stained with ethidium bromide, visualized with ultraviolet illumination, and imaged with the Gel Doc 2000 documentation system (Bio-Rad, Hercules, CA, USA). DNA sequencing of antibiotic resistance genes was performed by Macrogen Inc. (Seoul, Korea).
10.1371/journal.pone.0070887.t001
###### PCR primers targeting antibiotic resistance genes.
{#pone-0070887-t001-1}
Antibiotics Target gene Sequence 5′-3′ Amplicon size (bp) References
----------------- ------------- ------------------------------------- ------------------------------------- -------------------- ----------------------------
ampicillin *blaSHV* FW[\*](#nt101){ref-type="table-fn"} TTA TCT CCC TGT TAG CCA CC 796 [@pone.0070887-Prabu1]
RV GAT TTG CTG ATT TCG CTC GG
*blaOXA* FW ACC AGA TTC AAC TTT CAA 589
RV TCT TGG CTT TTA TGC TTG
*blaTEM* FW ATA AAA TTC TTG AAG AC 1,073
RV TTA CCA ATG CTT AAT CA
chloramphenicol *catA1* FW CGC CTG ATG AAT GCT CAT CCG 456 [@pone.0070887-Soge1]
RV CCT GCC ACT CAT CGC AGT AC
*catA2* FW ATG AAT TTT ACC AGA ATT GAT CTG AA 639
RV ATT TCA GTA TGT TAT CAC ACA TCA TCT
*catA3* FW AAA TTG GGT TCG CCG TGA 1,863
RV ATT TAC TGT TAC ACA ACT CTT GTA GCC
*catB3* FW TCA AAG GCA AGC TGC TTT CTG AGC 566
RV TAT TAG ACG AGC ACA GCA TGG GCA
erythromycin *ermA* FW TAT CTT ATC GTT GAG AAG GGA TT 138 [@pone.0070887-Martineau1]
RV CTA CAC TTG GCT TAG GAT GAA A
*ermB* FW CTA TCT GAT TGT TGA AGA AGG ATT 141
RV GTT TAC TCT TGG TTT AGG ATG AAA
*mefA* FW AGT ATC ATT AAT CAC TAG TGC 348 [@pone.0070887-Prabu1]
RV TTC TTC TGG TAC TAA AAG TGG
penicillin *pbp2a* FW CCG CTG ATC TTG ATT GAA TAG 355 [@pone.0070887-Sanbongi1]
RV ATG CGT TTT CAT CCC CTC TG
kanamycin *aphA-3* FW GGGACCACCTATGATGTGGAACG 600 [@pone.0070887-Gibreel1]
RV CAGGCTTGATCCCCAGTAAGTC
tetracycline *tetA* FW GTA ATT CTG AGC ACT GTC GC 956 [@pone.0070887-Lucarelli1]
RV CTG CCT GGA CAA CAT TGC TT
*tetB* FW ACG TTA CTC GAT GCC AT 1,169
RV AGC ACT TGT CTC CTG TT
*tetC* FW AAC AAT GCG CTC ATC GT 1,138
RV GGA GGC AGA CAA GGT AT
*tetG* FW CCG GTC TTA TGG GTG CTC TA 603
RV CCA GAA GAA CGA AGC CAG TC
vancomycin *vanA* FW GCT ATT CAG CTG TAC TC 783 [@pone.0070887-Depardieu1]
RV CAG CGG CCA TCA TAC GG
*vanB* FW CAT CGC CGT CCC CGA ATT TCA AA 297
RV GAT GCG GAA GAT ACC GTG GCT
FW, forward; RV, reverse.
Results {#s3}
=======
Culturable microbiota {#s3a}
---------------------
A total of 381 isolates, including 221 from *C. opilio*, 76 from *C. japonicus*, and 84 from *Chionoecetes* sp. were isolated using six different media ([Figure 1A](#pone-0070887-g001){ref-type="fig"}). [Figure 1](#pone-0070887-g001){ref-type="fig"} summarizes the phylogenetic distribution of the 16S rRNA gene sequences. The genera *Pseudomonas*, *Stenotrophomonas*, and *Acinetobacter* predominated the bacterial communities found in *Chionoecetes*, commonly representing more than 60% of the sequences isolated from the three crab species. The microbial isolates from *C. japonicus* and *C. opilio* included 19 and 20 genera, respectively, whereas the isolates of *Chionoecetes* sp. included 21 genera ([Figure 1A](#pone-0070887-g001){ref-type="fig"}). The greatest diversity of microbiota among the six different internal organs investigated was found in the gills; we identified 14, 15, and 17 genera in gills of *C. japonicus*, *C. opilio*, and *Chionoecetes* sp. respectively. Nine or fewer genera were identified in the other organs ([Figures 1B](#pone-0070887-g001){ref-type="fig"} and [S1](#pone.0070887.s001){ref-type="supplementary-material"}).
{#pone-0070887-g001}
Isolates from snow crab were predominantly *Acinetobacter* spp. ([Figure S1b](#pone.0070887.s001){ref-type="supplementary-material"}), *Pseudomonas* spp. ([Figure S1a](#pone.0070887.s001){ref-type="supplementary-material"}), *Bacillus* spp. ([Figure S1d](#pone.0070887.s001){ref-type="supplementary-material"}), and *Stenotrophomonas* spp. ([Figure S1e](#pone.0070887.s001){ref-type="supplementary-material"}), which collectively accounted for approximately 84% of the isolates (n = 186). Forty-six isolates from *C. japonicus* included either *Acinetobacter* spp. ([Figure S1b](#pone.0070887.s001){ref-type="supplementary-material"}), *Agreia* spp. ([Figure S1f](#pone.0070887.s001){ref-type="supplementary-material"}), *Bacillus* spp. ([Figure S1d](#pone.0070887.s001){ref-type="supplementary-material"}), or *Psychrobacter* spp. ([Figure S1f](#pone.0070887.s001){ref-type="supplementary-material"}), whereas 60% of isolates (n = 57) from *Chionoecetes* sp. were affiliated with *Agreia* spp., *Agrococcus* spp. ([Figure S1g](#pone.0070887.s001){ref-type="supplementary-material"}), *Bacillus* spp., *Microbacterium* spp. ([Figure S1g](#pone.0070887.s001){ref-type="supplementary-material"}), and *Rhodococcus* spp. ([Figure S1g](#pone.0070887.s001){ref-type="supplementary-material"}). *Bacillus* spp. were common in all three species of snow crabs. *Pseudomonas* spp. (n = 68 isolates) were predominantly found in *C. opilio*, while only three and two bacterial species were isolated from the other two *Chionoecetes* spp. ([Figures 1](#pone-0070887-g001){ref-type="fig"} and [S1a](#pone.0070887.s001){ref-type="supplementary-material"}). *Acinetobacter* spp. were predominant in *C. opilio* and *C. japonicus* (53 and 18 isolates, respectively), and only one bacterial isolate was found in *Chionoecetes* sp. ([Figures 1](#pone-0070887-g001){ref-type="fig"} and [S1b](#pone.0070887.s001){ref-type="supplementary-material"}). *Stenotrophomonas* spp. dominated in *C. opilio* (62 isolates), while only one bacterial species from this genus was isolated from each of *C. japonicus* and *Chionoecetes* sp. ([Figures 1](#pone-0070887-g001){ref-type="fig"} and [S1c](#pone.0070887.s001){ref-type="supplementary-material"}). Numbers of *Bacillus* spp. isolated included nine from *C. opilio*, nine from *C. japonicus*, and 26 from *Chionoecetes* sp. ([Figures 1](#pone-0070887-g001){ref-type="fig"} and [S1d](#pone.0070887.s001){ref-type="supplementary-material"}).
In *C. opilio*, 67% (nine isolates) of *Bacillus* spp. were localized in the heart, while 33% (nine isolates) were found in the gill and carapace in *C. japonicus*. *Bacillus* spp. from *Chionoecetes* sp. were found in the carapace juices and heart (26% and 30% of 26 isolates respectively). Some bacterial isolates were confined to particular parts of *Chionoecetes*. Some isolates were not amplified using 16S rRNA gene sequencing; therefore, *Rhodotorula* (one strain) and *Pichia* (three strains) were identified from the gills of *Chionoecetes* sp. using ITS gene sequencing.
Enumeration of total cultivable bacteria {#s3b}
----------------------------------------
As shown in [Figure 2](#pone-0070887-g002){ref-type="fig"}, aerobic bacteria counts numbered from 10^3^ to 10^5^ cells/g in gills and from 10^2^ to 10^3^ cells/g in carapace meat in the three snow crab species on TSA, R2A, MA, and NA media, whereas no aerobic bacteria or other prokaryotes appeared on YPD or PDA media ([Figures 2A and 2B](#pone-0070887-g002){ref-type="fig"}). In comparison with the other organs, gills of the three crab species contained relatively high aerobic bacterial populations (up to 10^5^) on TSA, R2A, MA and NA ([Figures 2A and 2C](#pone-0070887-g002){ref-type="fig"}).
{#pone-0070887-g002}
Enumeration of antibiotic-resistant bacteria {#s3c}
--------------------------------------------
More than 50% of the 221 *C. opilio* isolates were resistant to ampicillin, erythromycin, penicillin, ticarcillin, and vancomycin. Isolates from *C. japonicus* and *Chionoecetes* sp. were resistant to the nine antibiotics tested, representing a 15% and 30% resistance ratio respectively ([Figure 3](#pone-0070887-g003){ref-type="fig"} and [Table 2](#pone-0070887-t002){ref-type="table"}). In *C. opilio*, *Pseudomonas*, *Acinetobacter*, *Enterobacter*, *Psychrobacter*, *Stenotrophomonas*, and *Lactobacillus* spp. were resistant to the nine antibiotics tested (19 isolates); *Pseudomonas*, *Acinetobacter*, and *Stenotrophomonas* spp. were resistant to seven or eight of the nine antibiotics (12 isolates; Figures S1a, S1b, S1c and [Table 2](#pone-0070887-t002){ref-type="table"}). The MDR bacteria identified in our study were affiliated with *Agreia* and *Psychrobacter*; two isolates from *C. japonicus* (red-tanner crab; [Figure S1f](#pone.0070887.s001){ref-type="supplementary-material"} and [Table 2](#pone-0070887-t002){ref-type="table"}) and nine genera (14 isolates) from *Chionoecetes* sp. were affiliated with *Shewanella*, *Rhodococcus*, *Agrococcus*, *Leifsonia*, *Deinococcus*, *Staphylococcus*, and *Agreia* bacteria, and with *Rhodotorula* and *Pichia* yeasts ([Figure S1g](#pone.0070887.s001){ref-type="supplementary-material"} and [Table 2](#pone-0070887-t002){ref-type="table"}).
{#pone-0070887-g003}
10.1371/journal.pone.0070887.t002
###### The media used to isolate bacterial strains displayed the relationship of multidrug resistance (MDR) in bacteria from snow crab.
{#pone-0070887-t002-2}
Medium
------------------------------------------ -------------------- --------------------------------------------------------------------------- -------- ---------------------------------------- ------- ------- ------- -------
*Pseudomonas* 1 (1)[\#](#nt103){ref-type="table-fn"} 1 (0) 5 (2)
*Acinetobacter* 6 (6) 4 (3)
*Stenotrophomonas* 1 (1) 1 (0) 7 (1)
*C. opilio* *Psychrobacter* γ[7)](#nt110){ref-type="table-fn"} (GN)[11)](#nt114){ref-type="table-fn"} 1 (1)
(19/31)[\*](#nt102){ref-type="table-fn"} *Enterobacter* 1 (1)
*Leclercia* 1 (1)
*Lactobacillus* B[8)](#nt111){ref-type="table-fn"}(GP)[12)](#nt115){ref-type="table-fn"} 1 (1) 1 (1)
*C. japonicus* *Salinibacter* A[9)](#nt112){ref-type="table-fn"} (GP) 1 (1)
(2/2) *Psychrobacter* γ (GN) 1 (1)
*Agrococcus* 2 (2)
*Salinibacter* A (GP) 1 (1) 1 (1) 1 (1)
*Chionoecetes* sp. *Rhodococcus* 2 (2) 1 (1)
(14/14) *Deinococcus* D[10)](#nt113){ref-type="table-fn"} (GP) 1 (1)
*Staphylococcus* B (GP) 1 (1)
*Shewanella* γ (GN) 1 (1)
*Pichia* Yeast 1 (1) 1 (1)
*Rhodotorula* Yeast 1 (1)
(Numbers of nine antibiotic-resistant bacteria among more than seven antibiotic-resistant bacteria).
Parentheses indicated all resistant bacteria to tested nine antibiotics.
NA : Nutrient Agar,
YPD : Yeast Extract Peptone Dextrose,
PDA : Potato Dextrose Agar.
R2A Agar : Reasoner\'s 2A agar,
TSA ; Tryptic Soy Agar,
MA : Marine Agar.
γ : gamma Proteobacteria,
B : Bacilli,
A : Actinobacteria,
D : Deinococcus,
GN : gram-negative bacteria,
GP : gram-positive bacteria.
Phylogenetic distribution {#s3d}
-------------------------
Phylogenetic analysis revealed that isolates within *Pseudomonas* and *Stenotrophomonas* were grouped separately, representing 66 and 62 isolates from *C. opilio* (snow crab, C), while two and three isolates from *C. japonicus* (red-tanner crab, CJ) and *Chionoecetes* sp. (Neodo-Daege, B) were represented (Figures S1a and S1c). For *Acinetobacter* spp., 49, 14, and one isolate were found in *C. opilio*, *C. japonicus*, and *Chionoecetes* sp., respectively ([Figure S1b](#pone.0070887.s001){ref-type="supplementary-material"}). In *C. japonicus* three clusters formed with high similarly to *A. johnsonii* DSM 6963, *A. haemolyticus* DSM 6962, and *A. guillouiae* ATCC 11171 ([Figure S1b](#pone.0070887.s001){ref-type="supplementary-material"}). Up to 26 *Bacillus* spp. were isolated from *Chionoecetes* sp. and nine *Bacillus* spp. isolates were found in each of *C. opilio* and *C. japonicus* ([Figure S1d](#pone.0070887.s001){ref-type="supplementary-material"}). These isolates showed similarity with several comparative groups, suggesting that the relationship between the species of *Chionoecetes* and the isolated *Bacillus* spp. was not strong ([Figure S1d](#pone.0070887.s001){ref-type="supplementary-material"}). Other actinobacteria were found in *Chionoecetes* sp. and *C. japonicus* (34 and 21 isolates, respectively). Interestingly, 10 isolates of *Salinibacterium* from *C. japonicus* were clustered together (Figures S1f and S1g). Isolates from *Chionoecetes* sp. were affiliated with *Microbacterium* (eight isolates), *Rhodococcus* (nine isolates), and *Deinococcus* (two isolates; [Figure S1g](#pone.0070887.s001){ref-type="supplementary-material"}). Proteobacterial taxa were affiliated with 16 isolates of γ-proteobacteria from a total of 17 isolates from *C. japonicus* ([Figure S1f](#pone.0070887.s001){ref-type="supplementary-material"}). Isolates from *C. opilio* were affiliated with α- (seven isolates), β- (five isolates), and γ-proteobacteria (nine isolates; [Figure S1e](#pone.0070887.s001){ref-type="supplementary-material"}). Bacteroidetes taxa from *C. japonicus* were affiliated with *Flavobacterium* (two isolates) and *Chryseobacterium* (one isolate) in *Chionoecetes* sp. Bacilli, excluding the *Bacillus*, were isolated from *C. opilio* (five isolates), *C. japonicas* (five isolates), and *Chionoecetes* sp. (six isolates). Specifically, *Exiguobacterium* was isolated from *C. japonicus* ([Figure S1f](#pone.0070887.s001){ref-type="supplementary-material"}).
PCR detection of antibiotic resistance genes {#s3e}
--------------------------------------------
We tested the antibiotic multiresistant bacteria, i.e., *Acinetobacter* spp., *Leclercia* sp., *Pseudomonas* spp., *Stenotrophomonas* spp., *Lactobacillus* spp., and *Bacillus* sp. for the detection of antibiotic resistance genes, i.e., *bla~SHV~*, *bla~OXA~*, and *bla~TEM~* as ampicillin resistance genes; *catA1*, *catA2*, *catA3*, and *catB3* as chloramphenicol resistance genes; *ermA*, *ermB*, and *mefA* genes as erythromycin resistance genes; *pbp2a* as penicillin resistance gene; *aphA-3* as kanamycin resistance genes; *tetA*, *tetB*, *tetC*, and *tetG* as tetracycline resistance genes; and *vanA* and *vanB* as vancomycin resistance genes. The *catA1* gene was detected in all the tested bacteria. However, resistance genes against ampicillin, erythromycin, penicillin, and kanamycin were not found in the tested bacterial isolates. Interestingly, *Leclercia* sp. possessed *catB* and *Pseudomonas* sp. possessed *tetB*. Vancomycin resistance gene, *vanB* was detected in *Pseudomonas* spp. and *Stenotrophomonas* spp ([Table 3](#pone-0070887-t003){ref-type="table"}). Sequencing analysis of the PCR products showed that the sequences of *catA1* gene were identical, with 100% nucleotide homology in the tested isolates except for the gene of *Leclercia* spp. The *catA1* sequence of *Acinetobacter* sp. C-G-MA6 (AB826491) and *Pseudomonas* sp. C-D-MA7 (AB826493) showed that the gene represented 99% and 100% nucleotide identity to an antibiotic resistance gene of *Klebsiella pneumonia* subsp. *pneumonia* KPX plasmid pKPX-1 DNA. Moreover, amino acid sequences translated from the nucleotide sequences of the PCR products showed 100% identity with the amino acid sequence of chloramphenicol acetyltransferase. This result indicates that *catA1* gene is derived from the chloramphenicol resistance gene detected in several pathogenic bacteria.
10.1371/journal.pone.0070887.t003
###### The specific resistance of the multidrug resistance (MDR) bacterial strains.
{#pone-0070887-t003-3}
Isolates Genera Group Resistance phenotype PCR detection
---------------------------- ------------------ -------------------- ------------------------------------- ----------------------------------------- ---------------
C-D-PYD4 *Acinetobacter* GN[1)](#nt116){ref-type="table-fn"} Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
[C-G-MA6]{.ul} *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1*
C-G-MA4 *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-G-PYD9 *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-J-PYD3 *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-LS-MA1 *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-LS-PYD3 *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
[C-S-PYD1]{.ul} *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1*
C-S-PYD3 *Acinetobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-S-MA2 *Acinetobacter* GN Am, Chl, Em, Pen, Tet, Tc, Van
[C-G-MA1]{.ul} *Leclercia* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1, catB*
[C-D-MA4]{.ul} *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1, tetB*
[C-LS-PYD4]{.ul} *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1*
C-LS-MA4 *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
***C. opilio*** C-S-MA1 *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Tet, Tc, Van
C-S-MA7 *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Tet, Tc, Van
[C-D-MA7]{.ul} *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Tet, Tc, Van *catA1, vanB*
C-D-TSA1 *Pseudomonas* GN Am, Chl, Em, Pen, Rif, Te, Van
[C-G-MA5]{.ul} *Stenotrophomonas* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1, vanB*
[C-S-PYD2]{.ul} *Stenotrophomonas* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1*
C-LS-MA2 *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van
C-LS-MA5 *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van
C-LS-MA7 *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van
C-J-MA2 *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van
C-J-MA5 *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van
[C-G-MA2]{.ul} *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van *catA1*
C-S-PDA4 *Stenotrophomonas* GN Am, Em, Pen, Km, Tet, Tc, Van
C-J-MA7 *Enterobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-G-MA3 *Psychrobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
C-G-TSA3 *Lactobacillus* GP[2)](#nt117){ref-type="table-fn"} Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
[C-LS-PDA4]{.ul} *Lactobacillus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van *catA1*
***C. japonicus*** CJ-G-NA9 *Salinibacterium* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
CJ-S-NA3 *Psychrobacter* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-NA3 *Rhodococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-NA8 *Rhodococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-R2A1 *Rhodococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-R2A7 *Rhodococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-NA4 *Agrococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-NA10 *Agrococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
***Chionoecetes*** **sp.** B-G-NA5 *Leifsonia* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-R2A5 *Leifsonia* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-NA11 *Deinococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-R2A2 *Staphylococcus* GP Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-TSA8 *Shewanella* GN Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-NA7 *Pichia* Yeast Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-PYD12 *Pichia* Yeast Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
B-G-TSA1 *Rhodotorula* Yeast Am, Chl, Em, Pen, Rif, Km, Tet, Tc, Van
GN : Gram negative bacteria,
GP : Gram positive bacteria,
Abbreviations used: Am, ampicilline; Chl, chloramphenicol; Em, erythromycin; Pen, penicillin; Rif, rifampicin; Km, kanamycin;
Tet, tetracycline; Tc, ticarcillin; Van, vancomycin.
The underline indicates tested isolates for detection of MDR genes by PCR.
GN : gram-negative bacteria, GP : gram-positive bacteria.
Nucleotide sequence accession numbers {#s3f}
-------------------------------------
All sequences were deposited in GenBank under the following accession numbers: HM755454--HM755674 (*C. opilio*), HM584223--HM584298 (*C. japonicus*), HM629343--HM629422, and HM588762--HM588765 (*Chionoecetes* sp.).
Discussion {#s4}
==========
Until recently, the study of *Chionoecetes* was conducted mainly by artificial cultivation for the examination of disease [@pone.0070887-Hoskin1], [@pone.0070887-Benhalima1]. In contrast, the present study was performed to characterize the types of microbiota within *Chionoecetes*, as little is known about microbial dynamics within *Chionoecetes* [@pone.0070887-Hoskin1], [@pone.0070887-Benhalima1]. In this study, we provided ratios of antibiotic resistance and microbial community distribution in three *Chionoecetes* species. Our results indicate that future microbial studies of *Chionoecetes* in their natural ecosystems are necessary to assess and monitor potential human risk.
This study revealed four genera prevalent in *Chionoecetes*: *Pseudomonas*, *Acinetobacter*, *Stenotrophomonas*, and *Bacillus* ([Figure S1](#pone.0070887.s001){ref-type="supplementary-material"}). Microbial diversity was high in the gills of *C. opilio* ([Figure 2C](#pone-0070887-g002){ref-type="fig"}). Schuwerack et al. [@pone.0070887-Schuwerack1] reported that bacterial colonies enmeshed in polysaccharide-like films produced indentations in the gill cuticular surfaces and dissociation of microvillus membranes at the basal zone of epithelial cells of gill lamellae of the fresh crab *Potamonautes warren*.
The yeasts *Rhodotorula* and *Pichia, which* were identified from *Chionoecetes* sp. *Rhodotorula*, *Cryptococcus*, *Torulopsis*, *Candida*, *Trichosporon*, and *Aureobasidium*, have previously been isolated from the meat of Dungeness (*Cancer magister*) and King crabs (*Paralithodes camtschatica*) [@pone.0070887-Eklund1]. The discovery of yeast in *C. opilio* in the present study, as well as in Dungeness and King crabs [@pone.0070887-Eklund1], suggests that future ecological studies of yeast populations will be necessary as well.
While several studies show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressures of clinical antibiotics and agricultural use of antibiotics [@pone.0070887-Allen1]--[@pone.0070887-Thaller1], human activity has probably increased the prevalence of MDR bacteria in air, soil, and marine and freshwater ecosystems. Most antibiotic resistance genes are acquired through horizontal gene transfer [@pone.0070887-Forsberg1]. In this study, MDR bacteria from *Chionoecetes* demonstrated antibiotic resistance in nonclinical environments, suggesting an ecological role for antibiotics that warrants additional investigation.
Some *Bacillus* spp. (e.g., *Bacillus cereus*) are ubiquitous in nature and constitute a major portion of the microbial populations in contaminated food, causing food spoilage and poisoning to the detriment of the consumers [@pone.0070887-Okanlawon1]. Two *Bacillus* species are considered medically significant: *B. anthracis*, which causes anthrax, and *B. cereus*, which causes a food-borne illness [@pone.0070887-Okanlawon1]. Because we found *Bacillus* spp. in all *Chionoecetes*, the incidence and survival of *Bacillus* spp. is thought to be controlled by cooking *Chionoecetes* at high temperatures prior to consumption.
In terms of antibiotic-resistant *Acinetobacter* spp., the bacteria were found in *C. opilio* and *C. japonicus*; infections generally occur in hospitalized patients with weakened immune systems. Therefore, understanding antibiotic resistance in *C. opilio* isolates is clinically important for cases involving multidrug resistance (MDR) [@pone.0070887-BergogneBerezin1]. In this study, 54 isolates of *Acinetobacter* spp. were isolated from *C. opilio*, more than 60% of which were resistant to at least one of the antibiotics tested. Antibiotic resistance was high not only for the dominant *Pseudomonas*, *Acinetobacter*, and *Stenotrophomonas* (γ-proteobacteria), but was also high regardless of whether bacterial strains were gram negative or gram positive. γ-Proteobacteria demonstrated high antibiotic resistance to isolates from *C. opilio*, while the actinobacteria of *Chionoecetes* sp. were resistant to all of the nine antibiotics tested ([Table 2](#pone-0070887-t002){ref-type="table"}). Here, we revealed that actinobacteria were commonly isolated from *Chionoecetes* sp., including many multidrug-resistant strains ([Table 2](#pone-0070887-t002){ref-type="table"}). Moreover, clinical reports of secondary urinary or respiratory infections by *Pseudomonas* and *Enterobacter* spp. have been presented [@pone.0070887-Schaberg1], [@pone.0070887-Richards1]. While isolates of snow crabs rarely infect respiratory organs or the skin, proper heating of food prepared from *Chionoecetes* must be ensured in order to protect against infection.
Similar results for MDR bacteria have been observed in shrimp [@pone.0070887-Nawaz1]--[@pone.0070887-Sajjad1], chicken [@pone.0070887-Glenn1]--[@pone.0070887-Schwaiger2], fruit [@pone.0070887-Schwaiger1], vegetables [@pone.0070887-Schwaiger1], pork [@pone.0070887-Schwaiger1], [@pone.0070887-Schwaiger2], salad [@pone.0070887-Bakri1], drinking water [@pone.0070887-Defives1], fish [@pone.0070887-Ghosh1]--[@pone.0070887-VernerJeffreys1], and fish farms [@pone.0070887-Shah1], [@pone.0070887-Su1]. These commonalities also suggest that MDR bacteria should be investigated from many samples following standard methods described by the National Committee for Clinical Laboratory Standardization [@pone.0070887-Clinical1]. It is also necessary to replicate studies of microbiota and the inhibition zone diameter.
The results of the present genetic study showed that the *catA1* gene is widespread in many bacteria ([Table 3](#pone-0070887-t003){ref-type="table"}). These data indicated that this gene moved between species via horizontal gene transfer. However, whether the multiresistance of the bacteria could be derived from intrinsic characteristics of bacteria or from unknown mechanisms (e.g., uncharacterized specific genes and dissemination through unknown transposable elements) is an open question. Therefore, we suggest that further studies are necessary to elucidate whether the resistance gene of snow crabs is intrinsic or arises from horizontal gene transfer between the environmental and pathogenic resistomes. Additional research is required to determine how resistance genes become incorporated into a range of bacteria species. In the future, it is essential that the implications of MDR for human consumption of snow crabs be entirely understood and that the penetration of antibiotic resistance into natural environments be prevented.
In summary, we revealed for the first time a high level of microbial infiltration or inclusion in the internal organs of three *Chionoecetes* species. In addition, we isolated 381 microbial strains from three species of *Chionoecetes* spp.; unexpectedly, microbes with antibiotic resistance are widely distributed throughout the internal organs of wild, commercial snow crabs. In the future, additional research on antibiotic resistance and its mechanism and on microbial dynamics in the fishery industry will enhance further understanding of the clinical and ecological implications of these results.
Supporting Information {#s5}
======================
######
**Phylogenetic tree of dominant bacteria in three species of snow crab.** (a): *Pseudomonas* spp., (b): *Acinetobacter* spp., (c): *Stenotrophomonas* spp., (d): *Bacillus* spp., (e): the other of *C. opilio*, (f): the other of *C. japonicus* crab, (g): *Chionoecetes* sp. Bootstrap values represent the percentage of 1,000 replicates. Box indicates resistance to more than seven antibiotics. Dark star symbol (★) in boxes indicates resistance against tested nine antibiotics, light star symbol (☆) against eight antibiotics, and box without symbol against seven antibiotics. C: *Chionoecetes opilio*, CJ: *C. japonicus*, B: *Chionoecetes* sp.
(TIF)
######
Click here for additional data file.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: JSK NHP CGK. Performed the experiments: MSK THK SYJ SMJ. Analyzed the data: JSK SHC. Contributed reagents/materials/analysis tools: JSK. Wrote the paper: JSK.
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{#F1}
Dr Yvonne Edmonstone -- who died of breast cancer on 17 September 2015 at the age of 52 -- was the clinician responsible for the creation of the National Health Service (NHS) Highland Eating Disorder Service and one of the founder members of the North of Scotland Managed Clinical Network for Eating Disorders. In 2009 she helped pave the way for the opening of the Eden Unit, Scotland\'s first NHS in-patient unit for adults with eating disorders. As well as her enormous contribution to the field on a local and regional level, she was a key player on a national scale, as part of the core group which set up Eating Disorders Education and Training Scotland, and as a long-standing contributor to the Scottish Eating Disorders Interest Group. On her retirement, grateful patients produced a tribute video and -- in appreciation of her contribution to the field -- she received an award from BEAT, the UK charity supporting people with eating disorders.
Although the area of eating disorders was where she focused most of her professional energy, Yvonne had a broad interest in the application of psychotherapy to mental disorder. She was a valued supervisor for the South of Scotland Cognitive Behavioural Therapy Course and an early adopter of the interpersonal psychotherapy (IPT) model, her case reports being preserved as exemplars by IPT course accreditors. Yvonne was also a keen advocate of dialectical behaviour therapy and a key member of the Highland team working in that field. She was an expert psychotherapist whose advice was sought by other clinicians because of her impressive clinical acumen and compassionate, common-sense approach. As well as her clinical commitments, she contributed to more academically focused endeavours. She co-authored a chapter on psychotherapy in the award-winning manual Research Methods in Psychiatry^[@R1]^ and was an author of the Scottish Intercollegiate Guidelines Network guideline on the non-pharmaceutical management of depression in adults.
Born on 20 March 1963, Yvonne, as a schoolgirl, was torn between her artistic talents and passion for becoming a doctor. In adult life her creativity expressed itself not only through her psychotherapeutic practice but also in her talent for organising (and participating in!) social events. She enthusiastically planned and enjoyed holidays, lunches and trips to music festivals with family and friends until only weeks before her death.
Yvonne undertook dual training in general adult psychiatry and psychotherapy. She qualified in 1985 with an MBChB from the University of Aberdeen. Her special interest in eating disorders involved both clinical and research work in Edinburgh\'s Cullen Centre. On completion of her training, she moved back to her beloved Highlands, where she poured her heart and soul into building the Highland Eating Disorder Service. She became a Fellow of the Royal College of Psychiatrists in 2014.
Humane and thoughtful, Yvonne was held in the highest regard and affection by colleagues and patients alike. Her easy, yet infectiously enthusiastic manner and sense of humour were valued not only by those who knew her professionally, but also by everyone who came into contact with her socially.
Yvonne demonstrated extraordinary generosity of spirit, both professionally and personally. She was someone who really would go the extra mile. She encouraged, mentored and otherwise inspired many clinicians from different professional backgrounds to fulfil their potential. She always had time for her colleagues despite her frenetic schedule, while her engaging nature brightened many a dull day at work and many a social occasion besides.
Yvonne\'s impact on local, regional and national psychotherapy and eating disorders services cannot be underestimated. However, her greatest legacy is her beloved family -- the four children she raised with her husband Alistair and the close relationship she enjoyed with her parents. Her ability to maintain such a truly productive work--life balance is an example for us all. She is sadly missed.
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{#sp1 .266}
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Background {#s1}
==========
To start with, this disease is rare; the treatment was endovascular stenting, which is relatively a new approach, so it is important to know about the efficacy of this procedure in such scarce cases. After all, case reporting may be the only way to have an evidence-based approach for such rare cases.
Case presentation {#s2}
=================
A 38-year-old, otherwise healthy, woman presented with increasing mass of her right glutei for the last 3 months.
The patient reported a mild asymmetry between the two glutei since childhood, but after a moderate trauma (the patient fell on her buttocks), she noticed marked increase and mild discomfort of the affected side. No neural or sciatic nerve neuropathic symptoms.
Investigations {#s3}
==============
Ultrasound study showed heterogeneous mass which is high in vascularity possible liposarcoma.
MRI pelvis suggested cavernous haemangiosarcoma versus haemangioma ([figures 1--4](#F1 F2 F3 F4){ref-type="fig"}).
{#F1}
{#F2}
{#F3}
{#F4}
Angiogram ([figures 5--9](#F5 F6 F7 F8 F9){ref-type="fig"}).
{#F5}
{#F6}
{#F7}
{#F8}
{#F9}
Differential diagnosis {#s4}
======================
Soft-tissue sarcoma.
Treatment {#s5}
=========
Endovascular intervention.
Coils ([figures 10 and 11](#F10 F11){ref-type="fig"}) and stenting images ([figures 12--14](#F12 F13 F14){ref-type="fig"}).
{#F10}
{#F11}
{#F12}
{#F13}
{#F14}
Outcome and follow-up {#s6}
=====================
The patient had immediate relive of her discomfort. Within 1 week, she noticed around 50% reduction in the size of the mass. She shall have MRI film follow-up in 3 months.
Discussion {#s7}
==========
Persistent sciatic artery (PSA) is a rare vascular anomaly with estimated incidence of 0.03%--0.06%.
During early embryonic development, the sciatic artery (which usually supply fetal lower buds and caudal part) disappears when the superficial femoral artery develops properly and the lower limbs grow.
There are very few cases reported in literature around 45 cases from 1977 to 1994.[@R1] Most of these cases presented with catastrophic events such as acute limb ischaemia or gangrenous glutei. A systemic review reported 122 published patients collected in 43 years for the period 1964--2007. Ten cases in total had been treated by minimal endovascular techniques, all for stenotic lesions. Three to five cases were treated conservatively, one case with oral anticoagulant, less than 10% had regular surveillance for ischaemia and embolic manifestations. In total the amputation rate reached up to 22%.[@R2] In the Japanese literature, one case of PSA presented by gangrenous glutei.[@R3]
One may question the need for treatment for our case altogether. They may argue that femoral artery is present and it supplies the lower limb by popliteal artery. Nevertheless, we decided to intervene because: the sudden increase of the size, the discomfort feeling the patient experienced, the huge asymmetry which disfiguring, not to forget the big venous pool that may harbour clots (that my dislodge and endanger her life). Besides, the MRI showed the destruction of the fascia of the gluteus medius and maximus that may point on a pending rupture.
###### Patient's perspective
I was very pleased by both the diagnosis and how it was treated.
They told me in the beginning that this may be a type of cancer.
It turns to be related to a defect in my vessels since I was in the womb of my mom.
The treatment was with wires and needles introduced through my groin. They gave me local anaesthesia so I don't feel pain. I stayed overnight in the hospital.
Next morning I could notice the relief of the discomfort I used to have.
Within one week the mass regressed to less than half.
###### Learning points
- Interestingly the diagnosis was overlooked by MRI but easily picked up by conventional angiography.
- Endovascular procedures are proving their superiority on daily bases.
- Deferring the biopsy was a wise decision as it could have complicated the case and the diagnosis as well.
- This case questions if aneurysmal changes in persistent sciatic artery particularly are congenital or due to repeated trauma as it was thought earlier.
**Contributors:** HKA is the sole author of this manuscript.
**Funding:** The author has not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
**Competing interests:** None declared.
**Patient consent:** Obtained.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
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1. Introduction {#Section1}
===============
The loss of pRB function either by mutation or by association with viral oncoproteins is linked to uncontrolled cell proliferation and is accompanied by the release of transcription factors such as E2F-1 [@B1]-[@B5]. Over the past decade, the E2F family of transcription factors has emerged as central components of the regulatory machinery of cell growth and proliferation [@B6]. E2F DNA binding sites in the adenovirus E2 promoter were the first E2F binding sites to be identified [@B7].
Thereafter, E2F DNA binding sites have been identified in promoters of many genes intimately involved in the regulation of cell cycle progression. It was shown that around 1,000 genes are regulated by E2F-1 [@B8]-[@B10]. E2F proteins are encoded by at least eight genes, E2F-1 through E2F-8. While specific roles for individual E2Fs in mediating the effects of RB loss are emerging, it is also becoming clear that there are no simple divisions of labor among the E2F family. Instead, an individual E2F can function to activate or repress transcription, promote or impede cell cycle progression and enhance or inhibit cell death, dependent on the cellular context [@B6].
In the initial model of E2F-1 action, activation of E2F-1 dependent transcription was proposed to be caused by the unmasking of an activation domain after release of pRB in the E2F complex [@B2], [@B3]. Regarding the mechanism of E2F-mediated transactivation, it is believed that "free" E2F binds to its binding sites in the promoters of the target gene. So-called "free" E2F is the heterodimer formed between E2F family members and DP family members without making a complex with pRB or related proteins [@B11]. This view was supplemented by the discovery that E2F-1/pRB complexes can actively repress transcription [@B12] [@B13]. Therefore, there appears to be three generic types of E2F complexes: *activator E2F complexes ("free" E2F),* in which the E2F activation domain promotes transcription, *inhibited E2F complexes*, in which the activation domain is masked by pRB-family proteins to give a complex that is essentially inert, and *repressor E2F complexes*, in which RB family proteins that are recruited to the DNA by E2F assemble a repressor activity [@B5].
The discovery of the E2F-1/pRB repressor has raised a debate that is still not completely settled. Should E2F binding sites be thought of as activators of gene expression in S phase or as elements that confer cell-cycle regulated repression in G~0~/G~1~? Mutational analysis of putative E2F-binding sites in relatively small fragments of genomic DNA suggests that E2F-binding sites regulate gene expression in different ways in different promoter contexts [@B11] [@B14], [@B15]. For example, promoter mutation experiments have shown that alteration in the E2F-1 binding sites of the c-myc, cdc2 and human DHFR promoters resulted in 50-90% loss of transcription activity [@B16] [@B17], [@B18]. Here the E2F-1-binding sites appear to act as activator elements during the S phase of the cell cycle. Release of E2F-1 from pRB results in activation of gene expression during the S phase of the cell cycle. In contrast to the experiments described above, similar mutations of these sites in the promoters of other genes, such as b-myb [@B19] and mouse DHFR [@B20] caused increased transcriptional activity. Here the E2F-1-binding sites appear to act as repressor elements during G~0~/G~1~and gene expression results from the release of repression. It is still unclear how active repression by the E2F-1/pRB complex contributes to control of transcription, compared to pRB-mediated inhibition of transcriptional activation by free E2F-1 on individual target gene expression.
In this study, cell lines overexpressing E2F-1/wt and E2F-1/411, which doesn\'t complex with pRB [@B21], [@B22], were used to better understand the role of E2F-1/pRB interaction on cell phenotype and gene expression. This paper presents a novel finding that the E2F-1/pRB complex acts in different ways not only dependent on the individual target genes, but also on the same target genes in cycling cells as compared to cells re-entering the cell cycle. The findings show that E2F-1 acts as a part of the repression complex with pRB on the expression of DHFR, b-myb, TK and cdc2 in cycling cells; on the other hand, E2F-1 acts as an activator on the expression of the same genes of cells that are re-entering the cycle. DHFR, b-myb, TK and cdc2 are also actively growth regulated by E2F-1, and free E2F increased the expression of these target genes in all stages of the cells re-entering the cycle. However, c-myc and cyclin D1 are not significantly growth regulated by E2F-1 in any stage of the cell cycle. Rb is also growth regulated by E2F-1. Interestingly, free E2F-1 acts as a repressor of Rb expression at G~1~ and as an activator at S phase of the cell cycle.
2. MATERIALS AND METHODS {#Section2}
========================
2.1. Cells and viruses {#Section2.1}
----------------------
ψ-CRE, a murine fibroblast cell line was used in these experiments. The cells were grown in DMEM supplemented with 5% (vol/vol) fetal bovine serum and 5% calf serum in a 5% CO~2~ atmosphere at 37^0^C [@B23]. Retroviral vector, Linker Neo CMV E2F-1 was used to express E2F-1/wt and E2F-1/mutant genes as described below. Linker Neo CMV E2F-1 is identical to Linker CMV T [@B24]. except that the large T antigen gene from simian virus 40 was replaced by a cDNA, approximately 1326 bps long, encoding E2F-1/wt [@B25] or E2F-1/Y411A [@B2] (obtained from Dr. K. Helin). pX17 is a retroviral vector [@B24], [@B26] that encodes the neomycin resistance gene but does not encode E2F-1 gene. pX17 was used as negative control in the experiments. To produce infectious virus, the recombinant plasmids encoding Linker Neo CMV E2F-1s were transfected together with helper virus containing vector (SV-A-MLV-env) which encoded genes needed to complement the linker virus, but did not encode whole virus, [@B27] into Cos-7 packaging cells using the calcium-phosphate transfection method [@B24]. Two to three days later, virus containing medium was collected from these cells and used to infect established ψ-CRE murine cell lines [@B23].
2.2. Antibodies {#Section2.2}
---------------
The antibodies used in immunoblotting for detection of E2F-1, were E2F-1 (C-20), cdcp34 (cdc2; B-6), cyclin D1 (HD 11), c-myc (9E10) Santa Cruz Biotechnology, respectively. The antibodies used for immunoprecipitation of E2F-1 and pRb were KH95 (Santa Cruz Biotechnology) and G3-245 (Pharmingen; San Diego, CA) monoclonal antibodies, respectively
2.3. Immunoblotting {#Section2.3}
-------------------
For Western blotting, cells were lysed by addition of 1 ml of lysis buffer (50 mM Tris \[pH 8.0\], 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 1 mM PMSF and 0.01 mg of aprotinin per ml to the cell monolayer. Cell free extracts prepared from 4x10^4^ cells were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to Hybond ECL membranes (Amersham; Arlington Heights, IL). Antibodies described above were used for immunoblotting.
2.4. Immunoprecipitation {#Section2.4}
------------------------
Immunoprecipitations were performed using cell-free extract prepared as described in the immunoblotting procedure. The extracts were precleared by incubation with Protein G Plus-/Protein A-Agarose beads (Oncogene Science; Uniondale, NY) that had been treated with BSA. E2F-1 or pRB was immunoprecipitated from precleared extracts by adding 2 µg of specific antibodies and 20 µl of BSA treated Protein G Plus-/Protein A-Agarose beads. This mixture was incubated for 15 hours and centrifuged as described above. The immune complexes were washed 4 times with lysis buffer and then analyzed by SDS-PAGE and immunoblotted as described above.
2.5. Soft agar assays {#Section2.5}
---------------------
Five or ten thousand cells were plated in 1 ml of top agar composed of DMEM supplemented with 5% fetal bovine serum and 5% calf serum and containing 0.38% Noble agar. Dishes were maintained at 37°C in a humidified 5% CO~2~atmosphere. After 3 weeks, colonies were either photographed or counted. For photomicrographs, dishes were stained with p-iodonitrotetrazolium violet and photographed. For counting, colonies were visualized with a microscope using low power magnification and those greater than 50 µm in diameter were counted. Six microscope fields per dish were counted and used to calculate the total number of colonies on the entire dish (based on dish surface area). Colony counts were obtained from four dishes each cell line for per experiment. The data are expressed as the means of counts from five independent experiments.
2.6. Cell cycle synchronization {#Section2.6}
-------------------------------
To synchronize cells by serum starvation, 5 x 10^5^ cells were seeded into 10-cm dishes. Forty-eight hours later, cells were washed with PBS and DMEM supplemented with 0.5% fetal bovine serum was then added. After 60 h, cells were stimulated into the cell cycle by addition of complete medium (DMEM supplemented with 5% (vol/vol) fetal bovine serum and 5% calf serum). Samples for total RNA preparation and flow cytometry were collected at time 0 and appropriate intervals thereafter as indicated.
2.7. Total RNA isolation {#Section2.7}
------------------------
RNA STAT-60 Total RNA (TEL-TEST, INC.; Friendswood, TX) isolation reagent kit was used and its protocol followed for total RNA isolation. Cells in culture dishes were homogenized with RNA STAT-60, followed by RNA extraction using chloroform. RNA was precipitated with 0.5 vol. of isopropanol followed by washing with 75% ethanol. The quality of the samples was checked electrophoretically, and quantification was done spectrophotometrically. Two independent RNA preparations were used in the experiment.
2.8. Primers used in the MRT-PCR {#Section2.8}
--------------------------------
All primers for E2F-1 target genes were designed with the Primer3: WWW primer tool program (<http://biotools.umassmed.edu/bioapps/primer3_www.cgi>) and are shown in the table [1](#T1){ref-type="table"}. Primers were made by IDT, Inc. (Coralville, IA).
2.9. Semiquantitative Multiplex RT-PCR {#Section2.9}
--------------------------------------
Multiplex RT-PCR was performed using Promega\'s Access RT-PCR system. RT-PCR was used first to determine the linear range of target genes. Second, the linear range of the internal standard was determined using β-actin and β-actin competimer (QuantumRNA β-actin internal standards, Ambion). Conditions for MRT-PCR were optimized after determining the β-actin and β-actin competimer ratio. PCR cycles were optimized so that the target gene would be in the linear range. Reactions contained AMV-RT and Tfl DNA polymerase, AMV/Tfl Reaction Buffer, dNTP mix, specific upstream and downstream primers, β-actin and β-actin competimer primers, MgSO4, and RNA template. The PCR machine was programmed as follows: 48^0^C for 45 minutes to synthesize first strand cDNA; 94^0^C for 2 minutes to denature template; 94^0^C 45 seconds for denaturation; at 60^0^C for 45 seconds for annealing; and 68^0^C for 1 minute for extension. The PCR products were run in a 1.5% agarose gel and the gel was stained with EtBr. EtBr signals were analyzed by computer assisted densitometry.
2.10. Fluorescence-activated cell sorting (FACS) analysis {#Section2.10}
---------------------------------------------------------
To analyze cellular DNA content, cells were fixed and stained [@B28]. Briefly, 10^6^ trypsinized cells were washed three times with PBS and then fixed by incubation with 50 μl of 0.125% paraformaldehyde at 37^0^C for 5 minutes. Then 450 μl of ice-cold methanol was added to the sample. The fixed cells were washed three times with 0.1% Triton X-100 in PBS and treated with RNase A (0.04 Kunitz units) for 30 minutes. The cells were then stained with 50 μg of propidium iodide per ml. Cell analysis was performed using a Coulter EPICS Elite EPS flow cytometer (Coulter Electronics; Miami, FL). The fraction of the total cell population present in each of the G~1~, S and G~2~/M cell cycle phases was obtained from DNA histograms by mathematical modeling using MPLUS software (Phoenix Flow Systems; San Diego, CA [@B28]).
3. Results {#Section3}
==========
3.1. Overexpression of E2F-1/wt and E2F-1/411 genes in ψ-CRE cell lines changes the cell growth phenotype differentially depending on interaction with pRB {#Section3.1}
----------------------------------------------------------------------------------------------------------------------------------------------------------
For overexpression of E2Fs, the mouse fibroblast ψ-CRE cell line was used since it had the highest amount of pRB protein among the cell lines C3H10T1/2, BALB/c-3T3 and NIH 3T3 (data not shown) and we wanted to examine interaction between pRB and E2F-1. Using retroviral vectors, three cell lines were established that overexpress E2F-1/wt, E2F-1/411 or the control, pX17 (the retroviral vector with no insert, negative control). Cell clones were pooled from cells growing in the presence of G418. Figure [1](#F1){ref-type="fig"}A shows immunoblots of the cells expressing E2F-1. Multiple closely-migrating protein bands were seen. These are differentially-phosphorylated forms of E2F-1 protein [@B29]. In addition, cells infected with pX17 were used as negative controls. These cell lines were used for the remaining experiments of this study. The E2F-1/411 pRB interactions were described previously *in vitro* and in the yeast two-hybrid system and a single mutation of E2F-1 411 site decreased binding of E2F-1 to pRb without affecting E2F-1 transcription activity [@B21]. Therefore it would be important to show E2F-1/411 pRB binding acts the same way in the cell. To test the *in vivo* affinity of the E2F-1/411 for pRB, E2F-1 was immunoprecipitated from extracts made from asynchronously growing E2F-1/wt or E2F-1/411 cell lines using an anti-E2F-1-specific antibody as explained in detail in Material and Methods. It was found that E2F-1/411 pRB binding is five fold less than E2F-1/wt pRB binding (Fig. [1](#F1){ref-type="fig"}B).
3.2. Overexpression of E2F-1/wt and E2F-1/411 changes the cell cycle of asynchronously growing cells and increased cell transformation {#Section3.2}
--------------------------------------------------------------------------------------------------------------------------------------
Overexpression of E2F-1 is sufficient to induce S-phase in most quiescent cells, as reported previously [@B30], [@B31]. However, cycling E2F-1 null mutant cells showed no difference in the cell cycle compared to cycling normal cells [@B32]. Therefore, we first determined the effects of E2F-1/wt and free E2F-1 in cycling cells using flow cytometry. It was found that the overexpression of E2F-1/wt and E2F-1/411 significantly decreased the cell number in G~1~ phase compared to the control (pX17) cells. Furthermore, E2F-1/Y411A showed fewer cells in G~1~ as compared to E2F-1/wt-expressing cells (Fig. [1](#F1){ref-type="fig"}C and D).
As shown in this and some other laboratories previously [@B33], [@B34], overexpression of E2F-1 increases the transformation of cells. Therefore we aimed to assess how free E2F-1 (E2F-1/411) would change the transformation efficiency compared to E2F-1/wt. Anchorage-independent growth of E2F-1-overexpressing cells was tested by plating the cells in soft agar medium and assaying the ability of the cells to form colonies. E2F-1/wt- and E2F-1/411- overexpressing cell lines showed significant increases in transformation, compared to the control (pX17) cell line. The mutant E2F-1/Y411A showed a significant increase compared to E2F-1/wt overexpressing cell line (Fig. [1](#F1){ref-type="fig"}E). Therefore, free E2F-1, unbound to pRB, decreased the G~1~ phase and increased the transformation of ψ-CRE cell more than E2F-1 bound to pRB.
3.3. Overexpression of E2F-1/wt and E2F-1/411 changed the target gene expression in asynchronously growing cells {#Section3.3}
----------------------------------------------------------------------------------------------------------------
It is likely that the different phenotypic changes that occurred in the E2F-1/wt and E2F-1/411-overexpressing cells are a result of changes produced by alterations in the expression of E2F-1 target genes. E2F-1 target genes, such as RB, c-myc, b-myb, DHFR, TK, cdc2, cyclin D and others, are important cell cycle regulators. Therefore, the effects of E2F-1/wt and E2F-1/411 on target gene expression using semiquantitative multiplex RT-PCR (MRT-PCR) were compared.
Results obtained from asynchronized proliferating cells showed that overexpression of E2F-1/wt repressed the expression of DHFR, b-myb, cyclin D1, cdc2, c-myc and TK (Fig. [2](#F2){ref-type="fig"}). This finding correlates well with most of the previous studies [@B35], [@B36]. In addition, DHFR, b-myb, cyclin D1, cdc2, c-myc and TK gene expression were less repressed in cell lines overexpressing E2F-1/Y411A compared to cell lines overexpressing E2F-1/wt. However, none of these genes in E2F-1/411-overexpressing cells had expression levels exceeding those of the control (pX17). Therefore these data imply that in the asynchronously growing cells for DHFR, b-myb, cdc2, TK and cyclin D1 expression, the functional consequence of the pRB-E2F-1 interaction is not that E2F-1 acts as activator and is repressed by pRB binding. Instead, E2F-1 might have a role as a protein helping the E2F-1/pRB complex possibly bind to the promoter and actively repress transcription.
From this follows that, first, if E2F-1 acts as activator, target gene expression would be higher in E2F-1/wt- overexpressing cells compared to that of controls (pX17). Secondly and more importantly, target gene promoters will have only free E2F-1 (unbound to pRB) in E2F-1/411- overexpressing cells. Therefore, if E2F-1 acts as activator, target gene expression levels would be higher than the controls, pX17. However, the data shows that free E2F-1 did not increase target gene expression more than the control levels, and it only decreased the repression of the E2F-1/pRB complex by replacing E2F-1/pRB complex on the promoter. Therefore, E2F-1 does not act as activator, but it helps pRB repress the promoter of target genes including DHFR, b-myb, cyclin D1, cdc2, and TK in the asynchronously growing cells.
Very different results were observed with RB expression (Fig. [2](#F2){ref-type="fig"}) from those described for the other target genes. The data show that E2F-1 acts as activator for the expression of RB. First, overexpression of E2F-1, even bound to pRB, increased the expression of RB; second, E2F-1/411 (unbound to pRB) increased RB expression more than did E2F-1/wt. These data, therefore, show that E2F-1 has different roles for the expression of different target genes and pRB has a role in controlling the E2F-1 effects on all target genes that were examined in asynchronously growing cells.
3.4. The E2F-1/wt and E2F-1/411 overexpression changes the protein expression of E2F-1 target genes in asynchronously growing cells {#Section3.4}
-----------------------------------------------------------------------------------------------------------------------------------
Next, to see the effects of E2F-1/wt and 411 on target genes at the level of protein, the protein levels of some of the E2F target genes used in previous experiments were checked. Western Blot analysis (Fig. [3](#F3){ref-type="fig"}) showed similar results to RNA levels from RT-PCR. E2F-1/wt decreased the protein levels of cyclin D1, cdc2, c-myc and increased the pRB level. E2F-1/411 overexpression showed less repression compared to E2F-1/wt on the level of cdc2, c-myc and cyclin D1, and increased the pRB level more than E2F-1/wt. Difference in protein expression in different cells is more remarkable compared to the RNA expression analysis. This difference might be related with the sensitivity of the techniques.
3.5. E2F-1/wt and E2F-1/411 change G~0~/G~1~ checkpoint controls and S-phase entry {#Section3.5}
----------------------------------------------------------------------------------
The previous experiment showed that overexpression of E2F-1 in cells decreased the percentage of cells in the G~1~ phase in cycling cells (Fig. [1](#F1){ref-type="fig"}). In this experiment the effect of overexpression of E2F-1/wt and free E2F-1 on cells re-entering the cell cycle was examined. E2F-1/wt and 411 showed significantly less accumulation in G~0~/G~1~, compared to the control (pX17) cells after serum starvation (Fig. [4](#F4){ref-type="fig"}A and B). Furthermore, E2F-1/411 showed less accumulation in G~0~/G~1~ as compared to E2F-1/wt-expressing cells, similar to the result with cycling cells. Fig. [4](#F4){ref-type="fig"}C shows the time course of S phase entrance of E2F-1/wt- and mutant E2F-1/411- after resupplying cells with serum. The E2F-1/411 mutant overexpressing cells showed the fastest S-phase entry compared to the other cell lines.
3.6. E2F-1 and pRB have different roles for the growth regulation of the different target genes {#Section3.6}
-----------------------------------------------------------------------------------------------
To better understand the function of free E2F-1 and the E2F-1/pRB complex on individual target gene expression in different stages of the cell cycle, growth regulation of chosen target genes was investigated in control, E2F-1/wt and E2F-1/411 overexpressing cell lines. Growth regulation of these genes was expressed as the ratio of the expression levels of the target gene in S phase to that in G~0~/G~1~ phase [@B20].
RNA was extracted from cell lines arrested in G~0~/G~1~. After serum stimulation, RNA was extracted at various times. Expression of E2F-1 target genes was measured by MRT-PCR. Subsequently, the growth regulation of target genes in E2F-1/wt and E2F-1/411 overexpressing cell lines was compared in Fig. [5](#F5){ref-type="fig"}.
Target genes, including b-myb, DHFR, cdc2, and TK, showed similar results for growth regulation (Fig. [5](#F5){ref-type="fig"}). For these genes, expression in control (pX17) cells was similar to that in cells expressing E2F-1/wt in both G~0~/G~1~ and S phase. In contrast, to that, expression of these genes in the E2F-1/411 overexpressing cells in G~0~/G~1~ was generally 3-4-fold higher than that of controls (pX17) and E2F-1/wt cells. In S phase, expression of DHFR and TK was increased as compared to pX17 and E2F-1/wt cells. Expression of c-myc was decreased in cells expressing E2F-1/wt in G~0~/G~1~ as compared to cells expressing E2F-1/411. For RB, expression in E2F-1/411 expressing cells was increased in S phase as compared to cells expressing E2F-1/wt (Fig. [5](#F5){ref-type="fig"}).
4. Discussion {#Section4}
=============
It has been known that E2F-1 can be a transactivator and a transrepressor depending on the target genes and cell lines. Here, it is shown that E2F-1 could be both transactivator and transrepressor on the same target gene expression in the same cell depending on the cell cycle. Although cell cycle phenotypic effects of E2F-1 are the same on both cycling cells and cells re-entering the cell cycle (Fig. [1](#F1){ref-type="fig"}C and [4](#F4){ref-type="fig"}A and B), the target genes we examined are regulated differently in re-entering cells compared to cycling cells. It was found that on DHFR, b-myb, cdc2 and TK expression, the functional consequence of the pRB-E2F-1 interaction is that E2F acts as activator and is repressed by pRB binding in cells re-entering the cycle. During the re-entry into the cell cycle, free E2F-1 increases the transcription levels of the same genes more than 3-4 fold compared to cells in which the E2F-1/pRB complex was present. However, free E2F-1 (E2F-1/411- overexpressing cells) only de-repressed the expression of DHFR, b-myb, cdc-2, Cyclin D1, C-myc and TK up to the control (pX17) level in asynchronously growing cells. In addition, while there was no significant effect of E2F-1/wt over control (pX17) on the expression of DHFR, B-myb, cdc-2, TK, cyclin D1 and RB in cells re entering the cell cycle, overexpression of E2F-1/wt significantly repressed the expression of the DHFR, B-myb, cdc-2, Cyclin D1, C-myc and TK in cycling cells. Therefore, E2F-1 acts as part of the repression complex for the the expression of the DHFR, B-myb, cdc-2, Cyclin D1, C-myc and TK in cycling cells and as transactivator in cells re-entering the cycle on the expression of DHFR, b-myb, cdc-2, and TK. This study shows that free E2F-1 increased the expression of B-myb, DHFR, cdc2 and TK in cells re-entering the cell cycle and E2F-1 overexpression de-repressed the expression of the same genes in cycling cells.
It may be assumed in this study that E2F-1 overexpression may replace other E2F proteins which have a role for repression of cell cycle at G~0~/G~1~. In contrast to the activator E2Fs, E2F4 and E2F5 lack NLS nuclear localization signals (NLS) [@B37]. So, without the association with pRb2/p130 and pRb/p107, E2F4 and E2F5 can no longer access the cell nucleus [@B38]. In addition to this mechanism, there is a second mechanism that completely eliminates the possibility of forming pRb2/p130-E2F4/5 complexes. This second mechanism contributed by Skp2, which is the ubiquitin ligase of pRb2/p130 and belongs to the SCF (Skp1, Cullin, F-box protein) family. Skp2 recognizes hyperphosphorylated pRb2/p130, ubiquitin-ligases it and, therefore, causes the quick removal of pRb2/p130 by proteosomes [@B39], [@B40]. At this point, the promoter regions become vacant for the activator E2Fs, which, in turn, express the set of gene to initiate the S phase entry [@B41].
It is obvious that E2Fs and related proteins are not the only factors related with gene expression related with the cell proliferation. Some other factors or changes are different in synchronously growing and asynchronously growing cells that give the differences for the target gene expression in cycling cells versus cells re-entering the cycle. As previous reports indicate, some other elements besides pRB might be involved in regulation of the target genes. For example, in the c-myc promoter, transcription factors Sp1, ATF1, ATF2 and a novel factor distinct from E2F mediates C-MYC promoter activation through its E2F element during exit from quiescence, [@B42]-[@B44] for the b-myb promoter, CHR (cell cycle gene homology regions), which is also found in some other target genes, and CDE (cell cycle-dependent elements) have been shown to be regulatory factors [@B45] effecting E2F regulation**.**
Fig. [6](#F6){ref-type="fig"} is proposed to explain the data obtained in these studies. For the target genes DHFR, b-myb, cdc-2, Cyclin D1, C-myc and TK, in cycling cells, the model shows pRB brought to the DNA by E2F-1, and repressing transcription. In this model, E2F-1 without pRB (free E2F) has no effect on expression of these targets. For DHFR, b-myb, cdc-2, and TK, in G~0~/G~1~, the functional consequence of the pRB-E2F-1 interaction is that E2F acts as activator and is repressed by pRB binding in cells re-entering the cycle. In here, repression is due to the effect of pRB in repressing the transactivation function of E2F (because transcription is increased in cells expressing E2F-1/411). Therefore, the *inhibited E2F complexes* model most accurately describes the results from this group, showing that E2F-1 bound to pRB does not stimulate expression of these target genes. But, when pRB cannot bind E2F-1 (in the E2F-1/411 cells) it does stimulate target gene expression.
Interestingly, this study showed that B-myb, DHFR, cdc2 and TK expression was higher in cells overexpressing E2F-1/411 than in cells overexpressing E2F-1/wt not only in the G~0~/G~1~ but also in the S phases. Therefore, the E2F-1/pRB complex also regulated these genes during S phase.
Previously it was known that pRB is phosphorylated during S phase in the cells overexpressing E2F-1/wt, and therefore E2F-1 would not be complexed with pRB. This implies that pRB would not have a function in the regulation of target genes in S phase. Our results contradict most of the previous studies in which it was shown that during S phase, the E2F-1 target gene promoter was not protected by a protein complex [@B46], [@B47]. However, results of this study showing that DHFR, cdc-2, and TK are regulated during S phase, albeit to a lesser extent than in G~0~/G~1~ are mostly consistent with the finding of Wells et al. [@B48], [@B49]. They showed with chromosomal immunoprecipitation that some of the genes have protein complexes on the E2F-1 sites not only at G~0~/G~1~ but also at S phase.
The E2F-1/pRB complex regulates c-myc only during G~0~/G~1~ phase, and no significant effect of the E2F-1/pRB complex on c-myc is seen during S phase. Since c-myc expression is lower in E2F-1/wt and E2F-1/411-overexpressing cells compared to control, *active repressor complex* model is the most appropriate for regulation of c-myc by the E2F-1/pRB complex and E2F-1 (Fig. [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}).
Although cyclin D1 showed growth regulation in both cell lines overexpressing E2F-1/wt and E2F-1/Y411A, the E2F-1/pRB complex does not play any role in this regulation.
Regulation of RB gene expression was more complex. Contrary to previous reports, our study showed that E2F-1 activates RB expression in asynchronously growing cells. The E2F-1/411-mutant-overexpressing cells showed that in G~0~/G~1~, free E2F-1 decreased RB expression, but the opposite happened in S phase**.** This result may indicate that the RB expression was controlled by E2F-1/pRB and E2F-1 was inhibited by pRB during S phase but not in G~0~/G~1~. This fits well with the role of pRB in the cell cycle. During G~0~/G~1~, a cell needs pRB to repress genes involved in cell proliferation. However, in S phase, pRB effect should be reduced on the target gene promoter so that gene expression may occur. This might be another way of regulating transcription-related proteins by pRB in S phase, distinct from pRB phosphorylation.
It is possible that some additional factors may be involved with the E2F-1/pRB complex. For example, Udvadia et al. showed that pRB and Sp1 increased the activation of cyclin D1 [@B50]. It was presented previously [@B50] that RB promoter also has Sp1 site on promoter close to one of the E2F-1 site and deletion of Sp1 site decreased the activity of the promoter. Although, not significantly, our data also shows that cyclin D1 expression in the E2F-1/wt- expressing cell is higher than that of E2F-1/411 similar to the pRB expression in the cells re-entering the cycle. These may suggest that pRB may be an activator of certain promoters that contain Sp1 sites. Similar finding was also reported previously by Wells et all [@B48].
These novel findings about interaction of E2F-1 -pRb complex at different cell cycle provide new insight into the understanding of the molecular mechanism of pRB-E2F-1 interaction. In addition, this finding may represents a new characteristic of the transcription factors, which may act differently at different part of the cell cycle.
{#F1}
{#F2}
{#F3}
{#F4}
{#F5}
{#F6}
######
Oligonucleotides used in this study
Primer n-mer sequence PCR fragment size (bp)
------------------ ------- ------------------------------------------- ------------------------
FBmyb (forward) 21 5\'-GCC AGT CAC CCT TTT AAC CGC-3\' 713
RBmyb (reverse) 23 5\'-CAA ATT TAC TCA GGT CGC ACC AA-3\'
FDHFR (forward) 21 5\'-GAC CAT TGA ACT GCA TCG TCG-3\' 452
RDHFR (reverse) 25 5\'-AAT CAA TTT CTG GGA AAA ACG TGT C-3\'
FcycD1 (forward) 23 5\'-CTG ACA CCA ATC TCC TCA ACG AC-3\' 647
RcycD1 (reverse) 22 5\'-AAA GAA AGT GCG TTG TGC GGT A-3\'
Fcdc2 (forward) 23 5\'-AGA GCG TTT GGA ATA CCG ATA CG-3\' 423
Rcdc2 (reverse) 24 5\'-TGT CCA AGT CAT CAA AGT ACG GGT-3\'
Fc-myc (forward) 23 5\'-ATA AAA GAA GCT TTT CGG GCG TT-3\' 508
Rc-myc (reverse) 22 5\'-GGC TGT ACG GAG TCG TAG TCG A-3\'
FTK (forward) 21 5\'-GTA TGC CAA AGA CAC GCG CTA-3\' 395
RTK (reverse) 22 5\'-ACA CGG AGT GAT ACT TGT CGG C-3\'
FRb (forward) 25 5\'-GCG ATA TAA ACT TGG AGT CCG ATT G-3\' 569
RRb (reverse) 24 5\'-GCG TGC ACA GAG TGT ATT TAG TCG-3\'
[^1]: Conflict of Interest: There are no conflicts of interest and financial disclosure in the subject matter of this paper.
| {
"pile_set_name": "PubMed Central"
} |
In recent years, transarterial radioembolization (TARE) with Yttrium-90 (Y90) has emerged as a technique for treating malignant neoplasms in the liver. Compared with other locoregional therapies, such as transarterial chemoembolization (TACE), patients who underwent TARE with Y90 have higher tumor response rates and better outcomes. Moreover, no significant treatment-related complications or treatment-related deaths have been reported \[[@goz074-B1]\]. We here review the clinical application of TARE and its associated issues.
TARE is most frequently used to treat hepatocellular carcinoma (HCC). Salem *et al.* \[[@goz074-B2]\] performed a study to compare the effects of TACE and TARE in 179 patients with Barcelona Clinic Liver Cancer (BCLC) stages A or B HCC. They found similar tumor response rates in the two groups (74% in the TACE group vs 87% in the TARE group) (*P *=* *0.433). However, patients in the TARE group had a significantly longer median time to progression (\>26 months) than those in the TACE group (6.8 months) (*P *\<* *0.001). Furthermore, the occurrence rate of complications was lower in the TARE group. The most recent study by Salem *et al.* \[[@goz074-B3]\] presented overall survival (OS) outcomes in a 1,000-patient cohort acquired over a 15-year period. On the basis of these data, they decided to adopt TARE as the first-line transarterial locoregional therapy for patients with HCC. The only treatment recommended by the BCLC system for patients with advanced HCC and portal vein tumor thrombus (PVTT) or hepatic vein tumor thrombus is Sorafenib \[[@goz074-B4]\]. However, the management of advanced HCC is complex and still controversial. Recently, two phase III clinical trials compared TARE and Sorafenib and concluded that the results of TARE are not better than those of Sorafenib in terms of survival benefits \[[@goz074-B5]\]. However, this does not mean that TARE should not be used to treat advanced HCC. In fact, the difficulties and potential biases undermining trials that compare an interventional procedure (TARE) with a drug (Sorafenib) should be carefully analysed. Spreafico *et al.* \[[@goz074-B6]\] performed a single-center retrospective study that included 120 patients with advanced HCC and PVTT. These patients were all treated with TARE. Median OS was 14.1 months (95% confidence interval, 10.7--17.5) and 1-year and 3-year OS rates were 53.2% and 18.5%, respectively. These survival results seem to be better than those achieved by Sorafenib. The researchers further developed a prognostic score model to predict response to TARE for patients with HCC and PVTT. They identified three prognostic categories: favorable, intermediate, and dismal prognoses. Median OS in these three categories was 32.2, 14.9, and 7.8 months, respectively (*P *\<* *0.001) \[[@goz074-B6]\]. This model may help doctors to better identify patients who are suitable for TARE treatment.
Liver transplantation (LT) is a curative therapy option for patients with HCC who meet the Milan criteria. However, there is usually a 6-month to 1-year wait for an appropriate donor liver. During this period, interventional procedures are usually required to prevent tumor progression. In addition, the tumor stage of patients with HCC that does not meet the Milan criteria can be reduced by interventional therapy to the point of meeting these criteria. Interventional specialists may prefer TARE because of its better disease-free survival and OS and lower toxicity than with TACE. Ettorre *et al.* \[[@goz074-B7]\] performed TARE before LT in 22 patients with HCC, including 3 who met the Milan criteria prior to treatment with TARE and 19 who did not. Downstaging was observed in 78.9% of cases and bridging was achieved in 100% of cases. Liver resection (LR) is another important procedure for treating HCC. However, many patients with advanced HCC do not meet the criteria for radical resection on presentation. TARE with Y90 can serve as a safe bridge to LR by treating tumors and promoting hypertrophy of the future liver remnant.
A recent study has identified the immune effects of TARE in patients with HCC. This immunological impact can elicit a sustained therapeutic response characterized by regression of locally advanced HCC and delaying of disease progression. Chew *et al.* \[[@goz074-B8]\] detected immune activation in the local microenvironment of tumors treated with TARE. They identified potential biomarkers associated with positive clinical response and built a prediction model to identify sustained responders prior to TARE treatment. This model provides a new means of studying the relationship between Y90 and immune responses. A combination of Y90--TARE and immunotherapy may improve the clinical outcomes of patients with liver cancer.
TARE can be used to treat not only HCC, but also intrahepatic cholangiocarcinoma and colorectal cancer, neuroendocrine tumor, and breast-cancer liver metastases. The safety and efficacy of TARE in the treatment of these liver tumors have been confirmed previously \[[@goz074-B9]\].
Although the adverse effects of TARE are not significantly greater than those of TACE, they should not be ignored. The high-dose beta-radiation emitted in TARE penetrates only 2.5 mm from the source, thus limiting its effects to the site of delivery. However, the off-target diversion of Y90 microspheres to tissues other than the tumor may lead to complications; the most prominent of these complications are radiation gastritis and gastrointestinal ulcers, cholecystitis, radiation pneumonitis, and radioembolization-induced liver disease. Experts recommend that the TARE procedure be performed in accordance with appropriate quality-assurance standards and radiation doses be calculated strictly according to the manufacturer's recommendations. Patients who are sensitive to radiation damage or have reduced liver functional reserve are at high risk of significant tissue damage. Physicians should adopt the most appropriate strategies for the prevention, early diagnosis, and management of potential radiation injury to the liver and other organs \[[@goz074-B10]\].
In conclusion, TARE is an excellent treatment of patients with advanced hepatobiliary cancer who are not eligible for surgery. However, prospective randomized--controlled trials are still required to further demonstrate the role of TARE with Y90. With further study of the immunological effects of Y90, the authors believe that the combined application of Y90 and immunotherapy will show great power in the treatment of cancer.
Authors' contributions
======================
Y.L.M. proposed the study and revised the manuscript. H.Y.Y. and B.J. drafted the first manuscript. G.X., L.J.S., and S.D.D. made a writing suggestion and revised the first draft. All authors contributed to the design and interpretation of the study and to further drafts. Y.L.M. is the guarantor. All authors read and confirmed the final version of this paper.
Funding
=======
This study was supported by grants from the CAMS Innovation Fund for Medical Sciences (CIFMS) \[No. 2016-I2M-1--001\] and the National High-tech Research and Development Projects (863) \[No. 2015AA020303\].
Conflicts of interest
=====================
None declared.
[^1]: Hua-Yu Yang and Bao Jin contributed equally to this paper.
| {
"pile_set_name": "PubMed Central"
} |
Infertility affects 1 in 7 couples and is defined as the inability of a couple to achieve pregnancy after one year of unprotected intercourse[@b1]. Considered as a disease by the World Health Organisation (WHO)[@b2], infertility is known to arise from multi-factorial origins. Between 25 and 30% of cases relate to a male factor, while 20--35% relates to a female factor, and 25--40% arise from a combination of both male and female factors. Worryingly, 10--25% of cases remain idiopathic/unexplained (reviewed in ref. [@b3]).
Male factor infertility is frequently associated with abnormal semen quality parameters such as low sperm concentration (oligozoospermia), poor motility (asthenozoospermia), abnormal morphology (teratozoospermia) or even the complete absence of sperm (azoospermia). Such problems can be attributed to spermatogenic deficiencies and/or abnormal epididymal maturation, and may arise from either genetic or extrinsic factors[@b4]. In this context, advancing male age has been reported to be associated with a gradual decline in sperm quality, which may result in sub-fertility[@b5][@b6]. This reduction in sperm quality not only affects conventional parameters, such as semen volume and sperm motility[@b7][@b8][@b9], but is also related to increased proportions of sperm with either fragmented DNA[@b10][@b11][@b12][@b13], or chromosomal defects[@b14][@b15].
Upon ovulation, oocytes are held in metaphase-II arrest and can only complete meiosis-II when activated by the fertilizing sperm[@b16]. A mounting body of evidence from both basic and clinical research now provides clear support for phospholipase C zeta 1 (PLCζ) as the sperm-borne protein factor responsible for activating the oocyte upon gamete fusion[@b17][@b18][@b19]. Following diffusion into the ooplasm, PLCζ triggers a series of intracellular calcium oscillations that subsequently drives a cascade of biological events, including cortical granule exocytosis, prevention of polyspermy, polar body extrusion, cytoskeletal rearrangements and the formation of pronuclei[@b17][@b18][@b19][@b20][@b21].
Over recent years, a series of clinical studies have clearly related PLCζ deficiency to male infertility. For example, abnormal expression levels or genetic mutations may lead to oocyte activation deficiency (OAD) and total fertilisation failure (TFF)[@b22][@b23][@b24][@b25][@b26][@b27]. Furthermore, the proportions of sperm exhibiting PLCζ in a semen sample are correlated with fertilisation outcome following intracytoplasmic injection (ICSI) but not following *in vitro* fertilisation (IVF)[@b28]. In addition, although seven different PLCζ-localisation patterns have been identified in human sperm (acrosomal; post-acrosomal; equatorial; acrosomal and post-acrosomal; acrosomal and equatorial; post-acrosomal and equatorial; acrosomal, post-acrosomal and equatorial)[@b29], only the specific localisation of PLCζ in the equatorial and post-acrosomal regions are correlated with fertilisation rates following ICSI[@b28]. Moreover, while genetic causes, such as those underlying globozoospermia, may result in sperm which are devoid of PLCζ[@b25][@b26][@b30], the potential effects of other factors, including male age, have received far less attention[@b31]. Such considerations are becoming very relevant because, for various socio-economic factors, the age of fatherhood is progressively increasing. Indeed, the proportion of men aged 35--55 years of age and fathering a child has increased by approximately 15% over the last decade[@b32].
Against this background, the present study sought to determine whether paternal age exerts impact upon the expression or localisation of PLCζ in human semen samples, and to determine whether the influence of age differs between fertile controls and infertile patients.
Results
=======
Differences between age groups and between fertile controls and infertile patients in the proportions, total levels and localisation patterns of PLCζ
-----------------------------------------------------------------------------------------------------------------------------------------------------
When age was considered as a categorical factor, no significant (*P* \> 0.05) differences were detected between the two age groups (i.e. \<40 and ≥40) within either fertile control or infertile patient groups ([Fig. 1](#f1){ref-type="fig"}). In contrast, the proportions (%) of sperm exhibiting PLCζ were significantly (*P* \< 0.05) higher in controls than in patients, for both age groups. No interaction (*P* \> 0.05) between the classification of samples (fertile controls or infertile patients) and age groups were observed and, thus, the effects of age on the proportions of sperm exhibiting PLCζ were similar in control and patient groups. Similar findings were observed for the total levels of PLCζ, in which fertile controls presented significantly (*P* = 0.01) higher levels of PLCζ than infertile patients, but without significant differences (*P* \> 0.05) between the two age groups ([Fig. 2](#f2){ref-type="fig"}).
The proportion (%) of sperm exhibiting PLCζ in acrosomal (A), post-acrosomal (PA), acrosomal + post-acrosomal (A + PA), acrosomal + equatorial (A + E), post-acrosomal + equatorial (PA + E) and acrosomal + post-acrosomal + equatorial (A + PA + E) localisation patterns did not differ significantly, either between fertile controls and infertile patients, or between the two age groups ([Table 1](#t1){ref-type="table"}). In contrast, the proportion (%) of sperm exhibiting PLCζ in the equatorial region only were significantly higher, and those devoid of PLCζ significantly (*P* \< 0.05) lower, in fertile controls than in infertile patients. In spite of such differences, there were no significant differences between the age groups tested.
Correlation of male age with proportions, total levels and localisation patterns of PLCζ
----------------------------------------------------------------------------------------
None of the parameters evaluated for PLCζ (proportions, total levels or localisation patterns), either in fertile controls or infertile patients, was significantly correlated with male age ([Table 2](#t2){ref-type="table"}). The same result was obtained when samples from fertile controls and infertile patients were analysed collectively as one common group.
Clustering of data into fertile controls and infertile patients, and age groups
-------------------------------------------------------------------------------
All parameters used to evaluate PLCζ (proportions, total levels, and localisation patterns) were also used for additional cluster analysis. The optimal number of clusters was two and the descriptive parameters used for these two clusters are shown in [Table 3](#t3){ref-type="table"}. When logistic regression models were used to evaluate the effects of age (considered as both continuous and categorical factors), it was apparent that male age (χ^2^ Wald = 0.05; *P* \> 0.05) did not determine the pertinence of cases to any specific cluster. In contrast, the male pertinence to either fertile control or infertile patient groups was included as a factor in the model (χ^2^ Wald = 11.09; *P* \< 0.05), which indicated that pertinence of a given case to a specific cluster relied on whether the case was a fertile control or an infertile patient.
Relationship between age and other sperm quality parameters
-----------------------------------------------------------
Male age was significantly (*P* \< 0.05) associated with total sperm motile count and the proportion of sperm with progressive motility. This was not only observed when male age was considered as a categorical variable, but also when correlations were calculated with male age being considered as a continuous variable ([Table 4](#t4){ref-type="table"}). When correlations were calculated from individuals belonging to controls or patients groups, the age was correlated to motile count and progressive sperm motility.
Discussion
==========
Although advancing parental age is recognised as a risk factor for sub-fertility, the age of fatherhood has been steadily increasing over the last few decades, particularly in Western countries[@b32]. Thus far, the bulk of research attention has focused upon the consequences of advancing maternal age and the potential implications for mother and child[@b33][@b34]. In comparison, far less focus has been directed towards the potential effect of paternal age[@b5]. Within this limited body of research, previous work has established that advancing male age results in a steady reduction in sperm quality, increased proportions of sperm with fragmented DNA and an increased risk of infertility[@b6][@b9][@b10][@b13][@b35]. However, it remains to be determined whether male age has any other effects upon relevant sperm proteins, such as PLCζ.
One fundamental role of the sperm is to activate the oocyte upon gamete fusion at fertilisation. This vital signalling mechanism is initiated and regulated by PLCζ, a sperm-specific protein, and a number of previous publications have associated PLCζ-deficiency with oocyte activation deficiency or total fertilisation failure[@b22][@b23][@b25][@b26][@b28][@b36]. While biochemical and clinical evidence continues to accrue with regard to the implicit association between PLCζ and successful oocyte activation[@b3][@b17], very little is known as to how PLCζ-deficiency arises in the first place. The present study firstly confirmed that infertile patients presented lower proportions of sperm exhibiting PLCζ, as reported previously[@b28][@b36], but secondly, failed to find any correlation between male age and any of the PLCζ-related parameters tested (i.e. total levels, localisation patterns, or the proportions of sperm exhibiting PLCζ). We arrived at the same conclusion using three distinct and independent statistical approaches (ANOVA, correlation, cluster analysis and further logistic regression). In addition, the absence of age effects upon PLCζ-related parameters was not only observed when samples were separated as fertile controls and infertile patients, but also when these two groups were analysed collectively as one larger cohort.
Two of the three statistical approaches employed (ANOVA, and cluster analysis followed by logistic regression) considered male age as a categorical factor. The cut-off value for classifying patients into two different age groups was assigned to 40 years of age, concurring with previous studies that reported an apparent reduction in sperm quality upon reaching this particular chronological milestone[@b37][@b38]. In addition, considering male age as a categorical or as a continuous factor yielded the same results, thus providing further justification to support our cut-off age value. Finally, clustering the samples according to proportions, total levels and PLCζ-localisation patterns was not dependent on male age, either in fertile controls or in infertile patients.
In contrast to PLCζ-related parameters, male age was negatively correlated with motile sperm count and the proportion of sperm exhibiting progressive motility. These results are in agreement with earlier studies investigating the effects of age upon sperm[@b37][@b39]. The fact that none of the three statistical approaches adopted to examine the effects of age upon PLCζ allowed us to distinguish between fertile controls and infertile patients not only made our current study comparable with previous reports, but also allowed us to exclude any interaction between male age and fertility status. In this regard, it is worth mentioning that Plastira *et al.*[@b10] reported that while male age was negatively associated with sperm motility, morphology and DNA integrity in an infertile patient group, such age-effects were not evident in a fertile control group. In contrast to this study, the present work found a clear reduction of sperm motility with advancing age in both control and patient groups.
As mentioned earlier, increasing male age is related to reductions in semen volume, sperm motility and normal morphology, along with an increased likelihood of sperm DNA fragmentation[@b6][@b7][@b8][@b9][@b10][@b11][@b13]. It has been suggested that such reductions in sperm quality could arise from deterioration of the testicular tissue architecture which involves thickening of the basal membrane in seminiferous tubuli, reduction of the seminiferous epithelium and in the number of both Sertoli and Leydig cells, along with compromised testicular vascularisation[@b40]. An additional explanation could be related to changes in composition of seminal plasma occurring with advanced age[@b41].
Although the precise mechanisms underlying the observed differences between fertile controls and infertile patients in terms of the proportion, total levels, and localisation pattern of PLCζ found here, and in previous studies[@b28][@b36], are not yet fully understood, previous research has identified that genetic factors can underlie PLCζ deficiency, at least in some patients. For example, globozoospermia, an extreme case of teratozoospermia in which sperm can be totally devoid of PLCζ[@b25][@b30][@b42], is known to have a genetic cause[@b43]. In addition, two point mutations in the coding region of the PLCζ gene have been found in a non-globozoospermic, infertile patient. These mutations affected the X and Y domains of PLCζ (substitution of histidine for leucine at residue 233 \[H233L\], and substitution of histidine for proline at residue 398 \[H398P\]), and were directly responsible for oocyte activation failure[@b23][@b25][@b26]. In the present work, we have shown that proportions, localisation patterns and total PLCζ levels are independent from male age. Since genetic factors have only been related to a very small number of patients thus far, more research is warranted to address which other factors might underlie the differences between fertile controls and infertile patients observed here and in other previous studies[@b27][@b28][@b36].
The effect of paternal age on fertility outcome is far less consistent than those related to sperm quality. Indeed, while some studies indicate a positive correlation between age and infertility risk in men[@b9][@b35], it is prudent to consider that other factors could potentially confound our ability to evaluate the impact of paternal age upon fertilising ability. For example, maternal age, and a reduction in the frequency of intercourse with advancing age[@b38][@b44]. In fact, previous studies have failed to identify an association between male age and fertilisation outcomes following *in vitro* fertilisation (IVF), even when donated oocytes were used, and therefore, any maternal effect had been removed[@b45][@b46][@b47]. In addition, the fact that Spandofer *et al.*[@b48] failed to detect any significant effect of male age upon fertilisation outcomes following ICSI, is consistent with our present results, since proportions of sperm exhibiting PLCζ are known to be positively correlated with fertilisation rates[@b28]. Furthermore, confounding factors and covariates that are associated with age, such as vascular disease, obesity and factors related to lifestyle, may also mask the actual contribution of advancing age to the observed decline in sperm quality[@b49][@b50][@b51][@b52].
Since the contribution of sperm to events occurring post-fertilisation is not restricted to PLCζ, the findings reported herein do not exclude the possibility that advancing male age could influence other important sperm factors. For example, sperm chromatin integrity, which has been linked to total fertilisation failure and miscarriage, is related to male age[@b12][@b53][@b54][@b55].
In conclusion, the present study reports, for the first time, that the proportions, total levels and localisation patterns of PLCζ in human ejaculated sperm are not associated with male age. Consequently, while ageing is related to a clear reduction in sperm quality, it appears that the aging process does not affect all sperm proteins, but rather acts upon specific parameters. Therefore, patients with reduced sperm quality due to age may be advised to use IVF/ICSI, since advancing male age is unlikely to cause problems in terms of the sperm's fundamental ability to activate an oocyte and initiate the process of embryogenesis. Of course, this does not exclude that these processes may be influenced by the oocyte quality, and in particular the viability of oocyte proteins that might interact with PLCζ during gamete fusion.
Methods
=======
Ethics
------
Males were recruited from the Oxford Fertility Unit (OFU; Oxford, UK) and from the Assisted Conception Unit (ACU; Ninewells Hospital, Dundee, UK) with approval from the National Research Ethics Service (South Central Oxford Committee C; Reference number: 10/H0606/65) and the East of Scotland Research Ethics Service (EoSRES) (REC 1; Reference number: 13/ES/0091), respectively. Informed consent was obtained from all subjects involved in this study and all methods described in this section were carried out in accordance with the guidelines approved by the two Ethical Committees.
Sperm samples
-------------
Semen samples were provided following at least three days of abstinence. A total of 71 males were recruited into this study, ranging from 22 to 54 years of age (age, mean ± standard error of the mean, SEM: 36.4 ± 0.7). All males were classified into either a fertile control group or an infertile patient group, as described by Yelumalai *et al.*[@b28]. The control group (n = 44; age, mean ± SEM: 35.8 ± 1.0) included (i) men with normal semen analysis parameters that were able to successfully fertilise an oocyte following assisted reproductive technology (ART), (ii) men with no history of infertility or oocyte activation deficiency, and (iii) men who had previously fathered children via natural conception. These clinical data were provided by the respective clinical uit providing the sample. All other samples were classified as infertile patients (n = 27; age, mean ± SEM: 37.4 ± 1.1 years) based upon sperm quality analysis, including total motile count, the proportion of sperm exhibiting progressive motility, sperm concentration (sperm·mL^−1^), and morphological abnormalities (e.g. sperm presenting pin-shaped or round heads). A history of total fertilisation failure following IVF and/or ICSI was also used as a criterion to classify samples into the infertile patient group[@b28].
Preparation of sperm samples
----------------------------
Semen volume, pH, viscosity, sperm motility and sperm concentration were evaluated upon arrival in our laboratory, and were then subjected to density gradient washing (DGW) using PureSperm™ (PureSperm™ 40/80, Nidacon International AB, Gothenburg, Sweden), as described previously[@b56]. In brief, 2 mL of 80% PureSperm medium was layered onto the bottom of a 15 mL tube and overlaid with 2 mL of 40% PureSperm medium. Subsequently, 1.5 mL of liquefied semen sample was layered on top of the 40% PureSperm medium and samples centrifuged at 300×*g* (room temperature) for 20 min. Following the centrifugation step, most of the supernatant was discarded and approximately 0.5 mL of pellet remained at the bottom of the tube. This pellet was then transferred to a clean 15-mL tube containing 5 mL of PureSperm™ Wash medium (Nidacon International AB). This mixture was subsequently centrifuged at 500 × *g* (room temperature) for 10 min. The pellet was resuspended in 1 mL of PBS to evaluate sperm motility and 200 μL was used for PLCζ analysis. This latter aliquot was centrifuged at 800 × *g* (room temperature) for three min and the supernatant discarded without disturbing the remaining pellet. This pellet was resuspended with 100 μL 4% paraformaldehyde (Sigma-Aldrich; Gillingham, Dorset, UK) and incubated at room temperature for 10 min in order to fix sperm cells. Following a final centrifugation step, and removal of paraformaldehyde, the sperm pellet was re-suspended and stored in 200 μL PBS at 4 °C to await immunofluorescent analyses.
Evaluation of sperm quality
---------------------------
Sperm concentration and motility were assessed before and after DGW, as described in ref. [@b28], and according to WHO guidelines[@b57]. For the evaluation of sperm concentration, samples were diluted with sterile water (at a ratio of 1/20) and counted using a Neubauer chamber (Paul Marienfeld GmbH & Co. KG; Lauda-Königshofen, Germany). Two separate counts per sample were carried out.
Sperm motility was also evaluated in two independent replicates, in accordance with the latest WHO guidelines[@b57]. This analysis was performed taking into account the proportion of sperm exhibiting progressive sperm motility and the motile count, which represented the number of sperm exhibiting progressive motility.
Purification and specificity of the PLCζ antibody
-------------------------------------------------
Anti-human PLCζ antibody was synthesised by Cova-Lab (Cambridge, UK) against two immunogenic peptides identified in the human PLCζ amino acid sequence (C-RESKSYFNPSNIKE-coNH~2~ and C-ETHERKGSDKRGDN; Accession Number: AF532185). Before use, the crude antibody was purified with a SulfoLink Kit (Pierce Biotechnology, Rockford, USA), and the two original immunogenic peptides. Specificity of the PLCζ antibody was confirmed by competitive pre-incubation studies with excess peptides (data not shown), following the procedure described in ref. [@b29]. The PLCζ antibody was proven to be specific since no immunostaining was observed when samples were incubated with antibody in the presence of blocking peptides. It is worth noting that the same antibody has been used in several other studies arising from this laboratory, and others, and has provided a consistent pattern of immunostaining throughout[@b23][@b25][@b28][@b29][@b36][@b56].
Immunofluorescent detection of PLCζ
-----------------------------------
Sperm samples were immunostained for PLCζ using the protocol described by Grasa *et al.*[@b29]. First, a 100-μL drop of fixed sperm was placed onto a slide pre-coated with 0.01% Poly-L-Lysine and permeabilised with 0.5% Triton X-100 in PBS (Sigma Aldrich) overnight. Slides were subsequently incubated with 3% bovine serum albumin (BSA, Sigma-Aldrich) at room temperature for one hour, and then with 100 μL of anti-PLCζ antibody (25 μg·mL^−1^; Cova-Lab, UK) diluted in 0.05% BSA at 4 °C and overnight. After this step, samples were washed three times with PBS and then incubated with 5 μg·mL^−1^ of a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Invitrogen, Paisley, UK) at room temperature for 1 hour. After re-washing the slides with PBS a total of three times, 3 μL of Vectashield H-1200 mounting medium containing 4′-6′-Diamidini-2-phenylindole (DAPI) (Vector, UK) was added onto the top of the sample prior to covering with 20 mm × 20 mm glass cover slips.
Evaluation of samples was performed under a fluorescence microscope (Eclipse 80i, Nikon UK Ltd., Kingston upon Thames, UK) at 400× magnification. Since the excitation wavelength for the secondary antibody was 488 nm, a fluorescein isothiocyanate filter (FITC; Excitation filter: 465--495 nm; Dichroic Mirror: 505 nm: Barrier Filter: 515--550 nm) was used. All images were acquired with a Nikon DS-Ri1 camera (Nikon UK) at an exposure time of 400 ms. Illumination intensity and camera settings, such as quality (16-bit), resolution (1280 × 1024), and colour (high contrast), were set and standardised to ensure that they were the same for all images, following previously described protocols[@b28][@b36]. Prior to any evaluation, bright field images were used to select sperm heads for analyses and only those sperm that presented the head attached to the tail, and did not overlap with other sperm, were selected. Fluorescent illumination was then used to outline the sperm heads through the region of interest manager (ROI) tool from ImageJ software (Version 1.46a; National Institutes of Health, Bethesda, MD, USA). For each slide, separate fields were randomly captured to image 100 sperm per sample. Mean PLCζ levels per sperm cell (arbitrary units) were quantified using Image J software. This mean value was derived from both sperm exhibiting PLCζ-staining and those that were devoid of staining. In addition, the proportion of sperm exhibiting PLCζ (in any localisation pattern), as well as the PLCζ localisation profile in each sperm cell, were evaluated following the procedure described by Grasa *et al.*[@b29]. Thus, PLCζ expression was assessed in each of the following eight localities: acrosomal (A); post-acrosomal (PA); equatorial (E); acrosomal and post-acrosomal (A + PA); acrosomal and equatorial (A + E); post-acrosomal and equatorial (PA + E); acrosomal, post-acrosomal and equatorial (A + PA + E); or 'none' indicating sperm totally devoid of PLCζ.
Statistical analyses
--------------------
Data were managed using Microsoft Excel 2010 (Microsoft Corp., Redmond, Washington, USA) and IBM SPSS 21.0 for Windows; IBM Corp., Chicago, Illinois, USA) and the two graphs were prepared with Origin Pro 8.0 software (OriginLab Corp., Northampton, Massachusetts, USA).
Three different statistical approaches were taken after first checking the data for normality (Shapiro-Wilk test) and homogeneity of variance (homoscedasticity; Levene test). Since some of the data (x) in proportions did not fit with parametric assumptions, they were transformed using arcsine root transformation (arcsin √x) and, when this transformation did not correct data normality and variance homogeneity, alternative non-parametric tests were used. Data for sperm motile counts were log-transformed prior to analysis. \[log (x)\].
The first statistical approach considered male age as a categorical factor. Thus, the effects of age and group (fertile controls and infertile patients) on proportions, total levels and localisation patterns of PLCζ, total motile count and the proportion of progressive motile sperm, were evaluated by two-way analysis of variance (ANOVA) followed by a post-hoc *t*-test with Bonferroni adjustment for pair-wise comparisons (the two factors used were the (i) age and (ii) the control/patient grouping). Since data for total proportions of sperm exhibiting PLCζ, and proportions of sperm with PLCζ localisation in E, A + PA and A + E did not match parametric assumptions, even after transformation, a two-way, non-parametric ANOVA for ranked data (Scheirer-Ray-Hare approach) was performed[@b58]. This approach is an extension of the Kruskal-Wallis test for two or more factors and calculates *P* values both for factors and their interaction by calculating an 'H' statistic. This H statistic results from dividing the sum of squares of a given factor (SS) by the mean squares (MS) and is then tested as a chi-square variable using the degrees of freedom pertaining to each factor (SS). The Mann-Whitney test was used to compare controls and patients regardless of age grouping, and to compare the two age groups regardless of whether data came from controls or patients.
The second approach considered male age as a continuous variable. In this case, correlation coefficients were calculated between male age, PLCζ parameters, motile count and the proportion of progressive motile sperm. These correlations were calculated with and without separating the statistical groups between controls and patients. Pearson correlations were used for these variables that required, or did not require, transformation, matched with parametric assumptions. In all other cases, Spearman correlations were carried out.
In the third approach, cases were first clustered via two-step cluster analysis using proportions, levels and localisation patterns as continuous variables. The log-likelihood was utilised as a measure for distance and the clustering was performed following Schwarz's Bayesian Criterion (BIC). Descriptive statistics were calculated for the resulting clusters. The next step consisted of conducting logistic regression analyses using the number of clusters as the dependent variable and male age and grouping (controls vs. patients) as covariates. The method used was a forward stepwise protocol based on the likelihood ratio to determine the statistical significance of the factors as well as the order of importance. The probability-to-enter a variable into the model was 0.05 and the probability-to-remove was 0.10.
In all statistical tests, the significance level (two-tailed) was set at *P* ≤ 0.05. Data are expressed as mean ± standard error of the mean (SEM).
Additional Information
======================
**How to cite this article**: Yeste, M. *et al.* Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm? *Sci. Rep.* **6**, 27543; doi: 10.1038/srep27543 (2016).
M.Y. is funded by the European Commission, FP7-People Programme, Marie Curie-IEF (Grant Number: 626061). The authors would also like to thank Dr. Junaid Kashir and Dr. Walaa Ramadan for their earlier contributions to processing patient samples.
**Author Contributions** M.Y. conducted the laboratory assessments, analysed the data and wrote the manuscript. C.J., S.N.A. and S.Y. helped conduct laboratory analyses. G.M. and S.J.M.d.S. recruited the patients and provided clinical data. T.C. provided clinical expertise/data and critically revised the manuscript. The study was performed in KC's laboratory, as part of his ongoing research programme in the field of oocyte activation. C.J. and K.C. helped revise the manuscript and KC gave his final approval.
{#f1}
{#f2}
###### Proportions (%) of sperm exhibiting PLCζ (mean ± SEM) in fertile controls and infertile patients in two age groups.
PLCζ localisation Fertile Controls (n = 44) Infertile Patients (n = 27)
------------------- --------------------------- ----------------------------- ----------------- ----------------
A 1.4 ± 0.5 1.2 ± 0.6 2.4 ± 1.2 2.6 ± 1.2
PA 8.1 ± 2.1 8.0 ± 2.3 6.2 ± 2.0 7.6 ± 2.5
E 31.5 ± 5.3^a^ 23.3 ± 6.2^a,b^ 14.5 ± 3.9^b,c^ 10.1 ± 4.3^c^
A + PA 0.9 ± 0.2 0.8 ± 0.6 2.1 ± 0.9 3.5 ± 1.8
A+ E 4.7 ± 1.8 1.9 ± 0.7 0.9 ± 0.5 1.1 ± 0.5
PA+ E 22.2 ± 4.4 29.5 ± 7.4 24.1 ± 5.8 25.9 ± 9.8
A + PA +E 14.8 ± 3.1 19.3 ± 5.7 12.0 ± 4.5 12.4 ± 5.2
None 16.3 ± 2.8^a^ 16.0 ± 5.5^a^ 37.8 ± 7.7^b^ 36.7 ± 10.8^b^
Different superscripts (*a, b, c*) denote significant differences between columns within a given row. \[Specific localisation patterns of PLCζ are referred to as: A = acrosomal; E = equatorial; None = absence of PLCζ; PA = post-acrosomal. 'None' refers to sperm that were completely devoid of PLCζ\].
###### Correlation coefficients between age and different PLCζ patterns in fertile controls, in infertile patients, and without separating these two groups (overall).
PLCζ localisation Fertile Controls (n = 44) Infertile Patients (n = 27) Overall (n = 71)
--------------------------- --------------------------- ----------------------------- ------------------
\% Sperm exhibiting PLCζ 0.02 0.05 0.01
Total levels of PLCζ 0.14 −0.06 −0.02
Localisation patterns (%)
A 0.13 0.02 0.09
PA 0.19 −0.08 0.08
E −0.13 −0.21 −0.15
A + PA 0.17 0.11 0.14
A+ E −0.04 0.01 −0.04
PA+ E 0.10 −0.06 0.03
A + PA +E 0.05 0.05 0.02
None −0.14 0.32 0.07
No significant correlations were observed in any of the cases evaluated. \[Specific localisation patterns of PLCζ are referred to as: A = acrosomal; E = equatorial; None = absence of PLCζ; PA = post-acrosomal. 'None' refers to sperm that were completely devoid of PLCζ\].
###### Descriptive parameters (mean ± SEM) for the two clusters obtained following two-step cluster analysis using all sperm samples collectively.
PLCζ Cluster 1 Cluster 2
--------------------------- ------------ ------------
N 47 24
\% Sperm exhibiting PLCζ 88.9 ± 1.2 36.9 ± 5.5
Total levels of PLCζ 37.2 ± 2.3 22.6 ± 2.6
Localisation patterns (%)
A 1.4 ± 0.4 2.4 ± 1.0
PA 7.4 ± 1.4 7.9 ± 1.9
E 29.3 ± 3.8 10.8 ± 2.9
A + PA 1.4 ± 0.5 1.3 ± 0.6
A+ E 3.5 ± 1.1 1.0 ± 0.4
PA+ E 29.9 ± 3.9 8.5 ± 1.8
A + PA +E 15.7 ± 2.6 10.6 ± 3.0
None 11.3 ± 1.1 52.7 ± 5.8
\[Specific localisation patterns of PLCζ are referred to as: A = acrosomal; E = equatorial; None = absence of PLCζ; PA = post-acrosomal. 'None' refers to sperm that were completely devoid of PLCζ\].
###### Correlation coefficients between male age, % progressive motile sperm and total sperm motile count in fertile controls, infertile patients, and when both groups were analysed collectively (overall).
Parameter Fertile Controls (n = 44) Infertile Patients (n = 27) Overall (n = 71)
----------------------------- --------------------------- ----------------------------- ------------------
\% Progressive motile sperm −0.31\* −0.29\* −0.34\*
Total sperm motile count −0.28\* −0.27\* −0.30\*
Asterisk (\*) denotes significant correlation (*P* \< 0.05).
[^1]: Present address: Department of Biology, University of Girona, E-17071 Girona, Spain.
[^2]: Present address: Department of Obstetrics and Gynaecology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Actinic keratosis (AK) is a common skin disease caused by long-term sun exposure \[[@CR1]\], and typically forms on the face, neck, balding scalp, chest, shoulders, and the back of arms and hands of adults \[[@CR2]\]; 75% of all reported lesions present on the head, neck, and forearms \[[@CR3]\]. AK is characterized by the formation of keratotic macules, papules, or plaques with superficial scales on a red base, which are classified based on histological features (Table [1](#Tab1){ref-type="table"}). Lesions are often asymptomatic, but they can be sore or itchy \[[@CR4]\]. Due to the cumulative nature of the condition, the incidence of AK increases with age and is a common condition in the adult population aged over 50 years \[[@CR2]\].Table 1Classification of actinic keratosis based on histologic featuresVariantCharacteristicsHypertrophicPronounced hyperkeratosis with areas of parakeratosisThickened epidermisIrregular downward proliferationKeratinocytes in stratum malpighii (may show loss of polarity and pleomorphism)AtrophicGenerally atrophic epidermis with slight hyperkeratosisBasal layer shows cells with hyperchromatic nucleiCells may proliferate towards dermis as buds or duct-like structuresBowenoidDifficult to distinguish from Bowen diseaseFull thickness atypia presentAcantholyticIntercellular clefts present as result of anaplastic changes in base of epidermis that produce dyskeratotic cells with disrupted cellular bridgesEpidermolyticGranular degeneration or epidermolytic hyperkeratosisLichenoidDense dermal infiltrate of lymphocytes in papillary dermis that damages epidermis basal layerPigmentedExcessive amounts of melanin in basal epidermisNumerous melanophages in superficial dermisReproduced with permission from Rosen et al. \[[@CR7]\]
Some AK lesions can undergo malignant transformation and progress to invasive squamous cell carcinoma (SCC) \[[@CR5]\]. However, despite its seriousness, public awareness of this potential complication is generally poor. Treatment of AKs depends on the clinical presentation of the lesions: it may be targeted at specific lesions (lesion directed) or at multiple lesions over a large area (field directed), and sometimes both treatment approaches are combined. A number of treatment options are available for AK, although no universal standard has yet been established. Therapy options include cryosurgery, curettage, excision surgery, photodynamic therapy (PDT), and topical treatments (5-fluorouracil \[5-FU\] cream, diclofenac gel, imiquimod cream, and ingenol mebutate gel).
This review examines the awareness of AK among primary care physicians and the public, current therapeutic options and strategies, and comparison of the prevalence and management of AK around the world, especially Australia, Canada, and the United Kingdom (UK). The analysis in this article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by any of the authors.
Disease Awareness {#Sec2}
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Prevalence {#Sec3}
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AK is a global condition and its prevalence is significant; for example, it is the second most common diagnosis made by dermatologists in the USA \[[@CR1]\]. Recent reports have indicated an increase in AK rates \[[@CR6], [@CR7]\]. This is partly due to environmental factors, such as thinning of the ozone layer and more ultraviolet (UV) radiation reaching the Earth's surface \[[@CR8]\], and partly to lifestyle factors such as increased tanning (outside and sunbed) \[[@CR9]\]. The prevalence rates are highest in countries that have high sun exposure and have a large fair-skinned population (Fitzpatrick skin type I and II).
It is not surprising therefore that the highest AK prevalence rate has been documented in Australia---over 40% in individuals aged over 40 years \[[@CR10]\]. This rate is exacerbated by the much thinner layer of ozone above this country. In contrast, countries with low levels of sun exposure, such as the UK and Ireland, report much lower rates of 19--25%, in individuals aged over 60 years \[[@CR11]\]. Similar rates have been reported in the USA and Europe: 11--26% and 11--25%, respectively \[[@CR12], [@CR13]\]. It would therefore be advisable for areas with high levels of sun exposure to incorporate a skin examination as part of patients' annual check-up.
Risk Factors {#Sec4}
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The prevalence of AK is strongly linked to fair skin, advanced age, and gender. A study in England reported a prevalence rate of 15.4% and 5.9% in men and women, respectively. This rate increased to 34.1% and 18.2% in men and women aged above 70 years \[[@CR14]\]. The study found the incidence of AK was greater in individuals with red hair and freckles, which indicates Fitzpatrick skin type I. There was a similar, but more pronounced, increase reported in Australia; prevalence rates of AK were 22% and 8% for men and women aged 30--39 years, which increased to 83% and 64%, respectively, in adults aged 60--69 years \[[@CR15]\]. One reason for the greater occurrence rate in males could be they are more likely to work outdoors and receive more cumulative sun exposure.
Poor Awareness {#Sec5}
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Despite the high prevalence of AK, awareness of the disease and its relationship with SCC is generally poor \[[@CR16]\]. In a telephone survey of 1,500 Europeans aged 40--70 years, only 6% were aware of AK \[[@CR17]\]. Similarly, when 2,100 physicians from seven countries (USA, Australia, UK, Italy, France, Germany, and Spain) were surveyed about their awareness of non-melanoma skin cancer and AK, results showed that primary care physicians were more familiar with basal cell carcinoma (90%) than with AK (74%) \[[@CR18]\]. Of the primary care physicians who were aware of AK, only 40% had treated the condition \[[@CR18]\]. Over 700,000 new cases of SCC are diagnosed every year in the USA. Nevertheless, SCC is excluded from national cancer registries in the USA, making accurate prevalence rates, along with resulting metastases and deaths, difficult to estimate \[[@CR16]\].
The potential for malignant transformation is a reason for concern about the poor public awareness of AK. Intraepidermal proliferation of atypical keratinocytes can be seen on AK lesions, and studies have shown that up to 16% of AKs progress to invasive SCCs \[[@CR5]\]. One reason for the low level of AK awareness is the wide range of variations in its clinical presentation (Fig. [1](#Fig1){ref-type="fig"}), which include the cutaneous horn, the pink and pearly lichenoid AK, pigmented AK, and actinic cheilitis \[[@CR4]\]. The clinical variants of AKs are shown in Fig. [2](#Fig2){ref-type="fig"}.Fig. 1Signs and symptoms of AKs. **a** Sun-damaged skin: severe sun damage on lower legs due to chronic exposure to UV; an increased risk of developing AKs is expected. **b** Scaly and crusted patches on sun-damaged skin, an AK can usually be felt before it is seen. **c** Rough, *reddish*, raised bumps: most AKs look like raised, scaly, *red* bumps on the skin. **d** Thick, *discolored*, scaly and crusted skin with many growths: skin that has accumulated years of sun damage, such as the scalp, face, and arms can have many AKs. **e** Pigmented AK: the AKs on this man's face appear as pigmented skin or as *brown* patches. When AKs look like this, they can resemble melanoma. **f** Cutaneous horn: some AKs grow quickly and look like an animal's horn. Horns are more likely to progress to skin cancer; illustrated is a squamous cell carcinoma (keratoacanthoma). **g** *Whitish* scale on bottom lip: when an AK forms on the lip, the AK is called actinic cheilitis. If the patient has a rough scaly lip, splitting lips, or lips that always feel dry, they should see a dermatologist. **h** Squamous cell carcinoma on right temple: without treatment, some AKs progress to squamous cell carcinoma. *AK* Actinic keratosis. Images are published with permission from the New Zealand Dermatological Society IncorporatedFig. 2Clinical variants of AKs. **a** Hyperkeratotic AK. **b** AK with field change (*forehead and partial scalp*). **c** Classical AK (*milder degree of field change on the arm*). **d** AK with adjacent hyperpigmented areas. *AK* Actinic keratosis. Images are published with permission from the New Zealand Dermatological Society Incorporated
The Burden of AK {#Sec6}
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The burden and cost of AK on the patient and society is also underestimated. In 2005, the Society of Investigative Dermatology and the American Academy of Dermatology estimated that AK affected more than 58 million people in the USA \[[@CR19]\]. Furthermore, direct costs relating to AK were considerable: \$1.2 billion in 2004, with 92% of these costs due to physician office visits and 5% to prescription drugs \[[@CR19]\].
For patients, the burden can be both personal and financial. Patients with AK experience impairment to their quality of life in terms of physical symptoms such as itching, burning, tenderness, and dyspigmentation \[[@CR19], [@CR20]\]. Psychological impairment has also been reported, impacting on the patients' confidence and their sense of well-being \[[@CR20]\]. There is no inpatient hospital stay associated with a primary diagnosis of AK. However, the costs of services and treatment, and physician time to the patient, can be expensive, if they are not reimbursable \[[@CR19]\].
Therapeutic Options and Disease Management {#Sec7}
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A substantial proportion of skin cancers are preventable. Therefore, an important first step is education for the use of sunscreens, which have been shown to reduce AKs and SCC \[[@CR21]--[@CR23]\]. However, the current prevalence of AKs demonstrates that prevention alone is not sufficient.
Treatment of AKs is always recommended, as there are no reliable clinical predictors to distinguish an AK lesion from those that will become malignant and transform into SCC \[[@CR11], [@CR20]\]. If untreated, SCC can become locally invasive, leading to metastases and death.
Treatments for AK fall into two categories: Lesion directed or field directed. Both may also be combined sequentially. The optimal treatment is determined by the pathology of the AK lesion and patient considerations. The most common therapy options for AK are described in Table [2](#Tab2){ref-type="table"}.Table 2Summary of treatment options for AK \[[@CR24]\]TreatmentResponseRecurrence^a^Side effectsLesion-directed treatment Cryosurgery (liquid nitrogen)75--98%1.2--50%Pain, redness, edema, blistering, scarring, hypopigmentation Laser therapy\~90%10--15%Pain, inflammation, pigment changes, scarring, delayed healing, erythema Curettage/excision/shave biopsyUndocumentedUndocumentedPain, bleeding, scarringField-directed therapy Ingenol mebutate34.1--42.2%^b^44.6--67.6%^b^Erythema, flaking, scaling, crusting Topical 5-FU50%55%Severe dermatitis, wound infections, pruritus, pain, ulceration, scarring Chemical peeling\~75%25--35%Pain, inflammation, pigment changes, scarring Diclofenac 3% gel50--79%UndocumentedPruritus, erythema, dry skin Topical photodynamic therapy70--90%^c^UndocumentedApplication-site pain, photosensitivity, post-treatment inflammation Imiquimod 5%55--84%10%Erythema, itching, burning sensation, fatigue, nausea, influenza-like symptoms, myalgia Sun protectionNANANAAdapted with permission from Stockfleth et al. \[[@CR24]\], Table [1](#Tab1){ref-type="table"}*5*-*FU* 5-fluorouracil, *AK* actinic keratosis, *NA* not applicable^a^One-year recurrence^b^LEO: SMPC, ingenol mebutate^c^Response rates enhanced by curettage
Lesion-Directed Treatments {#Sec8}
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Lesion-directed treatments commonly involve focal ablative procedures \[[@CR24]\]. Cryosurgery, with the use of liquid nitrogen, is a standard first-line approach for the treatment of discrete lesions that can be carried out in a primary care setting. It is a rapid, low-cost approach \[[@CR24], [@CR25]\], and a 2- to 5-s freeze is reported to remove around 70% of AKs treated \[[@CR26]\]. Response to treatment appears to be related to the length of the freeze time \[[@CR25]\]. Side effects associated with cryosurgery include pain, redness, edema, blistering, and hypopigmentation \[[@CR24], [@CR27]\].
Laser therapy is another lesion-directed treatment, but is a higher cost option to cryosurgery and requires more specialized training. The associated side effects include pain, inflammation, pigment changes, and delayed healing. Curettage and excisional surgery can be an effective approach for some lesions, particularly hyperkeratotic lesions and/or where histological information is required (e.g., resistant lesions or to check for an occult SCC) \[[@CR24]\]. However, local anesthetic is necessary and the procedure can result in pain, bleeding, and scarring.
Field-Directed Treatments {#Sec9}
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### 5-FU {#Sec10}
5-FU 5% cream has been used as a topical approach for the treatment of AKs for many years \[[@CR25], [@CR26]\]. The cream is applied twice daily for up to 4--6 weeks \[[@CR25]\] and clearance rates of 50% have been reported \[[@CR24]\]. However, treatment with 5-FU cream can result in severe dermatitis, sometimes accompanied by wound infections, pruritus, pain, and ulceration, with scarring lasting throughout the duration of the treatment \[[@CR28]\]. The inflammation induced can be so severe that patients stop before adequate treatment has been received and the photosensitivity reaction also limits its use in the summer.
### Diclofenac {#Sec11}
Diclofenac 3% gel, used twice daily for 90 days, is associated with complete response rates of 34--47% of patients \[[@CR29]\]. Diclofenac is generally well tolerated; side effects include pruritus, erythema, and dry skin \[[@CR24]\]. Australian guidelines suggest the use of diclofenac in combination with cryosurgery for hypertrophic or resistant AKs \[[@CR25]\]. As a non-steroidal anti-inflammatory drug, diclofenac may cause photosensitivity reactions in rare cases that are usually attributed to UV radiation \[[@CR30]\]; however, contact dermatitis is a more common side effect \[[@CR31]\].
### Imiquimod {#Sec12}
In Australia, the approved indication for imiquimod is once daily, three times per week, for up to 16 weeks. However, in practice, most clinicians recommend applying it two to three times a week for two 4-week cycles \[[@CR25]\]. If necessary, cycles may be repeated after a month. Complete clearance rates of 44--46% have been reported \[[@CR32]\]. Side effects associated with imiquimod include erythema, itching, and a burning sensation \[[@CR24]\]. Systemic side effects of imiquimod include headache, fatigue, nausea, influenza-like symptoms, and myalgia \[[@CR33]\].
The beneficial effect of use of cryosurgery followed by field treatment with imiquimod 3.75% cream has been shown \[[@CR34]\]. Median total AK reductions of 86.5% were achieved with imiquimod compared with 50% for vehicle, with complete clearance rates of 30.2% and 3.3%, respectively.
### Ingenol Mebutate {#Sec13}
Ingenol mebutate gel is a recent addition to topical treatments for non-hyperkeratotic, non-hypertrophic AK in adults. Ingenol mebutate appears to have a dual mechanism of action; it preferentially causes cell death in transformed keratinocytes and induces an inflammatory reaction that kills the remaining cancerous cells \[[@CR35]\]. It is approved for the treatment of AKs located on the face, scalp, trunk, and extremities. It was approved by the US Food and Drug Administration (FDA) for AK in January 2012 as a 2- or 3-day course of therapy; it is also approved in Europe, Australia, Canada, and other countries. In a pooled analysis of trials on the face and scalp, a complete clearance rate of lesions of 42% and lesion reduction rate of 83% were reported at day 57 \[[@CR36]\]; at 12 months a sustained clearance rate of 46% and lesion reduction rate of 87% were achieved \[[@CR37]\]. Adherence to treatment was \>98%. As with all topical treatments, local skin responses include erythema, flaking, scaling, and crusting \[[@CR35]\]. Ingenol mebutate does not exhibit any phototoxic or photosensitizing properties and can therefore be used throughout the year \[[@CR38]\].
A beneficial effect on AK treatment of ingenol mebutate following cryosurgery compared with cryosurgery alone has been reported \[[@CR39], [@CR40]\]. Treatment with ingenol mebutate 0.015% gel for 3 consecutive days, 3 weeks after cryosurgery resulted in higher complete clearance rates than vehicle at both 11-week rates (60.5% vs. 49.4%) \[[@CR39]\] and 12 months (30.5% vs. 18.5%) \[[@CR40]\]. Complete clearance was sustained at 12 months with greater reduction of AKs compared with vehicle (68.2% vs. 54.1%) and fewer patients experiencing the emergence of new lesions \[[@CR39], [@CR40]\].
### PDT {#Sec14}
PDT, using the photosensitizing cream methyl aminolevulinate, is another effective topical treatment option for AKs. The cream is applied to the affected area and left for 3 h before a 7--9-min illumination of the area is carried out, usually in a specialized clinic. A single PDT treatment is sometimes sufficient, although multiple numbers are used \[[@CR25]\]. Efficacy rates of 70--90% have been reported \[[@CR24]\]. Side effects associated with PDT include intense application-site pain that requires management and hypersensitivity to daylight. There is a degree of post-treatment inflammation, which usually subsides within a week \[[@CR26]\].
Lesion Characteristics Determining Treatment {#Sec15}
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Lesion-directed treatment is usually a first-line approach for isolated lesions; a field-directed approach is used when there are multiple lesions present \[[@CR24]\]. In some cases, field-directed therapy is combined with cryosurgery. As lesion-directed treatment fails to take into account potential damage to the surrounding skin, field-directed treatment may be the more effective way of eradicating both evident and subclinical lesions \[[@CR24]\]. This may prevent the potential development of invasive SCC.
The anatomic location of AK lesions is a significant factor in the choice of the most appropriate treatment approach. Pharmacologic field-directed therapy is ideal for cosmetically sensitive or difficult to treat locations such as the face, chest, back of the hands, arms, and lower legs \[[@CR24], [@CR41]\]. The area around the eyes and mouth requires the use of a treatment with a minimal skin-irritating effect \[[@CR11]\]. The hyperkeratotic surface of the backs of the hands requires multiple, extended treatments or pre-treatment with 5% salicylic acid \[[@CR11]\]. Lesions below the knee have a propensity for ulceration and poor healing, will require close monitoring and may require elevation of the leg and compression bandaging.
The grade of the lesion affects the treatment response. Lower-grade lesions are generally more responsive than higher-grade lesions \[[@CR41]\], and consequently an important consideration for treatment.
Patient Factors for Treatment {#Sec16}
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Patients' willingness to seek diagnosis and treatment depends primarily on their attitude towards AK as a potentially malignant lesion. Young patients view skin cancer as a problem of adulthood, whereas adults think that their general self-assessment of good health limits their susceptibility to skin cancer \[[@CR42]\]. Moreover, the desire for a suntanned appearance and the erroneous belief that a suntan is protective against skin damage contributes to inadequate use of sun-protection behaviors \[[@CR42]\].
For treatment strategies to succeed, physicians and patients need to agree on a treatment plan that is understood by the patient to promote high adherence. For the physician, treatment choice will depend on disease-related factors: the patient profile (e.g., existing comorbidities); the cost of treatment; and patient preference. Understandably, both patients and physicians wish to combine high efficacy with minimal side effects. This is one reason why 5-FU cream, with its significant side effects, is less often used \[[@CR24]\]. In addition, the photosensitizing effects of some topical agents, such as 5-FU, will discourage some patients from selecting this approach. Nonetheless, it should be noted that patient tolerance of side effects can vary and depend on factors such as age, physical health, and presence of comorbidities. Consequently, it is imperative that patients are informed about the efficacy and side effects of the available treatments. The cosmetic effects of the treatment are also an important consideration for some patients, as the side effects can be very pronounced and visually obvious and consequently result in psychosocial difficulties (Fig. [3](#Fig3){ref-type="fig"}) \[[@CR43]\]. Therefore, treatment choice for patients depends both on the cost of the treatment and the length of its side effects.Fig. 3Pustulation in a patient with marked AK **a** pre-treated with retinoic acid and **b** followed by methyl aminolevulinate photodynamic therapy. *AK* Actinic keratosis. Reproduced with permission from Tran and Salmon \[[@CR43]\]
Pharmacologic therapy is a good choice for patients who understand and demonstrate high personal responsibility, as well as understand the risks of AK and the need for good adherence to treatment, as this treatment option can be undertaken conveniently in their home \[[@CR20]\]. It is speculated that there is a lower rate of treatment adherence with an agent that requires repeat application over a prolonged period of time, compared with a topical agent with short treatment duration such as ingenol mebutate \[[@CR36]\]; however, comparative studies have not been conducted \[[@CR41]\]. For example, imiquimod treatment duration can range 4--16 weeks, and is administered 2--3 times weekly. In addition, diclofenac in hyaluronic acid is used twice a day for 3 months, leading to adherence issues as well as financial costs. Other strategies that are likely to improve adherence are a good doctor--patient relationship, and patient education and follow-up by telephone or visits \[[@CR41]\].
Cryosurgery and PDT are alternative treatment options for patients who are not suitable for pharmacologic field-directed therapy, given their age, medical comorbidities, or compromised cognition. The main advantages of these procedures are their speed and adequate clearance of abnormal tissue. However, targeted approaches fail to address field cancerization. In addition, there are treatment-associated side effects such as scarring and hypopigmentation for cryosurgery; and application-site pain and photosensitivity for PDT.
Comparison of Global Primary Care Management of AK {#Sec17}
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Influence of Prevalence {#Sec18}
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The prevalence of AK is high in Australia because of its large proportion of fair-skinned people and higher sun exposure compared with Canada and the UK. The latitude gradient within Australia results in a larger occurrence of AK in lower latitude regions such as Queensland compared with other parts of Australia; there is also a greater incidence of AK in rural areas where jobs involve greater sun exposure \[[@CR25]\].
The higher prevalence of AK in Australia has resulted in a greater awareness of AK for both patient and physician than in countries with lower prevalence \[[@CR18]\]. Consequently, management of AKs in Australia is very comprehensive; there are established management strategies and detailed clinical practice guidelines written by the Cancer Council of Australia, although these are now several years old and do not describe treatments that have become available more recently \[[@CR25]\]. Given their higher familiarity with AK, Australian primary care physicians are more likely to treat the condition themselves than refer on to other specialists (44% for Australia vs. 91% for UK) \[[@CR18]\]. With the aging population and the subsequent rise in numbers of patients with AK, this may provide challenges to secondary care systems in countries where primary care physicians are less familiar with diagnosing the condition, underlining the continuing need for education about AK in this setting.
Treatment Trends {#Sec19}
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### Current Treatments {#Sec20}
A 2006 retrospective study of Australian general practitioners' medical records reported that the most commonly used treatments for AK lesions were cryosurgery (63%), excision (18%), and a mixture of excision, curettage, and cryosurgery (5%) \[[@CR44]\]. Topical agents (4%) were not commonly employed, although a new product, ingenol mebutate gel, which was developed in Australia, is now being used \[[@CR26]\] as well as 5-FU, imiquimod cream, and diclofenac gel \[[@CR25]\]. One reason for the low use of topical agents could be that the resultant therapeutic response can be unpredictable. Another more practical reason is that invasive treatment options are eligible for government rebate, whereas this is not necessarily the case with topical treatments \[[@CR44]\].
There are no recent treatment guidelines for Canada; however, a comprehensive five-step approach strategy for the treatment of AK has been proposed (Fig. [4](#Fig4){ref-type="fig"}) \[[@CR20]\]. This strategy proposes: (1) periodic dermatologic examinations; (2) field-directed therapy; (3) lesion-directed therapy; (4) patient education with regard to sun protection and the importance of AK treatment and its completion; and (5) regular skin self-exams. The choice of therapy is a decision that combines both the characteristics of the AK lesion and patient considerations (Table [3](#Tab3){ref-type="table"}).Fig. 4Multistep approach for evaluation and treatment of AK and photodamaged skin. *AK* Actinic keratosis, *PDT* photodynamic therapy, *UV* ultraviolet. Reproduced with permission from Ceilley and Jorizzo \[[@CR20]\]Table 3Summary of management for patients with AKCommentsPatient considerations Medical comorbidities Likelihood of adherence Seeking single treatment Financial capabilitiesLesion characteristics NumberLow number: amenable to destructive therapy; high number: requires field therapy Thin vs. hyperkeratoticHyperkeratotic: may require debridement before destructive treatment; biopsy should be considered when hyperkeratotic LocationFacial lesions: well treated by field pharmacotherapiesBacks of hands: more difficult to treatUse special care for high-risk locations such as periorbital area, lips, and below kneeTherapeutic considerations Destructive modalities CryosurgeryMost common because of ease of use good tolerability, efficacy for thin lesions Shave excisionConsider for single recurrent lesion CurettageInvasive; requires medical prophylaxis; not first-choice treatment for AK Dermabrasion and chemical peelsTopical pharmacotherapy 5-fluorouracilFrequently usedMultiple concentrations availableIntensity of localized skin reaction may limit tolerability in some patients Imiquimod Diclofenac Ingenol mebutateConvenient: very short duration of treatment (2--3 days)Photodynamic therapyHigher cost; requires specialized equipmentPatient educationCounseling from dermatology clinical team should include Nature of disease Proper use of treatments Expectations of treatments Use of sunscreens Skin self-examinationsReproduced with permission from Ceilley and Jorizzo \[[@CR20]\]*AK* actinic keratosis
In Europe, a simple treatment algorithm to assist clinicians in the management of AK and to standardize and improve patient care was developed in 2008 by the European Skin Academy \[[@CR24]\]. An update of these guidelines by the European Dermatology Forum indicated that there is a paucity of studies on the frequency and cost of AK treatment in Europe \[[@CR28]\]. Clinical guidelines from the UK Primary Care Dermatology Society state that patients should be managed in the community if at all possible, and only referred to a consultant dermatologist in cases of diagnostic uncertainty or if the damage is widespread or severe \[[@CR45]\]. These guidelines recommend both individual lesion treatment for few or widely spread lesions and field therapy for areas of skin with multiple AKs (Fig. [5](#Fig5){ref-type="fig"}). Cryosurgery is generally widely utilized within Europe and is an effective treatment for single AKs \[[@CR24]\]. Alternative, non-invasive topical treatments such as imiquimod, PDT, diclofenac 3% gel, and ingenol mebutate gel are seen as promising options for treating larger areas of field cancerization \[[@CR28], [@CR45]\].Fig. 5Management of AK in United Kingdom \[[@CR45]\]. *AK* Actinic keratosis, *GPwSI* general practitioner with a special interest, *SCC* squamous cell carcinoma, *UV* ultraviolet
### New Treatments {#Sec21}
The use of daylight PDT along with a suitable sunscreen following methyl aminolevulinate application can minimize pain associated with conventional PDT and may be a convenient alternative in geographical areas where appropriate daylight and weather conditions prevail \[[@CR46]\]. An international consensus concluded that daylight PDT is an effective and well-tolerated field therapy, particularly in those patients with large areas affected by AKs that can be exposed to daylight \[[@CR47]\], and suggested that this therapy is studied further with regard to appropriate seasonal and time-of-day use in different geographical locations. Experience with daylight PDT in UK, Canada, and Australia primary care is currently minimal.
The advent of new and efficacious topical treatments for AK has led to the instigation of new guidelines. The National Institute for Health and Care Excellence (NICE) recently published a guide "Actinic Keratosis: Ingenol Mebutate Gel" \[[@CR48]\] to update physicians following the approval of ingenol mebutate gel in the UK. The publication concluded that ingenol mebutate gel had similar efficacy to other field-directed treatment therapies, but with much shorter treatment duration; however, side effects between the therapies varied. The most common side effects for ingenol mebutate gel were skin responses at application site. In addition, the Scottish Medicines Consortium completed an assessment on ingenol mebutate gel in February 2013, and concluded that in the four randomized, double-blind, phase III studies, a significantly greater proportion of adults with AK achieved complete clearance when treated with ingenol mebutate gel \[[@CR49]\]. Furthermore, a 2012 Cochrane Database Systematic Review of 83 randomized controlled trials (*n* = 10,036) of the management of AK concluded that for individual lesions, PDT appeared more effective and had increasing reports of a better cosmetic outcome than cryosurgery \[[@CR15]\]. For field-directed treatments, diclofenac, 5-FU, imiquimod, and ingenol mebutate gel had similar efficacy, but their associated side effect and cosmetic outcomes were different \[[@CR15]\]. The authors conclude that direct head-to-head studies of these treatments are required to determine the optimal therapeutic approach for AK.
Management and Healthcare {#Sec22}
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### Australia {#Sec23}
Primary care physicians have a pivotal position in the Australian health care system, as they diagnose and manage a greater number of skin lesions as well as more serious ones, particularly in rural areas. However, 9% of AK cases are referred to non-dermatological specialists, of which 57% are then referred to a dermatologist \[[@CR44]\].
In Australia, although cryosurgery is subsidized for the general public, not all field therapies are, except for repatriated patients. Imiquimod is only subsidized for superficial basal cell carcinoma that is not amenable to surgery. There are some subsidies available for AK treatment depending on patient health care cover. Patients are not currently subsidized for newer treatments such as non-surgical or specialist treatments (e.g., PDT, both conventional or daylight). The availability of subsidies is likely to influence treatment choice, particularly if the patient has many lesions. In accordance with the current Australian health care system fee schedule, treatment of SCC in situ attracts a fee \[[@CR25]\], as does both cryosurgery for more than 10 AK lesions, and curettage/excision of an AK. Consequently, cost is also a factor in treatment choice for the patient.
### UK {#Sec24}
Prescription charges apply in the UK for the majority of the adult population (16--60 years of age). However, most patients with AK will be exempt from payment because of their age. In the UK, primary care physicians act as the gatekeepers to access specialist services. They see patients presenting with AK and refer them principally to dermatologists. A UK study in 2005 reported that confirming diagnosis (39%) and management (57%) are the primary reasons to refer patients to a specialist \[[@CR50]\].
Clinical Commissioning Groups are responsible for the total expenditure resulting from primary care, including referral to secondary care. One of their roles is to reduce referrals. A simple to use treatment for AK, along with further education for general practitioners to increase their diagnosis skills, is likely to be very attractive as it will reduce referral rates.
### Canada {#Sec25}
In Canada, primary care physicians play a vital role in reducing the overall burden of AK through diagnosis and management. Resistant and difficult to treat cases are referred to dermatologists; however, no exact national referral rates have been documented in current literature. The national Pharmacare program subsidizes cryosurgery, with the patient paying either individually or through extended third-party insurance for the remaining AK treatment options. The treatment option most often used in Canada for the management of AKs is cryosurgery followed by topical 5-FU. However, patients who have extended insurance now opt for ingenol mebutate because it is convenient to use due to its shorter treatment duration, and also for field treatment of subclinical AK lesions that are not identified and treated with cryosurgery.
Conclusion {#Sec26}
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The most important reason to identify and treat AK correctly is to reduce the incidence and burden of cutaneous SCC. There is therefore a need to increase awareness and knowledge about AK, including symptoms, prevention, and its associated risk of non-melanoma skin cancer, especially among the public. Primary care physicians are perfectly placed to convey this message to their patients and play a pivotal role in educating them on the importance of AK treatment and its role in disease progression.
For AK treatment to be successful, physicians and patients should agree on a treatment plan. The physician should be mindful of patient expectations and ensure patient understanding of the treatment and its possible side effects, consequently improving overall adherence. Because of the recurrent nature of AK, patients should maintain an ongoing dialog with their primary care physician or their dermatologist.
Better use of sun-protective products, increased awareness of sun-induced damage, and improved dermatologic screening and treatment can help reduce the harmful effects of chronic sun exposure, and help to prevent malignant transformation and progression to non-melanoma skin cancers.
Electronic supplementary material
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{#Sec27}
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Supplementary material 1 (PDF 200 kb)
Selected images are published with permission from the New Zealand Dermatological Society Incorporated. Scientific support was provided by John R. Zibert, employee of LEO Pharma A/S. Medical writing services were provided by Isabelle Leach, Rasila Vaghjiani and Barbara Francis of iMed Comms, Macclesfield, UK, and were funded by LEO Pharma A/S. Article processing charges were funded by LEO Pharma A/S. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval for the version to be published.
Conflict of interest {#d30e1419}
====================
Praven Chetty reports no conflicts of interest. Felix Choi has received honoraria from LEO Pharma A/S for speaking engagements. Timothy Mitchell has received honoraria from LEO Pharma A/S for consulting at advisory boards.
Compliance with ethics guidelines {#d30e1424}
=================================
The analysis in this article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by any of the authors.
Open Access {#d30e1429}
===========
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
| {
"pile_set_name": "PubMed Central"
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INTRODUCTION {#s1}
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The opportunistic pathogen Klebsiella pneumoniae is a major public health threat due to its propensity to become extensively drug resistant ([@B1], [@B2]), the emergence of hypervirulent strains ([@B3][@B4][@B5]), and the recent evolution and increasing prevalence of strains displaying both hypervirulence and extensive drug resistance ([@B6], [@B7]). Virulence in K. pneumoniae is multifactorial and depends on both core-encoded and horizontally acquired factors ([@B8], [@B9]). Capsule is a critical K. pneumoniae virulence determinant present in all clinical strains; mutants lacking capsule are avirulent, while overproduction of capsule is associated with hypervirulent strains and more severe disease in animal models ([@B10], [@B11]). More than 130 capsule locus types have been described in K. pneumoniae ([@B12]), and hypervirulent strains usually produce capsule type K1 or, less frequently, K2. Nine lipopolysaccharide (LPS) O-side-chain groups have been identified and characterized in K. pneumoniae ([@B13]); these moieties modulate innate immune signaling and may contribute to serum resistance. Horizontally acquired virulence genes include siderophores and capsule upregulators ([@B9], [@B14]). In general, understanding of K. pneumoniae pathogenesis is confounded by the phylogenetic breadth of infectious lineages, and by the diversity of the virulence factors themselves.
The complement system, comprising more than 20 proteins in serum and tissue fluids, is a first line of defense against bacterial invaders that have breached the host's epithelial barriers. Resistance to complement is strongly correlated with the capacity for systemic survival, multiplication, and spread of a wide range of Gram-negative pathogens ([@B15]), and is a major virulence trait that enables K. pneumoniae to elicit invasive infections ([@B16], [@B17]). The complement cascade can be activated via the classical, alternative, and lectin pathways, which each act in a precise sequence of reactions to facilitate C3b deposition onto the target bacterial surface. The classical pathway is initiated following recognition of antigen-antibody complexes on the bacterial cell surface by hexameric C1q, whereas the lectin pathway begins with detection of bacterial surfaces by pattern recognition molecules such as mannose-binding lectin or the ficolins ([@B15], [@B18], [@B19]). All pathways converge at C3 cleavage, with the larger cleavage product C3b covalently bound to the target surface. Accumulation of anchored C3b by amplification leads to the assembly of C5 convertases that generate the C5b cleavage product, which spontaneously associates with one molecule each of C6, C7, and C8 and with multiple copies of C9 to form the C5b-9 membrane attack complex. In complement-susceptible bacteria, C5b-9 complexes intercalate into the outer membrane (OM) bilayer and perturb the cytoplasmic membrane through an incompletely defined process ([@B20][@B21][@B22]).
Gram-negative bacterial resistance to complement can be due to failure of activation of any of the complement pathways; degradation of activated complement proteins; arrest of activated pathways by complement inhibitors such as C1-inhibitor protein (C1-Inh), factor H (fH), and C4 binding protein (C4bp); or the inability of C5b-9 complexes to assemble and insert into the OM (which can be a result of impedance by bacterial surface structures) ([@B15]). The basis of the complement resistance of K. pneumoniae is still poorly understood. Although it has been reported that limiting complement activation and C3b accumulation is the primary mode of resistance, both complement-resistant and -susceptible clinical isolates and mutants may activate complement cascades after exposure to human serum ([@B16], [@B23][@B24][@B25]). Multiple different factors can influence serum resistance in K. pneumoniae, including capsule type and amount, O-antigen type, and various surface proteins; capsules and O antigens have each been invoked as the main determinant of complement resistance ([@B9], [@B26]). However, a recent study of \>150 K. pneumoniae clinical isolates from Thailand with various complement susceptibilities concluded that susceptibility did not correlate with the presence of specific genes, particular capsule types, or even with the hypercapsulation phenotype of the isolates ([@B27]). This study highlighted the main limitation of collective studies on complement resistance in K. pneumoniae to date---that although many resistance factors are individually well characterized, there is very limited understanding of how their activities play out in different combinations or across diverse isolates of K. pneumoniae.
Untangling the mechanisms behind complement resistance in K. pneumoniae will lead to better understanding of the virulence of this bacterium and will provide avenues to target complement resistance in the clinic, particularly in view of growing interest in the targeting of capsule and other virulence factors as an anti-infective strategy for K. pneumoniae ([@B28][@B29][@B30]). In particular, developing generally applicable (rather than K type-specific) therapeutics that promote complement killing requires deeper knowledge of the activity of different complement resistance factors in diverse strains. In this study, we used functional genomic profiling by transposon-directed insertion site sequencing (TraDIS) to define the genetic basis of serum survival in four diverse strains of K. pneumoniae. We show that complement resistance is multifactorial and strain specific, and we identify RfaH and Lpp as shared K. pneumoniae resistance determinants. Two of the strains evaded complement killing by preventing C3b and C5b-9 accumulation at the cell surface, which was disrupted in Δ*rfaH* and Δ*lpp* mutants, while the remaining two strains were resistant to serum despite substantial C5b-9 deposition. Our results present a picture of at least two distinct modes of complement resistance in K. pneumoniae and point to RfaH and Lpp as potential targets for complement-sensitizing therapeutics.
RESULTS {#s2}
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Serum-resistant isolates of K. pneumoniae. {#s2.1}
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Three well-studied hypervirulent K. pneumoniae strains and one recently isolated classical strain were tested for survival in human serum ([Fig. 1A](#F1){ref-type="fig"}; see also Table S1 in the supplemental material). B5055 (sequence type 66 \[ST66\]) produces a type K2 capsule and was originally isolated from a sputum sample in the 1920s. NTUH-K2044 is a hypervirulent strain (sequence type 23) that produces a K1 capsule and was the first characterized liver abscess-causing K. pneumoniae isolate ([@B31]). ATCC 43816 is another K2 strain that is commonly used in mouse virulence studies ([@B32]). K. pneumoniae RH201207 is a colistin-resistant ST258 strain obtained from Public Health England in 2012 ([@B33]). These strains differ in their capsule production as determined by uronic acid assay, with B5055 and NTUH-K2044 producing copious capsule and ATCC 43816 and RH201207 producing less ([Fig. 1B](#F1){ref-type="fig"}). All four strains survived exposure to 66% normal human serum (a potent source of complement) over a 3-h incubation period at 37°C when an inoculum of 1 × 10^6^ cells was employed ([Fig. 1C](#F1){ref-type="fig"}); strains B5055, NTUH-K2044, and ATCC 43816 proliferated in serum, whereas viable counts for RH201207 did not change between 0 and 2 h but showed a slight reduction at 3 h. A sensitive control strain, Escherichia coli DH5α, showed no viability after a 30-min incubation, and killing of all strains was completely abrogated by heat inactivation of the serum (56°C, 30 min; data not shown).
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TraDIS analysis of complement resistance in K. pneumoniae isolates. {#s2.2}
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We performed transposon insertion sequencing of saturated mutant libraries exposed to serum to define the genes contributing to serum survival in each of the four K. pneumoniae strains. The K. pneumoniae B5055 library was constructed for this study by conjugative delivery of pDS1028 and contained 225,000 unique transposon insertions (see Materials and Methods and Table S2 in the supplemental material), while NTUH-K2044, ATCC 43816 and RH201207 mutant library construction has been previously described ([@B33], [@B34]). Our experimental strategy was similar to that used in previous work with E. coli ST131 ([@B35]), with libraries treated with either normal human serum or heat-inactivated serum for 90 min, outgrown for 2 h, and sequenced and mapped using the BioTraDIS pipeline ([@B36]). Putative serum resistance genes were defined as those with altered mutant abundance in the serum-treated libraries in comparison to that in the control libraries treated with heat-inactivated serum (normal serum versus heat-inactivated serum log~2~ fold change of less than −1 or more than 1; significance \[*q* value\] of \<0.005; see Table S3 in the supplemental material). Comparing to the heat-inactivated serum control, rather than the input library, minimizes the chance of spurious hits to mutants with general growth defects.
A total of 93 genes were identified that altered serum survival in one or more K. pneumoniae strains ([Fig. 2](#F2){ref-type="fig"}; see also Table S4 in the supplemental material), with the number of hits in each strain ranging from 22 (for B5055) to 54 (for RH201207). These genes included 43 core or soft core K. pneumoniae genes (present in \>99% or 95 to 99% of K. pneumoniae strains), 24 shell genes (15 to 95% of strains), and 26 cloud genes (\<15% of strains). Despite the high proportion of shell and cloud genes, 60 of the serum survival-related genes were present in all four of the strains examined. Putative serum resistance genes came from multiple functional categories that included synthesis of surface polysaccharides, metabolism, cell surface or membrane structure and function, and transcriptional regulation ([Fig. 3](#F3){ref-type="fig"}; and Table S4). Overall, there was an unexpected strain specificity in the exact genes identified; even among the 60 gene hits present in all four strains, the majority (35 genes) influenced serum survival in only one strain, 22 genes were hits in two or three strains, and only three genes affected serum survival in all four K. pneumoniae strains ([Fig. 2A](#F2){ref-type="fig"} and [Fig. 3](#F3){ref-type="fig"}).
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Contribution of capsule to serum resistance. {#s2.3}
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Capsule biosynthetic genes (*cps*) were among the putative serum survival factors in the four strains investigated. The proportion of *cps* locus genes contributing to complement resistance and the magnitude of the fitness changes involved varied between isolates ([Fig. 2B](#F2){ref-type="fig"} and [Fig. 3](#F3){ref-type="fig"}). Note that mutagenesis of genes of the capsule locus can cause secondary cell envelope defects (shown for *wza* and *wzi* \[[@B37]\]), and not all *cps* locus mutations entirely eliminate capsule production ([@B34], [@B38]); therefore, complete consistency of selection across all genes of the *cps* locus is not expected. With K. pneumoniae B5055, which produces copious amounts of K2 capsule, 11/18 genes of the *cps* locus were called as hits, accounting for half of the serum survival determinants of this strain. They included the exporter *wzi*, the sugar precursor genes *manBC* and *galF*, and the majority of K-type specific genes in the central operon of the K2 locus ([Fig. 3](#F3){ref-type="fig"}; see also Fig. S1 in the supplemental material). The majority of these genes were also required in the K2 strain ATCC 43816 ([Fig. 3](#F3){ref-type="fig"} and Fig. S1), with the exceptions of *wzi*, and the sugar precursor genes *manB* and *ugd* (which had too few reads in this strain for serum-specific effects to be measured). In NTUH-K2044, 8/20 *cps* genes were called as hits and in RH201207 this proportion was 9/19 ([Fig. 3](#F3){ref-type="fig"} and Fig. S1). Because the pDS1028 transposon has transcriptional readout from one end, transposon insertions are not predicted to dramatically disrupt downstream gene expression in the NTUH-K2044, B5055, and ATCC 43816 libraries. This effect is clear in NTUH-K2044, where transposon insertions in several genes of the *cps* locus (e.g., KP1_3714, KP1_3718 through KP1_3720) are counterselected by serum on one strand but unaffected on the other (Fig. S1). The RH201207 library was constructed using a different transposon, and transcriptional read-through is not expected in this library. Our TraDIS results indicate that all K. pneumoniae strains require capsule to some extent to withstand serum challenge. Known regulators of capsule biosynthesis also influenced serum survival, including the antiterminator gene *rfaH* in all four isolates ([@B39], [@B40]), *rmpC* (BN49_pII0025) in B5055 only ([@B41]), and *rcsB* in RH201207 ([@B42]). We hypothesize that the *rcsB* mutant showed a serum survival defect only in the RH201207 background because this strain produces less capsule than the other three strains, making it more sensitive to mutations that further reduce capsule expression. Mutation of the *rmpC* gene had no effect in NTUH-K2044; however, this strain encodes both chromosomal and plasmid copies of *rmpC*.
Contribution of LPS O side chains to serum resistance. {#s2.4}
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Enterobacteriaceae species lacking LPS O side chains are generally susceptible to C5b-9-mediated killing ([@B43]), and introduction of genes determining O side chains into a highly complement-sensitive rough Escherichia coli strain elicited a large increase in complement resistance ([@B44]). With our four K. pneumoniae strains, the majority of O-antigen genes showed a serum fitness defect when mutated ([Fig. 2B](#F2){ref-type="fig"} and [F](#F2){ref-type="fig"}ig. [3](#F3){ref-type="fig"}), with the exception of those of K. pneumoniae B5055. This is surprising because B5055 encodes the same O-antigen type (O1v1) as NTUH-K2044, in which O-antigen mutants showed a drastic fitness defect ([Fig. 2B](#F2){ref-type="fig"} and [F](#F2){ref-type="fig"}ig. [3](#F3){ref-type="fig"}). We suggest that the B5055 strain is almost completely protected from serum bactericidal activity by its thick K2 capsule, masking the additional protective activity of the O antigen. K. pneumoniae ATCC 43816 produces K2 capsule, albeit in smaller amounts than that produced by B5055, but still required O antigen for serum survival. These findings suggest that the K2 capsule is sufficient to completely protect from complement-mediated killing when produced in copious amounts, while the K1 capsule is not, at least in these isolates.
Lipopolysaccharide (LPS) core biosynthetic genes contributed to serum fitness in isolates ATCC 43816 and NTUH-K2044 (see Table S4 in the supplemental material), although the same genes were either essential or had no effect on resistance in B5055, and were also not identified as statistically significant hits in RH201207. Note that mutation of many LPS core genes causes a severe general fitness defect, so their specific contributions to serum resistance are not always easy to define. A small subset of the genes (*arnD* to *arnF*) in the *arn* (*pmr*) operon responsible for LPS lipid A modification showed complement resistance defects in one or more strains when mutated ([Fig. 2B](#F2){ref-type="fig"}). This was unexpected, as the [l]{.smallcaps}-Ara4N lipid A modification is rarely made *in vitro* and is not produced under rich medium conditions like those used in this study ([@B45]). Loss of any of the *arnDEF* genes was previously shown to reduce K. pneumoniae mucoviscosity in a genome-wide density-based screen ([@B34]), and we suggest that reduced capsule production underpins the serum survival defects seen here.
Other genes implicated in serum survival. {#s2.5}
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Mutation of several genes involved in cell membrane or cell wall structure and function resulted in fitness defects in serum. These genes included *dacA* (RH201207 only), which is involved in cell wall biosynthesis, the inner membrane protein *dedA* (in isolates B5055, NTUH-K2044, and ATCC 43816) which has a role in membrane integrity, and components of the *tol-pal* outer membrane transporter (NTUH-K2044 and ATCC 43816). Finally, the outer membrane lipoprotein Lpp was required for full serum resistance in all four strains. A number of metabolic genes were also implicated in serum survival, primarily those involved in pyrimidine metabolism, and in metabolism of carbohydrates ([Fig. 3](#F3){ref-type="fig"} and Table S4). Some of these genes (*pgi* and *pgm*) are involved in precursor molecule biosynthesis for capsule and LPS.
Increased serum survival genes in K. pneumoniae RH201207. {#s2.6}
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Five genes of K. pneumoniae RH201207, namely *csrD*, *fabR*, *wecB*, *wecC*, and *cyoA*, led to increased serum fitness when mutated. CsrD promotes degradation of the capsule-regulating small RNA CsrB; mutation of *csrD* can promote capsule production (as measured by density) ([@B34]), which may explain the enhanced serum survival of this mutant. FabR, WecB, and WecC are not predicted to affect capsule, but all three genes have roles relating to the cell envelope: FabR is a transcriptional regulator which controls the ratio of saturated to unsaturated fatty acids in the cell membrane, and WecB and WecC produce the second component of enterobacterial common antigen (*N-*acetyl*-* [d]{.smallcaps}-mannosaminuronic acid) and attach this to UDP-GlcNac. We speculate that loss of *wecB* and *wecC* increases the pool of UDP-GlcNac in the cell, which is then diverted into O-antigen biosynthesis (which also utilizes UDP-GlcNac) ([@B46]). The cytochrome ubiquinol oxidase component CyoA also resulted in increased serum survival when mutated through an unknown mechanism. The identification of mutants with increased serum fitness in RH201207, but not in the other K. pneumoniae strains, is consistent with the observation that serum survival of K. pneumoniae RH201207 is less dependent on capsule.
Confirmation of RfaH and Lpp as shared serum resistance factors in K. pneumoniae isolates. {#s2.7}
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Only the following three genes affected serum survival in all four strains tested: the LPS modification gene *arnD*, the outer membrane lipoprotein *lpp*, and the transcription antiterminator *rfaH* ([Fig. 2](#F2){ref-type="fig"} and [@B3]). We selected Lpp and RfaH for further characterization as potential core serum resistance factors of K. pneumoniae. ArnD was not selected for followup because we failed to detect the relevant LPS modification *in vitro* (which is consistent with previous reports that the modification is made *in vitro* only under very specific conditions \[[@B45]\]) and therefore presumed its activity was indirect, though the potential role of lipid A modifications in K. pneumoniae complement resistance may be of interest for a future study. Deletion mutants of *rfaH* and *lpp* were constructed in K. pneumoniae NTUH-K2044, B5055, and ATCC 43816 by allelic exchange. In isolate RH201207, an insertion mutant in *rfaH* was obtained, but an *Δlpp* mutant could not be generated despite multiple attempts. Serum survival assays were conducted with an inoculum of 10^6^ cells in 66% normal human serum, and bacterial counts were monitored for 3 h ([Fig. 4](#F4){ref-type="fig"}). Loss of *rfaH* caused a large reduction in serum survival in all four strains, and complementation with plasmid-encoded *rfaH* expressed from its native promoter restored wild-type survival, confirming the importance of RfaH in complement resistance ([Fig. 4](#F4){ref-type="fig"}). Loss of *lpp* caused a modest change in complement sensitivity ([Fig. 4](#F4){ref-type="fig"}); these mutants lost the ability to proliferate in serum (note that *lpp* disruption does not cause a general growth defect; see Fig. S2B and Table S3 in the supplemental material), and with the NTUH-K2044 Δ*lpp* mutant, delayed complement killing was observed. The *Δlpp* mutations could not be complemented by expression of *lpp* from its native promoter due to unexpected toxicity during cloning. Expression of *lpp* from an arabinose-inducible promoter also failed to complement the serum proliferation defect of the *Δlpp* mutants. We suspect that this was due to insufficient expression. In addition, proliferation of the vector-only control strains was impaired by addition of arabinose (data not shown). Although we were unable to find an appropriate system for complementation of the *Δlpp* mutants, their phenotypes align with the results of the genome-scale screens ([Fig. 2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}), as well as with published work on Lpp in K. pneumoniae NTUH-K2044 ([@B47]).
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We were intrigued by the varied serum fitness effects caused by different *cps* locus mutations seen in TraDIS, and several randomly isolated capsule locus mutants of ATC C43816 and RH201207 were also examined for serum survival in order to further validate the genome-scale screens (Fig. S2). Each of these mutants showed the phenotype predicted based on TraDIS screening, namely, ATCC 43816 i-*wcaJ*, which was not identified as a serum resistance gene, multiplied to the same extent as that of the wild type, ATCC 43816 i-*wza* did not proliferate in serum, and RH201207 i-*wzc* was rapidly susceptible. RH201207 i-*wcaJ*, which was not a hit, was viable after 90 min (our TraDIS time point) but showed a delayed susceptibility to serum. Note that *wcaJ* deletion in K. pneumoniae does not completely eliminate K2 capsule production and can also have pleiotropic effects that include rounded cell morphology and increased fitness under nutrient limitation ([@B37], [@B38])---therefore, the full resistance of ATCC 43816 i-*wcaJ* does not preclude a role for capsule in the complement resistance of this strain. Taken together, the results of serum survival assays with defined mutants show perfect agreement with the phenotypes predicted from TraDIS screens (for 11/11 mutants), and establish RfaH and Lpp as shared serum resistance factors in K. pneumoniae. These experiments also revealed additional subtleties in the serum resistance phenotypes of the mutants, with survival patterns roughly following the underlying resistance of the parent strain (for example, ATCC 43816 Δ*rfaH* and RH201207 Δ*rfaH* mutants), and some differences (e.g., RH201207 i-*wcaJ*) were only revealed at later stages of incubation.
Lpp influences capsule retention but not capsule production and requires lysine-78. {#s2.8}
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The antiterminator RfaH and the murein lipoprotein Lpp contributed to complement resistance in all four K. pneumoniae strains. Lpp is an extremely abundant protein that contributes to cell envelope integrity by connecting peptidoglycan to the cell outer membrane ([@B47], [@B48]). We observed that the Δ*lpp* mutant colonies were flat and unstructured in comparison to those of the wild type, although their opacity suggested that they still produced capsule. To examine the effect of the *lpp* mutation further, we measured total and cell-attached capsule using the uronic acid assay. All three K. pneumoniae Δ*lpp* mutants produced capsule at wild-type levels but showed moderate decreases in amounts of cell-associated capsule ([Fig. 5A](#F5){ref-type="fig"}). Mutants of *rfaH* showed dramatically reduced capsule amounts ([Fig. 5B](#F5){ref-type="fig"}). We then tested whether Lpp activity requires covalent linkage to peptidoglycan, mediated through the ε-amino group of the C-terminal lysine residue in Lpp and the meso-diaminopimelic acid residue on the peptidoglycan peptide stem ([@B49]). Expression of Lpp from an arabinose-inducible vector partially complemented the hypermucoid phenotype of NTUH-K2044 and B5055 ([Fig. 5C](#F5){ref-type="fig"}). Partial complementation was not seen with an Lpp-ΔK78 construct, confirming that the C-terminal lysine is required in order for Lpp to promote capsule retention in both K1 and K2 strains. The shared serum survival factors Lpp and RfaH therefore both appear to function at least partly through effects on capsule.
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Deposition of C3b and C5b-9 complexes. {#s2.9}
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Genome-scale screening revealed a very high degree of strain specificity in the serum resistance determinants across four K. pneumoniae strains. We decided to explore complement activation by these strains and how this is affected by the loss of *rfaH* or *lpp*. Activation of any or all complement pathways will lead to C3b generation and binding to the target bacterial surface; subsequent formation of C5 convertase complexes may lead to deposition of membrane attack complexes and cell death ([@B15], [@B19]). Surface C3b deposition and C5b-9 formation on K. pneumoniae strains and mutants during incubation with human serum are reported in [Fig. 6](#F6){ref-type="fig"} and [7](#F7){ref-type="fig"} and in Fig. S3 and S4 in the supplemental material. The three hypervirulent strains showed little to no C3b binding, while serum exposure of RH201207 led to a considerable increase in levels of C3b and C5b-9 over time ([Fig. 6A](#F6){ref-type="fig"} and [Fig. 7A](#F7){ref-type="fig"}; Fig. S3 and Fig. S4A). ATCC 43816 also showed C5b-9 binding at later time points, while B5055 and NTUH-K2044 did not. In all backgrounds, the deletion of *rfaH* led to significant levels of C3b and C5b-9 binding compared to that of the wild type, with a peak after 2 to 3 h of serum exposure ([Fig. 6A](#F6){ref-type="fig"} and [Fig. 7A](#F7){ref-type="fig"}), confirming that the mechanism of serum killing observed ([Fig. 4](#F4){ref-type="fig"}) is through formation of the membrane attack complex. With the B5055 Δ*rfaH* mutant, cells could not be examined after the 30 min time point due to cell lysis as determined by the release of cytoplasmic green fluorescent protein (GFP) from strain B5055 Δ*rfaH* pFLS21 (Fig. S4B). Imaging of the Δ*rfaH* mutants showed that C3b binding is evenly distributed over the cell surface and occurs within 5 min of serum exposure ([Fig. 6B](#F6){ref-type="fig"}), while C5-9 deposition is minimal at 5 min (except for ATCC 43816) and uniformly detected at 15 min ([Fig. 7B](#F7){ref-type="fig"}). Similarly, Δ*lpp* mutants were also found to significantly bind C3b and C5b-9 compared to the wild type, although to a lesser extent than the Δ*rfaH* mutants ([Fig. 6A](#F6){ref-type="fig"} and [Fig. 7A](#F7){ref-type="fig"}; Fig. S3 and S4). Most Δ*lpp* mutant cells maintained their rod shape following 15 min of serum exposure ([Fig. 6C](#F6){ref-type="fig"} and [7C](#F7){ref-type="fig"}), which correlates with increased serum susceptibility only after longer exposure times ([Fig. 4](#F4){ref-type="fig"}). By examining cell population dynamics, we observed that Δ*lpp* mutants showed a similar distribution to wild-type cells (Fig. S3 and S4A, third columns). In contrast, Δ*rfaH* mutants displayed a more compact distribution in the Q2 quadrant, suggesting that not only do more Δ*rfaH* cells bind C3b and C5b-9 over time, but that the level of binding to individual cells increases. These findings indicate that B5055, NTUH-K2044, ATCC 43816, and RH201207 activate the complement system to different extents and that loss of *lpp* or *rfaH* increases the recruitment of complement components.
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DISCUSSION {#s3}
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Resistance to killing by complement is an important yet incompletely understood feature of K. pneumoniae pathogenesis ([@B4], [@B8], [@B27]). The prominent polysaccharide capsule has been invoked as a key determinant of resistance by virtue of its capacity to limit C3b deposition or assembly of the membrane attack complex ([@B8], [@B26]), but it is clear that other factors also contribute to the complement-resistant phenotype ([@B27]). Resistance to serum killing is associated with K. pneumoniae hypervirulence, and we therefore selected three well-studied hypervirulent strains, as well as a recently isolated clinical strain, for our analyses. To our knowledge, this study represents the first multistrain functional genomics study of complement resistance in any bacterial species.
TraDIS identified 93 genes that impacted serum survival in one or more strains, but only three of these, *rfaH*, *lpp*, and *arnD*, were common to all four strains. All three genes influence the physical characteristics of the outer surface of K. pneumoniae. RfaH controls transcription of operons that direct synthesis, assembly, and export of the lipopolysaccharide core and capsular polysaccharide in E. coli and other Gram-negative bacteria ([@B40]), the abundant peptidoglycan-linked outer membrane protein Lpp is involved in the maintenance of cell envelope integrity and retention of capsule at the cell surface ([Fig. 5A](#F5){ref-type="fig"}) ([@B34], [@B49]), and the *arn* operon encodes proteins that participate in the addition of 4-amino-4-deoxy-[l]{.smallcaps}-arabinose to lipid A ([@B50]) and may also affect capsule levels through an unknown mechanism ([@B34]). Deletion of *rfaH* markedly increased complement susceptibility in all four strains, confirming the key contributions of capsule and LPS O side chains to the resistant phenotype. However, there were strain differences in the rate of complement killing; the K. pneumoniae RH201207 Δ*rfaH* mutant was rapidly killed, while ATCC 43816 and NTUH-K2044 displayed a delayed killing response typical of smooth (O-side-chain-replete) complement-susceptible Gram-negative bacteria ([@B51]). RH201207 possesses LPS O side chains but elaborates less capsule than the other three. These complement susceptibility profiles emphasize the interdependence of the various surface structures that contribute to serum resistance. Deletion of *lpp* in the three hypervirulent isolates modified the serum responses but to various degrees. The loss of proliferation in serum of strains ATCC 43816 and B5055 was not sufficient to convert them to full complement susceptibility, whereas the degree of complement killing of the K. pneumoniae NTUH-K2044 Δ*lpp* mutant was more pronounced. Although capsule retention is impaired in the *lpp* mutants, reducing the protective barrier against complement binding, the presence of large amounts of unattached polysaccharide is likely to have caused off-target complement activation ([@B25]) and depletion of complement components in the serum, resulting in less pronounced killing than that in *rfaH* mutants.
Removal of the capsule by deletion of *rfaH* ([Fig. 5B](#F5){ref-type="fig"}) led to significant deposition of C3b on the outer surface in all four strain backgrounds. Deletion of *rfaH* presumably caused loss of O side chains as well as capsule, as shown in E. coli and other Gram-negative bacteria, including Salmonella enterica and Yersinia enterocolitica ([@B40], [@B52], [@B53]). The formation of C5b-9 complexes at the cell surface and subsequent changes in cell morphology point to a loss in integrity of both the outer membrane and peptidoglycan layer, eventually leading to cell lysis, although the exact mechanism by which the inner membrane is disrupted is not yet understood. With the Δ*lpp* mutants, which have detached capsule ([Fig. 5](#F5){ref-type="fig"}) and increased membrane permeability but retain their O antigen ([@B47]), sufficient deposition of C3b and perturbation of the cell envelope by C5b-9 complexes occurred to prevent proliferation in serum, as seen in [Fig. 4](#F4){ref-type="fig"}. While B5055 and NTUH-K2044 did not show detectable C3b or C5b-9 levels by flow cytometry, the complement-resistant ATCC 43816 strain showed a limited increase in levels of C5b-9 complexes following serum exposure, despite these not functioning as bactericidal entities ([Fig. 6A](#F6){ref-type="fig"} and [Fig. 7A](#F7){ref-type="fig"}; Fig. S4A). Finally, although the classical isolate RH201207 survived 2- to 3-h serum incubation, both C3b and C5b-9 levels rose dramatically following incubation with serum.
These differences in the interplay between surface factors and the complement system are unlikely to be due to differences in strain-to-strain gene content. Around half of the hit genes were present in all four strains but contributed to complement resistance in only one or two (46 of 93 total genes, 60 of which were present in all strains), and this trend held when the classical RH201207 strain was excluded (of 36 hits in B5055-NTUH-K2044-ATCC 43816 shared genes, 12 were specific to one strain, 19 were hits for two strains, and only 5 were hits for 3 strains). However, the degree of strain specificity we found is broadly comparable to that observed for daptomycin resistance genes in two strains of Streptococcus pneumoniae, which showed only 50% overlap despite the two strains sharing 85% of their genes ([@B54]). Furthermore, bacteria such as Salmonella spp., Mycobacterium tuberculosis, and Pseudomonas aeruginosa have been shown by TraDIS/transposon insertion sequencing (Tn-Seq) methods to possess strain-specific essential gene sets ([@B55][@B56][@B57]).
Strain-specific effects are likely to be due to a combination of imperfect hit identification, functional divergence of genes in different strains, and context-dependent fitness contributions of genes with the same activity, due to either redundancy with other factors or differences in the relative contribution of each gene to overall bacterial surface architecture. For example, the O1v2-type O antigen produced by both NTUH-K2044 and B5055 contributed to serum resistance only in the former strain, presumably because in B5055 the protection from the capsule is so strong that other factors are not needed. We speculate that such context-dependent fitness effects may be a common feature in bacterial populations. Our finding that vastly different gene sets underpin serum survival in four strains supports the notion that serum resistance is determined by the overall biophysical properties of the cell surface, rather than by any single factor, and also shows that there are multiple routes by which a complement-resistant cell surface can be generated.
A limitation of our study is that K. pneumoniae is a highly genetically diverse species ([@B58]), and the four isolates that we studied do not cover the range of potential combinations of cell surface structures that may impact survival in serum. We did note that the classical strain RH201207 was markedly different from the three hypervirulent strains in terms of genes involved in complement resistance and complement binding patterns; it would be useful to explore the properties of other classical strains in future studies. Another limitation is that in order to maintain library diversity and provide enough material for sequencing, we based our TraDIS strategy on that used by Phan and coworkers ([@B35]), employing a library inoculum of 10^8^ CFU with only a single 90-min time point, which may have missed delayed or subtle effects on complement resistance. Although high-throughput mutagenesis studies such as ours are the only way to profile the contributions of all nonessential genes to serum survival, mutation or deletion of genes encoding major surface structures (such as capsule, LPS O side chains and abundant membrane proteins) may force a major reconfiguration of the cell surface as a compensatory mechanism to deal with envelope stress ([@B59]); conversion of complement-resistant cells to complement-susceptible cells could be caused by this compensatory response, rather than by loss of the structure itself. In particular, capsule locus mutations can have a range of secondary effects that include changes to cell envelope integrity, cell morphology, and growth rate, and some do not fully abolish capsule production ([@B37], [@B38]). Such effects cannot be detected or avoided by employing different mutagenesis strategies (e.g., gene deletion versus transposon insertion) or by complementation. While these findings fit with the range of serum resistance phenotypes that we observed among different *cps* locus mutants (Fig. S1 and Fig. S2A), they also suggest that any data derived from capsule mutant strains should be interpreted with caution. More direct information comes from recent studies using phage-derived capsule depolymerases, where K. pneumoniae strains are stripped of capsule prior to treatment with serum. In this way, at least eight different capsular types of K. pneumoniae have been confirmed to protect from serum to date, including type K1 (of NTUH-K2044) ([@B60][@B61][@B65]). The magnitude of the change in serum sensitivity following enzymatic capsule removal varies depending on both the strain and the capsule type. We are currently examining the impact of capsule removal on complement susceptibility in systematic fashion using enzymes selective for the most frequently isolated K. pneumoniae capsular serotypes. Despite its inherent limitations, our genome-scale screening gives a picture consistent with recent phage depolymerase work and collective molecular microbiological studies ([@B9], [@B26])---K. pneumoniae capsule can protect from serum killing, and the strength of this protection depends on capsule type, capsule thickness, and the strain background.
The data suggest that K. pneumoniae may adopt different strategies for evasion of complement-mediated attack. Isolates may fail to strongly activate complement pathways (B5055 and NTUH-K2044) or may activate one or more pathways but avoid C5b-9-mediated lethality (ATCC 43816). With either scenario, the capsule is likely to be critical. Implicit in the design of bactericidal assays is the assumption that normal human serum contains IgM or IgG subclasses directed against exposed bacterial surface antigens with the capacity to efficiently activate the classical pathway ([@B66]); this is certainly the case with much-studied E. coli strains but is less clear with K. pneumoniae. After activation, C5b-9 will engender lethal membrane damage only after disruption of lipid domains on the bacterial surface, resulting in a drastic change to membrane topology and architecture. complement-resistant bacteria may not only mask their cell surface from the initial recognition by the three complement pathways but may also inhibit later stages of the complement pathway by altering their surface configuration in response to envelope stress, preventing membrane insertion and MAC pore formation. Our findings that distinct K. pneumoniae strains can have distinct complement evasion mechanisms, underpinned by dramatically different gene sets, highlights the complexity associated with predicting serum resistance based on genome sequence or single virulence factors---an undertaking which is not yet possible for K. pneumoniae ([@B27]). A comprehensive understanding of the basis of complement resistance in Gram-negative bacteria will only be forthcoming when the behavior of such clinically relevant pathogens can be explained.
MATERIALS AND METHODS {#s4}
=====================
Construction of the K. pneumoniae B5055 TraDIS library. {#s4.1}
-------------------------------------------------------
The K. pneumoniae B5055 transposon mutant library was constructed by conjugation with E. coli β2163 pDS1028 as described previously ([@B34]), with selection of transposon-containing K. pneumoniae B5055 colonies performed at 25°C on LB agar supplemented with 25 μg/ml chloramphenicol. Approximately 600,000 colonies were scraped, pooled, and used as the final B5055 TraDIS library.
Serum challenge of TraDIS libraries. {#s4.2}
------------------------------------
Experiments were performed in biological triplicate. TraDIS libraries were grown overnight in 10 ml LB with an inoculum of 10 to 20 μl, which was sufficient to ensure representation of the entire mutant library. Overnight cultures were diluted 1:25, subcultured in 25 ml LB in a 250 ml flask, and grown at 37°C at 180 rpm on an orbital incubator to an optical density at 600 nm (OD~600~) of 1. A 1-ml aliquot of each culture was centrifuged for 2 min at 8,000 × *g* and resuspended in sterile phosphate-buffered saline (PBS). Bacterial suspension (500 μl) was added to 500 μl normal human serum (catalog no. S7023; Sigma) and incubated at 37°C for 90 min. Control reactions were performed in the same way, except that serum was heat-inactivated at 56°C for 30 min prior to use. Following incubation, serum reactions were centrifuged, the pellets suspended in 10 ml LB, and the surviving bacteria outgrown at 37°C for 2 h.
DNA extraction and next-generation sequencing. {#s4.3}
----------------------------------------------
Genomic DNA (gDNA) was purified by phenol-chloroform extraction; 1 to 2 μg DNA was used for the construction of the TraDIS sequencing libraries as described previously ([@B36]). Amplification of transposon junctions was performed using primer FS108 (NTUH-K2044, B5055, and ATCC 43816 libraries) or Tn5tetR_5PCR (RH201207 library). Libraries from the RH201207 strain were sequenced on the Illumina MiSeq platform using the primer Tn5tetR_5Seq. All other libraries were sequenced on the Illumina HiSeq platform using FS107. Sequencing was performed as described previously ([@B36]).
Analysis of TraDIS data. {#s4.4}
------------------------
TraDIS sequencing reads were analyzed using the BioTraDIS pipeline as described previously ([@B36], [@B67]), with the following parameters passed to the bacteria_tradis script: "-v -smalt_y 0.96 -smalt_r -1 -t TAAGAGACAG -mm -1." Reads and insertion sites were assigned to each gene using a custom script (available at <https://github.com/francesca-short/tradis_scripts/blob/master/tradis_insert_sites_FS.py>), with reads mapping to the 3′ 10% of the gene ignored. Output samples following treatment with serum or heat-inactivated serum were compared to the input sample and to each other using the tradis_comparison.R script without filtering. Hits were defined as those genes with a log~2~ fold change (log~2~FC) of less than −1 or more than 1 (*q* value \< 0.005). Values reported in the manuscript are for serum compared to heat-inactivated serum. A previously generated pangenome ([@B34]) from a global collection of 265 K. pneumoniae strains ([@B58]) was used to identify orthologs between strains and to classify genes as belonging to the K. pneumoniae core or accessory genome. Where needed, pathway information on specific genes was extracted from BioCyc ([@B46]).
Quantification of capsule. {#s4.5}
--------------------------
Capsule production was measured using an assay for uronic acids as described previously ([@B68]). Overnight cultures of K. pneumoniae were grown in LB at 37°C, and 500-μl aliquots were used directly in the assay. To examine cell-attached capsule, the 500-μl culture samples were centrifuged at 8,000 × *g* for 2 min, and cell pellets were then suspended in 500 μl fresh LB medium prior to uronic acid quantification. A standard curve of glucuronic acid (Sigma-Aldrich) was used to calculate uronic acid concentrations.
Serum survival assays. {#s4.6}
----------------------
Bacteria were grown overnight in LB, subcultured 1:100 in fresh LB medium, and grown to the late exponential phase (OD~600~ = 1). Cultures were then washed once in PBS and diluted 1:100 in sterile phosphate-buffered saline; 50 μl diluted culture was added to 100 μl prewarmed human serum (Sigma) and incubated at 37°C. Samples were taken at set time points, serially diluted, and plated for enumeration of viable bacteria.
Hypermucoidy assay. {#s4.7}
-------------------
Overnight cultures of the strains of interest carrying pBAD33-derived Lpp expression plasmids (see Table S1 in the supplemental material) were grown in LB supplemented with 25 μg/ml chloramphenicol, and subcultured for 5 h in LB plus 0.1% [l]{.smallcaps}-Ara to induce vector expression. Cultures were centrifuged at 1,000 × *g* for 5 min, and hypermucoidy was expressed as a ratio of OD~600~ of the supernatant/OD~600~ of the original culture.
Construction of mutants. {#s4.8}
------------------------
Clean single-gene deletion mutants in K. pneumoniae were constructed as described previously ([@B34]) using pKNG101Tc-derived allelic exchange vectors introduced by conjugation with E. coli β2163 as the donor strain. Details of the plasmids and oligonucleotides used in mutant construction are in Table S1. Defined transposon insertion mutants of ATCC 43816 and RH201207 were isolated by subjecting the relevant TraDIS library to two rounds of density gradient centrifugation ([@B69]). The noncapsulated fraction was grown as single colonies, and mutant locations were identified by randomly primed PCR as described previously ([@B70]) using primers FS57 to FS60 together with FS108 and FS109 for ATCC 43816 and FS346 and FS347 for RH201207 (Table S1). Complementation plasmids were constructed using the primers listed in Table S1 and introduced by electroporation.
Detection of surface-located complement components. {#s4.9}
---------------------------------------------------
Early mid-logarithmic-phase LB cultures (1 ml) were washed in gelatin veronal buffered saline containing Mg^2+^ and Ca^2+^ (pH 7.35) (GVB^2+^), and incubated in 66% prewarmed (37°C) pooled human serum (MP Biomedicals) for 15, 30, 60, 120, and 180 min ([@B1]). Prewarmed, heat-inactivated (56°C; 30 min) human pooled serum served to set the 0-min time point (*T*~0~). Human C3-deficient and C5-deficient serum (Sigma) were used as negative controls. For flow cytometric staining, after incubation the mixtures were washed in PBS and approximately 1 × 10^6^ cells were stained. C3b binding was detected with a mouse monoclonal allophycocyanin (APC) anti-C3b/iC3b antibody (BioLegend) (4 μl per 10^6^ cells), and C5b-9 formation was detected by indirectly staining cells with 8 μg/ml mouse anti-C5b-9 antibody \[aE11\] (abcam) as the primary antibody and 2.5 μg/ml Alexa Fluor 488 goat anti-mouse IgG H+L (abcam) as the secondary antibody. After 20 min of incubation at room temperature (RT), mixtures were washed and suspended in 200 μl PBS. Samples were acquired using a MACSQuant instrument (Miltenyi Biotec) within 60 min. Approximately 40,000 cell events were collected. Flow cytometry data analysis was carried out using FlowJo 10 Software. GraphPad 7.05 software was used for graph design and statistical analysis.
For microscopy, samples of early mid-logarithmic-phase LB cultures (equivalent to 1 ml at an OD~600~ of 0.5) were washed in GVB^2+^ and incubated with serum for 0, 5, or 15 min. Cells were then washed with PBS, separated into two aliquots, and stained with either 10 μg/ml mouse monoclonal APC anti-C3b/iC3b antibody (BioLegend) or 10 μg/ml mouse anti-C5b-9 antibody \[aE11\] (abcam) followed by 10 μg/ml Alexa Fluor 488 goat anti-mouse IgG H+L (abcam) for 10 min at RT. Cells were washed with PBS following each staining step, resuspended in PBS, and mounted on 1% PBS agarose pads for imaging. Highly inclined and laminated optical sheet (HILO) microscopy was performed using the Nanoimager S Mark II (Oxford Nanoimaging \[ONI\]) equipped with 473-nm/300-mW (10%) and 640-nm/300-mW (7%) lasers, dual emission channel split at 560 nm, 100× oil-immersion objective (numerical aperture \[NA\], 1.49; Olympus) and an ORCA-Flash4.0 v3 complementary metal-oxide-semiconductor CMOS camera (Hamamatsu). Images were acquired at an illumination angle of 51° with 100-ms exposures for \>40 frames and processed using FIJI software ([@B71]). In brief, transillumination images were generated as an average of 10 frames (total of 1 s of exposure) while fluorescence images were processed by averaging 40 images (total of 4 s of exposure). Brightness and contrast of images in [Fig. 6](#F6){ref-type="fig"} and [7](#F7){ref-type="fig"} are normalized.
Measurement of cytoplasmic marker release by the B5055 Δ*rfaH* mutant. {#s4.10}
----------------------------------------------------------------------
Bacteria containing pFLS21 (Table S1), a pDiGc ([@B72]) derivative that expresses GFP from the constitutive *rpsM* promoter, were grown to the early log-phase in LB medium and washed once in PBS. A 250-μl aliquot of undiluted cell suspension was combined with 500 μl serum or heat-inactivated serum and incubated at 37°C. The total GFP fluorescence of a 100-μl sample and the fluorescence of 100 μl of supernatant following centrifugation at 8,000 × *g* for 2 min were measured in a Pherastar fluorimeter at set time points following incubation. Values were calculated as percent supernatant fluorescence intensity/total fluorescence intensity, with background signal from 66% human serum with PBS subtracted.
Statistical analysis. {#s4.11}
---------------------
TraDIS comparisons were conducted using EdgeR as implemented in the BioTraDIS pipeline, for which the statistical approaches have been described in detail. The Benjamini-Hochberg correction for multiple testing was applied.
All quantitative experiments were performed in biological triplicate, with the exception of those shown in Fig. S2B (*n* = 2, 7 technical replicates) and Fig. S4B (*n* = 2) in the supplemental material. All graphs show mean ± 1 standard deviation (SD), and statistical significance is indicated throughout as follows: *\**, *P* \< 0.05; \**\**, *P* \< 0.01; \*\**\**, *P* \< 0.001; \*\*\**\**, *P* \< 0.0001. Serum survival data were compared between bacterial strains by two-factor repeated measures analysis of variance (ANOVA) on log~10~-transformed bacterial viable counts with Huynh and Feldt correction. Where the ANOVA indicated a significant time × strain interaction, viability at *t* = 180 was compared by one-way ANOVA with Dunnett's test for multiple comparisons. Uronic acid quantification and hypermucoidy data were compared between strains by one-way ANOVA on untransformed data followed by Dunnett's *post hoc* test to compare multiple strains to a single reference or by the Tukey-Kramer test for all-against-all comparisons. Complement binding time series data were tested for significance by two-factor repeated-measures ANOVA on untransformed data, followed by Fisher's protected least significant difference (LSD) test to compare mutant to wild-type at individual time points.
Data availability. {#s4.12}
------------------
The TraDIS sequencing data generated for this study have been deposited in the European Nucleotide Archive (ENA) under project number [PRJEB20200](https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJEB20200). Sample accession numbers are provided in Table S2 in the supplemental material.
Supplementary Material
======================
###### Supplemental file 1
###### Supplemental file 2
###### Supplemental file 3
###### Supplemental file 4
Supplemental material is available online only.
We thank Matt Mayho, Jacqui Brown, and the sequencing teams at the Wellcome Trust Sanger Institute for TraDIS sequencing and the Pathogen Informatics team for support with bioinformatic analysis. We thank Theresa Feltwell for technical support and Sebastian Bruchmann for critical reading of the manuscript. We thank Luca Guardabassi and Bimal Jana for providing the RH201207 library and Matthew Dorman for providing the RfaH complementation plasmid.
This work was supported by a Sir Henry Wellcome postdoctoral fellowship to F.L.S. (grant 106063/A/14/Z) and by the Wellcome Sanger Institute (grant 206194). This study also received support from the Medical Research Council, UK, through project grant MR/R009937/1.
[^1]: Present address: Francesca L. Short, Department of Molecular Sciences, Macquarie University, NSW, Australia.
[^2]: **Citation** Short FL, Di Sario G, Reichmann NT, Kleanthous C, Parkhill J, Taylor PW. 2020. Genomic profiling reveals distinct routes to complement resistance in *Klebsiella pneumoniae*. Infect Immun 88:e00043-20. <https://doi.org/10.1128/IAI.00043-20>.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Programmed cell death (PCD)[^2^](#FN2){ref-type="fn"} refers to any form of cell death mediated by an intracellular death program ([@B1]). PCD systems such as apoptosis ([@B2]) or autophagy ([@B3]) in eukaryotic multicellular organisms play significant roles in a variety of biological processes including development, cell homeostasis, and oncogenesis ([@B4]). Traditionally, PCD has been considered associated with only eukaryotic multicellular organisms; however, recently, PCDs have also been observed in bacteria ([@B1]). For most eubacteria and archaea, the most common mechanism involved in bacteria PCD is the toxin-antitoxin (TA) system ([@B5]).
TA systems are usually composed of a stable toxin that arrests cell growth and a labile antitoxin that counteracts the toxin. The former always exists as a stable protein, whereas the latter is either a protein (type II TA system) or an RNA species (type I or III TA system) ([@B6]). In the type I TA system, the antitoxins are small antisense RNAs forming duplexes with the toxin mRNAs, which repress translation of the toxin genes ([@B7]). In the type II system, the antitoxins are proteins that neutralize the toxins by a direct protein-protein interaction with the toxins ([@B8]). In type III TA systems, small RNA antitoxin combines with and neutralizes protein toxins ([@B9], [@B10]). In most cases, toxin and antitoxin genes are in an operon where the antitoxin gene often locates upstream of the toxin gene ([@B6]), usually overlapping or being separated by a small intergenic region, so that TA genes are co-transcribed. In type II TA systems, TA operons are usually negatively autoregulated at the transcription level by antitoxins and TA complexes, which bind to the TA locus promoters ([@B8]).
Several TA systems have been discovered in bacterial plasmids and on chromosomes. Plasmid-borne TA systems are involved in plasmid stabilization through toxin-mediated post-segregation killing of plasmid-free progeny cells ([@B11]): Cells that do not inherit a copy of a plasmid upon division are killed because the unstable antitoxin is degraded faster than the more stable toxin. On the contrary, the biological roles of chromosomal TA systems have been the subject of widespread discussion and speculation. Recently, nine possible functions including genomic junk, stabilization of genomic parasites, selfish alleles, gene regulation, growth control, and production of persisters were suggested ([@B12]), but a flexible response of a bacterial cell to stress conditions such as starvation is regarded to be the major function of chromosomal TA systems. Moreover, TA systems can contribute to the formation of persistent cells during exposure to antibiotics, and recently, persistence was suggested as a common function of TA systems ([@B13], [@B14]).
The *Escherichia coli* K12 chromosome possesses at least eight relatively well characterized TA systems: MazF-MazE, RelE-RelB, ChpBK-ChpBI, YafQ-DinJ, YoeB-YefM, HipA-HipB, YafO-YafN, and MqsR-MqsA ([@B6]). Among the toxins of these modules, the RelE, YafQ, and YoeB toxins are classified within the same family (RelE family) due to low but significant sequence similarities among them ([@B8]). These toxins function by inhibiting translation through mRNA cleavage ([@B8]). Among those toxins, RelE is a ribosome-dependent endoribonuclease that is only active when associating with a ribosome ([@B15]--[@B17]), whereas the others have intrinsic ribonuclease (RNase) activity ([@B18], [@B19]). However, YoeB and YafQ also associate with ribosome and are regarded to function as ribosome-dependent mRNA interferases, too ([@B6], [@B19], [@B20]).
*Helicobacter pylori* is a Gram-negative neutralophile that multiplies in an environmental pH from 6.0 to 8.0 with optimal growth close to pH 7.0 ([@B21], [@B22]). However, it has a unique ability to survive in an acidic environment of human stomach and to colonize the gastric mucosa ([@B21]). To live in the extremely acidic environment of the stomach, it has developed acid resistance mechanisms ([@B21]). For example, urease of *H. pylori*, which has optimum activity at neutral pH, produces NH~3~ from gastric juice urea, and neutralizes acidity around the bacterium ([@B21]). Because of these acid resistance mechanisms, *H. pylori* is thought to possess an intracellular pH near neutrality even in the acidic environment of stomach ([@B23], [@B24]). It can cause diverse gastric diseases such as peptic ulcers, chronic gastritis, mucosa-associated lymphoid tissue lymphoma, and gastric cancer ([@B25]--[@B27]). *H. pylori* is one of the most common chronic bacterial infections of humans, infecting more than half of the world population.
In our previous study ([@B28]), we found that HP0894 (a YafQ-homologous toxin) and HP0895 (an antitoxin for HP0894) proteins of *H. pylori 26695* strain form a TA system. In another study of ours ([@B29]), we reported the solution structure of the conserved hypothetical protein HP0892 from this strain and found that its structure is very similar to those of HP0894 and other RelE family TA toxins. Those findings about HP0892 prompted us to speculate that HP0892 might also act as a TA toxin with intrinsic RNase activity like HP0894. In this study, we identified the biological function of HP0892 as a TA toxin and characterized some structural aspects of its toxin-antitoxin binding.
EXPERIMENTAL PROCEDURES
=======================
###
#### Molecular Cloning
Subcloning was carried out as described previously for HP0892 ([@B29]). The same procedure was used to prepare the cells harboring the recombinant plasmids for mutated HP0892. All resulting HP0892 constructs contained eight non-native residues (LEHHHHHH) at the C terminus (referred to as HP0892-His). Those residues facilitated subsequent protein purification. Additionally, the same procedure with minor change was used for HP0892 without any purification tag or non-native residues (in vector pET-21a; Novagen, Madison, WI) and for glutathione *S*-transferase (GST) fusion HP0893 (referred to as GST-HP0893) (in vector pGEX-4T-1; GE Healthcare) and HP0893 with 20 non-native residues including a His tag at the N terminus (referred to as His-HP0893) (in vector pET-28a; Novagen).
#### Preparation of Protein Samples
Overexpression was carried out as described previously ([@B29]) for HP0892-His. For the production of ^15^N- or ^15^N,^13^C-labeled proteins, M9 medium containing ^15^NH~4~Cl and/or \[^13^C\]glucose as stable isotope sources were used. The protein samples were purified using a Ni^2+^-affinity column following standard protocol. The same procedures were used to prepare His-HP0893 and mutated HP0892-His. For His-HP0893, because it is a putative DNA-binding protein, further purification using polyethyleneimine was carried out. Polyethyleneimine was added to the previously purified His-HP0893 solution at a final concentration of 0.08%, which was determined by a titration test. After ultracentrifugation, the supernatant was collected, dialyzed against a 50 m[m]{.smallcaps} borate buffer (pH 11), and loaded onto a disposable PD-10 column (GE Healthcare) packed with Q-Sepharose fast flow resin (GE Healthcare). The column was washed with 100 ml of the same buffer, and then the protein was eluted with a 50 m[m]{.smallcaps} borate buffer (pH 11) containing 1 [m]{.smallcaps} NaCl.
#### Construction of HP0892/HP0893 Co-expression Systems
The full-length *hp0892* and *hp0893* genes were amplified from *H. pylori* genomic DNA by PCR as different DNA fragments and were subsequently subcloned under the T7 promoter in pET-21a (ampicillin-resistant) and pET-29a (kanamycin-resistant) vectors (Novagen), respectively. These two vectors were co-transformed into *E. coli* BL21 (DE3) (HP0892/HP0893 co-expression system I). In this case, the HP0893 construct contained eight non-native residues including a His tag at the C terminus (referred to as HP0893-His). The HP0892 construct contained no additional residues. Using the same procedure, another co-expression system composed of HP0892 (in vector pET-21a) and HP0893 (in pET-28a) was constructed (HP0892/HP0893 co-expression system II). In this case, the HP0893 construct contained 20 non-native residues including a His tag at the N terminus (referred to as His-HP0893). The HP0892 construct contained no additional residues.
#### Pulldown Assay for HP0892/HP0893 Binding
*E. coli* BL21 (DE3) cells harboring GST-HP0893 expression plasmid (vector pGEX-4T-1) and HP0892-His expression plasmid (vector pET-21a) were cultured and expressed with 0.5 m[m]{.smallcaps} isopropyl-β-[d]{.smallcaps}-thiogalactopyranoside, separately. The cells were harvested by centrifugation and resuspended with cell lysis buffer (20 m[m]{.smallcaps} sodium phosphate buffer (pH 7.3)), separately. The two cell cultures were mixed and disrupted by sonication. The lysate was centrifuged at 10,000 rpm, 4 °C for 1 h, and the supernatant was loaded onto a glutathione-Sepharose column. The column was washed with the same cell lysis buffer until the UV peak stabilized. The adsorbed protein was eluted with a glutathione buffer (3.08 g of reduced glutathione in 1 liter of 50 m[m]{.smallcaps} Tris-HCl buffer (pH 8.0)). The eluted solution was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing the target proteins were pooled, and buffer exchange was carried out with 20 m[m]{.smallcaps} Tris-HCl buffer (pH 7.9) containing 0.5 [m]{.smallcaps} NaCl. The solution was applied to a Ni^2+^-affinity column. The column was washed with the 20 m[m]{.smallcaps} Tris-HCl buffer (pH 7.9) containing 60 m[m]{.smallcaps} imidazole and 0.5 [m]{.smallcaps} NaCl. The adsorbed proteins were eluted with the 20 m[m]{.smallcaps} Tris-HCl (pH 7.9) buffer containing 0.5 [m]{.smallcaps} NaCl and a linear imidazole gradient (0∼500 m[m]{.smallcaps} imidazole) and analyzed by SDS-PAGE.
#### Co-expression and Co-purification of HP0892/HP0893
HP0892 and HP0893 were co-expressed and co-purified using the HP0892/HP0893 co-expression system I described above. Bacteria were grown at 37 °C in LB broth containing antibiotics (ampicillin 50 μg/ml and kanamycin 40 μg/ml). Induction, cell harvest, cell lysis, and purification with a Ni^2+^-affinity column were done following standard protocols. The fractions eluted from the Ni^2+^-affinity column (using stepwise imidazole gradient) were analyzed by SDS-PAGE.
#### Toxicity of HP0892 in E. coli
*E. coli* BL21 (DE3) cells harboring the HP0892 expression plasmids (HP0892/pET21a) and the HP0892/HP0893 co-expression plasmids (HP0892/HP0893 co-expression system II) were grown at 37 °C in 100 ml of LB broth containing antibiotics (50 μg/ml ampicillin for pET21a and HP0892/pET21a, and 50 μg/ml ampicillin and 40 μg/ml kanamycin for HP0892/HP0893 co-expression system II). At an *A*~600~ of approximately 0.3, 1 m[m]{.smallcaps} isopropyl-β-[d]{.smallcaps}-thiogalactopyranoside was added. Subsequently, samples for viable counts and SDS-PAGE analyses were taken at several time points. Viable counts were made by plating dilutions of the cultures onto LB plates containing the appropriate antibiotics. In addition, the cells harvested from 125 μl of the samples taken at each time point were analyzed by SDS-PAGE.
#### In Vitro RNA Cleavage by HP0892
A DNA fragment containing the T7 promoter and the *hp0893* gene was obtained by PCR amplification of *H. pylori* genomic DNA. The *hp0893* mRNA was synthesized *in vitro* from this DNA fragment using the T7-MEGAshortscript kit (Ambion, Austin, TX). Mixtures of *hp0893* mRNA (∼310 bases, 300 ng), HP0892, HP0892 mutants, and/or HP0893 were prepared in several ratios in 20 m[m]{.smallcaps} Tris-HCl (pH 7.4) buffer containing 150 m[m]{.smallcaps} NaCl (total volume, 5 μl). The reaction was done at 37 °C for 60 min and was stopped by adding 5 μl of the sequencing loading buffer (Ambion). The samples were run on a 1.5% agarose gel containing ethidium bromide with Tris borate/EDTA buffer.
#### Primer Extension
The *hp0893* mRNA was used as a primer template. Two different DNA primers were synthesized and 5′-labeled with \[γ-^32^P\]ATP using T4 polynucleotide kinase (New England Biolabs). Primers (0.8 pmol) were annealed to each template (1 pmol) in a 10-μl mixture samples by incubation at 90 °C for 5 min followed by slow cooling to room temperature. The reaction mixture was digested by adding 1 μl of various amounts of HP0892 at 37 °C for 1 h. Subsequent primer extension reactions were performed by adding 4 μl of 5× buffer (250 m[m]{.smallcaps} Tris-HCl (pH 8.3), 375 m[m]{.smallcaps} KCl, and 15 m[m]{.smallcaps} MgCl~2~), 5 m[m]{.smallcaps} DTT, 0.5 m[m]{.smallcaps} dNTP, and 20 units of SuperScript III reverse transcriptase (Invitrogen) for a final reaction volume of 20 μl. The cDNA was synthesized at 55 °C for 30 min and then purified by phenol/chloroform extraction and ethanol precipitation. The products were then subjected to 8% denaturing PAGE and analyzed by autoradiography.
#### NMR Spectroscopy and Titrations
For HP0893Ctp titration against HP0892, a 30-residue peptide corresponding to the C-terminal region (Glu-66 to Leu-95) of HP0893 was commercially synthesized (Anygen, Kwangju, Korea) and purified (96% purity). The two-dimensional ^1^H,^15^N TROSY spectra of ^15^N-labeled HP0892 alone (final concentration 0.1 m[m]{.smallcaps}) and with HP0893Ctp added incrementally (0.05, 0.1, and 0.2 m[m]{.smallcaps}) to the sample were obtained on a Bruker Avance 600 spectrometer. The averaged chemical shift changes were calculated by ([@B30]).
For backbone resonance assignments of HP0892 bound with HP0893Ctp, the three-dimensional TROSY-type HNCA (trHNCA) of ^13^C,^15^N-labeled HP0892 (0.2 m[m]{.smallcaps}) mixed with HP0893Ctp (0.3 m[m]{.smallcaps}) was obtained on a Varian VNMRS 900 spectrometer. All NMR samples were dissolved in 20 m[m]{.smallcaps} sodium phosphate (pH 6.0) buffer without any additional salt. The NMR samples contained 10% D~2~O for the lock signal. All spectra were processed and analyzed using NMRPipe/NMRDraw ([@B31]) and NMRView ([@B32]), respectively.
#### Gel Filtration Chromatography
A mixture of HP0892 with HP0893 was prepared by co-lysis of cells harboring HP0892-His and His-HP0893, respectively, and subsequently, Ni^2+^-affinity column purification followed. The mixture and molecular mass markers were fractionated on a Superdex 75, 10/300 GL column (GE Healthcare) using an AKTA FPLC system (GE Healthcare). The mobile phase consisted of 50 m[m]{.smallcaps} sodium phosphate (pH 7.4) buffer containing 100 m[m]{.smallcaps} NaCl. The void volume was measured with blue dextran 2000 (2000 kDa). The proteins were eluted at flow rates of 0.5 ml/min and were monitored using UV light at 280 nm.
RESULTS
=======
###
#### HP0892 and HP0893 Proteins Form a Stable Complex
Generally, the antitoxin gene is usually located directly upstream of the toxin gene in TA systems. On the *H. pylori* chromosome, the *hp0893* gene is located directly upstream of the *hp0892* gene ([Fig. 1](#F1){ref-type="fig"}). Although HP0893 has been known to be a hypothetical protein, we expected that if HP0892 is a TA toxin, then HP0893 would bind to HP0892, as a putative TA antitoxin of *H. pylori*. To identify the potential interaction between HP0892 and HP0893, samples of protein mixtures including HP0893 fused with GST (GST-HP0893) and HP0892 fused with His tag at the C terminus (HP0892-His) were purified by the pulldown assay method (see "Experimental Procedures") using a glutathione-Sepharose and subsequently, Ni^2+^-affinity column. As shown in [Fig. 2](#F2){ref-type="fig"}*A*, the SDS-PAGE analysis revealed the presence of both proteins, HP0892-His and GST-HP0893, in the same eluted fractions from the latter Ni^2+^-affinity column. For a control, we also monitored the interaction between HP0892-His and GST alone by the same pulldown assay method, but no interaction was observed (data not shown). To confirm the interaction between HP0892 and HP0893, both proteins were co-expressed from the HP0892/HP0893 co-expression system I (see "Experimental Procedures") and then co-purified. For detection and purification, HP0893 was expressed as a C-terminal His tag fusion (HP0893-His) and HP0892 was expressed without a purification tag. SDS-PAGE analysis under a range of imidazole treatments revealed the presence of both expressed proteins (HP0893-His and HP0892) in the same eluted fractions from the Ni^2+^-affinity column ([Fig. 2](#F2){ref-type="fig"}*B*). These results indicate that there is a stable and strong interaction between the HP0892 and HP0893 proteins.
{#F1}
{#F2}
#### Inhibitory Effect of HP0892 on E. coli Cell Growth Is Neutralized by HP0893
The potential of HP0892 as a toxin and of HP0893 as an antitoxin was examined by measuring their effects on the viability of *E. coli* cells grown in the presence of isopropyl-β-[d]{.smallcaps}-thiogalactopyranoside. As shown in [Fig. 3](#F3){ref-type="fig"}, the number of viable *E. coli* cells decreased significantly with expression of HP0892, but co-expression of HP0893 neutralized the detrimental effect of HP0892 on *E. coli* cell growth.
{#F3}
#### In Vitro RNase Activity of HP0892 Is Inhibited by HP0893
To determine whether HP0892 has intrinsic RNase activity, we tested the *in vitro* RNase activity of HP0892 and the inhibitory effect of HP0893. As mentioned previously in the Introduction, *H. pylori* is very likely to possess an intracellular pH near neutrality in even acidic environment of stomach, so this test and the following primer extension test were conducted at a neutral pH condition (pH 7.4). As shown in [Fig. 4](#F4){ref-type="fig"}, *in vitro* synthesized mRNA was digested into smaller fragments following incubation with HP0892, whereas addition of HP0893 inhibited mRNA decay by HP0892. HP0893 alone did not digest mRNA. In addition, the RNase activity of a single mutant HP0892 (H86A) was markedly reduced, indicating that RNA cleavage by HP0892 was not due to contamination of RNases during purification and that the His-86 residue is a key catalytic residue of HP0892.
![**Cleavage of *hp0893* mRNA by HP0892 and the inhibitory effect of HP0893.** *In vitro* transcribed mRNA was incubated at 37 °C for 60 min with various combinations of HP0892, HP0892 mutant (H86A), and/or HP0893. *Lanes*: *1*, DNA ladder; *2*, mRNA (300 ng, control); *3*, mRNA and 4 μ[m]{.smallcaps} HP0892; *4*, mRNA and 4 μ[m]{.smallcaps} HP0892 mutant (H86A); *5* and *6*, mRNA, 4 μ[m]{.smallcaps} HP0892 and 2 or 4 μ[m]{.smallcaps} HP0893, respectively; *7*, mRNA and 4 μ[m]{.smallcaps} HP0893.](zbc0121340950004){#F4}
#### Primer Extension
To identify the sequence of HP0892 cleavage site on mRNA, HP0892-mediated cleavage of *hp0893* mRNA was analyzed by primer extension experiments. As shown in [Fig. 5](#F5){ref-type="fig"}, cleavage occurred preferentially at the 5′ end of adenine (A) or guanine (G) residues, although not all of A or G, and AG-rich sequence regions were relatively more sensitive.
![**Primer extension analyses of mRNA cleavage by HP0892.** *hp0893* mRNA with different DNA primers was used to produce *A* and *B. Lanes 1--4*, mRNA. *Lane 5*, 2.5, 1.25, or 0 μ[m]{.smallcaps}, respectively, HP0892. DNA sequence ladders are shown at the *left* with *upper lane labels* for the sense strand. Major cleavage sites are indicated by *sequential numbers* along the *right side* of the images. The RNA recognition sequences for *A* and *B*, shown below, were derived from the RNA complement of the DNA sequencing ladder in the above images. The *numbers* and *inverse triangles* on the sequences correspond to the sequentially numbered major cleavage sites in the above images.](zbc0121340950005){#F5}
#### Binding Sites of HP0892 and HP0893
Because HP0893 exists as an aggregated form with a high molecular mass and is easily precipitated in solution at NMR scale concentrations, we were not able to get well dispersed NMR spectra of ^15^N-labeled HP0893 and could not investigate the binding aspect between HP0892 and HP0893 with the NMR titration method using full HP0893 protein sample. In the *H. pylori* HP0894-HP0895 TA complex, the 30-residue C-terminal region of the HP0895 antitoxin is responsible for binding with the HP0894 toxin ([@B28]), which is a structural and sequential homologue close to HP0892. In addition, in *E. coli* YoeB-YefM and RelE-RelB TA complexes, approximately a 30-residue C-terminal region of YefM or RelB antitoxin, also mainly takes part in the binding with YoeB or RelE toxin ([@B18], [@B33]), respectively, which are also structural homologues of HP0892. Therefore, we hypothesized that the C-terminal region of HP0893 is also responsible for binding with HP0892 and investigated its binding with the C-terminal region of HP0893 instead of the full-length HP0893.
To confirm the binding of this C-terminal region of HP0893 with HP0892, we synthesized a 30-residue peptide, HP0893Ctp (Glu-66 to Leu-95), corresponding to the C-terminal region of HP0893, and investigated the interaction between this peptide and HP0892 with the NMR titration method. A series of two-dimensional ^1^H,^15^N TROSY spectra of ^15^N-labeled HP0892 were recorded with successive additions of unlabeled HP0893Ctp (0, 0.5, 1, and 2 molar equivalents). As shown in [Fig. 6](#F6){ref-type="fig"}, obvious chemical shift changes in slow or intermediate exchange mode on the NMR time scale were observed for a lot of residues of HP0892, which implies that the dissociation constants (*K~d~*) of HP0893Ctp (and consequently HP0893) with HP0892 are relatively small (∼10^−8^ [m]{.smallcaps}) ([@B34]) and their binding is strong. Chemical shift changes from the residues in slow exchange mode were completed with an equimolar amount of HP0893Ctp. These results and the relative intensities of the protein bands corresponding to HP0892 and HP0893 in the SDS-PAGE gel from the previous pulldown assay ([Fig. 2](#F2){ref-type="fig"}*B*) indicate that the binding stoichiometry of HP0892 and HP0893 is 1:1. Considering the overall results, we can reasonably suggest that the C-terminal region of HP0893 is responsible for its binding with HP0892.
![**Chemical shift perturbations of HP0892 residues by HP0893Ctp binding.** *A*, two-dimensional ^1^H,^15^N TROSY spectra of 100 μ[m]{.smallcaps} ^15^N-labeled HP0892 with HP0893Ctp added at 0 (*black*) and 1 (*red*) molar ratios of HP0893Ctp/HP0892 are superimposed. A central spectral region is *enlarged* aside. The residues whose resonances disappeared (intermediate exchange mode) in the spectrum at 1 molar ratio are labeled in *green. B*, averaged chemical shift changes (Δδ~ave~) of individual residues of HP0892 upon binding with HP0893Ctp (1:1 molar ratio). The changes of the residues in the obvious slow exchange mode (with Δδ~ave~ more than 1.0 ppm) are colored in *dark blue*, those in the intermediate exchange mode in *light red*, and those with Δδ~ave~ \<1.0 ppm in *light blue. C*, ribbon and surface displays of HP0892 structure colored according to chemical shift perturbations. Residues whose chemical shift perturbations are in the obvious slow or intermediate exchange mode are colored in *red*. The program PyMOL ([@B35]) was used to visualize the structures.](zbc0121340950006){#F6}
A plot of the chemical shift changes against the residues of HP0892 is shown in [Fig. 6](#F6){ref-type="fig"}*B*. For this plot, the backbone N and HN resonances of uncomplexed HP0892 were assigned from the data obtained in an earlier study ([@B29]). Regarding backbone resonance assignments for HP0893Ctp-bound HP0892, sequential Cα connections determined from three-dimensional trHNCA of ^13^C,^15^N-labeled HP0892 in complex with HP0893Ctp and comparison of Cα resonance values between uncomplexed and HP0893Ctp-complexed HP0892 were used. The N-terminal residues Thr-3 to Asn-19, Asp-23, Lys-25, Leu-27 to Val-30, Gly-32, Leu-34, Thr-35, and Gln-38, and the middle range residues Leu-49, Lys-50, Leu-53, Lys-54, and Phe-56, and the C-terminal residues Leu-66, Ile-71, Lys-73, Glu-75, Thr-77, Phe-79 to Leu-83, Ser-85, Ser-87, and Phe-88 showed obvious chemical shift changes in the slow or intermediate exchange mode. These three residue groups in the HP0892 sequence could be mapped structurally into a nearly consecutive region on a three-dimensional HP0892 NMR structure ([Fig. 6](#F6){ref-type="fig"}*C*). It is reasonably suggested that those residue groups form the binding site for HP0893. Overall, the chemical shift changes of the residues in the N-terminal secondary structure elements (one β-strand and two α-helixes in Leu-2 to Gln-38) were more substantial than those of the other residues ([Fig. 6](#F6){ref-type="fig"}*B*), which indicates that this N-terminal region of HP0892 is the main region responsible for its binding with HP0893.
#### Oligomerization State of HP0892-HP0893 Complex
To evaluate the oligomerization state of the HP0892-HP0893 complex, gel filtration chromatography was performed on a mixture of the HP0892-His and His-HP0893 (nearly the same amount of HP0892-His and His-HP0893 was in the mixture) using 20 m[m]{.smallcaps} sodium phosphate buffer (pH 7.4) containing 100 m[m]{.smallcaps} NaCl as a mobile phase. As shown in [Fig. 7](#F7){ref-type="fig"}, the HP0892-HP0893 (molar ratio 1:1) complex (HP0892-His, 11.48 kDa; His-HP0893, 13.52 kDa) eluted as a single peak with an apparent molecular mass of 73.69 kDa, which fit well with an oligomerization state of (HP0892-HP0893)~3~ heterohexamer (74.99 kDa).
{#F7}
DISCUSSION
==========
In our previous study, based on the structural and sequential similarities between *H. pylori* HP0892 and other RelE family toxin molecules such as *E. coli* YoeB, *Pyrococcus horikoshii* aRelE (the archaeal homologues of *E. coli* RelE), and *H. pylori* HP0894, we reported that there is a reasonable possibility that HP0892 may be a TA toxin. In TA systems, the antitoxin gene is usually located directly upstream of the toxin gene, and on the *H. pylori* chromosome, the *hp0893* gene is located directly upstream of the *hp0892* gene ([Fig. 1](#F1){ref-type="fig"}) in an operon. Therefore, we expected that if HP0892 is a TA toxin, it would bind to HP0893. The 95-residue *H. pylori*-specific hypothetical protein HP0893 from *H. pylori* 26695 shares no detectable sequence similarity with other antitoxins. However, as revealed in the present study, HP0892 strongly binds to HP0893, with presumably a 1:1 binding stoichiometry. HP0892 has RNase activity on mRNA, and HP0893 inhibits the decay of mRNA by HP0892. Furthermore, His-86 of HP0892, which is homologous to key catalytic residues of His-87 of YafQ ([@B19]), His-83 of YoeB ([@B18]), Arg-81 of RelE ([@B33]), Arg-85 of aRelE ([@B36]), and His-84 of HP0894 ([@B28]), is also essential for its RNase activity. HP0892 expression has a toxic effect on *E. coli* cell growth, but the co-expression of HP0893 neutralizes the cell toxicity of HP0892. Especially, HP0892 shares detectable and relatively high sequence similarity with YafQ toxin among the three *E. coli* RelE family toxins ([Fig. 8](#F8){ref-type="fig"}). These results, including sequential and structural analyses, suggest that HP0892 is a YafQ-homologous toxin with intrinsic RNase activity and HP0893 is the antitoxin against HP0892.
![**Sequence homology of HP0892.** *A*, multiple sequence alignments of *H. pylori* HP0892, *H. pylori* HP0894, and *E. coli* YafQ. *B*, average distance tree of *H. pylori* HP0892, *H. pylori* HP0894, *E. coli* YafQ, *E. coli* YoeB, and *E. coli* RelE. Sequence alignment was done with ClustalW ([@B37]) and colored according to Blosum scores. The figure was generated by JalView ([@B38]). The *red rectangles* in *A* indicate the residues forming putative catalytic triad of HP0892 and HP0894 ([@B28]) and the functionally important catalytic residues of YafQ revealed by a mutagenesis analysis ([@B39]).](zbc0121340950008){#F8}
The comparison of HP0892 with its structural homologues harboring intrinsic RNase activity indicated that some of the key catalytic (or putative catalytic) residues involved in the RNase activity in RNase Sa, *E. coli* YoeB, and *H. pylori* HP0894 are conserved in HP0892 ([Fig. 9](#F9){ref-type="fig"}). His-85 and Glu-54 of RNase Sa ([@B42]), His-83 and Glu-46 of YoeB ([@B18]), and His-84 and Glu-58 in HP0894 ([@B28]) are replaced with His-86 and Glu-58 in HP0892, respectively. However, Arg-69 of RNase Sa ([@B42]) and Arg-65 of YoeB ([@B18]), which form catalytic triads with the above two residues, respectively, are not conserved in HP0892 or HP0894 ([@B28]). Instead, Arg-82 of HP0892, like Arg-80 of HP0894 ([@B28]), seems to play the role corresponding to those of Arg-69 of RNase Sa or Arg-65 of YoeB.
) ([@B29]) (*A*); *H. pylori* HP0894 (PDB ID [1Z8M](1Z8M)) ([@B40]) (*B*); *E. coli* YoeB (PDB ID [2A6R](2A6R)) ([@B18]) (*C*); and *Streptomyces aureofaciens* RNase Sa (PDB ID [1RGE](1RGE)) ([@B41], [@B42]) (*D*) are shown. The labeled functional or predicted key residues (see "Discussion") are colored *red*. The structures were visualized by the program PyMOL ([@B35]).](zbc0121340950009){#F9}
By contrast, in the sequence homology analysis among HP0892, HP0894, and YafQ, the residues forming putative catalytic triad of HP0892 (Glu-58, Arg-82, and His-86) or HP0894 (Glu-58, Arg-80, and His-84) are relatively well conserved in YafQ (Asp-61, Arg-83, and His-87). However, whereas the Arg and His residues are absolutely conserved among them, the Glu residue, which seems to play the role of general base, of HP0892 or HP0894 is replaced with a slightly different residue, Asp-61 in YafQ. According to a mutagenesis study on *E. coli* YafQ toxin by Armalyate *et al.* ([@B39]), Asp-61 of YafQ plays a moderate role for its catalytic activity. On the contrary, Asp-67 and His-50 were shown to be critical for the catalytic activity of YafQ; thus, both or either of the two residues was proposed to play the role of general base instead of Asp-61. The above change of catalytic residues in YafQ compared with HP0892, HP0894, YoeB, and RNase Sa might be due to the lack of absolute conservation of Glu as the general base residue. In the study by Armalyate *et al.* ([@B39]), other catalytic residues of YafQ were also presented ([Fig. 8](#F8){ref-type="fig"}). Overall, despite the small differences in local organization, the amino acid residues forming active site (catalytic residues and residues around them, some of which can take part in the recognition of specific RNA sequence ([@B29])) of YafQ are well conserved in HP0892 and HP0894 ([Fig. 8](#F8){ref-type="fig"}). Therefore, there is a high probability that HP0892 and YafQ share nearly the same functional mechanism. Moreover, HP0892, HP0894, and YafQ commonly display a preference for purine immediately downstream from the cleavage site when cleaving mRNAs *in vitro* ([@B19], [@B28], [@B39]), which can explain the result of the similar active site organizations among them. These similarities in the active site organization and the sequence specificity for RNA cleavage confirm that HP0892 is a type of YafQ-homologous toxin, similar to HP0894.
Our results show that the direct interaction of the HP0892-HP0893 TA pair occurs mainly between the 30-residue C-terminal tail of HP0893 and the N-terminal secondary structure elements and an adjacent C-terminal region of HP0892. In addition, some residues in the core region of HP0892 also take part in the interaction. Similar interaction modes are seen in *H. pylori* HP0894-HP0895, *E. coli* RelE-RelB, and YoeB-YefM complexes ([@B18], [@B28], [@B33]). Overall, anchoring of the C-terminal region of the antitoxin to the N-terminal secondary structure of the toxin ([@B33]) is the common critical factor for strong binding between the toxin and the antitoxin in RelE family TA complexes such as *E. coli* RelE-RelB, YoeB-YefM, *H. pylori* HP0894-HP0895, and HP0892-HP0893 complexes.
However, there is a difference in the oligomeric state between HP0892-HP0893 and other TA complex. In other TA complexes such as RelE-RelB ([@B43]), YoeB-YefM ([@B18]), YafQ-DinJ ([@B44]), or MazF-MazE ([@B45]) TA pairs in *E. coli*, the antitoxin molecules exist in dimeric form. The dimeric form of the antitoxins is related to the DNA binding mode of those TA complexes ([@B36], [@B45]). By contrast, in the HP0892-HP0893 complex, it seems that HP0893 antitoxin exists in trimeric form, and the overall complex has a heterohexameric arrangement ((HP0892-HP0893)~3~). Therefore, the HP0892-HP0893 TA complex can bind to their promoter region on the chromosomal DNA with a different mode than other TA complexes in the process of the negative autoregulation of the transcription of their genes.
According to a study by Graham *et al.* ([@B46]), in common with *hp0895* (the antitoxin gene in the HP0894/HP0895 TA system) ([@B28]), *hp0893* is also one of the *H. pylori* genomic open reading frames that correspond to genes that are potentially expressed in response to interactions with the human gastric mucosa. In addition, according to gene comparison studies by Terry *et al.* ([@B47]), both *hp0892* and *hp0893* belong to *H. pylori* genes absent from a set of five-cag pathogenicity island-negative strains, and may represent a marker for the identification of virulent strains or novel virulence factors. Interestingly, until now, there have been no other TA pairs in *H. pylori* 26695 except HP0892-HP0893 and HP0894-HP0895 TA pairs, which have been predicted from bioinformatics studies or identified experimentally ([@B48]). These indicate that the TA systems in *H. pylori*, especially the HP0892-HP0893 pair, are related to the status of infections of *H. pylori* in the human gastric mucosa, probably through negative regulation of the toxin molecules by the antitoxins.
Because TA systems exist only in prokaryote and are directly involved in cell death, TA systems are promising antibiotic drug targets. Thus, the HP0892-HP0893 TA pair in this study as well as the HP0894-HP0895 TA pair may be an appropriate new target for antibacterial agents for *H. pylori*. The information in this study on the binding aspect of these two proteins may be helpful in the design and development of new antibiotic drugs.
This work was supported by National Research Foundation of Korea Grants 20110001207 and 2012R1A2A1A01003569 funded by the Korean Government (MEST); Korea Healthcare Technology R&D Project, Ministry for Health, Welfare, and Family Affairs, Republic of Korea Grants A084420 and A092006; 2011 BK21 Project for Medicine, Dentistry, and Pharmacy; and Korea Institute of Science and Technology Grant 2V02900.
The abbreviations used are: PCDprogrammed cell deathPDBProtein Data BankTAtoxin-antitoxintrHNCATROSY-type HNCATROSYtransverse relaxation optimized spectroscopy.
We thank the National Center for Inter-University Research Facilities at Seoul National University and Korea Institute of Science and Technology for providing high field NMR equipment.
| {
"pile_set_name": "PubMed Central"
} |
Soil sample information, phylotype abundance tables, and bacterial and fungal representative sequences are publicly available in FigShare ([10.6084/m9.figshare.3113125](http://dx.doi.org/10.6084/m9.figshare.3113125)).
Introduction {#sec001}
============
Plant communities are typically composed of a combination of native and non-native species. The majority of these non-native species are benign, demonstrating little to no negative effect on neighboring organisms. However, a small fraction of these non-native plants are characterized as invasive because they are able to profoundly modify local plant and animal communities, nutrient cycling, hydrological regimes and fire frequency \[[@pone.0163930.ref001]--[@pone.0163930.ref002]\]. Not only do these impacts erode biodiversity and devalue ecosystem services, but they can also enhance further invasion by con- and heterospecific exotics (e.g. \[[@pone.0163930.ref003]\]).
Soil microbial communities might mediate relationships between invasive plant species and their ecosystem impacts \[[@pone.0163930.ref004]--[@pone.0163930.ref006]\]. Soil microbial communities, which are typically dominated by fungi and bacteria, can be altered by invasive plants directly through growth facilitation or inhibition near the root zone \[[@pone.0163930.ref007]\], and indirectly through changes in abiotic conditions (e.g. pH or nutrient availability) that occur in tandem with weed establishment \[[@pone.0163930.ref008]\]. For example, species-specific effects of non-native grasses on soil nutrients have been shown to subsequently modify soil microbial community composition, biomass, and bacterial:fungal ratios \[[@pone.0163930.ref009]\]. In addition to being vulnerable to impacts from aboveground plant dynamics, soil microbial communities may also play an important role in mediating the success of plant invasions \[[@pone.0163930.ref004]\]. For example, extant soil biota have been shown to enhance invasion success of some of the world's most noxious invasive plants, such as exotic knotweeds (*Fallopia* spp.) \[[@pone.0163930.ref010]\]. In general, it has been concluded that invasive species may be differentially affected by soil bacterial or fungal pathogens as compared to native plant species \[[@pone.0163930.ref011], [@pone.0163930.ref008]\], but see \[[@pone.0163930.ref012]\], which could be important for developing reliable control strategies for invasive plants that demonstrate resistance to current management efforts.
The invasive annual winter grass medusahead (*Taeniatherum caput-medusae* \[L.\] Nevski) has invaded much of the western USA and has been shown to decrease soil carbon stocks, reduce native plant diversity, and enhance fire frequency \[[@pone.0163930.ref013]\]. A recent meta-analysis of medusahead control outcomes in annual grassland and intermountain regions identified large variance in the effectiveness of conventional approaches for managing medusahead \[[@pone.0163930.ref014]\], suggesting that underlying variables, such as habitat type and seedbank density, might mediate control efforts. Despite increasing recognition that bacterial and fungal communities can influence plant invasion dynamics, only two published studies have investigated the direct relationship between medusahead and soil microbial communities \[[@pone.0163930.ref015]--[@pone.0163930.ref016]\]. These studies have conflicting results, suggesting both that the interaction between medusahead and soil microorganisms might or might not enhance its own invasion. Understanding medusahead effects on the soil microbial community is critical for enhancing predictions of invasion effects and for developing effective management strategies.
We investigated how soil microbial communities differ across the invasion front of medusahead in experimental plots in open grassland and oak woodland habitat in the Sierra Foothill region of California, USA. We attempted to understand (1) if medusahead modifies soil microbial communities across the invasion front (simulated by differences in seed density) within systems; and (2) how soil microbial communities differ between areas where medusahead invasion is successful (open grassland habitat) and not successful (oak woodland habitat), and the factors that could be responsible for these differences. We hypothesized that medusahead would modify the soil microbial communities within each habitat. We expected this for two reasons. First, early work on this species \[[@pone.0163930.ref015]\], as well as more recent work on other invasive annual grasses with similar invasion dynamics to medusahead, have demonstrated linkages between the soil microbes and invasion success \[[@pone.0163930.ref017]\]. Second, plant-soil interactions are common in the savannah/oak woodlands of California \[[@pone.0163930.ref018]\], so we would expect strong effects from the extant soil microorganisms. We also hypothesized that soil microbial communities (in particular symbiotic and pathogenic fungi) would differ between areas where medusahead invasion is typically successful (open grasslands) and typically not successful (oak woodlands). In California, invasion of winter annual grasses can be strongly limited within oak canopies \[[@pone.0163930.ref019]\], possibly due to the different microbial communities associated with oak trees compared to adjacent open grasslands \[[@pone.0163930.ref020]--[@pone.0163930.ref021]\]. Through shading, litter input, and hydraulic lift, Mediterranean oak trees can also modify a wide variety of soil edaphic factors, such as pH and organic matter concentrations, which directly influence soil microbial communities (e.g. \[[@pone.0163930.ref022]\]). At present, we do not know what role, if any, soil microbial communities play in mediating the likelihood of successful medusahead invasions.
Materials and Methods {#sec002}
=====================
Study site and experiment {#sec003}
-------------------------
The study site was located on a research reserve in Yuba County, California (39°14´N, 121°18´W), which experiences a Mediterranean climate of hot, dry summers and cool, wet winters. Permission to perform this experiment was granted by the owner of the property, the University of California Division of Agriculture and Natural Resources. The field study did not involve endangered or protected species.
Mean annual precipitation is 75cm and mean annual temperature is 17.8°C. Soils at the site are fine-loamy, mixed, superactive Ultic Haploxeralfs and fine, mixed, superactive Typic Rhodoxeralfs. Soil pH ranges between 5.7 and 6.2. The experiment is located in an annual grassland system that is irregularly interrupted by small patches of winter-deciduous blue oak (*Quercus douglasii*), and evergreen interior live oak (*Q*. *wislizeni*) that provide approximately 40% shade. The area has experienced seasonal low intensity grazing by livestock since the 1960's.
The experimental set-up is described fully in \[[@pone.0163930.ref019], [@pone.0163930.ref023]\]. Briefly, plots (1m^2^, separated by 2m) were installed in open grassland habitat and paired oak woodland habitat. The two habitats differ in the identity of dominant herbaceous species and the presence of leaf litter \[[@pone.0163930.ref019]\]. Average soil temperatures and soil moisture in the top 5cm of soil were also lower in oak woodland plots (18.4°C and 1.6%, respectively) compared to open grassland plots (22.2°C and 6.4%, respectively).
The experimental site was mowed, solarized to enhance seedbank germination and then sprayed with glyphosate herbicide to kill existing and newly germinated plants. In September 2013, fully replicated (n = 4) plots were hand seeded with one of five densities of field-collected medusahead (0, 100, 1000, 10000, and 50000 seeds/m^2^), mixed in with 500 grams of medusahead thatch. Immediately following the addition of medusahead seed, 6,000 seeds each of neighboring grass species (annual rye, and Blando brome) and 4,000 seeds of a clover mix were added---for a total of 16,000 neighbor seeds---to maintain a realistic competitive environment. Medusahead tiller density the following season reflected the seeding rate. This treatment was expected to simulate differences in medusahead invasion intensity from low to high infestation \[[@pone.0163930.ref024]\].
A defoliation treatment was applied to half the plots in April 2014. This treatment was intended to simulate a mowing or grazing regime included in a typical management program. Defoliation was applied when 75% of the medusahead tillers were in the 'boot' stage within a plot. All standing biomass in treatment plots was clipped using electronic shears positioned approximately 15cm above the soil surface.
To understand how factors associated with the oak woodland habitat might contribute to medusahead invasion, an additional set of replicated 1m^2^ plots were installed in the open grassland sites. Treatments were deployed in order to simulate environmental factors associated with the oak woodland habitat, and included the presence of shading, the presence of oak litter, and the presence of both shading and litter. Shading was applied via 50% shade cloth suspended over the plots, and litter was applied by collecting 500g of litter from under paired oak canopies and distributing it evenly over treatment plots. Medusahead seeds (at a density of 50000 seeds/m^2^) were then introduced to these plots.
Soil sampling {#sec004}
-------------
In April 2015, we collected surface soil cores (7cm depth), where the majority of fungal and biomass is present \[[@pone.0163930.ref025]\] in four random locations in each plot. Soil from each plot was mixed together in the field, sieved and placed in a plastic bag. Soil samples were frozen in the field and transported at -20°C to the University of Colorado, Boulder for microbial analyses.
Molecular analyses {#sec005}
------------------
Microbial diversity was assessed using high-throughput sequencing methods to describe the composition of taxonomic marker gene sequences. For bacterial analyses, we sequenced the V4 hypervariable region of the 16S rRNA gene, using the 515-F (`GTGCCAGCMGCCGCGGTAA`) and 806-R (`GGACTACHVGGGTWTCTAAT`) primer pair \[[@pone.0163930.ref026]\]. For the fungal analyses, we sequenced the first internal transcribed spacer (ITS1) region of the rRNA operon, using the ITS1-F (`CTTGGTCATTTAGAGGAAGTAA`) and ITS2 (`GCTGCGTTCTTCATCGATGC`) primer pair \[[@pone.0163930.ref027]\]. The primers included Illumina adapters and an error-correcting 12-bp barcode unique to each sample. PCR products were quantified using the PicoGreen dsDNA assay, and pooled together in equimolar concentrations for sequencing on an Illumina MiSeq instrument. All sequencing runs were conducted at the University of Colorado Next Generation Sequencing Facility.
Reads were demultiplexed using a custom Python script (<https://github.com/leffj/helper-code-for-uparse>), with quality filtering and phylotype clustering conducted using UPARSE \[[@pone.0163930.ref028]\]. For quality filtering, we used a maxee value of 0.5 (that is, a maximum of 0.5 nucleotides incorrectly assigned in every sequence). Singleton sequences were removed prior to phylotype clustering. Quality filtered sequence reads were then mapped to phylotypes at the 97% similarity threshold. Phylotype taxonomy was assigned using the Ribosomal Database Project (RDP) classifier with a confidence threshold of 0.5 \[[@pone.0163930.ref029]\] trained on the 16S rRNA Greengenes database \[[@pone.0163930.ref030]\] or the ITS rRNA UNITE database \[[@pone.0163930.ref031]\], for bacteria and fungi respectively. Sequences representing any phylotypes unclassified at the domain level or classified as mitochondria, chloroplasts, archaea or protists were removed. Subsequently, we removed potential contaminants (i.e. phylotypes with abundances greater than 1% in the blanks and no-template controls \[[@pone.0163930.ref032]\]), and we normalized the sequence counts using a cumulative-sum scaling approach \[[@pone.0163930.ref033]\]. We used FUNGuild to identify fungi functional guilds \[[@pone.0163930.ref034]\]. Soil sample information, phylotype abundance tables, and bacterial and fungal representative sequences are publicly available in FigShare ([10.6084/m9.figshare.3113125](http://dx.doi.org/10.6084/m9.figshare.3113125)).
Statistical analyses {#sec006}
--------------------
Soil microbial community similarity patterns were represented by non-metric multidimensional scaling (NMDS) using the Bray-Curtis distance metric. We used nested analysis of variance (ANOVA) and permutational multivariate analysis of variance (PERMANOVA) based on 1,000 permutations \[[@pone.0163930.ref035]\] to assess the explanatory power of the different treatments on soil microbial richness and community similarity patterns, respectively. Differences in the proportion of taxa and fungal functional guilds were tested using non-parametric Wilcoxon tests after false discovery rate (FDR) correction \[[@pone.0163930.ref036]\]. All multivariate statistical analyses were implemented in the R environment ([www.r-project.org](http://www.r-project.org)) using the vegan package (vegan.r-forge.r-project.org).
Results {#sec007}
=======
The total number of phylotypes across all soil samples was 5604 and 4349, for bacteria and fungi respectively ([S1 Fig](#pone.0163930.s001){ref-type="supplementary-material"}). The average number of phylotypes per soil sample was 1781 and 687, for bacteria and fungi respectively. Oak woodland soil samples tended to harbor more bacterial and fungal phylotypes than open grassland samples (although the differences were not statistically significant; ANOVA P \> 0.05; [Fig 1A and 1B](#pone.0163930.g001){ref-type="fig"} respectively; see [S2 Fig](#pone.0163930.s002){ref-type="supplementary-material"} for Shannon diversity). Both bacterial and fungal soil community composition were significantly different between open grasslands and oak woodlands (PERMANOVA R^2^ = 0.24, P \< 0.001, R^2^ = 0.28, P \< 0.001, respectively; [Fig 1C and 1D](#pone.0163930.g001){ref-type="fig"} respectively). Bacteria from the proteobacterial classes alpha, beta, gamma and delta, and Acidobacteria subgroup 6 as well as fungi from the Pezizomycetes, Agaricomycetes and Eurotiomycetes classes were more abundant in oak soils than in grassland soils (Wilcoxon test P \< 0.01 after FDR; [S3 Fig](#pone.0163930.s003){ref-type="supplementary-material"}; see [S1 Table](#pone.0163930.s005){ref-type="supplementary-material"} for results at the genus level). Acidobacteria from the classes Solibacteres and Acidobacteriia, Spartobacteria, Planctomycetia and Phycisphaerae, and fungi from the classes Dothideomycetes, Sordariomycetes and Glomeromycetes were more abundant in open grassland soils (Wilcoxon test P \< 0.01 after FDR; [S3 Fig](#pone.0163930.s003){ref-type="supplementary-material"}; see [S1 Table](#pone.0163930.s005){ref-type="supplementary-material"} for results at the genus level). As expected from the contrasted fungal taxonomic composition, oak woodlands were different from open grasslands based on the overall abundance of fungal functional guilds ([S4 Fig](#pone.0163930.s004){ref-type="supplementary-material"}). Ectomycorrhizal fungi (in particular, species from the *Tuber* and *Tomentella* genera) were significantly more abundant in oak woodlands while arbuscular mycorrhizal fungi and plant pathogens (in particular, *Drechslera* and *Fusarium* species) were more abundant in open grasslands (Wilcoxon test P \< 0.01 after FDR; [Fig 2](#pone.0163930.g002){ref-type="fig"}).
{#pone.0163930.g001}
{#pone.0163930.g002}
We found no significant effects of seed density treatments or clipping treatments on soil microbial richness (ANOVA P \> 0.05 for both bacteria and fungi; [Fig 1A and 1B](#pone.0163930.g001){ref-type="fig"}) or microbial community composition (PERMANOVA P \> 0.05 for both bacteria and fungi; [Fig 1C and 1D](#pone.0163930.g001){ref-type="fig"}) within each habitat type. Likewise, for those plots where we simulated the oak environment, we observed no significant effects of the litter or shade treatments on the richness of soil bacterial communities (Wilcoxon test P \> 0.05; [Fig 3A](#pone.0163930.g003){ref-type="fig"}) or bacterial community composition (PERMANOVA P \> 0.05; [Fig 3C](#pone.0163930.g003){ref-type="fig"}). For fungi, we detected weak significant effects of litter on richness (Wilcoxon test P \< 0.01; [Fig 3B](#pone.0163930.g003){ref-type="fig"}) and on fungal community composition (PERMANOVA R^2^ = 0.09 P \< 0.01; [Fig 3D](#pone.0163930.g003){ref-type="fig"}).
{#pone.0163930.g003}
Discussion {#sec008}
==========
Soil biota has been implicated in the facilitation of invasive plant dominance \[[@pone.0163930.ref005], [@pone.0163930.ref037]\]. However, not all invasive species support plant-soil microbe feedbacks as a driver of invasion \[[@pone.0163930.ref038]--[@pone.0163930.ref039]\]. We attempted to identify how relationships between the weedy annual grass medusahead and soil microorganisms might mediate invasion success. Unexpectedly, we did not find evidence for medusahead density effects on the soil microbial communities across a simulated invasion front in either habitat. This supports other studies that have documented instances where soil communities are unresponsive to the presence of invasive weeds in arid grasslands \[[@pone.0163930.ref040]\] and in other systems \[[@pone.0163930.ref041]\]. However, several aspects of our experiment could hinder our ability to capture an existing relationship between medusahead seed density and soil microbial communities. First, we assessed the relationship between medusahead seed density and bulk soil microbial communities and not rhizosphere communities. Rhizosphere microbial communities are different from bulk soil communities \[[@pone.0163930.ref042]\], so the potential effects of medusahead invasion intensity on microbial communities may be observable at smaller spatial scales in the rhizosphere (but see \[[@pone.0163930.ref043]\]). Second, although this study was conducted across two growing seasons, there may have been insufficient time for soil microbial communities to respond to different seed densities of medusahead \[[@pone.0163930.ref044]\]. Although microbial communities have been shown to respond to changes in aboveground plant communities in as little as a month \[[@pone.0163930.ref045]\], these communities could be especially slow to respond to the presence of weeds in environments where soil edaphic factors are slow to change in response to invasion. Moreover, in California grasslands, soil appears to be particularly buffered from aboveground changes \[[@pone.0163930.ref040]\]. Third, extracellular microbial DNA and DNA from dead cells can persist in soils for years and thus, obscure DNA-based present estimates of soil microbial composition \[[@pone.0163930.ref046]\]. Finally, as this study only assessed the composition of the microbial communities, we cannot eliminate the possibility that medusahead seed density can influence the activity and function of belowground soil communities.
Using reciprocal soil transplant experiments, \[[@pone.0163930.ref047]\] reported higher medusahead biomass in introduced soil than in native soil, which suggests that medusahead success is partially due to release from native soil pathogens \[[@pone.0163930.ref048]--[@pone.0163930.ref049]\]. In addition to escaping from soil pathogens, certain plant invasive species have been shown to accumulate local pathogens \[[@pone.0163930.ref011], [@pone.0163930.ref049]\]. Although previous studies have reported that medusahead is sensitive to antagonistic fungi \[[@pone.0163930.ref050]--[@pone.0163930.ref051]\], we observed important differences in bacterial and fungal community composition between open grassland sites where medusahead is typically found in high densities and oak woodland sites where medusahead is typically found in low densities. Specifically, we found significantly higher abundances of fungal pathogens in open grasslands compared to oak woodland habitats. Environmental conditions in the grassland habitat are likely more ideal for both soil and foliar fugal pathogens, which can be important drivers of above ground plant dynamics (e.g. \[[@pone.0163930.ref052]\]). Indeed, these pathogens have been documented in grasslands in other studies (e.g. \[[@pone.0163930.ref053]\]). These results collectively highlight the potential contribution of microbial mechanisms (e.g., via pathogen accumulation) to medusahead dominance in California grasslands. Because the differences in bacterial and fungal communities exist in the absence of medusahead, it is more likely that favorable grassland soil microbial communities facilitate medusahead establishment instead of resulting from the invasion itself.
In addition to negative interactions, a large number of plant species establish symbiotic associations with soil microorganisms (in particular with mycorrhizal fungi and nitrogen-fixing bacteria, \[[@pone.0163930.ref006], [@pone.0163930.ref054]\]). In this experiment we detected a higher proportion of ectomycorrhizal fungi in soil samples from oak woodland habitats. This result is expected as ectomycorrhizal fungi are important to oak trees for acquiring nutrients and for increasing root absorptive area \[[@pone.0163930.ref018], [@pone.0163930.ref055]\]. Our results also show that oak litter (rather than shade) influence overall soil fungal community composition and richness, but not soil bacterial community composition and richness. Although plant litter inputs can change important environmental conditions for soil bacteria such as pH and base cation content \[[@pone.0163930.ref042]\], soil fungi are key decomposers of plant necromass and depend more directly on leaf litter than bacteria \[[@pone.0163930.ref056]\]. We also observed a significantly higher proportion of arbuscular mycorrhizal fungi in open grasslands. Given the generally non-specific interactions with arbuscular mycorrhizal fungi, it has been proposed that several invasive plants make use of these fungi to enhance their success \[[@pone.0163930.ref057]--[@pone.0163930.ref060]\]; but see \[[@pone.0163930.ref061]\]. Other invasive plants (for example, the garlic mustard *Alliaria petiolata*) inhibit mycorrhizal fungi on which natives depend \[[@pone.0163930.ref062]\]. Collectively, this work suggests that biocontrol and management initiatives should consider the potentially beneficial plant-microbe interactions rather than just focusing on antagonistic relationships.
The context dependency associated with invasion success and weed management efficiency is well documented for both medusahead as well as other weedy species (e.g. \[[@pone.0163930.ref063]\]). The presence and abundance of bacterial and fungal groups potentially underlie this context dependency in several instances. Environmental changes, such as exacerbated drought conditions, might modify suitability of oak woodland habitat and perhaps enhance invasibility of previously resistant systems. Therefore, given the complex relationships between aboveground and belowground biota \[[@pone.0163930.ref064]\], understanding the potential mechanisms mediating the association between invasive plant species and soil microorganisms could provide practical information for developing effective management strategies, as well as insight into the ecology of plant-soil food webs and diversity.
Supporting Information {#sec009}
======================
###### Sample-based phylotype accumulation curves for bacterial and fungal soil communities.
(EPS)
######
Click here for additional data file.
###### Shannon diversity for bacterial and fungal soil communities (A, B).
Differences between open grassland samples and oak woodland samples were statistically significant (ANOVA P \< 0.05).
(EPS)
######
Click here for additional data file.
###### Significant differences (Wilcoxon test P \< 0.01 after false discovery rate correction) in the abundance of bacterial and fungal classes between oak woodland soil samples (green) and open grassland soil samples (blue).
(EPS)
######
Click here for additional data file.
###### Abundance of fungal functional guilds between oak woodland soil samples (green) and open grassland soil samples (blue).
Note that the y axis is squared.
(PDF)
######
Click here for additional data file.
###### Significant differences (Wilcoxon test P \< 0.01 after false discovery rate correction) in the abundance of bacterial and fungal genera between oak woodland soil samples and open grassland soil samples.
(DOCX)
######
Click here for additional data file.
We are thankful to Xavier Rojas for lab mentoring.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: **Conceptualization:** ESG NF AB.**Data curation:** ESG AB.**Formal analysis:** AB.**Funding acquisition:** ESG NF AB.**Investigation:** ESG NF AB.**Methodology:** ESG NF AB.**Project administration:** ESG NF AB.**Resources:** NF AB.**Software:** NF AB.**Supervision:** ESG NF AB.**Validation:** NF AB.**Visualization:** AB.**Writing -- original draft:** ESG AB.**Writing -- review & editing:** ESG NF AB.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Country-specific patient rare disease registries are rarely used to make international comparisons because of protocol discrepancies in data collation. Here, we attempt to overcome this limitation by using the inherited disease Cystic Fibrosis (CF) as a paradigm. CF provides a good example because its common form (homozygous F508del-CFTR) occurs across all European social strata appearing frequently but randomly thus providing an opportunity to measure health outcomes.
Methods
=======
Country-specific CF Registries were combined cross-sectionally using a common data protocol (<http://www.eurocarecf.eu>) to compare patient demographics between the European Union (EU) and non-EU countries using EU membership in 2003 as a reference base. We tested the hypothesis that the nine-fold higher resources within the EU would translate into better outcomes.
Findings
========
Data were collected on age, age at diagnosis and CF genotype from 29,025 CF patients registered in 35 European countries. Median age was 16.3 years but was \~4.9 years older in EU countries (17.0 years) than non-EU countries (12.1 years; p\<0.001; CI for the difference was 4-5.1 years, a significant difference (OR 2.4, 95% CI 1.9 - 3.0). Under-ascertainment was unlikely because the relative paucity of F508del-homozygous patients outside the EU was also significant (95% present clinically in childhood). We estimate that the current CF population of non-EU countries would rise by 84% if they had a CF demographic profile comparable to those of the EU countries who were already EU members in 2003.
Interpretation
==============
Given that neither the CF carrier frequency nor the relative territorial population size is significantly different between the EU and non-EU participants, the reasons for this apparent deficit in CF patients of a common genotype in non-EU countries require explanation. It may be that under diagnosis and premature childhood mortality are the main drivers of the relative paucity of CF in non EU states.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Chronic periodontitis (CP), the most common periodontal disease, is an inflammatory disease leading to the destruction of connective tissue and loss of the adjacent supporting bone. The initiation and progression of CP is a consequence of interaction between oral bacteria and the host immune responses. It is well known that the immune response to bacterial products such as lipopolysaccharide, proteolytic enzymes, and subsequent production of proinflammatory cytokines could also evoke an inflammatory reaction in periodontal tissue.
Periodontal treatment involves mechanical cleaning of tooth surfaces to remove calculus and dental biofilm, and strict control of biofilm prevents recolonization of the subgingival area. In some cases, scaling and root planing (SRP) is insufficient to solve periodontal infection. CP is caused by pathogens called the red complex, which includes *Porphyromonas gingivalis* \[P.g.\], *Tannerella forsythia* \[T.f.\], and *Treponema denticola* \[T.d.\]. These pathogens dominate the subgingival layers and are recognized as the most important pathogens in adult periodontal disease, currently thought to be closely associated with CP.[@b1-tcrm-13-307],[@b2-tcrm-13-307]
The last 20 years have seen the emergence of a range of adjunctive antimicrobial regimens designed to aid the mechanical methods of dealing with subgingival plaque. Local application of antibiotic directly at the subgingival area (into the periodontal pocket) has become an alternative. SRP combined with local antibiotics has been shown to yield better results than SRP only.[@b3-tcrm-13-307]--[@b6-tcrm-13-307] The local application of minocycline in the treatment of periodontal disease has promising results compared with other nonsurgical therapies.[@b7-tcrm-13-307]--[@b10-tcrm-13-307] Local administration can avoid many of the side effects associated with systemic antibiotic therapy by limiting the agent to the periodontal pocket, minimizing systemic absorption. A stable, sustained-action formulation of minocycline gel has been developed for subgingival use. This formulation, extensively evaluated in clinical trials conducted in Japan, contains 2% minocycline, a concentration selected on clinical, bacteriological, and gingival crevicular criteria. It also demonstrated that repeated subgingival administration of minocycline ointment in the treatment of CP leads to significant adjunctive improvement after subgingival instrumentation. It possesses widespread bacteriostatic properties and acts slowly by binding to the tooth surface, inhibiting the activity of collagenase at low concentrations and thus preventing periodontal tissue destruction.[@b11-tcrm-13-307],[@b12-tcrm-13-307] Application of minocycline ointment as an adjunct to periodontal flap surgery in generalized CP demonstrated that there was significant reduction in the clinical parameters with improvement in the periodontal status.[@b13-tcrm-13-307] Thus, a direct approach using antibacterial agents by topical administration has become an important part of periodontal disease management.[@b3-tcrm-13-307],[@b14-tcrm-13-307],[@b15-tcrm-13-307] However, it has not been reported that local application of minocycline HCl 2% gel, used as an adjunct to SRP, has effectively suppressed the regrowth of periodontal pathogens.
The objective of the present study was to establish the efficacy of suppressing regrowth of periodontal pathogens and risk of recurrent periodontal pathogens of locally applied minocycline HCl 2% gel when used as an adjunct to SRP without repeated administration for 6 months after initial treatment.
Materials and methods
=====================
Type of study
-------------
A prospective randomized open blinded study was conducted in a single center (Periodontic Clinic, Faculty of Dentistry, Universitas Indonesia) from November 2013 to November 2014. Ethical approval was given by the Research Ethical Committee, Faculty of Dentistry, Universitas Indonesia (KEPKG 2013). Written informed consent was given and signed by subjects prior to the study. Subjects aged \<16 years were not allowed and not included in this study.
Subjects
--------
Patients were aged 30--55 years with localized CP, who had 4--6 mm proximal pocket depth (PD), clinical attachment loss (CAL) equal to or greater than 4 mm, and gingival bleeding on probing (BOP). Patients were included if they did not take any antibiotics in the last 3 months and had no periodontal treatment in the last 6 months. Patients were excluded if they were suffering from a systemic disease, allergic to minocycline hydrochloride, had proximal tooth restorations, or had proximal and cervical caries; pregnant or breast-feeding women and smokers were also excluded, as were also those with poor oral hygiene or malocclusion and patients on continuous medication.
Treatment regimen
-----------------
The drugs were applied locally into the gingival pocket by inserting the gel on the periodontal pocket base and then slowly pulling the ends of the syringe while continuing the injection. The microcapsule gel containing 2% minocycline HCl (Periocline, Sunstar, Osaka, Japan) was commercially packed (0.5 g) to achieve a dose of 20 mg of minocycline HCl per subject. Subjects that fulfilled inclusion and exclusion criteria were randomized and examined. Periodontal examinations included oral hygiene, bleeding scores, PD, attachment loss, and doing the SRP at day 0 of this study (baseline). The subjects were evaluated 1 month before receiving treatment; if the PD was still 4 mm, they were recruited into the study. A total of 84 subjects with CP and with baseline CAL above 4 mm were randomly assigned to receive SRP alone (control group) or SRP followed by four times of local application of minocycline HCl 2% gel (Periocline) once a week (test group). Subgingival plaque samples were collected using a sterile curette at days 1 (baseline), 2, 3, and 6 months before SRP (baseline) or scaling, for bacteriological testing. The outline of this study design is described in [Figure 1](#f1-tcrm-13-307){ref-type="fig"}.
Clinical measurements
---------------------
In this study, supra gingival SRP were performed in all subjects at baseline or day 1. In the minocycline group (test group), subgingival minocycline HCl gel 2% was added. The same procedures were repeated at days 7, 14, and 21. Oral hygiene instruction was given to the patients after each procedure. At days 14 and 21, the amount of plaque was recorded with Loe and Sillness index.[@b16-tcrm-13-307] Papilla bleeding index (PBI) by Muhlemann and Son[@b17-tcrm-13-307] modification index was scored. PD and CAL were examined using bite registration for probing. At month 2, we measured the bleeding scores, PD, and attachment loss in both groups. The subjects were followed up for 6 months. At months 3 and 6, subgingival plaque samples were taken for microbiologic testing before clinical examinations. The plaque index (PLI), PBI, PD, and loss of attachment were scored during the follow-up.
PLI, using an index of Loe and Silness, was measured on teeth 16, 12, 24, 36, 32, and 44.[@b16-tcrm-13-307] The labial or buccal surface (divided into facial, mesio-facial, and disto-facial) was examined, as was palatal or lingual, considered as single surface. A probe was passed along the cervical portion of the tooth surface. Score 0 was given if no plaque adhered to the tip of the probe. Score 1 was given if a film of plaque adhered to the tip of the explorer. Score 2 was given for thin to moderate accumulation of plaque seen on the tooth surface at the cervical portion of the crown. Score 3 was given when an abundance of plaque was seen on the tooth surface at the cervical portion of the crown. The individual scores of all the teeth were then added and divided by the total number of teeth examined.
PBI by Muhlemann and Son[@b17-tcrm-13-307] modification was measured by a Hu-Fredy periodontal probe. PBI of mesial surface was measured from the labial/buccal site, while PBI of distal surface was measured from the palatal/lingual site by carefully inserting the probe into the margin of the gingival sulcus. The PBI was scored as follows: 0, equals to no bleeding; 1, equals to bleeding in the form of a point; 2, equals to bleeding in the form of a line; 3, equals to bleeding in the form of a triangle; and 4, equals to widespread bleeding.
Periodontal PD was measured from the direction of the labial/buccal and the palatal/lingual. CAL was measured from the boundary between the cementum-enamel junction to the basic pocket. Increased tissue attachment loss is defined as the difference between the distance before and after treatment. We categorized the CAL, based on the cutoff point of 4 mm, as mild attachment loss and moderate to severe attachment loss.
The subjects were followed up for 6 months. At 2, 3, and 6 months, subgingival plaque samples for microbiologic testing were taken before clinical examinations. The PLI, PBI, PD, and loss of attachment were scored during the follow-up. In a prospective clinical study, threshold levels of P.g. and T.d. at a periodontal site as measured by reverse transcription--polymerase chain reaction (PCR) could predict CAL at that site over the following 3 months, suggesting that P.g. and T.d. were important pathogens in the progression of CP.[@b18-tcrm-13-307]
Treatments
----------
In this study, supragingival SRP was performed in all subjects at baseline or day 1. In the minocycline group, subgingival minocycline HCl gel 2% was added. The same procedures were repeated at days 7, 14, and 21. Oral hygiene instruction was given to the patients after each procedure. Chemotherapeutic agents is reported to effectively eliminate the bacteria harbored at the bottom of deeper pockets or in dentine tubules that can not be removed with mechanical treatment only.[@b19-tcrm-13-307]
Quantification of periodontal pathogens by real-time PCR
--------------------------------------------------------
Bacterial quantification was carried out using real-time PCR (7500 Fast RT-PCR System, Applied Biosystems, Tokyo, Japan). The bacterial genomic DNA was extracted separately from each sample using the QIAamp DNA Mini Kit (QIAGEN). The extracted DNA was diluted with distilled water to a concentration of 100 ng/μL. Quantification of target species from unknown plaques was achieved by projecting them to standard curves of targeted bacteria based on counts of pure bacterial cultures with serial tenfold dilution from 102 to 108 cell copies.
Bacteria-specific primer pairs ([Table 1](#t1-tcrm-13-307){ref-type="table"}), according to the literature,[@b20-tcrm-13-307]--[@b22-tcrm-13-307] were used to qualify each target bacterium. All samples were run in duplicate in 96-well plates in 7500 Fast RT-PCR System (Applied Biosystems). RT-PCR amplification was performed in a 20 μL reaction mixture containing 2 μL template DNA, 10 μL ds DNA-binding dye SYBR^®^ Green MIX (Invitrogen, Tokyo, Japan), 2 μL, 2.5 μM bacterium-specific primer pair, and 4 μL distilled water. The amplification cycling conditions were 95°C for 10 minutes and then 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. Melting curve analysis was performed for each run to evaluate the specificity of the PCR products. The detection limits of all three bacteria were less than log 10^2^.
Real-time PCR was performed with the ds DNA-binding dye SYBR^®^ Green MIX (Invitrogen) using species-specific primers ([Table 1](#t1-tcrm-13-307){ref-type="table"}).
Statistical procedure
---------------------
In this study, the sample size was calculated based on power of 80% and alpha level of 5% using standard deviation of 0.8 mm and precision of 0.3736.[@b23-tcrm-13-307] We got the minimal sample size of 36 subjects for each group.
For the clinical analysis, statisticians were blinded on the treatment arms. Differences between means of both groups at each time point were tested using Student's *t*-test for numerical data (PD, CAL) and Wilcoxon rank-sum test for nonparametric, categorical data (PLI, bleeding index, calculus index). Changes between different time frames (baseline vs month 3 vs month 6) were tested using Wilcoxon signed-rank test. Differences between means of both groups were tested using analysis of variance (ANOVA) and Friedmann's two-way ANOVA. Data were analyzed using Statistical Package for the Social Sciences (SPSS) 20 (StataCorp LP, College Station, TX, USA).
For the microbiological analysis, the results were analyzed using IBM SPSS software.23 (StataCorp LP); nonparametric tests were selected. In order to analyze the microbiological counts within the group from baseline to 6 months after treatment, a Friedman test followed by a post hoc test (signed test with Bonferroni correction) was adopted. The changes in bacterial counts were compared between the two groups, on a patient basis, using Mann--Whitney *U*-test. The results are presented as the mean difference with a 95% confidence interval and are regarded as statistically significant when *P*\<0.05, unless otherwise stated.
Results
=======
We recruited 84 patients into the clinical study. We analyzed 42 patients for the test group (age =43.67±6.88, men/women =9/33, PLI score =1.04±0.54, calculus index score =1.26±0.64, oral hygiene index score =2.31±1.10) and 39 patients for the control group (age =44.00±6.93, men/women =9/30, PLI score =1.08±0.49, calculus index score =1.44±0.84, oral hygiene index score =2.53±1.13), who were eligible for microbiological assay.
The P.g. bacterial counts were significantly reduced in both groups at 2 months when compared with the baseline ([Table 2](#t2-tcrm-13-307){ref-type="table"}). In the control group, a significant regrowth of P.g., T.d., and T.f. was observed from 2 to 6 months and of P.g. and T.f. from 3 to 6 months ([Table 2](#t2-tcrm-13-307){ref-type="table"}). P.g. in the control group had reduced significantly (*P*\<0.05) after therapy observed in 2 months (0.00±0.17 log~10~ copies/mL); later on, increased slightly in 3 months (0.17±0.72 log~10~ copies/mL); in 6 months the P.g. is back (1.11±1.81 log~10~ copies/mL) similar to baseline. P.g. in test group (minocycline applied) had reduced significantly (*P*\<0.05) after therapy observed in 2 months (0.40±1.28 log~10~ copies/mL); later on increased slightly in 3 months (0.51±1.47 log~10~ copies/mL); in 6 months the P.g. returned (0.83±1.68 log~10~ copies/mL) similar to baseline. T.f. in the control group tended to be reduced after therapy observed in 2 months (0.09±0.55 log~10~ copies/mL); it increased significantly later on (*P*\<0.05) in 3 months (0.55±1.13 log~10~ copies/mL); in 6 months, the T.f. returned higher than baseline (1.41±1.81 log~10~ copies/mL). T.f. in the test group (minocycline applied) tended to be reduced after therapy observed in 2 months (0.88±1.71 log~10~ copies/mL); later on stable in 3 months (0.81±1.75 log~10~ copies/mL); in 6 months, even though the T.f. is increased (1.26±1.94 log~10~ copies/mL), it is still lower than baseline. T.d. in the control group had reduced significantly (*P*\<0.05) after therapy observed in 2 months (0.63±1.40 log~10~ copies/mL); later it increased slightly in 3 months (1.16±1.72 log~10~ copies/mL); in 6 months the T.d. returned (2.08±2.02 log~10~ copies/mL) twice significantly higher than baseline. T.d. in the test group (minocycline applied) had tended to be reduced after therapy observed in 2 months (1.30±1.93 log~10~ copies/mL); later it reduced slightly in 3 months (1.10±1.97 log~10~ copies/mL); in 6 months the T.d. is back (1.70±2.13 log~10~ copies/mL) similar to baseline. On the other hand, in the test group, regrowth of P.g., T.d., and T.f. was not significantly observed from 2 to 6 months ([Table 2](#t2-tcrm-13-307){ref-type="table"}).
The changes in T.d. and T.f. counts from 2 to 6 months were found to be significantly higher in the control group than in the test group ([Table 3](#t3-tcrm-13-307){ref-type="table"}). The comparison of P.g. in the control group and in the test group in a time-dependent manner had no significant differences (*P*\<0.05) after therapy observed in 2, 3, and 6 months. A comparison of T.f. in the control group and in the test group in a time-dependent manner showed a significant difference (*P*\<0.05) after therapy observed in 2 and 6 months, 1.32±1.71 log~10~ copies/mL and 0.37±2.16 log~10~ copies/mL, respectively (*P*=0.033). Also, a comparison of T.d. in the control group and in the test group in a time-dependent manner showed a significant difference (*P*\<0.05) after therapy observed in 2 and 6 months, 1.45±1.99 log~10~ copies/mL and 0.39±2.15 log~10~ copies/mL, respectively (*P*=0.034).
In clinical parameters, the change in mean CAL at 2 months from baseline was significantly (*P*\<0.001) reduced in the test group (−1.58±0.57 mm) when compared with the control group (−0.76±0.99 mm), although the changes in PD and PBI were not significantly different between the two groups. The changes in PD from 2 to 3 months were significantly improved (*P*\<0.05) in the control group (−0.33±0.76 mm) when compared with the test group (−0.01±0.58 mm), (*P*=0.036). Moreover, the PBI was also observed to be significantly reduced (*P*\<0.05) in the control group (−0.06±0.14 mm) compared with the test group (−0.01±0.06 mm), (*P*=0.03) ([Table 4](#t4-tcrm-13-307){ref-type="table"}).
Discussion
==========
SRP is recognized as the gold standard of periodontal therapy. Conventional mechanical debridement sometimes cannot reach the site with the deep periodontal pocket or difficult positions of the tooth, resulting in the recurrence of periodontal pathogen. The local application of minocycline in the treatment of periodontal disease as an adjunct to SRP has promising results compared with other nonsurgical therapies.[@b7-tcrm-13-307]--[@b10-tcrm-13-307] Local administration of antibiotic can limit the agent to the periodontal pocket, minimizing systemic absorption and avoiding the side effects associated with systemic antibiotic therapy. The choice of antibacterial agents is based on the bacterial etiology of the infection. As we know, the subgingival pocket contains many pathogens rather than a single pathogenic species, and it is resistant to many antibacterial therapies.[@b24-tcrm-13-307]
Minocycline gel, a semisynthetic derivative of tetracycline, has a wide spectrum of action against anaerobic as well as aerobic bacteria. Local delivery of antimicrobial agents can be used systemically or topically as an adjunct to SRP in an attempt to further reduce the number of periodontal pathogens, thereby improving the periodontal condition. In past years, these agents have been extensively researched.
As [Table 3](#t3-tcrm-13-307){ref-type="table"} shows, the clinical parameters analyzed in this study show that clinical attachment levels increased rapidly in the minocycline group and are significantly different compared with those in the control group. The improvement in the clinical attachment levels in the minocycline group was consistent with the decrease in inflammation in the deep pocket. This condition showed that the differences were related to local administration of antibiotic gel minocycline rather than to clinical procedures or oral hygiene. The PBI in the minocycline group was decreased and significantly different compared with that in the control group and remained stable until 6 months of follow-up. These results are similar to two short-term, double-blind, parallel studies by Nakagawa et al[@b6-tcrm-13-307] and Steenberghe et al[@b5-tcrm-13-307] that evaluated the effect of subgingivally administered 2% minocycline in addition to mechanical debridement.[@b5-tcrm-13-307],[@b6-tcrm-13-307] Their studies showed that the treatment group had a better response than patients in the placebo group.
In this study, the pocket reduction in both groups was not significant; however, the mean reduction in probing depth from the 21st day to the 6th month was more in the test group compared with that in the control group. This is in accordance with the studies done by Timmerman et al[@b4-tcrm-13-307] and Cortelli et al[@b25-tcrm-13-307] and could be due to supragingival SRP on both groups every 1 month following mechanical debridement, and causing the formation of the fully epithelialized gingival crevice in both groups. The result of this study are similar to those of Timmerman et al,[@b4-tcrm-13-307] showing no statistically significant differences between the test and the control groups in probing depth and attachment level.
In the control group, the number of three bacteria was significantly increased at 6 months. Although SRP was effective in bringing about a temporary reduction of bacteria, it alone may not suppress the regrowth of three periodontopathic bacteria without additional periodontal treatment for 6 months. This study result is consistent with that pertaining to other groups in that the bacterial counts including P.g. were higher than 6 months after SRP.[@b26-tcrm-13-307],[@b27-tcrm-13-307] The efficacy of SRP can be limited in cases with less access to deep pockets and furcations. In addition, there are well-documented secondary effects, such as gingival recession, loss of tooth substance, and dentin hypersensitivity. SRP alone suggested that the risk of recurrent CP was higher than in the case where local application of minocycline HCl 2% gel was used as an adjunct to SRP. On the other hand, in the test group, the number of three bacteria was not significantly higher during the 6-month period. SRP adjunctive local minocycline ointment may suppress the regrowth of three periodontopathic bacteria for 6 months without retreatment.
Sugi et al reported that in recurrent periodontitis patients, IgG titer to Gram-negative obligative anaerobe (*Prevotella intermedia*, T.d., and *C. rectus*) was significantly higher than in the control group.[@b28-tcrm-13-307] The study assumed that these bacteria were one of the causes of recurrence in periodontitis. Matesanz-Pérez et al[@b29-tcrm-13-307] in his meta-analysis concluded that the effect of the subgingival application of antimicrobials was statistically significant (*P*=0.000) for changes in both periodontal pocket depth (0.407 mm) and CAL (0.310 mm).[@b29-tcrm-13-307] The goal to reduce the incidence of recurrence of periodontitis caused by the return of bacteria was achieved. Previous studies have demonstrated that repeated subgingival administration of minocycline ointment in the treatment of CP leads to a significant adjunctive effect for suppression of regrowth of red complex in combination with SRP. The changes (2--6 months) in T.d. and T.f. counts in the test group were significantly lower than those in the control group. It is suggested that local application of minocycline changes the composition of subgingival microbiota and controls regrowth on the subgingival plaque. An adjunctive therapy of local minocycline ointment may reduce the risk of recurrent periodontal pathogens compared with SRP only.
Conclusion
==========
Red complex periodontal bacteria are one of the important etiologies contributing to the activity of periodontitis. Although SRP is recognized as the gold standard of periodontal therapy, occasionally mechanical debridement cannot effectively reach the site with deep periodontal pocket or difficult position of the tooth and resulting residual periodontal pathogen. The results showed that local application of minocycline HCl 2% gel as an adjunct to SRP was effective for suppressing regrowth of periodontal pathogens, suggesting risk reduction of recurrent periodontal pathogens in CP compared with SRP only. With a view to obtaining clearer insights, the author is planning, in future studies, to expand this experiment by investigating human cytokines responses, microbial alteration based on clinical and radiographic findings.
A financial award from Sunstar and a grant from The International Collaboration of Research by Directorate of Research and Community Services, Universitas Indonesia (DRPM-UI) 2015, were used to conduct this study.
**Author contributions**
Y Soeroso, H Sunarto, Y Kemal, R Salim, M Octavia, A Viandita, J Setiawan, and BM Bachtiar contributed as researchers in this study. The coauthor T Akase (Sunstar Company) reviewed the paper. All authors contributed toward data analysis, drafting and critically revising the paper and agree to be accountable for all aspects of the work.
**Disclosure**
The authors report no conflicts of interest in this work.
{#f1-tcrm-13-307}
######
PCR primer sequences for red complex periodontal pathogens
Bacteria Primers (F = forward; R = reverse) References
---------- ------------------------------------ -------------------
P.g. F: 5′-CCCTACGTGTACGGACAGAGCTATA-3′ [@b1-tcrm-13-307]
R: 5′-AGGATCGCTCAGCGTAGCATT-3′
T.f. F: 5′-GGGTGAGTAACGCGTATGTAACCT-3′ [@b2-tcrm-13-307]
R: 5′-GCCCATCCGCAACCAATAAA-3′
T.d. F: 5′-AGAGCAAGCTCTCCCTTACCGT-3′ [@b3-tcrm-13-307]
R: 5′-TAAGGGCGGCTTGAAATAATGA-3′
**Abbreviations:** PCR, polymerase chain reaction; P.g., *Porphyromonas gingivalis*; T.f., *Tannerella forsythia*; T.d., *Treponema denticola*.
######
Mean bacterial counts (log~10~, copies/mL)
--
--
**Notes:** Comparison between the two groups (Mann--Whitney *U*-test). Data are presented as mean ± SD. \**P*\<0.05, ^\*\*^*P*\<0.01.
**Abbreviations:** P.g., *Porphyromonas gingivalis*; T.f., *Tannerella forsythia*; T.d., *Treponema denticola*; SD, standard deviation.
######
Mean changes in bacterial counts (log~10~, copies/mL)
*Δ*(BL to 2 months) *Δ*(2 to 3 months) *Δ*(2 to 6 months) *Δ*(3 to 6 months)
------------ --------------------- -------------------- --------------------------------------------------- --------------------
P.g.
Control −1.43±2.20 0.16±0.72 1.11±1.81 0.94±1.65
Test −0.93±2.20 0.12±1.80 0.44±1.17 0.32±1.87
*P*-value 0.504 0.552 0.134 0.084
T.f.
Control −0.87±1.54 0.47±1.10 1.32±1.71 0.85±1.50
Test −0.51±2.23 −0.07±2.42 0.37±2.16 0.45±2.62
*P*-value 0.37 0.157 0.033[\*](#tfn5-tcrm-13-307){ref-type="table-fn"} 0.287
T.d.
Control −0.54±2.00 0.53±1.90 1.45±1.99 0.91±1.90
Test −0.40±2.84 −0.20±2.60 0.39±2.15 0.60±3.03
*P*-value 0.858 0.218 0.034[\*](#tfn5-tcrm-13-307){ref-type="table-fn"} 0.631
**Notes:** Comparison between the two groups (Mann--Whitney *U*-test). Data are presented as mean ± SD.
*P*\<0.05.
**Abbreviations:** BL, baseline; P.g., *Porphyromonas gingivalis*; T.f., *Tannerella forsythia*; T.d., *Treponema denticola*; SD, standard deviation.
######
Mean changes in clinical parameters
*Δ*(BL to 2 months) *Δ*(2 to 3 months) *Δ*(2 to 6 months) *Δ*(3 to 6 months)
------------ ----------------------------------------------------- ----------------------------------------------------- -------------------- --------------------
CAL
Control −0.76±0.99 −0.26±0.78 −0.13±0.98 0.13±0.82
Test −1.58±0.57 −0.01±0.58 0.01±0.68 0.02±0.60
*P*-value \<0.001[\*](#tfn8-tcrm-13-307){ref-type="table-fn"} 0.115 0.461 0.516
PD
Control −1.40±0.95 −0.33±0.76 −0.27±0.88 0.06±0.70
Test −1.58±0.57 −0.01±0.58 0.01±0.68 0.02±0.60
*P*-value 0.296 0.036[\*\*](#tfn9-tcrm-13-307){ref-type="table-fn"} 0.109 0.782
PBI
Control −0.43±0.42 −0.06±0.14 −0.01±0.17 0.05±0.15
Test −0.30±0.33 −0.01±0.06 0.03±0.08 0.04±0.09
*P*-value 0.123 0.033[\*\*](#tfn9-tcrm-13-307){ref-type="table-fn"} 0.162 0.682
**Notes:** Comparison between the two groups (paired *t*-test). Data are presented as mean ± SD.
*P*\<0.01,
*P*\<0.05.
**Abbreviations:** BL, baseline; CAL, clinical attachment loss; PD, pocket depth; PBI, papilla bleeding index; SD, standard deviation.
| {
"pile_set_name": "PubMed Central"
} |
1.. Introduction {#s1}
================
The concept of citizen science may have started in the early twentieth century when large amounts of data were generated for the National Audubon Society\'s Christmas Bird Count \[[@RSOS181871C1]\]. Over the last two decades, citizen science has seen a sharp rise in popularity in several fields. Recent projects include the monitoring of marine litter on coastal areas \[[@RSOS181871C2],[@RSOS181871C3]\], identification of endangered bird and frog species \[[@RSOS181871C4]\], measuring levels of environmental exposure following oil spills \[[@RSOS181871C5]\] and evaluating the occurrence of Fibonacci spirals of sunflowers \[[@RSOS181871C6]\].
Fewer studies have shown the involvement of citizens in the monitoring of water resources \[[@RSOS181871C7]--[@RSOS181871C9]\]. Nevertheless, there are several citizen-based water resource monitoring programmes that have been initiated over the last couple of decades. The literature shows that there were/are several objectives for initiating these programmes and these include to monitor water quality and identify environmental problems \[[@RSOS181871C10]--[@RSOS181871C14]\] to initiate social change and/or legislation \[[@RSOS181871C15]--[@RSOS181871C17]\], and to provide environmental education and awareness \[[@RSOS181871C18]--[@RSOS181871C22]\].
Datasets generated by citizen science programmes were used to initiate crucial management actions against surface water quality deterioration by eutrophication and climate change or to show surface water quality improvement arising from advanced treatment or reduced run-off. Measurements of water clarity (Secchi disc) were recorded across eight states in the USA over a 74-year period \[[@RSOS181871C23]\]. Another study reported 23 years of temperature and water transparency (Secchi test) values by the lay community in Lake George, NY, to characterize long-term trends linking water clarity to climatic, chemical and recreational parameters \[[@RSOS181871C11]\]. A long-term citizen science project spanning a 22-year period of summertime water quality monitoring by volunteers (temperature, pH, salinity and dissolved oxygen), from 122 stations in 17 embayments in Buzzards Bay, Massachusetts, followed the effect of climate change on water eutrophication \[[@RSOS181871C14]\]. In France, 18 years\' worth of citizen-generated data on river nutrients were analysed to determine whether nutrient concentrations were reduced during the intervening period \[[@RSOS181871C10]\]. Other citizen science projects investigated the impact of universities and professors in terms of their ability to establish a plan for an environmental monitoring organization and for educational purposes. These programmes targeted school children, undergraduates and graduates, as well as environmental group representatives. They aimed at engaging citizens in improving environmental conditions through connecting policy, science and action. Savan *et al*. \[[@RSOS181871C22]\] monitored biological ecosystem health parameters, mainly benthic invertebrates, lichen and *Escherichia coli* from 1997 to 2001; chemical indicator monitoring kits were expensive and thus not used; however, participants were taught about chemical water quality parameters and how these were affected by pollutants \[[@RSOS181871C22]\]. Sharpe & Conrad \[[@RSOS181871C24]\] emphasized the gap between 'monitoring' and 'management', i.e. when scientific data are collected but little is done concerning the optimization of the watersheds\' management. The study addressed the case of Canadian province Nova Scotia, where community groups managed to collect water data starting in 1990, getting samples from more than 200 sites and 10 watersheds \[[@RSOS181871C24]\]. Water management concerns also engaged citizens and lay community in different educational programmes in Nevada \[[@RSOS181871C25]\], Michigan \[[@RSOS181871C26]\] and South Africa \[[@RSOS181871C21]\].
[Table 1](#RSOS181871TB1){ref-type="table"} summarizes citizen science water quality studies that have appeared in the literature over the past 20 years where it can be seen that the vast majority of studies were conducted in North America and Europe and where the quality of surface (lakes, rivers, and estuaries) and groundwater were investigated. Most of these studies measure more than one water quality parameter but, with one or two exceptions, are limited to no more than six parameters, which are usually a combination of physical, chemical and biological tests. It can also be seen from [table 1](#RSOS181871TB1){ref-type="table"} that for the vast majority of studies, the citizen scientists' role in water quality projects was restricted to monitoring and data collection; the experts, whether scientists, university researchers or qualified laboratories undertook the water quality testing. In a small number of more recent studies, however, the role of the citizen scientists was expanded to include independent and simultaneous water quality testing, albeit for a very small number of water quality parameters. This approach, if properly implemented, will serve to empower community citizens with the skills needed to monitor their water quality long-term. It is also worth noting that a relatively small number of water quality projects were designed to include follow-up mitigation efforts, as the vast majority of these studies were intended for characterization and monitoring purposes. This issue is particularly relevant in the developing world where, in contrast to the developed world, where mitigation steps are usually implemented and monitored by regulated private/public sector entities, mitigation of documented water quality deterioration is rarely, if ever, implemented. Table 1.Summary of citizen science projects related to water quality measurement.referencelocationwater bodycitizen scientists' rolewater quality parameters measuredObrecht *et al*. \[[@RSOS181871C13]\]USA (Missouri)lakessample collectiontotal phosphorus, total nitrogen, water transparency (Secchi disc), chlorophyll, temperature and total suspended solidsAu *et al*. \[[@RSOS181871C15]\]Canadacreeksample collectioncoliforms, *E. coli*, phosphate, ammonia, pH, dissolved oxygen, hardnessCanfield *et al*. \[[@RSOS181871C27]\]USA (Florida)lakesample collectiontotal phosphorus, total nitrogen, water transparency (Secchi disc), chlorophyll, pH, alkalinity, electrical conductivityNicholson *et al*. \[[@RSOS181871C12]\]Australiariversample collectionturbidity, electrical conductivity, pH, total phosphorusSavan *et al*. \[[@RSOS181871C22]\]Canadalakes and streamssample collectionbenthic invertebrates, lichen, *E. coli*Boylen *et al*. \[[@RSOS181871C11]\]USA (New York)lakes and pondssample collectionwater transparency (Secchi disc), temperatureSharpe & Conrad \[[@RSOS181871C24]\]Canadalakes, streams, rivers, estuariessample collectiontemperature, total suspended solids, faecal coliforms, dissolved oxygenRoa Garcia & Brown \[[@RSOS181871C28]\]Colombiastream, headwater and household faucetssupervised testingelectrical conductivity, pH, total dissolved solids, dissolved oxygen, temperature, nitrate, phosphate, hardness, faecal and total coliformsLoperfido *et al*. \[[@RSOS181871C29]\]USA (Iowa)riversupervised testingnitrate, nitrite, total phosphorus, temperature, transparencyStepenuck *et al*. \[[@RSOS181871C30]\]USA (6 states)recreationalindependent testing*E. coli*Hoyer *et al*. \[[@RSOS181871C31]\]USA (Florida)lakesupervised testingtotal phosphorus, total nitrogen and chlorophyllPeckenham *et al*. \[[@RSOS181871C32]\]USA (Maine)groundwaterindependent testingelectrical conductivity, chloride, nitrate, pH, hardnessBurgos *et al*. \[[@RSOS181871C16]\]Mexicoriversample collectiontemperature, pH, hardness, alkalinity, turbidity, *E. coli*, coliformsLatimore & Steen \[[@RSOS181871C26]\]USA (Michigan)lakes, streamssample collectionwater transparency (Secchi disc), total phosphorus, dissolved oxygen, temperatureRheuban *et al*. \[[@RSOS181871C14]\]USA (Massachusetts)coastal water (estuary)sample collectiontemperature, pH, salinity, dissolved oxygenStorey *et al*. \[[@RSOS181871C33]\]New Zealandstreamsindependent testingwater transparency, electrical conductivity, nitrates, dissolved oxygen, *E. coli*, temperatureFarnham *et al*. \[[@RSOS181871C34]\]USA (NYC)waterwayssample collectionfaecal indicator bacteriaJollymore at al. \[[@RSOS181871C35]\]Canadastreams and riversindependent testingtotal suspended solids, nitrate, dissolved organic carbonMcGoff *et al*. \[[@RSOS181871C36]\]UKponds, lakes and riversindependent testingnitrate, phosphateScott and Frost \[[@RSOS181871C37]\]Canadastorm water pondindependent testingturbidity, phosphate, nitrateThornhill *et al*. \[[@RSOS181871C17]\]Chinastreamssupervised testingturbidity, phosphate, nitrateAbbott *et al*. \[[@RSOS181871C10]\]Franceriver estuarysample collectionphosphate, nitrateBrouwer *et al*. \[[@RSOS181871C18]\]Hollandtap waterindependent testingpresence of bacteriaWilson *et al*. \[[@RSOS181871C38]\]USA (Alaska)riversample collectionpH, dissolved oxygen, conductivity, temperature, dissolved organic carbon, dissolved greenhouse gases, major ions, nutrients, trace metals, stable water isotopes
Lebanon suffers from high levels of water pollution, depletion of water resources and limited wastewater infrastructure and treatment facilities \[[@RSOS181871C39]\]. Most urban centres and villages in Lebanon depend on septic tanks or sewers with direct discharge into the environment \[[@RSOS181871C40],[@RSOS181871C41]\]. High counts of faecal, total coliforms and diverse pollutants were reported \[[@RSOS181871C42],[@RSOS181871C43]\] leading to the pollution of surface water and groundwater \[[@RSOS181871C44]\]. Lebanon also suffers from poor water management and governance practices and effluent streams data remain insufficient and poorly analysed \[[@RSOS181871C39]\]. Furthermore, water resource policy and planning are restricted by scarce, often unreliable data \[[@RSOS181871C45]\] and the absence of well-maintained and operated monitoring networks \[[@RSOS181871C9]\].
The specific objectives of this work were to (i) assess the interest of *citizens* in conducting water quality measurements, (ii) instigate a citizen-centric monitoring campaign to determine the quality of groundwater, (iii) compare the values obtained by citizen scientists and experts to assess the accuracy of the results, and (iv) analyse whether this participatory approach will lead to concrete actions in water resource management. This case study, to the authors\' best knowledge, is the first of its kind in the Middle East and one of the very few from the developing world where the citizen science approach is used to gather water quality data from groundwater sources in a medium-sized village.
2.. Material and methods {#s2}
========================
2.1.. Study area {#s2a}
----------------
The selected area for this study is a village located in South Lebanon that occupies an area of approximately 12 km^2^ at an elevation of 400 m above sea level. No recent population census has been conducted but the population is estimated to be around 40 000. The village does not have a sewer network and wastewater is discharged to cesspools and septic tanks. As a result, septic tank infiltration may pose a serious problem on groundwater supplies given the increase in population and the scarcity in water supplies. Public water supply in the village is regulated by South Lebanon Water Establishment which distributes water from three artesian wells. Recently, one of these wells has been closed by the Ministry of Health after samples tested positive for bacterial contamination. To compensate for the water shortage, residents have resorted to privately owned wells and several well owners have been selling water from their private wells.
Lebanon has a Mediterranean climate characterized by rainy winters and hot dry summers. Most, if not all, of the precipitation falls during the cold season (between December and March) with an average daily high temperature below 18°C. This leaves the warm season (between June and October) with dry and humid weather with an average daily high temperature above 28°C.
2.2.. Citizen participation {#s2b}
---------------------------
### 2.2.1.. Research ethics approval {#s2b1}
The research proposal, oral informed consent documentation, presentations, data collection forms, questionnaires and surveys related to this project were reviewed by the American University of Beirut (AUB) Institutional Review Board (IRB). In addition, all researchers involved in this study took the Social & Behavioral Research---Basic Course which is a Web-based course offered by the Collaborative Institutions Training Initiatives (CITI) Programme to provide research ethics education to the research community.
### 2.2.2.. Citizens' involvement {#s2b2}
The framework adopted for citizen involvement is outlined in Talhouk *et al*. \[[@RSOS181871C46]\]. In 2014, the Nature Conservation Center (NCC), of the American University of Beirut, established a community outreach programme where participatory mapping was conducted by a university team with stakeholders in 50 villages to identify local natural and cultural landmarks. One of the outcomes of this work was the identification of clean water as a top environmental priority for one of the villages in the study group. Representatives from this village were contacted by the university team in early 2015 to verify interest in the project. In view of the university team\'s previous knowledge, and after the identification of various stakeholders (municipality, well owners, non-governmental organizations, community centres, etc.), meetings were arranged between representatives of the research team and the stakeholders. The village local community was invited through the municipality to attend a project introductory presentation on 31 October 2015. In this presentation, the project objectives and proposed analysis methods were outlined.
After the presentation, a call for participation was opened and citizens showing interest in participation were asked to complete a perception questionnaire about water quality. The water perception questionnaire answered by the citizens on the day of project introduction was made up of eight questions that examined their knowledge concerning household water at their residence, the health implications of using polluted water and the general sources of water pollution. Participation was open to all; however, the vast majority of volunteers were women. This gender imbalance may be in response to the composition of the university team which consisted mostly of women scientists. Another possibility may be that women leading a local women club took the initiative to recruit participants. The university team did not influence participation and hence did not enforce gender balance among the citizen scientist volunteers.
Several meetings and workshops were arranged with the participating/volunteer citizens. These meetings/workshops were used to explain various aspects of water quality (standards, parameters, significance of these parameters, testing procedures) and the participating citizens were trained to perform the various water quality tests. The presentation and workshops were planned to accommodate local interpretive capabilities of citizen scientists; fact sheets and infographics were prepared in Arabic for each water quality parameter and included background information, illustrated methodology and safety information.
2.3.. Sampling and water quality testing {#s2c}
----------------------------------------
The location of the water sources was determined using a collaborative effort between representatives from the university, the local municipality, the water authority, private well owners and volunteers ([figure 1](#RSOS181871F1){ref-type="fig"}). Water samples were taken from three public wells, three private wells and two water storage tanks. The number of collected samples from each water source is summarized in electronic supplementary material, table S1. Sampling was conducted by both trained citizens and researchers following the recommendations of the US EPA (United States Environmental Protection Agency) \[[@RSOS181871C47]\]. Figure 1.A Google Earth aerial map of the sampling site.
Prior to every sampling campaign, university researchers would contact the local village representative to arrange a mutually convenient time for the citizen scientists and university team to meet. The village representative would then contact the well owners and the municipality to advise them that sampling will take place at the agreed-upon time. The pre-sampling meetings were held at the local municipality and a team consisting of two university researchers and several citizen scientists (the numbers varied but there were a minimum of three and a maximum of five) would set out together to collect the samples from each of the locations shown in [figure 1](#RSOS181871F1){ref-type="fig"}. During the winter campaigns, the collected and labelled samples were stored in the municipality\'s dispensary overnight; the analysis was then conducted the following day. On the other hand, during the summer campaigns, the collection and analysis took place during the same day.
Water samples were tested for a total of nine physical and chemical (pH, electrical conductivity, turbidity, hardness, alkalinity, nitrates, nitrites, ammonia and phosphates) and three biological parameters (total coliforms, faecal coliforms and *E. coli*). For this purpose, simple field testing kits and laboratory supplies were purchased or assembled by the team. The selection criteria for the operational methods of the kits included ease of use, short testing duration and moderate sensitivity. A summary of the tested water quality parameters, designated instruments or kits used and method is presented in [table 2](#RSOS181871TB2){ref-type="table"}. Table 2.Summary of the water quality parameters, instrument used and testing method.parametersinstrument/test kitmethodphysical PHHach HQ40D-IntelliCALTM PHC101electrode method conductivityHach HQ40D-IntelliCALTM CDC401electrode method turbidityEutech TN100nephelometrychemical alkalinityHach AL-AP Test kitdrop count titration/sulfuric acid hardnessHach HA-71A Test kitdrop count titration/EDTA nitratesHACH DR900 Colorimeter/Hach NitraVer 5 Reagent Powder Pillowscadmium reduction method nitritesHACH DR900 Colorimeter/Hach NitriVer 3 Reagent Powder Pillowsdiazotization method phosphorusHACH DR900 Colorimeter/Hach PhosVer 3 Reagent Powder Pillowsascorbic acid method ammoniaHACH DR900 Colorimeter/Hach Ammonia Reagent setsalicylate methodbiological total coliformsHach m-ColiBlue24 Broth & Endo nutrient pad setsmembrane filtration method faecal coliformsm FC broth & m FC nutrient pad setsmembrane filtration method
Conductivity, pH and dissolved oxygen were measured by submerging the respective electrode of the Hach HQ40D meter in the water sample after rinsing it with deionized water. For turbidity measurements, the sample vial was rinsed with the sample three times, and then it was filled to line mark, wiped with a lint-free cloth and placed in the meter to be measured. All electrodes of the Hach HQ40D and the turbidity meter were calibrated prior to use. Alkalinity was measured using drop count titration by sulfuric acid after the addition of phenolphthalein and Bromcresol Green-Methyl Red indicators, respectively. Water total hardness was also measured using drop count titration by EDTA (Ethylene-diaminetetraacetic acid) after the addition of buffer and Calmagit indicator. Nitrogen-nitrate (NO~3~-N), nitrogen-nitrite (NO~2~-N), nitrogen-ammonia (NH~3~-N) and orthophosphate were measured by colorimetric assays using the Hach DR900 handheld colorimeter. For comparison purposes with the water quality guidelines, NO~3~-N, NO~2~-N and NH~3~-N were converted to $NO_{3}^{-}$ , $NO_{2}^{-}$ and $NH_{4}^{+}$ by multiplying each value by 4.4, 3.3 and 1.3, respectively.
For the summer and winter campaigns, all the physical and chemical water quality parameters were tested independently by both the citizens and laboratory technicians. However, bacteriological tests for winter samples were conducted by university technicians because the sterilizing filtration equipment was not available at the village. For the summer samples, bacteriological tests were performed by citizens using sterile disposable filtration units and nutrient pad sets.
3.. Results {#s3}
===========
3.1.. Community participation {#s3a}
-----------------------------
After the project introductory presentation, 46 citizens were interested in participating in the project of which 65.2% were adult females and 34.8% were young school students (6th grade); however, this number dropped to a total of 26 citizens, predominantly women, who participated in the water quality testing over six sessions. The age of the participants varied between 16 and 62 and averaged 35 years old. Their educational level also varied between primary, secondary and tertiary education. It is worth noting that female participants were mostly interested in conducting water test analysis, while the male participants were more engaged in the sample collection. This observation may be attributed to the local culture where men usually work outdoors while women partake in indoor work activities.
The results of the questionnaire are summarized in electronic supplementary material, table S2. Interestingly only 16% of the citizens who answered the questionnaire relied on municipal water while the remaining citizens purchased water from local wells or water tankers. Forty-three per cent of the participants were not aware of the quality of their household water and whether it was fit for the intended purpose (drinking and cooking). Most of the respondents (43%) stated that the water at their household was not potable because it contained microbes (13%), it had a non-desirable taste (11%), or it was contaminated with chemicals (7%). Most of the participants (44%) did not answer the question related to potential sources of water pollution.
During the water sampling and testing campaigns, some new citizen scientists joined in the middle of the project while others left, but there was always a core group which persisted throughout (and beyond) the duration of the project and this proved crucial for peer training newcomers. Throughout the project, the university team served as a knowledge resource for the citizen scientists and responded to all water quality-related inquiries, whether they were directly related to the project or not.
The water test results, generated by the university team and the citizen scientists, were shared in a public seminar organized by the local authorities and attended by all stakeholders including citizen scientists, private well owners, representatives from the municipality, representatives from a local school and concerned residents. Recognition and pride in local resident participation were expressed by all attendees. Women scientists, now better informed about the water quality in the village voiced their opinion and concerns to the local authorities during the public session.
3.2.. Groundwater quality {#s3b}
-------------------------
[Table 3](#RSOS181871TB3){ref-type="table"} lists the total average results for all nine physical and chemical water quality parameters recorded by the citizen scientists and university researchers. The test results were compared with the Lebanese water quality standard to identify non-conforming values. For five out of the nine parameters (pH, conductivity, turbidity, hardness and alkalinity), all of the test results for both the summer and winter campaigns were within the values recommended by the Lebanese standards. For nitrate, nitrite, ammonia and phosphate, the vast majority of the test results were within the recommended values; the exceptions were for well 7 where all four parameters exceeded the Lebanese standard values during two testing campaigns and for well 2 where the nitrite and phosphate levels exceeded the standard for one of the testing campaigns. Table 3.Average results for the water quality parameters recorded by citizen scientists and university researchers.parameter*N*citizenuniversity researcherspH397.71 ± 0.207.68 ± 0.30conductivity (µS/cm)39550.6 ± 93.9540.9 ± 93.8turbidity (NTU)331.86 ± 3.801.87 ± 5.20hardness (mg l^−1^)39330 ± 57315 ± 48alkalinity (mg l^−1^ as CaCO~3~)39284 ± 59284 ± 51nitrates (mg l^−1^)3915.7 ± 12.915.1 ± 11.6nitrites (mg l^−1^)390.08 ± 0.300.09 ± 0.30ammonia (mg l^−1^)390.15 ± 0.100.11 ± 0.10phosphates (mg l^−1^)390.35 ± 0.300.52 ± 0.40
3.3.. Comparison between citizens\' data and expert personnel\'s data {#s3c}
---------------------------------------------------------------------
A comparison between data generated by citizen scientists and those by university researchers for the nine physical and chemical water quality parameters was established based on paired *T*-test analysis for the dry/summer and wet/winter campaigns (these are summarized in electronic supplementary material, table S3). There was no significant difference between the citizen scientists\' data and the university researchers' data for seven of the nine water quality parameters: pH, conductivity, turbidity, alkalinity, nitrates, nitrites and ammonia. However, there were significant differences for the hardness and phosphate data ([table 3](#RSOS181871TB3){ref-type="table"}). [Table 4](#RSOS181871TB4){ref-type="table"} lists the results recorded by the citizen scientists during the summer and winter campaigns. Table 4.Citizen measurements (average ± s.d.) of nine water quality parameters at eight sites.siteseasonpHconductivity (µS/cm)turbidity (NTU)hardness (mg l^−1^)alkalinity (mg l^−1^ as CaCO~3~)nitrates (mg l^−1^)nitrites (mg l^−1^)ammonia (mg l^−1^)phosphates (mg l^−1^)1winter7.3163319.97393.328014.1700.130.44summer7.4 ± 0.06611 ± 62.235.54 ±342 ± 72.55300 ± 56.5723.02 ± 3.760.01 ± 0.010.14 ± 0.010.48 ± 0.492winter8.03 ± 0.11433 ± 18.010.25 ± 0.08250.8 ± 54.97206.7 ± 57.746.64 ± 1.170.01 ± 0.010.09 ± 0.020.24 ± 0.1summer7.83 ± 0.14420 ± 1.530.06 ± 0.08250.8 ± 26.12273.3 ± 70.244.43 ± 2.690.01 ± 0.010.33 ± 0.380.77 ± 0.763winter7.62 ± 0.32586 ± 20.651.54 ± 1.27393.3 ± 17.1333.3 ± 23.098.71 ± 0.260.01 ± 0.020.1 ± 00.29 ± 0.15summer7.66 ± 0.03590 ± 1.530.14 ± 0.09330.6 ± 54.97333.3 ± 61.17.08 ± 1.930.03 ± 0.010.12 ± 0.010.13 ± 0.024winter7.96 ± 0.04396 ± 27.580.28 ± 0.02333.5 ± 84.64220 ± 56.577.75 ± 0.310.01 ± 00.1 ± 0.010.42 ± 0.33summer7.86 ± 0.07482 ± 100.781.13 ± 1.57273.6 ± 17.1246.7 ± 50.335.02 ± 0.920.01 ± 0.010.11 ± 0.010.42 ± 0.345winter7.48 ± 0.13543 ± 39.890.93 ± 0.59330.6 ± 9.87246.7 ± 57.7420.07 ± 0.680.02 ± 00.12 ± 0.010.39 ± 0.28summer7.4 ± 0.15582 ± 6.360.14 ± 0.19350.6 ± 12.09350 ± 14.1417.93 ± 11.580.02 ± 00.12 ± 0.020.37 ± 0.466winter7.93 ± 0.12538 ± 42.770.45 ± 0.13364.8 ± 54.97293.3 ± 23.0919.04 ± 2.340.02 ± 0.010.09 ± 0.010.17 ± 0.17summer7.74 ± 0.11584 ± 15.370.1 ± 0.14324.9 ± 29.62293.3 ± 92.3822.58 ± 2.030.02 ± 0.010.12 ± 0.010.14 ± 0.057winter7.79 ± 0.27727 ± 41.245.59 ± 3.76381.9 ± 26.12293.3 ± 50.3351.21 ± 12.790.78 ± 0.640.34 ± 0.20.4 ± 0.318winter7.69 ± 0.1553 ± 10.691.17 ± 0.38353.4 ± 64.74313.3 ± 30.558.41 ± 0.890.03 ± 0.010.1 ± 0.010.28 ± 0.08
Bacteriological test, represented by total coliforms, faecal coliforms and *E. coli*, performed by university researchers during the winter (wet/cold) campaign are presented in [table 5](#RSOS181871TB5){ref-type="table"}. The biological test results for the summer campaign as recorded by citizens and university researchers are also shown in [table 5](#RSOS181871TB5){ref-type="table"}. For the winter campaign, faecal coliforms were not found in any of the tested samples while *E. coli* was only found in one sample from site 1 and another from site 8, and, with the exception of sites 3 and 4 (which were devoid of all forms of bacterial contamination), all the other sites exhibited total coliforms contamination to varying levels and occurrence with sites 1 and 7 exhibiting the highest counts. On the other hand, during the summer campaign, total coliform counts were found in all six sites and faecal coliforms were found in sites that did not show any contamination during the winter campaign, namely sites 3 and 6. It can also be seen from [table 5](#RSOS181871TB5){ref-type="table"} that there are several discrepancies between citizen scientists\' results and those recorded by the university researchers. Table 5.Bacteriological test results during the winter and summer campaigns. Y: bacteria was detected; N: no bacteria was detected; --: no results.sitewintersummerdateResults generated by university researchers onlydateuniversity researcherscitizen scientiststotal coliforms (CFU/100 ml)faecal coliforms (CFU/100)*E. coli* (CFU/100 ml)total coliformsfaecal coliformstotal coliformsfaecal coliforms125 Nov 1528003 Aug 16YNYN19 Dec 15320017 Aug 16--------117 Feb 1654601810 Sep 16YNYN225 Nov 150003 Aug 16YNYN29 Dec 152880017 Aug 16NNYY217 Feb 1600010 Sep 16YNY--325 Nov 150003 Aug 16YYYN39 Dec 1500017 Aug 16YYYN317 Feb 1600010 Sep 16YY----425 Nov 150003 Aug 16YNYN49 Dec 15------17 Aug 16YNYN417 Feb 1600010 Sep 16YNY--525 Nov 150003 Aug 16NNYN59 Dec 1580017 Aug 16YNYN517 Feb 1600010 Sep 16--------625 Nov 1548003 Aug 16YYYY69 Dec 15840017 Aug 16YYYN617 Feb 1620010 Sep 16YYY--7^a^25 Nov 15616003 Aug 16--------7^a^9 Dec 155800017 Aug 16--------7^a^17 Feb 16680010 Sep 16--------8^a^25 Nov 1584003 Aug 16--------8^a^9 Dec 152320817 Aug 16--------8^a^17 Feb 16120010 Sep 16--------[^3]
4.. Discussion {#s4}
==============
This study required assembling a mobile laboratory, developing and translating experimental protocols, preparing data forms and administering pre- and post-test presentations. These tools contributed to building local capacity, combining knowledge into actions, and ensuring the development of sustainable solutions. Although the initial objectives of the project were grounded in the citizen science framework, the project evolved to lead to (i) the formation of a local water committee whose mandate is to follow up on water testing at the village, (ii) the initiation of corrective measures such as the replacement of several interconnecting pipes for one of the contaminated storage tanks, and (iii) the use of low-cost disinfection treatment to the water wells that exhibited low level of bacterial contamination. The project, in terms of training, data collection and data analysis, occurred during the period January 2015 to December 2016. Remedial measures (piping modifications and disinfection equipment), initiated and procured by the local municipality in coordination with the water committee/university researchers were implemented in February 2017 and subsequent tests during March 2017 confirmed the effectiveness of these measures. The project was 'officially' concluded in May 2017. A 'concluding meeting' was held at the village on the 13 May 2017 where university researchers and the citizen scientists (as well as several members of the local municipality and water authority) were in attendance. Assurances were given during the meeting that water monitoring by the members of the water committee, and other volunteers, will continue and additional financial implications (i.e. new kits and consumables) will be factored into the local municipality\'s budget. It should be noted that the university team handed over a complete set of testing kits which would have been adequate for several testing campaigns. Follow-up by the university team over the period May 2017--May 2018 confirmed that partial, semi-regular water sampling and testing (bacteria, turbidity and hardness) was conducted at various locations and on a bi-monthly basis and that the quality of the water was consistent with previous tests. However, in a more recent follow-up discussion (early December 2018), members of the water committee confirmed that testing has not occurred for almost two months as a result of a dispute between the water committee and the municipality over the results of one of the sites. It appears that bacteriological contamination was found in one of the wells and the owner/municipality refused to accept the result and hence to proceed with remedial action. In addition, the water committee members intimated that future testing may be in jeopardy due to unavailability of testing kits (budgetary constraints have hindered the procurement process). It is anticipated that these set-backs will be resolved in due course as members of the committee have stated that both parties have agreed, in principle, to resolve this issue by seeking the opinion of the water authority.
The key components that ensured the success of the project are the long-time trust between the community and the university (American University of Beirut-Nature Conservation Center), the flexibility in setting the university-staff visits according to the local residents\' schedule, and the respect of the local culture and community dynamics. Research and community development in the context of an academic project is usually a high priority to the academic investigators. However, the same situation does not apply to citizens who are engaged in the project as volunteers. This creates an imbalance in the work progress because both parties are equally involved in the project. As such, citizen science projects conducted in a public and private participatory approach puts the conventional methodology that is usually bound to timeline and clear deliverables into question. Moreover, the university was keen on building the trust of the community by sharing the non-complying results with the stakeholders and discussing with them possible remedial solutions before involving the public at large. All findings were disseminated in public seminars, which served as a platform for discussion among citizens, the local authority and the academic experts. One additional outcome of the study is the development of a roadmap for testing the water in the village or at home, to identify possible quick remedial solutions and to establish links with the Water Authority personnel. Contrary to other studies where citizens were mainly involved as sample \[[@RSOS181871C48]\] or data \[[@RSOS181871C9]\] collectors, this project allowed citizens to play a major role in analysing the data, discussing corrective measures and ensuring sustainable solutions.
Lebanese municipal water, often sourced from groundwater, is generally perceived to be unsafe to drink, primarily because of contamination in pipelines or in local storage tanks. Most Lebanese households use bottled water for drinking purposes. However, municipal water is continuously monitored by governmental authorities and should in principle comply with the Lebanese drinking water standard. Results show that for a number of wells, the levels of nitrate, nitrite and phosphates exceeded the Lebanese standard and that the presence of faecal coliforms was also observed in a number of locations. The elevated chemical levels and the bacteriological contamination may have resulted from agricultural water run-off and/or septic tank infiltration into the local groundwater. It is important to note that the chemical and bacteriological findings confirm the general perception of local citizens that were assessed during the preliminary stage of the project. The results of this work also highlight the ability of citizens to produce reliable and repeatable data for a substantial number of water quality indicators. As reported earlier, the citizen scientists' results and those generated by the university researchers for seven out of the nine physical and chemical parameters, namely conductivity, pH, turbidity, alkalinity, nitrate, nitrite and ammonia, were in good agreement. Two out of the nine physical/chemical tests, namely hardness and phosphate, and all three bacteriological tests showed discrepancies between the citizens and the university researchers. This could be ascribed to the difficulty in discerning the change of the colour indicator in the hardness test, the sensitivity of the experiment in the case of phosphate and the complexity of the bacteriological tests. Agreement between data produced by citizen scientists and university researchers for most of the physical and chemical parameters considered in this work has been reported by several authors \[[@RSOS181871C27],[@RSOS181871C32],[@RSOS181871C33],[@RSOS181871C35],[@RSOS181871C37]\]. However, for the case of the phosphate and bacteriological tests, the literature is rather contradictory where some workers have reported agreement \[[@RSOS181871C31],[@RSOS181871C36]\] and others have reported divergence \[[@RSOS181871C13],[@RSOS181871C29]\] between the datasets. Other citizen 'biases' emanating from previous spatial or temporal events \[[@RSOS181871C9],[@RSOS181871C35],[@RSOS181871C49]\] do not apply to this study because citizen scientists were conducting the tests on coded water samples that were doubly blinded.
In summary, and despite some challenges faced throughout the project duration and beyond, the evolved methodology/framework of this work involved several sequential and iterative tasks which were performed either separately or synergistically by the university team and the citizen scientists. The key tasks may be summarized chronologically: those performed during the conception stage, those performed during the project implementation stage and those performed (and are still being performed) after the end date of the project. During the 'conception stage', the following activities were completed: (i) formation of university 'outreach team' to coordinate and maintain strong and long-term relations with the local community, (ii) site visits to reach consensus with the local stakeholders on an action/implementation plan, (iii) formation of a local 'citizen scientists committee' to implement the project, and (iv) selection of chemical, physical and bacteriological tests to ensure ease of use and reproducibility. During the 'implementation stage', the following tasks were performed: (i) finalization of the sampling locations, (ii) training workshops for the 'citizen science committee' on water quality testing (these were conducted prior to the actual sampling and testing), (iii) simultaneous sampling and testing by the university team and the citizen scientists, (iv) repeating the tests, using the same samples, at the university\'s certified laboratory for verification purposes, and (v) open discussion between the university team, the local citizen committee and all other stakeholders regarding the water testing results. After the project completion and in order to ensure sustainability beyond the end date, several actions were implemented including stakeholder meetings to discuss short- and long-term water treatment solutions and the formation of a 'water committee' to continue the water quality testing at the village level and to follow up the implementation of short/long-term treatment solutions.
5.. Conclusion {#s5}
==============
In this work, a citizen science approach was undertaken to determine the quality of commonly used water sources in a Lebanese village. Twelve physical, chemical and bacteriological water quality indicators were tested. The results showed no statistical differences in 7 out of the 11 tests. Trained citizens showed that they are as capable as university researchers in providing reliable, repeatable and accurate physical and chemical water quality test data. Discrepancies were attributed to the sensitivity and the complexity of certain chemical and biological procedures. Test results indicated that agricultural run-off and the preponderance of septic tanks in the area combined with the absence of wastewater treatment may have inadvertently resulted in groundwater pollution. The results were shared with the citizens in presentations followed by active discussions that focused mainly on corrective measures. Ultimately, the success of the project was primarily due to the collaborative efforts put forth by the community and the university stakeholders. Major outcomes included the formation of a water committee at the village, the development of a roadmap that allows citizens to either conduct their own water tests or report to the public authority, and the establishment of a local laboratory at the municipality that will ensure sustainability of water quality monitoring.
Supplementary Material
======================
###### Supplementary Tables
Supplementary Material
======================
###### Citizen Science Data 2
The authors would like to acknowledge the Open and Collaborative Science for Development network (OCSDnet) for the financial support. The authors would also like to thank all of the citizens of the village especially those who participated in the water quality study.
Research ethics {#s6}
===============
The study was reviewed and approved by the Institutional Review Board (IRB) of the American University of Beirut. Informed consent for all the participants to participate in the study was received.
Animal ethics {#s7}
=============
We were not required to complete an animal\'s ethics section from the Institutional Review Board of the American University of Beirut.
Permission to carry out fieldwork {#s8}
=================================
No permissions and licenses were required prior to conducting our research.
Data accessibility {#s9}
==================
All data used in this paper have been uploaded as part of the electronic supplementary material.
Authors\' contributions {#s10}
=======================
S.N.T., N.A.S. and M.A.H. designed the study. R.B., S.H.A. and W.K. project-managed the study, prepared relevant parts of the manuscript, coordinated data collection and provided feedback on several drafts of the manuscript. S.N.T. provided critical input on several drafts of the manuscript. N.A.S. and M.A.H. carried out the analysis and led the writing and revision of the manuscript. All authors approved the final version of the manuscript.
Competing interests {#s11}
===================
We declare we have no competing interests.
Funding {#s12}
=======
Funding from OCSDnet, supported by DFID and IDRC, Project \#107650-001.
[^1]: This article has been edited by the Royal Society of Chemistry, including the commissioning, peer review process and editorial aspects up to the point of acceptance.
[^2]: Electronic supplementary material is available online at <https://doi.org/10.6084/m9.figshare.c.4398749>.
[^3]: ^a^During the summer campaign, well 7 was shut off by the private owner and well 8\'s main outlet was directed to a swimming pool preventing water sampling activities.
| {
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1. Introduction {#sec1}
===============
The current increase in the use of embryos selected for their genetic merit and the need for high fertility recipients \[[@B1], [@B2]\] has made the improvement of pregnancy rates upon embryo transfer (ET) a major objective in cattle farming. Currently, the selection of cow embryos for ET takes place on the basis of morphology and development stage. However, morphological evaluation is a nonobjective method that leads to discrepant judgments among evaluators \[[@B3]\]. Equivocal classification can alter pregnancy rates \[[@B4]\]. Therefore, improving embryo viability prediction would increase the efficiency of ET programs.
Assessment of embryonic quality requests noninvasive or minimally invasive techniques that do not interfere with embryonic development to term. These conditions restrict the number of procedures with practical application for ET programs. In cattle, embryonic cell biopsy is barely used to evaluate incidence of chromosomal abnormality, while it is used to detect and quantify expression of some genes associated with developmental competence \[[@B5]--[@B7]\]. However, gene expression techniques are not extensively used on field, and biopsy, when associated with freezing in particular, may compromise embryonic viability \[[@B1], [@B8], [@B9]\]. The analysis of the culture medium (CM) that surrounds the embryo represents a noninvasive alternative in the search for markers associated with embryo viability. Single molecules measured in CM in correlation with embryo viability include glucose, lactate and pyruvate \[[@B10]--[@B12]\], oxygen \[[@B13], [@B14]\], amino acids \[[@B15], [@B16]\], and the proteome profiling \[[@B17], [@B18]\]. Recently, a noninvasive combined measurement of developmental kinetics and morphology with oxygen consumption allowed a reliable prediction of pregnancy rates from IVP bovine embryos \[[@B19]\].
Together with the particular limitations exposed, the above methods are technically difficult to perform, time-consuming, and expensive or require qualified operators \[[@B10]--[@B19]\]. Therefore, more objective and simple approaches are required to accurately predict embryonic viability in cattle.
Classically less attention has been paid to recipient selection in the ET field. In practice, selection of recipients is based on assessment of corpus luteum (CL) function, by rectal palpation or ultrasonography, and progesterone (P4) measurement. Such selection procedures help to increase pregnancy rates but also often exclude too many intrinsically fertile animals \[[@B20]--[@B24]\]. Therefore, developing efficient and systematic methods for recipient selection is a challenging and pertinent novel objective for cattle ET technology. Metabolic analysis of serum or plasma provides a global profile of the metabolic status. Blood perfuses essentially all living cells in the body and it carries information on virtually every cell type. Metabolic changes affect nutrient transport from blood to oviductal and uterine fluids \[[@B25]\]. Thus, some compounds of plasma could reflect the ability of a female to act as a high quality embryo-recipient.
A variety of spectral and analytical approaches may allow determination of the metabolites associated with embryo viability and pregnancy outcome \[[@B26]\]. One of the core approaches of metabolomics is metabolic fingerprinting (MF), a spectroscopy profile directly dependent on metabolites present in a sample that can anticipate the likelihood for an event or defined state to occur \[[@B27], [@B28]\]. In human in vitro fertilization (IVF), after analyzing culture medium by Raman and near-infrared spectroscopy (NIR), the MF obtained predicted viability in oocytes and embryos \[[@B29]--[@B32]\]. The results seemed to be not affected by differences in CM, laboratories, and days of embryo development nor by fertilization type (i.e., intracytoplasmic sperm injection versus insemination) \[[@B30], [@B32]\]. This indicates that IVP embryos bearing a high reproductive potential alter their CM differently compared to embryos that do not lead to pregnancy, although the use of NIR did not lead to increased pregnancy rates in randomized prospective trials \[[@B33], [@B34]\].
By using another spectroscopy technique, Fourier transform infrared spectroscopy (FTIR), we recently developed a noninvasive system that successfully predicted embryonic sex \[[@B35]\].
In the present study we analyzed plasma by FTIR metabolomics to predict pregnancy viability in recipients of superovulated embryos. In vivo embryo transfer accounts for approximately 65% of the total embryos transferred worldwide (source: IETS Newsletter, Dec. 2013). Therefore improving pregnancy rates by a better selection of embryos and recipients may contribute to increasing significantly the efficiency of selection schemes. The "gold standard" in vivo embryos used were singly cultured in vitro for 24 h and their CM also analyzed, in order to compare the predictive value of recipients and embryos (i.e., plasma versus CM, resp.). On the basis of previous studies with human embryos replicated in different laboratories and culture conditions \[[@B30], [@B32]\], we carried out our experiments in two laboratories with distinct work procedures.
2. Materials and Methods {#sec2}
========================
All experimental procedures were carried out in accordance with the European Community Directive 86/609/EC and were sanctioned by the Animal Research Ethics Committee of SERIDA (licensed 30/01/09).
Animal experiments with in vivo embryo production and ET were performed in independent laboratories from France (UNCEIA, Station Experimentale de Chateauvillain) and Spain (UTE-Bos).
All reagents were purchased from SIGMA (Madrid, Spain) unless otherwise stated.
2.1. Production and In Vitro Culture of In Vivo Embryos {#sec2.1}
-------------------------------------------------------
In vivo embryo production methods differed between Spanish and French laboratories involved.
In UTE-Bos (Spain), cyclic, Holstein donor cows were synchronized in estrus. Briefly, a progestagen device (PRID; Ceva, France) was inserted in the vagina. On Day (−5), 8 FSH (Folltropin, Bioniche, Canada) decreasing doses were given every 12 h apart. On Days (−3) pm and (−2) am, cows received prostaglandin F~2~ *α* analogue (2 mL Dalmazin; Fatro Ibérica, Spain). On Day (−2) the progestagen device was removed and donors were injected with 2 mL GnRH (Dalmarelin, Fatro Iberica, Spain). On Day 0, animals showing estrus were inseminated twice at 12 h intervals. Inseminations were performed with *n* = 4 bulls.
In UNCEIA (France) cyclic, lactating Holstein donor cows housed in station were synchronized in estrus by using progestagen implants and prostaglandin F~2~ *α* (PG), as prescribed by the manufacturer (Crestar method, Intervet, France). The animals came into heat 48 h after the implant removal (=Day 0). Starting on Day 10, animals were superovulated with a total dose of 500 *μ*g FSH (Stimufol, Reprobiol, Belgium) given as twice daily injections in a decreasing dose 4-day schedule. At the 5th FSH injection, a PG injection was given. Animals showing estrus 48 h after prostaglandin F~2~ *α* injection were inseminated twice at 12 h intervals. Inseminations were performed with *n* = 6 bulls.
In both the UTE-Bos and UNCEIA laboratories embryo recovery was performed by flushing the uterine horns on Day 6 in the cycle. Recovered embryos were in vitro cultured in synthetic oviduct fluid containing amino-acids, citrate, and myo-inositol supplemented with 6 g/L BSA (SOFaaci) in single 12 *μ*L drops for 24 h. Atmosphere conditions were 5% CO~2~ in air (UTE-Bos) and 5% CO~2~, 5% O~2~in air in UNCEIA. The SOFaaci compounds used were the same in the two laboratories involved, and culture medium in UTE-Bos was prepared in SERIDA. At the end of the culture period (Day 7), embryos were loaded in straws for ET.
2.2. Estrus Synchronization of Recipients {#sec2.2}
-----------------------------------------
In UTE-Bos, heifers were synchronized in estrus with intravaginal progestagen device (PRID ALPHA, Ceva, Barcelona, Spain) for 8 days and a PG analogue (Dalmazin) injected 24 h before progestagen removal. A GnRH analogue (Dalmarelin) was injected at the time of progestagen insertion and on Day 0.
In UNCEIA, heifers were synchronized in estrus with progestagen implant (Crestar, Intervet, France) for 10 days combined with a PG analogue (Estrumate, Intervet, France) injected 24 h before progestagen removal.
2.3. Culture Media and Embryo Recovery for Viability Analysis and Embryo Transfer {#sec2.3}
---------------------------------------------------------------------------------
Spent culture media (10 *μ*L) and blank controls (i.e., droplets incubated without embryos in them; up to 4 blank controls per batch of embryos cultured simultaneously) were collected on Day 7 and stored frozen at −80°C up to FTIR analysis. On Day 7, single embryos were nonsurgically transferred to recipients in the cranial third of the uterine horn ipsilateral to CL under epidural anesthesia.
2.4. Recipient Blood Sampling for Plasma Viability Analysis {#sec2.4}
-----------------------------------------------------------
Blood plasma samples from recipients were taken in EDTA-vacuum tubes from coccygeal vein puncture. Samples were taken at the time of standing estrus (Day 0) and on Day 7 (2--4 h before the ET time). Blood tubes were immediately refrigerated at +4°C, and centrifuged at 2,000 ×g, not later than 30 min after recovery. Supernatant plasma was aliquoted and stored at −80°C up to FTIR analysis.
2.5. Pregnancy Diagnosis {#sec2.5}
------------------------
In both places and all recipients, pregnancy was diagnosed by transrectal ultrasound scanning on Days 60 ± 2, and birth date registered.
2.6. FTIR Metabolomic Analysis {#sec2.6}
------------------------------
Spent CM and blank samples were analyzed using a Golden-Gate ATR device (diamond crystal) mounted on a Varian 620-IR FTIR spectrophotometer running Varian Resolutions Pro software version 5.0.0.700. 5 *μ*L of the sample was dropped on the ATR diamond and evaporated under a dry N~2~ flow until the FTIR spectrum was stable and different from that of the water. FTIR spectra (16 measurements per sample) were collected in the spectral range between 600 and 4000 cm^−1^, at 5 kHz speed and 4 cm^−1^ resolution. The relative standard deviation was lower than 3% at every wavelength in the range between 600 and 3500 cm^−1^ (excluding the CO~2~ zone).
2.7. Spectral Model Development {#sec2.7}
-------------------------------
The FTIR spectra obtained from CM and blood plasma were uploaded to Matlab programming environment (R2011b; The MathWorks, Natick, MA) for data analysis and predictive model development. Two separate datasets were generated considering Day 60 pregnancy and birth endpoints. In each dataset, samples were labeled as 1 and −1 associated with positive and negative outcomes, respectively. Binary (two-class) classification experiments were performed for model development.
The overall study population included spectra obtained from CM of embryos transferred (*n* = 26 in France and *n* = 23 in Spain). Individual spectral profiles were normalized to the control medium to account for possible impact of variations in the culture conditions. Data corresponding to CO~2~ frequency band (2285--2400 cm^−1^) were removed from the analysis. Each sample was then represented as a row vector of spectra data and the corresponding class label. Spectra from Day 0 and Day 7 plasma obtained from recipients transferred were also analyzed.
In a recent study, we performed a benchmarking experiment to assess discrimination capability of a variety of classification algorithms on prediction of embryonic sex using CM spectra (submitted). Among the classifiers tested, k-nearest neighbor (k-NN) provided the highest prediction accuracy. Therefore, we applied k-NN method for viability prediction in this study.
In the distance based local k-NN model the class label of a test sample is decided to be the same as the most frequent class among its *k* neighborhood. k-NN method provides local solutions assuming that samples which are close together in the feature space will belong to the same class. The distances of each test sample to all training samples are calculated and sorted ascendingly. The majority of the class among shortest *k* distances is chosen as the class of the test sample. As the most general distance metric of k-NN algorithm, Euclidean distance was used in the experiments. The Euclidean distance *d*(*p*, *q*) between the two points *p* and *q* in *N* dimensional space is $$\begin{matrix}
{d\left( {p,q} \right) = sqrt\left( {\sum\limits_{i = 1}^{N}\left( {p_{i} - q_{i}} \right)^{2}} \right).} \\
\end{matrix}$$
Specifically, a weighted k-NN approach was applied where the contributions of neighbors to the class choice were weighted by the inverse of distances to the test sample.
The study dataset is a typical example of high dimension low sample size (HDLSS) problem with 49 samples of CM, 96 samples from plasma, and 1704 features, obtained from spectroscopy analysis. We utilized principal component analysis (PCA) to spectra data for dimensionality reduction.
2.8. Training and Testing Strategy {#sec2.8}
----------------------------------
We applied 10-fold cross validation training-testing strategy in the classification experiments. The entire dataset was randomly divided into 10 bins. The predictive model was developed on the 9 bins (training samples) and the performance of prediction was assessed on the remaining bin (test samples). In order to overcome sampling bias, the training-testing procedure was repeated 10 times replacing the test samples with a bin from the training samples. The average results obtained from the repeated tests are presented.
The discrimination capability of the classifiers was assessed in terms of accuracy, sensitivity and specificity (i.e., the proportion of correctly detected positive and negative pregnancy outcomes, resp.), and receiver operating characteristics curve (ROC) analysis \[[@B36]\]. The ROC curve plots the*sensitivity* versus*1-specificity* by adjusting the decision threshold of classification. ROC analysis enables comparison of classifiers using area under the ROC curve (AUC) as the single performance measure where the classifier with the largest AUC dominates the others.
2.9. Experimental Design {#sec2.9}
------------------------
In Experiment 1, we obtained the metabolomic profile of individually cultured embryos, upon FTIR analysis of frozen/thawed CM samples of 24 h in vitro-cultured, in vivo-derived fresh embryos. Values were normalized versus those of blank samples cultured without embryos. Predictive models compared pregnant versus nonpregnant animals on Day 60 and at birth within all categories of embryos analyzed.
In Experiment 2, we predicted pregnancy success from recipient plasma metabolomic analysis. For normalization purposes, in the absence of blank controls, we included two days of plasma sampling. Our aim was that subtraction of Day 0 and Day 7 plasma values could be an appropriate tool to normalize recipient data between laboratories. Therefore, plasma spectral values were analyzed on Day 0 and Day 7, each being an independent prediction day, and normalized (i.e., Day 7 and Day 0).
2.10. Statistics {#sec2.10}
----------------
All spectral model development, data preprocessing steps, and postprediction statistical analysis were performed using Matlab (R2011b; The MathWorks, Natick, MA). Classifier benchmarking tasks were conducted using Weka (Waikato Environment for Knowledge Analysis), an open-source data mining system \[[@B37]\]. The significance of the differences between the predictive spectral models tested was assessed by comparing the associated AUC values using ANOVA or *t*-test when appropriate. A risk alpha of \<0.05 was considered significant for the comparisons.
3. Results {#sec3}
==========
3.1. Embryo Transfer and Pregnancies {#sec3.1}
------------------------------------
Day 6 in vivo recovered early morulae (*n* = 51) were individually cultured for 24 h. Only embryos that developed in vitro up to late morula to expanded blastocyst stages (*n* = 49) were transferred fresh on Day 7 to synchronized recipients in two experimental herds (UNCEIA, *n* = 26, and UTE-Bos, *n* = 23) ([Table 3](#tab3){ref-type="table"}). Samples of CM from all embryos transferred and the corresponding plasma from recipients were recovered and processed. Day 60 pregnancy and birth rates were \>50% ([Table 3](#tab3){ref-type="table"}).
3.2. Pregnancy Predictions with CM and Recipient Plasma {#sec3.2}
-------------------------------------------------------
Day 60 pregnancies in each individual laboratory involved and within cumulative data were higher than predictions at birth ([Table 1](#tab1){ref-type="table"}). However, cumulative predictions at Day 60 from CM were lower than individual laboratory predictions (France: accuracy = 74.6 ± 5.5, AUC = 0.751 ± 0.039; Spain: accuracy: 74.8 ± 3.9, AUC = 0.718 ± 0.024; cumulative: accuracy = 64.4 ± 1.4, AUC = 0.604 ± 0.029).
In contrast, cumulative analysis improved predictions with plasma ([Table 2](#tab2){ref-type="table"}) on Day 60 and at birth when compared to individual results (birth predictions with Day 0 plasma from France: accuracy = 66.4 ± 7.1, AUC = 0.690 ± 0.044; Spain: accuracy and AUC \< 0.55; cumulative: accuracy = 72.1 ± 2.0, AUC = 0.747 ± 0.032). Spectra profiles of birth predictions from Day 0 plasma are visualized in a 3D principal component space ([Figure 1](#fig1){ref-type="fig"}).
Plasma Day 0 gave AUC and accuracy cumulative values higher than those from Day 0 to Day 7 normalized.
4. Discussion {#sec4}
=============
We show here that CM metabolome reflects viability of in vivo embryos and that the metabolic fingerprint of recipient plasma provides robust information on the likelihood of pregnancy and birth. The embryo produced in vitro shows capacity to modify its environment early in development. Thus, in the cow uterus, IVP early embryos trigger detectable maternal responses on Day 8 in the uterine fluid \[[@B38]\], which also changes according to the embryonic sex \[[@B39]\]. Using IVP embryos, sex differences were also captured in the CM with the same FTIR techniques used in this work \[[@B35]\]. In this work, the MF of CM was affected by dissimilar laboratorial procedures, different embryo-donor management, or both. In contrast, results from recipient plasma overcame management differences between the two recipient herds used, leading to accuracy and AUC values, both on Day 60 and at birth (all of them \>0.72), higher than those from the two laboratories analyzed separately.
The present findings suggest that the predictive value of CM is limited with in vivo embryos, and future work should consider gaining insight into normalization procedures. Normalization should include classification by embryonic stages with higher sample numbers, and probably culture conditions and time periods other than 24 h.
We did not estimate the effects of 24 h in vitro culture on in vivo embryos after transfer. However, Grade 3 morulae that recovered from superovulated animals yield pregnancy rates similar to noncultured Grade 1 morulae after 24 h in vitro culture \[[@B40]\]. This is consistent with the information from these authors showing that 24 h in vitro culture can be a successful choice for low quality and/or delayed embryos that recovered from flushing.
CM from in vivo embryos was less predictive for birth than for Day 60 pregnancy rates. This is surprising, as recipients carrying in vivo-derived embryos usually show less than 5% embryonic losses from the second month of pregnancy to term \[[@B41]\], a lower rate than IVP embryos \[[@B41]--[@B43]\]. Further research is needed so as to investigate whether CM can be representative of these differences between both types of embryos.
The viability profiles of CM with in vivo embryos were not more efficient than conventional selection of embryos. Interestingly, the plasma recipient was generally more predictive of pregnancy success than the CM profile. Within cumulative results, plasma Day 0 gave AUC and accuracy values higher than those from Day 0 to Day 7 normalized, indicating that (combinations of) factors exist in single Day 0 samples able to act as internal controls. Information from recipient (plasma) was generally more predictive of pregnancy success than the embryo CM. Recipient plasma can be affected by different feeding, management, and environmental conditions. However, Day 0 plasma enabled identification of common predictive profiles between recipient herds with an AUC higher than those from each laboratory analyzed apart. These data suggest that two-day sampling could not be necessary, once single plasma samples on Day 0 may provide endogenous normalization.
The ability of a recipient to reach birth has been suggested to be a source of variation higher than the ability of the embryo to survive to term \[[@B44]--[@B46]\]. In addition, variation in recipient quality has been shown not to be an important contribution to fetal loss from Day 60 to term \[[@B44]\]. Effective identification of the higher recipient variability by FTIR could explain the superior predictive ability of the animals versus the embryo. It has been predicted that there are intrinsically superior recipients within individual herds \[[@B46]\]. Superior recipient heifers may show changes in endometrial expression of genes and proteins \[[@B7], [@B47]--[@B49]\] affecting major metabolic pathways and immune response. Some ET practitioners are becoming aware of this and retain recipients that successfully delivered calves after ET for future transfers.
To our knowledge, the association of embryos with specific recipients has been not yet studied, and it could help optimize the use of recipient herds. Promising research lines can investigate recipients that are usually discarded for ET using conventional selection criteria. In addition, as recipients and embryos can enter into an early dialogue into which immunological concerns are prominent \[[@B38], [@B39]\], compatibility between specific embryos (CM) and recipients (plasma) may exist and should be further investigated.
5. Concluding Remarks {#sec5}
=====================
Selection of in vivo embryos by FTIR analysis of CM to increase pregnancy rates may be performed. Although this approach did not improve the results obtained with conventional selection of in vivo embryos, it is likely that establishing more homogeneous procedures may allow normalization between laboratories leading to improved prediction rates. In contrast, cumulative analysis of recipient plasma from both laboratories identified pregnancy predictive profiles with an AUC higher than those from each laboratory analyzed apart, suggesting that FTIR can be an interesting, simple tool to select recipients on field in conventional MOET programs.
FTIR analysis of CM provides a noninvasive, rapid, and inexpensive method compatible with the highest sanitary standards and international exchanges of embryos. Using embryos and recipients with improved viability indexes will significantly increase pregnancy rates and economic benefit in the cattle breeding industry.
The authors thank F. Goyache for valuable scientific comments. Project support was provided by the Spanish Ministry of Economy and Competitiveness, Project AGL2012-37772 and FEDER. Marta Muñoz, Eva Correia, and Beatriz Trigal are supported by MICINN-RYC08-03454, MEC-FPU-AP2009-5265, and Cajastur, respectively. Emre Seli is supported by Award R01HD059909 from the National Institute of Health (NIH), USA. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception Environment.
Conflict of Interests
=====================
The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors\' Contribution
======================
Marta Muñoz and Asli Uyar contributed equally to this paper.
{#fig1}
######
Day 60 pregnancy and birth predictions on PCA transformed spectra data derived from metabolomic analysis of spent culture medium (CM) of Day 6 in vivo embryos cultured in vitro for 24 h and transferred fresh to recipients on Day 7 in two laboratories (UNCEIA, France, and UTE-Bos, Spain).
Laboratory Sample analyzed Pregnancy endpoint *N* Positive Negative *k* PCA Accuracy Sensitivity Specificity AUC
---------------- ----------------- -------------------- ----- ---------- ---------- ------------ ------------ ------------ --------------- ------------- ---------------
France Embryo CM Day 60 26 16 10 3 \+ 74.6 ± 5.5 75.0 ± 4.2 74.0 ± 8.4 0.751 ± 0.039
Birth 26 14 12 3 \+ 68.5 ± 7.6 69.3 ± 7.6 67.5 ± 9.2 0.655 ± 0.075
Spain Embryo CM Day 60 23 13 10 3 \+ 74.8 ± 3.9 67.7 ± 7.1 84.0 ± 6.9 0.718 ± 0.024
Birth 23 12 11 3 \+ 66.9 ± 4.2 58.3 ± 6.8 76.3 ± 4.7 0.625 ± 0.032
France + Spain Embryo CM Day 60 49 29 20 3 − 64.4 ± 1.4 57.5 ± 2.7 74.4 ± 5.8 0.604 ± 0.029
Birth 49 26 23 3 − \<0.6
*N*: culture medium or plasma samples analyzed. Positive: samples that correspond to pregnancy/birth. Negative: samples that do not correspond to pregnancy/birth.
*k*: adjustable model parameter of *k*-nearest neighbor classification algorithm.
PCA: principal component analysis (+: applied; −: did not improve the results when applied).
######
Day 60 pregnancy and birth predictions on PCA transformed spectra data derived from metabolomic analysis of plasma recovered on Day 0 and Day 7 from recipients prior to transfer on Day 7 of embryos that had been cultured in vitro for 24 h in two laboratories (UNCEIA, France, and UTE-Bos, Spain).
Laboratory Plasma analyzed Pregnancy endpoint *N* Positive Negative *k* PCA Accuracy Sensitivity Specificity AUC
---------------- ----------------- -------------------- ----- ---------- ---------- ------------ ------------- ------------ --------------- ------------- ---------------
France Day 0 Day 60 25 15 10 3 − 59.6 ± 4.4 58.7 ± 6.9 61.0 ± 5.7 0.657 ± 0.033
Birth 25 13 12 3 − 66.4 ± 7.1 73.1 ± 15.5 59.1 ± 9.2 0.690 ± 0.044
France Day 7--Day 0 Day 60 25 15 10 3 − \<0.6
Birth 25 13 12 3 − 72.0 ± 3.8 69.7 ± 10.2 75.0 ± 8.8 0.789 ± 0.032
Spain Day 0 Day 60 23 13 10 1 − 69.1 ± 5.2 72.8 ± 9.1 62.0 ± 6.3 0.671 ± 0.067
Birth 23 12 11 1 − \<0.55
Spain Day 7--Day 0 Day 60 23 13 10 3 \+ 68.3 ± 9.6 73.8 ± 15.5 61.0 ± 3.2 0.639 ± 0.047
Birth 23 12 11 3 \+ 67.8 ± 3.0 69.2 ± 5.6 66.3 ± 4.4 0.662 ± 0.021
France + Spain Day 0 Day 60 48 28 20 1 \+ 74.2 ± 1.1 78.2 ± 3.1 70.7 ± 1.1 0.766 ± 0.014
Birth 48 25 23 1 \+ 72.1 ± 2.0 79.2 ± 7.3 64.4 ± 7.0 0.747 ± 0.032
France + Spain Day 7--Day 0 Day 60 48 28 20 3 \+ \<0.6
Birth 48 25 23 3 \+ 69.8 ± 6.0 58.4 ± 3.4 82.2 ± 9.5 0.657 ± 0.033
*N*: culture medium or plasma samples analyzed. Positive: samples that correspond to pregnancy/birth. Negative: samples that do not correspond to pregnancy/birth.
*k*: adjustable model parameter of *k*-nearest neighbor classification algorithm.
PCA: principal component analysis (+: applied; −: did not improve the results when applied).
Day 7 plasma alone (not represented) yields \<0.6 AUC.
######
In vitro development and pregnancy rates of Day 6 in vivo embryos that recovered from superovulated, artificially inseminated cows in UNCEIA (France) and UTE-Bos (Spain) followed by a 24 h individual culture step in SOF + 6 g BSA/L prior to embryo transfer.
Development rates
-------- ---- ---- ---- ------------------- ----------
France 27 26 26 17 (65%) 16 (61%)
Spain 24 23 23 13 (56%) 12 (52%)
Total 51 49 49 30 (59%) 28 (55%)
*N*1: early embryos, morulae, flushed and cultured in vitro.
*N*2: embryos developed to a transferable stage after a 24 h in vitro culture.
*N*3: embryos transferred to recipients (used once for ET).
France: all embryos were transferred in a single herd (*n* = 5 bulls).
Spain: embryos transferred in 2 herds (*n* = 4 bulls).
[^1]: Academic Editor: Luigi Barberini
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Although the capacity bounds of two-way (TW) communication channel were first studied and calculated in the 1960s \[[@B1]\], various cooperative strategies have been extended to transmit over TW relay channels \[[@B2]--[@B6]\] only since the advent of network coding (NC) \[[@B7], [@B8]\] in recent years. In general, TW NC is consisted of multiaccess (MA) and broadcasting (BC) phases, and in the existing literatures, there are three main NC schemes that were studied in MA phase, which are XOR NC \[[@B8]\], amplify-and-forward (ANF) NC \[[@B9]\], and denoise-and-forward (DNF) NC \[[@B10]\]. Among these schemes, DNF is one of the most promising ones due to its very high spectral efficiency, since the TW communication can be accomplished within 2 time slots (TS), while 3 TS are required for XOR NC and ANF NC. This merit makes DNF NC very attractive in both industrial and academic areas in recent years. Despite the potential advantage, the performance loss caused by error propagation is also apparent. As shown in \[[@B11]\], fixed DNF without error check can offer almost no diversity gain in large signal-noise-ratio (SNR) region, while in \[[@B12], [@B13]\], the results similarly indicate that the signal reliability in MA phase dominates the performance of the whole system and any error occurred will be severely propagated among all the cooperative users. Hence, to mitigate error propagation effectively, DNF is always employed jointly with adaptive signal reliability check at relay. For example, \[[@B11], [@B14]\] utilize the metric based on SNR or square amplitude of channel gain to represent signal reliability. For accuracy improvement, \[[@B15]\] proposes the log-likelihood ratio- (LLR-) based threshold to replace the SNR-based one in the cooperative multiple access channels. In \[[@B12]\], this type of threshold is introduced into TW NC to evaluate the reliability of network coded bits, named as NC bit threshold. Besides these uncoded methods, \[[@B16], [@B17]\] employ cyclic redundancy code (CRC) check at relay for error bit identification. In all the above schemes, the detection or check is always operated separately; in other words, each signal from different cooperative users is checked independently, and the relay can decode and forward these signals only when all of them pass the check. Different from these former works, \[[@B18]\] first proposes joint-CRC (JCRC) check in space-time-block coded cooperation system, in which CRC check is applied to the mixed signals. However, the performance evaluation of this type check is only presented by simulations individually, not by theoretical derivation, nor compared with separate CRC (SCRC) check either. Besides, the impact of JCRC check on pairwise error probability (PEP) performance is also not analyzed theoretically. These two points motivate the work presented in this paper.
The contribution of this paper is threefold.Several theoretical bounds are derived and compared with SCRC check to evaluate the effectiveness of JCRC check in TW relay channels.3 JCRC aided DNF NC schemes are proposed, and with the obtained bounds mentioned above, the PEP performances of the proposed schemes are also derived.The effectiveness of the derived bounds and the proposed schemes are confirmed by the simulation results.
The rest of this paper is organized as follows. [Section 2](#sec2){ref-type="sec"} presents the system model and the theoretical bounds of detection reliability probability for JCRC check. [Section 3](#sec3){ref-type="sec"} describes 3 JCRC aided DNF schemes, while the corresponding PEP analysis is presented in [Section 4](#sec4){ref-type="sec"}. Simulation results are shown in [Section 5](#sec5){ref-type="sec"}. Finally, [Section 6](#sec6){ref-type="sec"} concludes the paper.
2. System Model {#sec2}
===============
2.1. System Model {#sec2.1}
-----------------
We consider a TW relay channel, where two source nodes *S* ~1~ and *S* ~2~ exchange information, with the aid of a relay node *R* in between them, as is shown in [Figure 1](#fig1){ref-type="fig"}. All the nodes are equipped with signal antenna and communicate over the same frequency band. To meet the practice situation, we assume that each node cannot transmit and receive signal at the same time, while time division multiplexing (TDM) is employed for channel access.
As depicted in Figures [1(a)](#fig1){ref-type="fig"} and [1(b)](#fig1){ref-type="fig"}, the whole transmission consists of MA and BC phases. In MA phase, each source node converts its own information into the CRC codeword and broadcasts it to each other during the first 2 TS; so the received signals at both relay and source nodes can be written as $$\begin{matrix}
{\mathbf{r}_{iR} = \sqrt{E_{s}}\mathbf{h}_{iR} \cdot \mathbf{s}_{i} + \mathbf{n}_{R},\quad i = 1,2,} \\
\end{matrix}$$ $$\begin{matrix}
{\mathbf{r}_{ij} = \sqrt{E_{s}}\mathbf{h}_{ij} \cdot \mathbf{s}_{i} + \mathbf{n}_{j},\quad i,j \in \left\{ 1,2 \right\},\,\, i \neq j,} \\
\end{matrix}$$ where **r** ~*iR*~ and **r** ~*ij*~ are the received signals at *R* and *S* ~*j*~, respectively, from *S* ~*i*~. It is assumed that *S* ~1~ and *S* ~2~ employ and transmit the same length of (*n*, *k*) CRC codeword, so **r** ~*iR*~ and **r** ~*ij*~ are both *n*-length vectors. **s** ~*i*~ is the CRC codeword transmitted by *S* ~*i*~, and it can be expressed as $$\begin{matrix}
{\mathbf{s}_{i} = \left\lbrack {s_{i}^{(1)},s_{i}^{(2)}\cdots s_{i}^{(n)}} \right\rbrack^{T},\quad s_{i}^{(j)} \in \left\{ + 1, - 1 \right\},} \\
\end{matrix}$$ where binary phase shift keying (BPSK) modulation is assumed. Let **h** ~*iR*~ capture the effects of frequency nonselective multipath fading between *S* ~*i*~ and *R*, and it can be written as $$\begin{matrix}
{\mathbf{h}_{iR} = \left\lbrack {\left| h^{(1)} \right|e^{j\theta^{(1)}},\left| h^{(2)} \right|e^{j\theta^{(2)}},\ldots,\left| h^{(n)} \right|e^{j\theta^{(n)}}} \right\rbrack^{T},} \\
\end{matrix}$$ where *h* ^(*i*)^ is modeled as zero-mean, independent, and circular-symmetric complex Gaussian random variable with variances *σ* ~*iR*~ ^2^ and *θ* ^(*i*)^ is the corresponding phase variable with uniform distribution. It should be noted \|*h* ^(*i*)^\|^2^ follows exponential distribution, where the distribution parameter *λ* ~*iR*~ = 1/*σ* ~*iR*~ ^2^, and is dependent on the distance between *S* ~*i*~ and *R*. Similarly, **h** ~*ij*~ is the channel gain between *S* ~*i*~ and *S* ~*j*~, and we assume that the uplink and downlink channels between two nodes are reciprocal; that is, **h** ~*ij*~ = **h** ~*ji*~. Let **n** ~*R*~ and **n** ~*j*~ capture the effects of receiver noise at *R* and *S* ~*j*~, respectively, and are modeled as zero-mean complex Gaussian random sequences with variance *N* ~0~. We denote *E* ~*s*~ as the average transmitting power, and *E* ~*s*~/*N* ~0~ represents the signal-noise-ratio (SNR) throughout this paper. After MA phase, relay *R* tries to decode each **s** ~*i*~ from **r** ~*iR*~ with maximum likelihood detection (MLD) as $$\begin{matrix}
{{\hat{\mathbf{s}}}_{i} = \arg\,\underset{\mathbf{s}_{i} \in W}{\min}\left| {{Re}\left( {\mathbf{h}_{iR}^{\ast} \cdot \mathbf{r}_{iR}} \right) - \sqrt{E_{s}}\left| \mathbf{h}_{iR} \right|^{2} \cdot \mathbf{s}_{i}} \right|,\quad i = 1,2,} \\
\end{matrix}$$ in which ${\hat{\mathbf{s}}}_{i}$ is the estimation of **s** ~*i*~ and *W* is the transmit codebook. *Re*(*z*) denotes the real part of a complex number *z*, and (·)\* indicates the complex conjugation.
Based on that, SCRC is employed to identify error bits; that is, each ${\hat{\mathbf{s}}}_{i}$ is checked by CRC function and generate the corresponding check remainder $c({\hat{\mathbf{s}}}_{i})$. It should be noted that according to the CRC check principle \[[@B19]\], $c({\hat{\mathbf{s}}}_{i})$ is generated by the division of ${\hat{\mathbf{s}}}_{i}$ and primitive polynomial, and if $c({\hat{\mathbf{s}}}_{i}) = 0$, ${\hat{\mathbf{s}}}_{i}$ can be considered as error-free with high probability. Hence, when both $c({\hat{\mathbf{s}}}_{i}) = 0$, *i* = 1,2, relay *R* will combine and forward ${\hat{\mathbf{s}}}_{1}$ and ${\hat{\mathbf{s}}}_{2}$ as $\mathbf{s}_{R} = f({\hat{\mathbf{s}}}_{1},{\hat{\mathbf{s}}}_{2})$, where *f*(·) is the NC mapping function. Although there exist different protocols for NC mapping, including bit-level XOR \[[@B8]\] and electromagnetic wave-level remapping \[[@B10]\], the actual effects are all equivalent to mod-2 operation in bit-level; that is, $\mathbf{s}_{R} = {\hat{\mathbf{s}}}_{1} \oplus {\hat{\mathbf{s}}}_{2}$.
In BC phase, *R* broadcasts **s** ~*R*~ to both *S* ~1~ and *S* ~2~; so the received signals at *S* ~*i*~ can be expressed as $$\begin{matrix}
{\mathbf{r}_{Ri} = \sqrt{E_{s}}\mathbf{h}_{Ri} \cdot \mathbf{s}_{R} + \mathbf{n}_{i},\quad i = 1,2.} \\
\end{matrix}$$ We assume that perfect channel state information (CSI) can be achieved at all the receivers; so *S* ~*i*~ first decode **s** ~*R*~ form **r** ~*Ri*~ through MLD as $$\begin{matrix}
{{\hat{\mathbf{s}}}_{R} = \arg\,\underset{\mathbf{s}_{i} \in W}{\min}\left| {{Re}\left( {\mathbf{h}_{Ri}^{\ast} \cdot \mathbf{r}_{Ri}} \right) - \sqrt{E_{\text{s}}}\left| \mathbf{h}_{Ri} \right|^{2} \cdot \mathbf{s}_{i}} \right|,} \\
\end{matrix}$$ in which ${\hat{\mathbf{s}}}_{R}$ is estimation of **s** ~*R*~. After that, maximal ratio combination (MRC) is employed to estimate **s** ~*j*~ as $$\begin{matrix}
{{\hat{\mathbf{s}}}_{j} = \arg\,\underset{\mathbf{s}_{i} \in {\,\,}W}{\min}\left| {{Re}\left( \mathbf{h}_{Ri}^{\ast} \cdot \mathbf{r}_{Ri} \cdot f^{- 1}\left( {\hat{\mathbf{s}}}_{R},\mathbf{s}_{i} \right) + \mathbf{h}_{ij}^{\ast} \cdot \mathbf{r}_{ij} \right)} \right.} \\
{\quad\quad\quad\quad\quad\left. {- \sqrt{E_{s}}\left( {\left| \mathbf{h}_{Ri} \right|^{2} + \left| \mathbf{h}_{ij} \right|^{2}} \right) \cdot \mathbf{s}_{i}} \right|,} \\
\end{matrix}$$ where *f* ^−1^(·) is the NC inverse mapping function, while **s** ~*i*~ is preknown *S* ~*i*~. Besides, if $c({\hat{\mathbf{s}}}_{i}) \neq 0$, *R* will keep silent in BC phase, and **s** ~*j*~ is estimated by substituting **r** ~*Ri*~ = 0 into ([8](#EEq8){ref-type="disp-formula"}).
2.2. Theoretical Bounds of JCRC Check {#sec2.2}
-------------------------------------
This paper aims to introduce JCRC check into TW DNF NC for effective error mitigation. Firstly, the equivalence of JCRC and SCRC check in TW relay channel is proved by the following theorem.
Theorem 2.2 I.In the above TW relay model, as SNR increases, there is $\Pr(c({\hat{\mathbf{s}}}_{R}) = 0) \approx \Pr(c({\hat{\mathbf{s}}}_{1}) = 0) \cdot \Pr(c({\hat{\mathbf{s}}}_{2}) = 0) \approx 1$.
ProofAccording to the property of CRC codeword \[[@B19]\], there is **s** ~*R*~ = **s** ~1~ ⊕ **s** ~2~ ∈ *W* \[[@B16]\] when **s** ~1~, **s** ~2~ ∈ *W*. Hence, it can be deduced that $c({\hat{\mathbf{s}}}_{R}) = c({\hat{\mathbf{s}}}_{1} \oplus {\hat{\mathbf{s}}}_{2}) = c({\hat{\mathbf{s}}}_{1}) \oplus c({\hat{\mathbf{s}}}_{2})$. The above equation indicates that $c({\hat{\mathbf{s}}}_{R}) = 0$ only when *a* happens, where *a* ∈ *A*, and the set *A* can be written as $$\begin{matrix}
{A = \left\{ {c\left( {\hat{\mathbf{s}}}_{1} \right) = 0} \right\} \cap \left\{ {c\left( {{\hat{\mathbf{s}}}_{2} = 0} \right)} \right\} \cup {\sum\limits_{i = 1}^{n}{\left\{ {{\hat{s}}_{1}^{(i)} \neq s_{1}^{(i)}} \right\} \cap \left\{ {{\hat{s}}_{2}^{(i)} \neq s_{2}^{(i)}} \right\}}}.} \\
\end{matrix}$$ Let *γ* ~*m*~ denote the error bit number in ${\hat{\mathbf{s}}}_{m}$, *m* = 1,2; then the detection probability $\Pr(c({\hat{\mathbf{s}}}_{m}) = 0)$ can be bounded as $$\begin{matrix}
{\Pr\left( {c\left( {\hat{\mathbf{s}}}_{m} \right) = 0} \right) = \Pr\left( c\left( {\hat{\mathbf{s}}}_{m} \right) = 0,\gamma_{m} \neq 0 \right)} \\
{\quad + \Pr\left( c\left( {\hat{\mathbf{s}}}_{m} \right) = 0,\gamma_{m} = 0 \right)} \\
{\geq \Pr\left( c\left( {\hat{\mathbf{s}}}_{m} \right) = 0,\gamma_{m} = 0 \right).} \\
\end{matrix}$$ According to Theorems 5--11 in \[[@B19]\], $\Pr(c({\hat{\mathbf{s}}}_{m}) = 0,\gamma_{m} = 0)$ can be further bounded as $$\begin{matrix}
{\Pr\left( {c\left( {\hat{\mathbf{s}}}_{m} \right) = 0,\gamma_{m} \neq 0} \right) \leq {\sum\limits_{i = 1}^{n - k - 1}{{\Pr}\left( {\gamma_{m} = i} \right)}}} \\
{\quad + {\sum\limits_{j = n - k}^{n}{\Pr\left( {\gamma_{m} = j} \right) \cdot \left( 1 - 2^{- (n - k - 1)} \right)}}.} \\
\end{matrix}$$ We assume that $P_{bm} = \Pr({\hat{s}}_{m}^{(i)} \neq s_{m}^{(i)})$ denotes the average bit error probability (BEP) of BPSK symbol over Rayleigh fading channel plus zero-mean unit-variance additive white Gaussian noise (AWGN), and it is easily deduced as \[[@B20], equation (14.3-7)\] $$\begin{matrix}
{P_{bm} = \frac{\left( {1 - \sqrt{\left( {E_{s}/{\lambda_{m}N_{0}}} \right)/\left( {1 + {E_{s}/{\lambda_{m}N_{0}}}} \right)}} \right)}{2},} \\
\end{matrix}$$ in which *λ* ~*m*~ = 1/*σ* ~*mR*~ ^2^. Hence, it can be derived that $$\begin{matrix}
{\Pr\left( {\gamma_{m} = i} \right) = \begin{pmatrix}
n \\
i \\
\end{pmatrix}\left( P_{bm} \right)^{i} \times \left( {1 - P_{bm}} \right)^{n - i},\quad i = 1\ldots n,} \\
{\Pr\left( {c\left( {\hat{\mathbf{s}}}_{m} \right) = 0,\gamma_{m} = 0} \right) = \left( {1 - P_{bm}} \right)^{n}.} \\
\end{matrix}$$ Substituting ([11](#EEq11){ref-type="disp-formula"})--([13](#EEq14){ref-type="disp-formula"}) into ([10](#EEq10){ref-type="disp-formula"}), the upper and lower bounds of $\Pr(c({\hat{\mathbf{s}}}_{m}) = 0)$ can be obtained as $$\begin{matrix}
{B_{1} \leq \Pr\left( c\left( {\hat{\mathbf{s}}}_{m} \right) = 0 \right) \leq B_{2},} \\
\end{matrix}$$ in which $$\begin{matrix}
{B_{1} = \left( {\frac{1}{2} + \frac{\sqrt{\left( {\left. {E_{s}/{\lambda_{m}N_{0}}} \right)/\left( {1 + {E_{s}/{\lambda_{m}N_{0}}}} \right)} \right.}}{2}} \right)^{n},} \\
\end{matrix}$$ $$\begin{matrix}
{B_{2} = B_{1} + {\sum\limits_{i = 1}^{n - k - 1}{\begin{pmatrix}
n \\
i \\
\end{pmatrix}\left( P_{bm} \right)^{i} \times \left( {1 - P_{bm}} \right)^{n - i}}}} \\
{\quad + {\sum\limits_{j = n - k}^{n}{\begin{pmatrix}
n \\
j \\
\end{pmatrix}\left( P_{bm} \right)^{j} \times \left( {1 - P_{bm}} \right)^{n - j} \times}}\left( {1 - 2^{- ({n - k - 1})}} \right).} \\
\end{matrix}$$ With the above results, the upper and lower bounds of detection reliability for SCRC check can be calculated as $$\begin{matrix}
{{\prod\limits_{m = 1}^{2}{B_{1}\left( m \right)}} \leq \Pr\left( {c\left( {\hat{\mathbf{s}}}_{1} \right) = 0,c\left( {\hat{\mathbf{s}}}_{2} \right) = 0} \right) \leq {\prod\limits_{m = 1}^{2}{B_{2}\left( m \right)}},} \\
\end{matrix}$$ in which *B* ~*i*~ is written as the function form *B* ~*i*~(*m*), *i* = 1,2, and can be calculated by ([15](#EEq16){ref-type="disp-formula"}).For JCRC check, according to the definition of the set *A* and ([10](#EEq10){ref-type="disp-formula"}), $\Pr(c({\hat{\mathbf{s}}}_{R}) = 0)$ can be calculated as $$\begin{matrix}
{\Pr\left( {c\left( {\hat{\mathbf{s}}}_{R} \right) = 0} \right) = \Pr\left( c\left( {\hat{\mathbf{s}}}_{1} \right) = 0,c\left( {\hat{\mathbf{s}}}_{2} \right) = 0 \right)} \\
{\quad + \Pr\left( {\sum\limits_{i = 1}^{n}{\left\{ {{\hat{s}}_{1}^{(i)} \neq s_{1}^{(i)}} \right\} \cap \left\{ {{\hat{s}}_{2}^{(i)} \neq s_{2}^{(i)}} \right\}}} \right),} \\
\end{matrix}$$ $$\begin{matrix}
{\Pr\left( {\sum\limits_{i = 1}^{n}{\left\{ {\hat{s}}_{1}^{(i)} \neq s_{1}^{(i)} \right\} \cap \left\{ {\hat{s}}_{2}^{(i)} \neq s_{2}^{(i)} \right\}}} \right)} \\
{\quad = {\sum\limits_{i = 1}^{n}{\Pr\left( \gamma_{1} = i \right) \times}}\left( P_{b2} \right)^{i} \times \left( {1 - P_{b2}} \right)^{n - i}.} \\
\end{matrix}$$ Note that ([19](#EEq20){ref-type="disp-formula"}) is due to the fact that, when the error bits coincidentally occur in the same position of ${\hat{\mathbf{s}}}_{1}$ and ${\hat{\mathbf{s}}}_{2}$, it will not be identified by JCRC check, because the mod-2 operation of two error bits can still obtain the correct bit. Hence, ([19](#EEq20){ref-type="disp-formula"}) indicates that JCRC will bring more NC opportunities compared with SCRC check. Substituting ([12](#EEq12){ref-type="disp-formula"}), ([17](#EEq18){ref-type="disp-formula"})--([19](#EEq20){ref-type="disp-formula"}) into ([18](#EEq19){ref-type="disp-formula"}), the detection reliability bounds of JCRC can be calculated as $$\begin{matrix}
{{\prod\limits_{m = 1}^{2}{B_{3}\left( m \right)}} \leq \Pr\left( c\left( {\hat{\mathbf{s}}}_{R} \right) = 0 \right) \leq {\prod\limits_{m = 1}^{2}{B_{4}\left( m \right)}},} \\
\end{matrix}$$ in which $$\begin{matrix}
{B_{m} = B_{m - 2} + {\sum\limits_{i = 1}^{n}{\Pr\left( \gamma_{1} = i \right) \times}}\left( P_{b2} \right)^{i} \times \left( {1 - P_{b2}} \right)^{n - i},} \\
{m = 3,4.} \\
\end{matrix}$$ Since there is always *B* ~*m*~ \> *B* ~*m*−2~, it can be easily concluded that $$\begin{matrix}
{\Pr\left( c\left( {\hat{\mathbf{s}}}_{R} \right) = 0 \right) > \Pr\left( c\left( {\hat{\mathbf{s}}}_{1} \right) = 0 \right) \times \Pr\left( c\left( {\hat{\mathbf{s}}}_{2} \right) = 0 \right).} \\
\end{matrix}$$ According to ([18](#EEq19){ref-type="disp-formula"}), it can be seen that as SNR increases, $\Pr(c({\hat{\mathbf{s}}}_{R}) = 0) \approx \Pr(c({\hat{\mathbf{s}}}_{1}) = 0) \cdot \Pr(c({\hat{\mathbf{s}}}_{2}) = 0)$ because ([19](#EEq20){ref-type="disp-formula"}) tends to 0. At the same time, *P* ~*bm*~ tends to 0, which indicates that *B* ~*m*~ tends to *B* ~*m*−2~ while *B* ~1~ tends to 1; thus, Theorem I is obtained.
3. JCRC Aided DNF NC Protocol {#sec3}
=============================
On the basis of Theorem I, 3 JCRC aided DNF NC schemes are proposed in TW relay channels in this section.
3.1. 3 TS Decode-JCRC-Forward (3T JCRC) {#sec3.1}
---------------------------------------
In the first 2 TS, ${\hat{\mathbf{s}}}_{1}$ and ${\hat{\mathbf{s}}}_{2}$ are gotten at *R* from coherent reception of **r** ~1*R*~ and **r** ~2*R*~, respectively. Then NC is accomplished at bit-level; that is, $\mathbf{s}_{R} = {\hat{\mathbf{s}}}_{1} \oplus {\hat{\mathbf{s}}}_{2}$. After that, **s** ~*R*~ is checked by JCRC, and if *c*(**s** ~*R*~) = 0, *R* will forward **s** ~*R*~ to *S* ~1~ and *S* ~2~ at BC phase. Otherwise, *R* keeps silent. Note that there are two main differences between this scheme and former TW denoise-XOR-forward CRC NC \[[@B8]\].XOR is operated before CRC check in this scheme while in the former one, the bits are XORed only when both the codewords pass the CRC check.Only once JCRC check is operated to **s** ~*R*~ in this scheme, while in the former one, 2 SCRC checks are required for ${\hat{\mathbf{s}}}_{1}$ and ${\hat{\mathbf{s}}}_{2}$, respectively.
3.2. 2 TS Decode-JCRC-Forward with Phase Synchronization (2T JCRC-P) {#sec3.2}
--------------------------------------------------------------------
It is proposed in \[[@B10]\] that the transmitted signals are precoded when CSI is known in the source nodes, so that it allows the direct merger of two signals from the source nodes in the form of electromagnetic waves at relay simultaneously. Relay then remaps the merged signals into one NC signal and broadcast it in BC phase. Only 2 TS are consumed for TW communication by this so-called physical layer NC (PNC). This paper introduces JCRC check into PNC system to judge the quality of the remapped signals. Assuming that only the phase synchronization is operated in MA phase, the received and remapped signals at relay can be written as $$\begin{matrix}
{\mathbf{r} = \sqrt{E_{s}}\left( \left| \mathbf{h}_{1R} \right| \cdot \mathbf{s}_{1} + \left| \mathbf{h}_{2R} \right| \cdot \mathbf{s}_{2} \right) + \mathbf{n}_{R},} \\
\end{matrix}$$ $$\begin{matrix}
{\mathbf{s}_{R} = f\left( \mathbf{r} \right),} \\
\end{matrix}$$ in which $$\begin{matrix}
{f\left( x^{(i)} \right) = \begin{cases}
{- 1,} & {\text{if}{\,\,}\left| x^{(i)} \right| > \sqrt{E_{s}}\max\left\{ \left| h_{1R}^{(i)} \right|,\left| h_{2R}^{(i)} \right| \right\}} \\
{1,} & {\text{if}{\,\,}\left| x^{(i)} \right| < \sqrt{E_{s}}\max\left\{ {\left| h_{1R}^{(i)} \right|,\left| h_{2R}^{(i)} \right|} \right\}.} \\
\end{cases}} \\
\end{matrix}$$ After that, the remapped signal **s** ~*R*~ is detected by JCRC check, and if *c*(**s** ~*R*~) = 0, relay will forward **s** ~*R*~ at BC phase.
3.3. 2 TS Decode-JCRC-Forward with Phase and Amplitude Synchronization (2T JCRC-PA) {#sec3.3}
-----------------------------------------------------------------------------------
Assuming both phase and amplitude synchronization are ideally achieved at *S* ~1~ and *S* ~2~ in MA phase, the received and remapped signals at relay can be expressed as $$\begin{matrix}
{\mathbf{r} = \sqrt{E_{s}}\left( \mathbf{s}_{1} + \mathbf{s}_{2} \right) + \mathbf{n}_{R}.} \\
\end{matrix}$$ Note that this is the special case of ([23](#EEq24){ref-type="disp-formula"}) when \|*h* ~1*R*~ ^(*i*)^ \| = \|*h* ~2*R*~ ^(*i*)^ \| = 1, so the remapping operation is similar to the scheme mentioned above, and *f*(·) in ([25](#EEq26){ref-type="disp-formula"}) becomes the normal BPSK demodulation.
4. PEP Analysis {#sec4}
===============
In this section, to evaluate the performance of the proposed schemes, we derive the PEP with the obtained theoretical bounds in Theorem I.
Let *d* ~min~ denote the minimal Hamming distance of a (*n*, *k*) system CRC codebook, and according to the principle of error correction \[[@B19]\], any received codeword that belongs to the codebook is able to correct at most ⌊*d* ~min~ − 1⌋/2 random error bits itself. In general, the PEP of the denoise-JCRC-forward scheme can be written as follows: $$\begin{matrix}
{P_{e} = \Pr\left( c\left( {\hat{\mathbf{s}}}_{R} \right) = 0 \right) \times \Pr\left( {\gamma_{\text{MRC}} > \frac{\left\lfloor {d_{\min} - 1} \right\rfloor}{2}} \right)} \\
{\quad + \Pr\left( c\left( {\hat{\mathbf{s}}}_{R} \right) \neq 0 \right) \times \Pr\left( {\gamma_{D} > \frac{\left\lfloor {d_{\min} - 1} \right\rfloor}{2}} \right)} \\
{\approx \Pr\left( c\left( {\hat{\mathbf{s}}}_{R} \right) = 0 \right) \times \Pr\left( {\gamma_{\text{MRC}} > \frac{\left\lfloor {d_{\min} - 1} \right\rfloor}{2}} \right),} \\
\end{matrix}$$ in which *γ* ~MRC~ and *γ* ~*D*~ denote the error bit number of the signal estimation at *S* ~*i*~ when *R* broadcasts **s** ~*R*~ and keeps silent, respectively. The approximation in ([27](#EEq28){ref-type="disp-formula"}) is due to the fact that *Pr*(*c*(**s** ~*R*~ ≠ 0)) ≈ 0 when SNR increases, which has been proved in Theorem I. *Pr*(*γ* ~MRC~ \> ⌊*d* ~min~ − 1⌋/2) can be expressed as $$\begin{matrix}
{\Pr\left( {\gamma_{\text{MRC}} > \frac{\left\lfloor {d_{\min} - 1} \right\rfloor}{2}} \right) = {\sum\limits_{i = {\lfloor{d_{\min} - 1}\rfloor}/2}^{n}\begin{pmatrix}
n \\
i \\
\end{pmatrix}}\left( P_{R} \right)^{i}\left( {1 - P_{R}} \right)^{n - i},} \\
\end{matrix}$$ in which the BEP *P* ~*R*~ can be obtained with the results deduced by \[[@B15]\] as$$\begin{matrix}
P_{R} \\
{= \begin{cases}
{\frac{\left( {1 - \sqrt{\left( {{\lambda_{sR}N_{0}}/E_{s}} \right)/\left( {1 + {{\lambda_{sR}N_{0}}/E_{s}}} \right)}} \right)^{2}\left( {2 + \sqrt{\left( {{\lambda_{sR}N_{0}}/E_{s}} \right)/\left( {1 + {{\lambda_{sR}N_{0}}/E_{s}}} \right)}} \right)}{4},{\,\,}} & {\text{if}{\,\,}\lambda_{sD} = \lambda_{sR}} \\
{\frac{\lambda_{sD}}{2\left( \lambda_{sD} - \lambda_{sR} \right)}\left\lbrack {1 - \sqrt{\frac{1}{1 + {{\lambda_{sD}N_{0}}/E_{s}}}}} \right\rbrack - \frac{\lambda_{sR}}{2\left( {\lambda_{sD} - \lambda_{sR}} \right)}\left\lbrack {1 - \sqrt{\frac{1}{1 + {{\lambda_{sR}N_{0}}/E_{s}}}}} \right\rbrack,} & {\text{if}{\,\,}\lambda_{sD} \neq \lambda_{sR},} \\
\end{cases}} \\
\end{matrix}$$in which *λ* ~*sD*~ and *λ* ~*sR*~ are the distribution parameters of **h** ~*iR*~ and **h** ~*ij*~, respectively.
By comparing ([1](#EEq1){ref-type="disp-formula"}) and ([23](#EEq24){ref-type="disp-formula"}), it can be concluded that 2T JCRC-P and 3T JCRC will achieve similar PEP, because the only difference between these two schemes is the variance of receiver noise at *R*. Since $\Pr(c({\hat{\mathbf{s}}}_{R}) = 0)$ can be approximated by the lower bound of ([17](#EEq18){ref-type="disp-formula"}), the PEP of these two schemes can be calculated by substituting ([17](#EEq18){ref-type="disp-formula"}), ([28](#EEq29){ref-type="disp-formula"}), and ([29](#EEq30){ref-type="disp-formula"}) into ([27](#EEq28){ref-type="disp-formula"}): $$\begin{matrix}
P_{\underset{(2\text{T}\,\text{JCRC-P})}{e\, 3\text{T}\,\text{JCRC}}} \\
{\quad \approx \left( {\frac{1}{2} + \frac{\sqrt{\left( {E_{s}/{N_{0}\lambda_{m}}} \right)/\left( {1 + {E_{s}/{N_{0}\lambda_{m}}}} \right)}}{2}} \right)^{n}} \\
{\quad\quad \times {\sum\limits_{i = \lfloor d_{\min} - 1\rfloor/2}^{n}\begin{pmatrix}
n \\
i \\
\end{pmatrix}}\left( P_{R} \right)^{i}\left( {1 - P_{R}} \right)^{n - i}.} \\
\end{matrix}$$ For 2T JCRC-PA, $\Pr(c({\hat{\mathbf{s}}}_{R}) = 0)$ can be approximated by the lower bound of ([20](#EEq21){ref-type="disp-formula"}), in which *P* ~*bm*~ can be written according to the results of \[[@B21]\] as $$\begin{matrix}
{P_{bm} = \frac{1}{2\pi}\left( {{\int\limits_{0}^{\pi/2}{3e^{- ({E_{s}/{2\sigma^{2}N_{0}\sin\theta}})} -}}e^{- ({{9E_{s}}/{2\sigma^{2}N_{0}\sin\theta}})}} \right)d\theta.} \\
\end{matrix}$$ Hence, the corresponding PEP can be calculated as $$\begin{matrix}
P_{e\, 2\text{T}\,\text{JCRC-PA}} \\
{\quad \approx \left\lbrack {\frac{1}{2\pi}\left( {\int\limits_{0}^{\pi/2}{2\pi - 3e^{- ({E_{s}/{2\sigma^{2}N_{0}\sin\theta}})}}} \right.} \right.} \\
{\quad\quad\quad\quad\quad\quad\left. {\left. {+ e^{- ({{9E_{s}}/{2\sigma^{2}N_{0}\sin\theta}})}} \right)d\theta} \right\rbrack^{n}} \\
{\quad\quad \times {\sum\limits_{i = \lfloor d_{\min} - 1\rfloor/2}^{n}\begin{pmatrix}
n \\
i \\
\end{pmatrix}}\left( P_{R} \right)^{i}\left( {1 - P_{R}} \right)^{n - i}.} \\
\end{matrix}$$
5. Simulation Results {#sec5}
=====================
In this section, the Monte-Carlo simulation is employed to estimate the detection probability of JCRC check and the PEP performance of the three proposed schemes. Besides, the performance of LLR-based TW NC algorithm in \[[@B12]\] is also simulated as the baseline. The simulation parameters are as follows: the simulation length involved in all points is fixed at 10 million BPSK-modulated codewords. *S* ~1~, *S* ~2~, and *R* are all equipped with single antenna. SNR denotes *E* ~*s*~/*N* ~0~, and we assume that all the nodes transmit with the same power. The path loss effects are modeled and parameterized as follows: the reference distance is *d* ~0~, and the channel gain parameter can be calculated as *λ* ~*i*~ = *λ* ~0~ · (*d* ~*i*~/*d* ~0~)^*γ*^, in which *γ* = 3.5 (typical urban). The system model is as shown in [Figure 1](#fig1){ref-type="fig"}, and we assume that *λ* ~1*R*~ = *λ* ~2*R*~ = 1, *λ* ~12~ = 2. A (7, 4) system CRC code is employed, which is generated by the origin polynomial with the minimum Hamming distance *d* ~min~ = 3. MLD is applied to estimate the signals at all the terminals. Since, in \[[@B12]\], LLR threshold is bit-oriented, the information rate is fixed as 1 bit/s/channel, and so its PEP equals the BEP. In all the simulations, the LLR threshold is set by the target BEP of 1%, and LLR-PA denotes that the checked signals are perfectly synchronized with both phase and amplitude.
[Figure 2](#fig2){ref-type="fig"} depicts the detection probability comparisons of JCRC and SCRC check at *R*. It is verified from the figure that the derived bounds in ([15](#EEq16){ref-type="disp-formula"}), ([16](#EEq17){ref-type="disp-formula"}), and ([21](#EEq22){ref-type="disp-formula"}) coincide well with the simulation results. Compared with SCRC, it can be seen that there is slight higher detection probability for JCRC in the regions of low and medium SNR. This is caused by the more loose restrictive conditions of JCRC check and that brings more opportunities to the relay to code and forward the received signals. However, this merit is not very obvious and gradually disappears when SNR increases. Compared with 3T JCRC and 2T JCRC-P, a distinct advantage can be achieved for 2T JCRC-PA in all SNR regions. As shown in the figure, when SNR \> 6 dB, the detection probability is close to 1. This is mainly caused by two reasons: (1) with the phase and amplitude compensation, the PNC signals at *R* are of good quality. (2) JCRC check matches the PNC signals well. This advantage enables 2T JCRC-PA to have more opportunities to exploit relay channel to obtain superior PEP performance.
[Figure 3](#fig3){ref-type="fig"} shows the NC opportunities comparison of the schemes at *R*, in which NC opportunity is defined as the probability that $c({\hat{\mathbf{s}}}_{R}) = 0$, because the relay *R* is able to combine the received signals through NC if that happens. As is depicted, when SNR increases, the NC opportunities tend to 1 for all the compared schemes, among which 2T JCRC-PA is optimal. Besides, the metric is also close to 95% for LLR-based check, when SNR \> 6 dB. The results show that 2T JCRC-PA is superior to LLR-based check in all SNR regions, which indicates that 2T JCRC-PA is able to fully exploit the relay channel to improve PEP performance. Moreover, the performance gaps are not obvious among 3T SCRC, 3T JCRC, and 2T JCRC-P, which indicates that these schemes will have similar opportunity to exploit relay channel through NC. However, it cannot be inferred that they achieve similar PEP performance, which will be discussed as follows.
[Figure 4](#fig4){ref-type="fig"} shows the correct rate of the signals which has successfully passed the check. As can be seen in the figure, CRC-based and LLR-based checks are both effective measures to evaluate the reliability of the signals, because when SNR \> 4 dB, all the compared check accuracies tend to 96%, and even when SNR = 0 dB, most of the compared accuracies are higher than 95%, except 3T JCRC and 2T JCRC-P. In general, CRC-based checks are superior to LLR-based one, but the performance gap is slight. In addition, in low SNR region, 3T SCRC and 2T JCRC-P are less efficient than other schemes, which indicates that JCRC is inferior to SCRC in low SNR region. This result indicates that although JCRC can enable the relay to obtain more opportunities to code and forward received signals, the relative low correct rate will degenerate the PEP performance in low SNR region, which make the gain less obvious compared with SCRC.
[Figure 5](#fig5){ref-type="fig"} depicts the PEP comparisons of the proposed schemes. It can be observed that the theoretical results derived by ([30](#EEq31){ref-type="disp-formula"}) and ([31](#EEq32){ref-type="disp-formula"}) correspond well with the simulations, which verify the effectiveness of Theorem I and the analysis presented in [Section 4](#sec4){ref-type="sec"}. The figure shows that 2T JCRC-PA outperforms the other compared schemes, which also corresponds well with the results in [Figure 3](#fig3){ref-type="fig"}, because 2T JCRC-PA enables the relay to obtain more opportunities to exploit the relay channel. Besides, it can be observed that the PEP gap is not obvious among 3T SCRC, 3T JCRC, and 2T JCRC-P. On the one hand, this result verifies the effectiveness of JCRC check; on the other hand, it also confirms that the PEP of 2T JCRC-P and 3T JCRC can be approximated as ([30](#EEq31){ref-type="disp-formula"}). Moreover, the figure also indicates that 2T JCRC-P outperforms LLR-P scheme in \[[@B12]\] with only phase synchronization, while 2T JCRC-PA is superior to LLR-PA. In the former case, the SNR gain is 2 dB when the target PEP is 10^−3^, while in the latter case, it grows to 4 dB. This gain growth is due to the error correction of CRC code, because for the simulated (7, 4) CRC, any single random error in the codeword can be recovered, which cannot be obtained for LLR check \[[@B12]\]. This advantage makes JCRC check more efficient to mitigate error propagation. Meanwhile, it should be noted that, for the proposed schemes, the spectral efficiency is sacrificed as the cost since the information rate = 4/7 bit/s/channel, while for LLR-based scheme, the metric equals 1 bit/s/channel.
6. Conclusion {#sec6}
=============
In this paper, we introduced JCRC check into the TW DNF NC system to evaluate the reliability of NC signals. Firstly, the detection probability bounds of JCRC are theoretically derived to prove its effectiveness. On the basis of that, three JCRC aided TW DNF NC schemes are proposed, and the corresponding PEP performances are also derived. Theoretical and simulation results indicate that the proposed JCRC schemes outperform conventional SCRC check in TW relay communications with only half complexity, because JCRC enables the relay node to have more opportunities to exploit the relay channel, which is helpful for PEP improvement. Moreover, it also shows that JCRC check is superior to the LLR-based scheme \[[@B12]\] in terms of PEP, at the cost of spectral efficiency. In future work, it is of significance to introduce JCRC check into cooperative multiple access channels for error mitigation.
This work was supported by the National Basic Research Program of China (973 Program) (2013CB329104), the National Natural Science Foundations of China (61372124 and 61171093), the Key Projects of Natural Science Foundations of Jiangsu University (11KJA510001), the National Science and Technology Key Projects (2011ZX03005-004-003), and Jiangsu 973 Projects (BK2011027).
Conflict of Interests
=====================
The authors declare that there is no conflict of interests regarding the publication of this paper.
{#fig1}
{#fig2}
{#fig3}
{#fig4}
{#fig5}
[^1]: Academic Editor: Branimir Reljin
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
Renal neoplasms are composed of heterogeneous groups including clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, collecting duct carcinoma, oncocytoma, and others. In most cases, these categories are easily differentiated from each other on the basis of histologic features alone. Occasionally, however, there are overlapping morphological characteristics between such tumors as well as histologic heterogeneity within a single tumor. In those cases, it may be difficult to make an accurate subtyping of the tumors.
A diagnostic problem can arise in distinguishing chromophobe RCC from oncocytoma. These two types of tumors have a common origin, distal nephrons, and similar phenotype, so it may be difficult to distinguish these tumors in the practice of diagnostic pathology. Chromophobe RCC and oncocytoma should be distinguished from each other because both tumors have different behaviors and clinical outcomes. Oncocytoma is a benign tumor, whereas chromophobe RCC is a subtype of RCC, namely, malignant. In recent years, several studies have been carried out to find helpful ancillary means to distinguish chromophobe RCC from oncocytoma. However, the results of these studies are inconsistent and still unsatisfactory \[[@B1]-[@B8]\].
Caveolin-1, a 24-kd membrane protein, is a major structural and functional protein component of caveolae, endocytic structures of the cell membrane \[[@B9],[@B10]\]. Caveolin-1 plays important roles in membrane traffic, lipid traffic, and signal transduction \[[@B9],[@B10]\]. Several studies have shown that the expression of caveolin-1 is elevated in various types of malignancies such as prostate, breast, colon, esophagus, and urinary bladder cancers \[[@B11]-[@B14]\], but the precise roles of caveolin-1 in carcinogenesis are still unclear.
MOC-31 is a kind of cell surface glycoprotein and is expressed in most benign and malignant epithelia. It is known that MOC-31 is useful for distinguishing adenocarcinoma from mesothelioma as well as hepatocellular carcinoma from cholangiocarcinoma and metastatic adenocarcinoma in the liver \[[@B15],[@B16]\].
In this study, we intended to identify whether there were any differences in immunohistochemical reactivity for caveolin-1 and MOC-31 between chromophobe RCC and oncocytoma and to determine whether the expression of these proteins has a benefit as useful immunohistochemical markers in distinguishing the tumors. In addition, clear cell RCC, particularly the granular cell type, may also have a phenotype similar to the previously mentioned two types of tumors and cause a diagnostic problem. Accordingly, we also investigated the immunohistochemical reactivity of clear cell RCC for caveolin-1 and MOC-31 and evaluated the differences among clear cell RCC, chromophobe RCC, and oncocytoma.
MATERIALS AND METHODS
=====================
1. Case selection
-----------------
Twenty-three chromophobe RCCs and 8 oncocytomas were retrieved from the surgical pathology archives between 1997 and 2006 at three institutes. Twenty-five clear cell RCCs, including 10 cases of clear cell type and 15 cases of granular cell type, were also selected from the surgical pathology files of one institute. Those cases were composed of only radical surgical specimens. Two pathologists, one of whom has expertise in genitourinary pathology, reviewed all cases and achieved consensus on classification according to the recommendations of the International Union Against Cancer \[[@B17]\] and the World Health Organization Classification of Tumors \[[@B18]\]. Most tumors were categorized on the basis of light microscopic histology. Electron microscopic examination was performed for 9 cases because they showed obscure and overlapping histologic features on light microscopic, histochemical, and immunohistochemical examinations. Two cases having unclear features despite the electron microscopic examination were excluded from this study. Staging of chromophobe RCC was performed according to the recently updated staging system (7th edition) of the American Joint Committee on Cancer (AJCC) (7th edition) and the Union Internationale Contre le Cancer (UICC) \[[@B19]\]. The clinicopathologic data of all chromophobe RCCs and oncocytomas are summarized in [Table 1](#T1){ref-type="table"}.
2. Immunohistochemistry
-----------------------
The immunohistochemical staining was performed with the DAKO EnVision Kit (Dako, Denmark) on sections prepared from formalin-fixed and paraffin-embedded specimens that were dewaxed and rehydrated with graded concentrations of alcohol. Endogenous peroxidase was blocked by dipping the sections in 3% aqueous hydrogen peroxide for 10 min, and antigen retrieval was performed with a 10-min microwave treatment in 10 mmol/l citrate buffer, pH 6.0. For immunohistochemical staining of MOC-31, Target Retrieval Solution (Dako, Denmark) was used for antigen retrieval. Diluted primary antibodies for caveolin-1 (1:200, Transduction Laboratories, US) and MOC-31 (1:50, Dako, Denmark) were treated at room temperature for 30 min and 1 h, respectively. After the primary antibody incubation, the sections were incubated with the secondary antibody. The sections were lightly counterstained with hematoxylin.
Immunoreactivity for caveolin-1 and MOC-31 was considered as diffuse positive when 50% or more of the tumor cells were stained, as focal positive when 10% to 49% of the tumor cells were stained, and as negative when less than 10% of the tumor cells were stained. Internal positive controls were endothelial cells and distal tubular epithelial cells for caveolin-1 and MOC-31, respectively.
3. Statistical analysis
-----------------------
Data were analyzed by the Fisher\'s exact test with SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA) to evaluate correlation between the tumor types and expression of immunohistochemical stain for caveolin-1 and MOC-31. p-values less than 0.05 were considered statistically significant.
RESULTS
=======
1. Immunoreactivity for Caveolin-1
----------------------------------
In normal kidney tissue, expression of caveolin-1 was seen in parietal epithelia of Bowman capsules, endothelial cells, and smooth muscle cells of blood vessels but not in endothelial cells of glomeruli or any other epithelia ([Table 2](#T2){ref-type="table"}, [Fig. 1A](#F1){ref-type="fig"}).
In chromophobe RCCs, 20 (87%) of 23 cases showed diffuse positivity for caveolin-1. All positive cases showed expression of caveolin-1 in 90% or more of the tumor cells and moderate to strong intensity except for one case that had relatively weak intensity. The pattern of immunohistochemical staining was diffusely cytoplasmic and granular. In most of the cases, the cell membrane was also stained and the staining intensity was stronger in the membrane than in the cytoplasm ([Fig. 2A](#F2){ref-type="fig"}). In the three negative cases, none of the tumor cells expressed caveolin-1.
Oncocytomas showed negative results for caveolin-1 in all eight cases ([Fig. 2B](#F2){ref-type="fig"}). None of the tumor cells expressed caveolin-1 except for one case in which caveolin-1 was expressed in only 5% or less of the tumor cells.
Clear cell RCCs were diffuse positive in 19, focal positive in 2, and negative in 4. The total number of positive cases was 21 (84%) of 25 clear cell RCCs. All 10 cases of the clear cell type were diffuse positive, whereas 9 (60%) of 15 cases of the granular cell type were diffuse positive. There was a slight difference in the pattern of immunohistochemical staining between the clear and granular cell types. All cases of the clear cell type showed dominantly membranous staining without cytoplasmic staining ([Fig. 3A](#F3){ref-type="fig"}), whereas most cases of the granular cell type exhibited a similar staining pattern to that of chromophobe RCC, namely, a diffusely cytoplasmic and granular pattern with focal membranous condensation ([Fig. 3B](#F3){ref-type="fig"}).
The incidence of caveolin-1 expression showed statistically significant differences between chromophobe RCCs and oncocytomas (p\<0.001) and between clear cell RCCs and oncocytomas (p\<0.001) but not between chromophobe and clear cell RCCs (p=0.466) ([Table 3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}).
2. Immunoreactivity for MOC-31
------------------------------
MOC-31 was positive in distal tubules and collecting ducts of normal kidney tissue but negative in other epithelial and mesenchymal tissues ([Table 2](#T2){ref-type="table"}, [Fig. 1B](#F1){ref-type="fig"}).
MOC-31 was diffuse positive in 22 (96%) of 23 chromophobe RCCs, showing a strong membranous pattern of immunohistochemical staining ([Fig. 4A](#F4){ref-type="fig"}), and negative in only 1 case.
In oncocytomas, MOC-31 was negative in 6 of 8 cases and diffuse positive in the other 2 cases (25%) ([Fig. 4B](#F4){ref-type="fig"}). Three of the six negative cases expressed MOC-31 in less than 10% of the tumor cells, and the other three cases showed no staining. In the former three cases, MOC-31 was characteristically expressed in the tumor cells showing tubular structures but not solid nests or trabecular structures. In addition, two oncocytomas showing diffuse expression of MOC-31 were composed of predominantly tubular structures, and solid or trabecular structures were only a minor portion. MOC-31 was expressed in such tubular structures rather than in solid or trabecular structures ([Fig. 5](#F5){ref-type="fig"}).
Clear cell RCCs showed relatively variable MOC31 staining results. Clear cell RCCs were diffuse positive for MOC-31 in 12, focal positive in 2, and negative in 11. The total number of positive cases was 14 (56%) of 25 cases. In particular, the granular cell type of clear cell RCC showed less positivity for MOC-31 than did the clear cell type. Only 6 (40%) of 15 cases of the granular cell type were positive for MOC-31. All positive cases showed a membranous staining pattern as for the chromophobe RCCs.
The incidence of MOC-31 expression showed statistically significant differences between chromophobe RCCs and oncocytomas (p\<0.001) and between chromophobe and clear cell RCCs (p\<0.001) but not between clear cell RCCs and oncocytomas (p=0.215) ([Table 3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}).
DISCUSSION
==========
Subtypes of renal cell tumors are various and complex. Although each renal cell tumor has unique characteristics, they may occasionally have overlapping histologic features that make it difficult to make a proper diagnosis. One of the most problematic differential diagnoses is to distinguish chromophobe RCC from oncocytoma.
Chromophobe RCC was first described by Thoenes et al in 1985 \[[@B20]\]. Chromophobe RCC consists of tumor cells showing abundant pale cytoplasm with a perinuclear halo and peripheral condensation and hyperchromatic, irregular, wrinkled nuclei. By contrast, oncocytomas have tumor cells showing abundant eosinophilic granular cytoplasm and regular and round nuclei. Both tumors can be distinguished on the basis of these histologic features. However, occasionally, when the tumors show obscure microscopic findings, it can be difficult to make a differential diagnosis. Therefore, many investigators have searched for helpful ancillary means to discriminate these two types of renal tumors.
Electron microscopic examination is an important and useful tool in the differential diagnosis of chromophobe RCC and oncocytoma. However, electron microscopic examination is technically difficult, expensive, and not available in many laboratories. Several studies have suggested that Hale\'s colloidal iron stain is helpful for the differential diagnosis of chromophobe RCC from oncocytoma \[[@B1]\]. However, Hale\'s colloidal iron stain is also technically demanding and difficult to interpret, and therefore reproducibility is relatively low \[[@B2]\].
Several studies with various immunohistochemical markers, including kidney-specific cadherin, CK7, EMA, CD10, parvalbumin, RCC, c-KIT, and RON proto-oncogene, have been performed to find useful markers for distinguishing chromophobe RCC from oncocytoma. However, the results of these studies are inconsistent or turned out to be not satisfactory. Accordingly, the use of these markers in differential diagnosis is limited \[[@B2]-[@B8],[@B21],[@B22]\].
Caveolin-1 is a major protein component of caveolae, which are endocytic structures of the cell membrane, and is localized to the cytosolic surface of caveolae. Caveolin-1 interacts with both lipids and lipid anchors on proteins; therefore, it has multiple functions in signal transduction, membrane traffic, and intracellular and extracellular lipid transport \[[@B9],[@B10]\]. Expression of this molecule has been studied in various types of tumors, including prostate, breast, colon, esophagus, and urinary bladder cancers \[[@B11]-[@B14]\]. However, only a small number of studies have recently reported a relationship between the expression of caveolin-1 and renal neoplasms, including chromophobe RCC and oncocytoma \[[@B23]-[@B25]\].
In the present study, caveolin-1 was expressed in 20 (87%) of 23 chromophobe RCCs but in 0 of 8 oncocytomas. All positive cases of chromophobe RCC showed diffuse, moderate to strong staining in 90% or more of tumor cells and cytoplasmic and granular staining with membranous condensation. This clear discrimination enables the use of caveolin-1 for differential diagnosis between chromophobe RCC and oncocytoma (p\<0.001).
Carrion et al and Mete et al reported that most oncocytomas express caveolin-1 with a diffuse cytoplasmic staining pattern and that chromophobe RCCs show a relatively decreased expression of caveolin-1 \[[@B23],[@B24]\]. This difference probably originated from the artifact that they did not use a blocking step for endogenous biotin despite using an avidin-biotin peroxidase kit. Endogenous biotin in oncocytomas may cause a false-positive result. Nevertheless, there was a difference in staining pattern between chromophobe RCCs and oncocytomas in their studies. According to them, chromophobe RCCs showed peripheral cytoplasmic staining, whereas oncocytomas showed diffuse cytoplasmic staining. There was an obvious difference in the incidence and staining pattern of caveolin-1 expression between both tumors.
In contrast with these findings, Garcia and Li reported that caveolin-1 was positive in all 21 chromophobe RCCs but was focal positive, with staining in less than 20% of the tumor cells, in only 3 of 26 oncocytomas \[[@B25]\]. This result is consistent with and supports our result.
Occasionally, it may also be difficult to distinguish clear cell RCC, particularly the granular cell type, from chromophobe RCC or oncocytoma in practice. Therefore, our present study also included clear cell RCCs. Twenty-one (84%) of 25 clear cell RCCs were positive for caveolin-1, including 2 focal positive cases. Granular cell type tumors of clear cell RCCs were very similar to chromophobe RCC in staining pattern as well as extent, whereas clear cell type tumors differed from chromophobe RCC, that is, showing membranous staining. On the basis of these results, the granular cell type of clear cell RCC could be differentiated from oncocytoma (p\<0.001) but not chromophobe RCC (p=0.466).
MOC-31, a kind of cell surface glycoprotein, is expressed in various benign and malignant epithelia, but its function is still unclear. MOC-31 has been studied for differential diagnosis between hepatocellular carcinoma and cholangiocarcinoma or metastatic adenocarcinoma in the liver or between mesothelioma and adenocarcinoma \[[@B15],[@B16]\]. Studies associated with renal neoplasms have rarely been performed.
In our study, MOC-31 showed diffuse, strong, and membranous staining in 22 (96%) of 23 chromophobe RCCs and diffuse cytoplasmic staining in only 2 (25%) of 8 oncocytomas. The other six cases of oncocytomas were considered to be negative, but three of them expressed MOC31 with cytoplasmic staining in less than 10% of the tumor cells. In these three cases and the diffuse positive two cases of oncocytomas, MOC-31 was predominantly expressed in the tumor cells showing tubular differentiation rather than solid or trabecular structures. This seems to be related to the fact that MOC-31 is expressed in distal tubular cells of normal kidney. Although most oncocytomas do not express MOC-31, some oncocytomas may express MOC-31 if they are differentiated toward tubules and have similar structural features to normal distal tubules. Further molecular analysis is needed to uncover the precise function of MOC-31 in tubular differentiation and tumorigenesis.
According to our result for MOC31 expression, diffuse membranous expression of MOC-31 is a feature of chromophobe RCC rather than of oncocytoma, particularly not showing tubular differentiation (p\<0.001). Pan et al showed that 23 (82%) of 28 chromophobe RCCs were positive for MOC-31, whereas all 7 oncocytomas were completely negative \[[@B7]\]. This result is consistent with ours.
Clear cell RCCs expressed MOC-31 in 14 (56%) of 25 cases, including 2 focal positive cases. Considering only the granular cell type, 6 (40%) of 15 cases were positive for MOC-31. As previously mentioned, clear cell RCC had a statistically significant difference in the expression of MOC-31 from chromophobe RCC (p\<0.001) but not oncocytoma (p=0.215). For example, if a tumor is negative for MOC-31, it is more likely to be clear cell RCC than chromophobe RCC.
CONCLUSIONS
===========
The results of the present study showed an obvious discrimination between chromophobe RCC and oncocytoma in the expression of caveolin-1 and MOC-31. In other words, these immunohistochemical markers are thought to be considerably useful for distinguishing these tumors. In addition, clear cell RCC also showed a different expression of caveolin-1 and MOC-31 from oncocytoma and chromophobe RCC, respectively. If used together with other known markers such as c-KIT or RCC marker, both caveolin-1 and MOC-31 may be helpful in the differentiation of clear cell RCC from chromophobe RCC or oncocytoma.
The authors have nothing to disclose.
{#F1}
{#F2}
{#F3}
{#F4}
{#F5}
######
Clinicopathologic data of chromophobe RCC and oncocytoma
RCC: renal cell carcinoma, EM: electron microscopic examination, M: male, F: female
######
Expression of caveolin-1 and MOC-31 in normal kidney tissue
######
Results of immunohistochemical staining for caveolin-1 and MOC-31
RCC: renal cell carcinoma
######
Statistical results of the Fisher\'s exact test
RCC: renal cell carcinoma
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Air pollution is considered a real problem both nationally and internationally ([@B1]). Scientific findings indicate several adverse effects of air pollution on human health ([@B2] - [@B4]). The most prevalent side effects of air pollution appertain to the respiratory system ([@B5] - [@B6]). There is also a significant relationship between air pollution and premature birth, infant death and lung capacity reduction ([@B7] - [@B10]). The adverse effects are not limited to the respiratory system; for example, excess lead exposure due to air pollution can cause nervous system complications in children ([@B11]), induce an increase in blood pressure among adults ([@B12]), and have a negative impact on the cardiovascular system ([@B13]).
Air pollution has adverse effects on the reproductive system, pregnancy, fetuses, and infants ([@B14]-[@B16]). Genetic studies demonstrate the harmful effects of air pollution ([@B17], [@B18]), and there are evidences that it may also result in infertility in men ([@B19]).
Certain symptoms may be direct indicators of exposure to air pollution, such as nausea, chest pain, dyspnea, itchy throat, eye irritation, headache, palpitation, sputum, coughs, , and excessive fatigue ([@B20]). Moreover, several studies have demonstrated that air pollution raises the rate of hospitalization significantly ([@B21] - [@B23]) and others have pointed to an increased number of emergency visits when pollution prevails ([@B24]).
It is evident that all the costs and side effects ensuing from air pollution appertain to the healthcare system. Thus, the healthcare system and health service providers should have a special ethical sensitivity to environmental problems due to their responsibility to protect people's physical and mental health; hence, their ethical liability toward the environment is more noticeable, which naturally creates certain roles for them.
Based on the aforementioned points, physicians' sensitivity, awareness and attitude with respect to their ethical responsibility play a major part. Studies also demonstrate the influence of physicians' attitude toward air pollution on changing the public view of the subject ([@B25] - [@B28]). Moreover, due to their status in the society, physicians' conduct can provide a proper model to contribute to a change of public attitude toward environmental problems, which are basically created by man.
Air pollution is an example of an environmental problem resulting from activities such as transportation, energy production (transformation), and other energy-related industrial activities, which are the major producers of pollutants ([@B29]). Thus, changing the behavior and attitude of the people in a society will be a significant contribution to the reduction of air pollution.
Physicians' role in this issue may be different depending on their approach to the subject. Few studies have been conducted on physicians' moral responsibility regarding air pollution and its status in physicians' personal and professional priorities, or the attitude of this significant social class toward their role in reducing environmental crises. Consequently, it is of utmost importance to investigate the above-mentioned issues in order to provide the opportunity for interventions (for instance through altering or consolidating physicians' influential attitude), and create the grounds for conducting further studies to discover the obstacles and deterrents. Therefore, this study endeavors to elicit the ideas of physicians and experts as professionals who are notably involved in the issue, and solicit their opinions on their own ethical role and responsibility regarding the reduction of air pollution.
Method
======
This study was conducted between March 2014 and February 2016 in Tehran University of Medical Sciences. It investigated physicians' attitude toward their role in reducing air pollution and presented the personal and public solutions offered by this social group. In order to discover the profound and diverse aspects of the issue, it seemed necessary to apply a qualitative methodology, and therefore the methodology used in this research was "content analysis" ([@B30], [@B31]).
The data collection method consisted of semi-structured interviews and focus group interviews. First, a group discussion took place with the presence of 21 physicians, including 7 pediatricians, 2 neonatologists, 2 fellowship specialists in high risk pregnancy and perinatology, 3 medical ethics experts, and 6 asthma specialists and allergists. The session started with the statement of the problem by the researcher followed by a real medical case; then, several questions were asked and the subject matters were discussed. After implementing and evaluating the session, in-depth interviews were conducted in order to complete the data, as the researcher inferred that more comprehensive results could be achieved by spending more time and conducting personal interviews. Subjects were selected by purposeful sampling with maximum variation and grouped based on gender, age, specialty and rate of clinical experience. These four indicators were chosen because they were easy to identify and evaluate, and not only affected the objective of the study, but also had sufficient variation among the participants. After the interviews began, more subjects were added through theoretical sampling. Since the authors aimed at studying different opinions, the samples were selected out of four groups: pediatricians, pulmonologists, medical ethics experts, and internists. Thus, based on the reference review mentioned in the introduction, those specialists who were more involved in air pollution and the resulting side effects and costs were selected as samples. Prior to the interview, a text containing the guideline to questions and a summary of research objectives were sent to the participants via email. All interviews were recorded with participants' consent and permission from the ethics committee of Tehran University of Medical Sciences (No. 1686-1395). Each interview began with 5 open questions. The questions were designed in a general and unbiased way in order to encourage the participants mentioning any item they considered valuable in this field ([table 1](#T1){ref-type="table"}); then the follow-up question were asked based on the information provided by the participants to clarify the construct. Questions for further interviews were prepared based on extracted classifications. Sampling continued until saturation, i.e. when the researcher felt certain that no new data could be added anymore, and continued up to 13 samples (3 internists, 2 pulmonologists, 1 dermatologist, 4 pediatricians, and 3 medical ethics experts). The last two interviews contained similar information, and the researcher was satisfied with adequacy of interviews.
######
General questions
----------------------------------------------------------------------------------------------------------
Concepts of Questions
The relationship between health and environment
Physicians' role in air pollution and the related side effects, and the obstacles to the realization of\
their role in air pollution reduction
The role of policymakers and administrators in reducing air pollution
----------------------------------------------------------------------------------------------------------
To analyze the data, the present research used "the Corbin & Strauss constant comparative method" consisting of three stages of open coding, axial coding, and selective coding ([@B32], [@B33]). During the open coding stage, the texts were first implemented and then reviewed and reread multiple times. Researchers examined the data and extracted the major points and constructs, then gave a special code to each sentence. Lincoln and Guba's evaluative criteria method was applied to validate the data ([@B34]) and the validity of all eight criteria of this qualitative research was confirmed.
The researcher's long-term involvement in the research atmosphere and constant observations were considered. The researcher spent fourteen months collecting evidence from various resources, while external research was controlled by a third party arbitration, that is, the medical ethics expert who was not involved in the research. Triangulation and negative case analysis were performed by the researcher, and the discussions were presented using thick description. As a final stage, member check was applied to increase validation.
Results
=======
*Participants and procedure*
Twenty-one physicians (55% male and 45% female) participated in focus groups and 13 in interviews. Their average age was 40, and they had an average of 18 years (±10) of clinical experience. The focus group sessions lasted 100 minutes, and the average duration of the interviews was 30 - 45 minutes. Researchers coded each transcript independently, which resulted in extraction of 887 codes from the focus groups, and 105 codes from the interviews. A thematic analysis was performed after each interview and focus group session.
Interpretation of the coded texts enabled classification of the codes and establishment of the relationships between the various codes or categories. The codes were compared and discussed until agreement was reached. After classification, various categories were identified on the basis of descriptive hypotheses, and 4 themes and 20 subthemes were extracted overall.
######
Evaluation of physicians' role in mitigating air pollution and environmental problems
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Themes** **Subthemes** **Codes**
--------------------------------------------------------------- -------------------------------------------------------------------- -------------------------------------------------------------------
**Physicians' role as**\ Observing environmental issues as\ Physicians are ordinary citizens, and are effective and\
**ordinary citizens** ordinary citizens influential in their role as members of the society. Since air\
pollution and the environment are public concerns, physicians\
ought to observe these issues as other individuals, for instance\
use their personal cars less frequently, use public\
transportation, avoid smoking, contribute to tree planting,\
observe regulations such as vehicle inspection, and so on.
**Physicians' role as**\ Physicians must have an appropriate\ Physicians have a distinctive position as special citizens and\
**special citizens**\ conduct toward the environment role models.\
**and role models** Physicians are role models in the society and can influence the\
perspective of the general public.\
Therefore, they have greater individual and social obligations,\
and are more influential in culture making.\
People believe in physicians.\
As individuals, physicians have two roles: a) improving\
behavior b) teaching.
They can teach others directly If physicians are concerned with the environment, they can\
make contributions through activities such as making posters\
conveying environmental messages, separating dry and wet
They can teach others implicitly (by\ waste in the clinic, and avoiding improper methods of garbage\
providing role models) disposal. Such acts have a promoting and educational effect\
on patients. Physicians' professional conduct is especially\
significant; if physicians separate their wastes or have a\
healthy conduct, they can influence their patients' and others'\
outlook. This is also a form of education for patients and a way\
of culture building for the society, since such physicians not\
only behave properly and professionally, but also educate the\
society. As an example, physicians' use of public\
transportation has a tremendous educational effect on other\
members of the society.
**Physicians' role as**\ Writing papers on air pollution and\ Physicians are influential because they can publish health-\
**professionals with**\ its adverse effects on human health related articles. For example, articles showing accurate\
**special ethical**\ statistics about the dangers of air pollution in various\
**liabilities and**\ organizations.\
**sensitivities, both**\ They can also attract the attention of policy makers and\
**personal and**\ managers to this important issue.
**social**
Presenting real statistics and reports\ Reliable, scientific and updated articles, statistics and reports\
for informing the healthcare system,\ can attract the attention of associations and scientific centers\
the society, and the public now more than ever.
Conducting relevant research\ Physicians and other health care providers must understand\
projects about air pollution and its\ the need for research in this field\
harmful effects on health Informing\ Reliable scientific articles with real statistics can cause the\
and raising awareness in three\ public to look into the issue. This raises awareness both in the\
levels: public associations, policy-\ scientific community and across the whole society. It can also\
making, and the society have a significant impact on the general outlook and even\
direct it.
Monitoring air pollution in a more\ Provision of monthly and annual detailed statistics to the\
systematic way, for instance on a\ Ministry of Health and other related organizations can be very\
monthly or daily basis effective in making changes in the present state of affairs.
There are few scientific papers and detailed statistics on air\
pollution and its harms to the environment.\
It is important that physicians be concerned about this issue.
Showing sensitivity to prevalent\ Monitoring environmental problems and responding to them\
environmental crises; in general,\ is essential. It is important that physicians be concerned about\
professional physicians should have\ the environment, especially air pollution. Factories that are\
a comprehensive insight and show\ built around large cities all contribute to pollution. Such\
sensitivity to environmental crises problems arise when people care only about economic\
interests and plan without first conducting the necessary\
environmental studies.
Observing some points by\ Likewise, employment and development are important, but all\
physicians and the healthcare\ should be in line with the environment.\
community (in the clinic, hospital,\ Deforestation, irresponsible urban development, car\
or laboratory) which decrease air\ manufacture, production of non-standard cars and fuels, and\
pollution. All physicians should\ destruction of the environment are very important issues, and\
evaluate the circumstances in their\ the medical community should also show sensitivity towards\
clinic or hospital in order to reduce\ them.
air pollution in their professional\
domain
Social commitments based on the\ Physicians and health care providers should start from\
attention of health organizations to\ themselves.\
the importance of the subject, and\ Every physician should evaluate issues in their workplace and\
designing the necessary models for\ address problems that can be resolved by themselves or the\
environment-protecting clinics,\ hospital. Gynecologists stated that the incubator machine\
laboratories, or hospitals produces a type of gas harmful to children, and the older the\
incubator, the higher the produced gas rate will be, an item\
mostly neglected by physicians. Moreover, physicians have\
some frequent unnecessary visitors, an issue that can be taken\
care of by telemedicine, or phone and internet communication.\
Repetitive unnecessary visits to physicians can be reduced\
through tele-medicine via phone or the internet.
Green hospitals and developing organizational guides for the\
staff to promote environmental behavior in the workplace is\
very effective.
Prevention is also another responsibility of physicians, and\
solving environmental problems is related to this issue.
Professional commitment to\ Treating patients is a major duty, but the harms and damages\
treatment and follow-up of the\ that air pollution induces on health should also be prevented.\
patients Drinking milk, for instance, can prevent the adverse effects\
due to the calcium in milk, or vitamin C intake is useful\
because of its antioxidant feature. Providing information on\
such subjects by physicians is very important for patients and\
the society alike.
Physicians can offer solutions for vulnerable groups to help\
them become less affected by air pollution, for instance by\
introducing a type of nutrition beneficial to children, or\
providing masks to pregnant women.
Professional commitment to paying\ As another vulnerable group, women should not to be exposed\
special attention to the health of\ to air pollution, and must be encouraged to drink milk to\
vulnerable groups protect them from the adverse effects. Likewise, the elderly\
should stay at home or avoid exercising in polluted air.
**Physicians' role as**\ Creating codes and directives on\ In this regard, physicians and hospitals must take serious\
**administrators of**\ green hospitals, laboratories and\ actions.\
**the healthcare**\ faculties\ Green hospitals can contribute to environmental protection.\
**system, and their**\ Having Green hospitals and developing organizational guide\
**environmental**\ the staff to have effective environmental behavior in the\
**responsibilities** hospital. This strategy is very effective.
Consulting with experts and patients\ Physicians should address the psychological and social\
and attracting the beneficiaries'\ aspects of the issue, which often necessitates consultation with\
participation colleagues and talking with patients and their relatives. It is\
also important to reach an agreement and get help from others\
if necessary and obtain the participation of all stakeholders.
Teaching environment protecting\ Education is very important for medical students, patients and\
behavior by university authorities\ other people, and physicians and health care managers can\
Supporting environment-related\ contribute. Culturalization begins with universities and\
research (on air pollution): if\ academic environments. Therefore, professors and university\
healthcare authorities support\ presidents can educate the students in environmental issues.\
research projects, physicians and\ The faculty and the department must support air pollution\
experts will pay heed to such\ research and ask physicians to present related articles and\
activities more carefully reports. Follow-ups by the authorities help and create an urge\
for physicians and experts in the field. Healthcare providers\
and health policy makers are responsible for making the health\
industry green. They should implement clean and green\
policies in hospitals, health centers, laboratories, and\
university departments. For example, green hospitals have\
specific codes. In general, health system actions on the policy-\
making level must be environmentally friendly. Health\
authorities should constantly think and function in\
environmental terms.
A serious request from experts and\
physicians to present reports and\
scientific papers in the field
Physicians should review research plans that may potentially\
harm the environment.
Reviewing all research plans in\
terms of environmental threats\
(specialty committees)
Healthcare system administrators\ Physicians may submit reasonable requests to related centers,\
should expect answers and\ such as public transportation or environment organizations,\
explanations regarding\ and urge activities to reduce air pollution. They may even go\
environmental crises from\ as far as observing environmental problems and showing\
authorities proper and timely reaction to environmental degradation\
(factory building, deforestation, drying the lakes and rivers).\
Furthermore, they can acquire a comprehensive insight and\
study the effects and outcomes of environmental degradation.
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*First theme: Physicians' role as ordinary citizens:*
One subtheme was extracted in this regard, which was observing environmental points as ordinary citizens. More explanations have been provided in [table 2](#T2){ref-type="table"}.
*Second theme: Physicians' role as special citizens and role models:*
There are three main subthemes in this item: a) a physician must have an appropriate conduct toward the environment; b) he/she can directly teach others; c) he/she can implicitly teach others ([Table 2](#T2){ref-type="table"}).
*Third theme: Physicians' role as professionals with special ethical liabilities and sensitivities, both personal and social:*
a. Physicians should write relevant papers regarding air pollution and its adverse effects on human health. This issue was considered particularly valuable and was pointed out in most interviews.
b. Physicians can present real statistics and reports. This is very useful for informing the healthcare system, society, and public, as the statistics given by this social class will be scientifically accurate, and appropriate solutions can be based on them.
c. Conducting relevant research projects on air pollution and its harmful effects on health will be extremely helpful.
d. Informing should take place on three levels: public associations, policy-making, and the society. Physicians have certain social commitments and should be sensitive to affairs that jeopardize the public health. Informing can be useful at all levels, either at the social or policy-making level. In addition to statistics and papers mentioned earlier, informing can be done in the form of interviews and technical meetings with the media, social networks, and so on.
e. Monitoring air pollution in a more systematic way, for instance on a monthly or daily basis, is especially important. Those who are engaged in environmental activities, particularly physicians, are expected to monitor environmental issues more systematically since they are much more involved in air pollution costs and harms thanks to the facilities and possibilities available for them.
f. Physicians need to be sensitive to prevalent environmental crises; in general, professional physicians should have a comprehensive insight into such issues, because the majority of environmental crises have a direct or indirect impact on human health.
g. In clinics, hospitals and laboratories, physicians and members of the healthcare community must observe certain details regarding environmental protection. More specifically, physicians should evaluate measures to be taken in the clinic or hospital in order to reduce air pollution in their professional domain ([Table 2](#T2){ref-type="table"}).
h. Physicians have social environmental commitments based on the attention of health organizations to the importance of the subject, and they can help design the necessary models for environment-protecting clinics, laboratories, or hospitals ([Table 2](#T2){ref-type="table"}).
i. Physicians have the professional commitment to treat and follow up their patients ([Table 2](#T2){ref-type="table"}).
j. Another professional commitment of physicians concerns the provision of special care to the health of vulnerable groups ([Table 2](#T2){ref-type="table"}).
*Fourth theme: Physicians' role as administrators of the healthcare system, and their environmental responsibilities:*
a. Physicians are required to create codes and directives on green hospitals, laboratories and faculties. There is great need for such establishments, and writing organizational guides on the staff's conduct in relation to environmental protection is also useful.
b. Physicians can consult experts and patients and attract the beneficiaries' participation in environmental affairs. They may consider the mental and social aspects and problems and consult their colleagues, patients and their families to reach an agreement and resolve the issues; furthermore, they can ask other people for help if necessary, and try to attract the beneficiaries' help.
c. University authorities should also teach and promote environment protecting behavior. Instructing patients, medical students and other members of the society is very important, and physicians and healthcare system administrators can participate in the process. Culturalization initiates from universities and academic institutions; therefore, professors and heads of universities can educate their students on environment-related issues and principles.
d. If healthcare system authorities support environment-related research projects (in this case on air pollution), physicians and experts will pay heed to such activities more often. Participants' examples are presented in [Table 2](#T2){ref-type="table"}.
e. There is serious request from experts and physicians to present reports and scientific papers on the field.
f. Specialty committees should review all research plans in terms of potential threats to the environment. For instance, ethical committees should consider the constructive points in research projects that contribute to protect the environment.
g. Healthcare system administrators should expect answers and explanations regarding environmental crises from government authorities. Moreover, they may request environment-related action from all centers involved. Participants' examples are presented in [Table 2](#T2){ref-type="table"}.
Discussion
==========
As health service providers, physicians face diseases and complications that result from environmental crises such as air pollution every day. As citizens in general and as professionals in particular, they are expected to assume responsibility for environmental crises. The present research was conducted to examine how they can participate in the process of reducing air pollution. This study showed that physicians believe they can be effective in this field on four levels:
**Level 1:** In their role as ordinary citizens, physicians resemble other citizens in that they are clearly expected to perform their duty to protect the environment. Other studies have mentioned this role as well, for instance, Lorenzoni et al. stated that those employed in the healthcare system are ethically responsible for the environment as individuals, both in their personal and social life ([@B35]). Islamic ethics also frequently recommends the protection and reconstruction of the environment. Influential Islamic doctrine emphasizes that the nature belongs to God, so human beings are always present in the divine land, even if they are not aware of it. According to Islamic teachings, environmental crises are the outcome of people's negligence in their responsibilities toward the nature, and their wrong belief that the environment is separate from the divine land. Thus, Islamic thoughts always urge citizens to protect and preserve the environment ([@B36] - [@B38]).
**Level 2:** A physician is also a special citizen and a role model whose conduct is emulated and followed by the society. A physician's conduct can be educational for the patients and others, both directly and indirectly.
**Level 3:**The next level mentioned in this research was a physician's role as a professional with special personal and social commitments. This role is of utmost importance, as WHO also defined the responsibility of the healthcare system and hospitals beyond mere treatment and prevention of diseases in 2009, stating that hospitals should align their policies with environmental and climate changes due to the effect of such factors on public health ([@B39]). The physicians in the present research also mentioned supplementary endeavors that show significant professional commitment to the subject. Instances included writing papers, presenting reports, and informing the public, all of which may take place on three levels: in scientific centers, policy-making, and the society. Global studies also indicate that health service providers should be ethically sensitive to environmental problems because of their immediate and direct impact on human health. For instance, climate change and the related issues have a serious effect on people's well-being, so the healthcare system is professionally expected to consider them ([@B40]). The common point between the present research and other studies was the importance of healthcare providers' direct and indirect role in environmental problems that affect not only the general health but also the environment ([@B41]). The next point achieved in the research was showing sensitivity to these problems and taking a comprehensive view on the matter. This topic has been discussed and agreed on in some studies, albeit in a different light; for example, a qualitative study conducted in 2015 on 18 nurses demonstrated that the nurses' view of their duties and responsibilities is mostly confined to their job-related tasks. They turned out to prioritize hygienic issues such as infections, and did not have a comprehensive insight on climate change or environmental problems, or at least did not prioritize them even when aware of their importance ([@B42]). Meanwhile, a 2005 study conducted in Paris insisted on the necessity of healthcare providers' comprehensive concern for environment, since environmental issues can play a major part in every individual's health in general ([@B43]). A 2015 study in the UK showed that those in the medical profession display their individual and organizational training and leadership roles in issues such as release of penicillin, tobacco control and enforcement of vehicle safety belt laws. Relying on such instances, the present study refers to physicians' important role in resolving social problems and urges a similar role in environmental issues ([@B44]).
Thus, a medical treatment team with heightened sensitivity to the environment may not only direct their professional conduct toward environmental protection, but also will look at environmental problems in a professional light and present their views on public health to policymakers. In fact, therapists are one of the best groups to observe and study environmental problems and provide policymakers with appropriate data.
**Level 4:** Physicians also have the role of caretakers of the health and education system. This role can be categorized into two groups: reforming educational policies and directing them toward the attitudes resulting in proper conduct, and appropriate policymaking to protect the environment as much as possible.
This study reviewed a new concept of environmental responsibility within the framework of physicians' social responsibility, presenting it as physicians' role in environmental issues. These roles are summarized as follows:
*The first role*
Teaching environmental ethics to students by university authorities was a pivotal point mentioned in this case. As centers of education and culturalization, faculties are highly important. Informing the students will contribute greatly to changing their attitude and behavior, and consequently those of the society. A 2011 study on senior medical students of medicine examined their attitude toward climate change and demonstrated that their opinions were changeable and resulted from the general attitude of the healthcare system toward environmental problems ([@B45]). Thus, the attitude and conduct of authorities will significantly affect the students' behavior.
*The second role*
Three domains can be mentioned in this respect: presenting proper executive policies, supporting research projects, and review and supervision. One of the subjects discussed here was regulation of codes and directives for green hospitals, laboratories and faculties. This is extremely useful and fundamental, as many renowned medical faculties all over the world have regulated related ethical codes and directives ([@B46], [@B47]). Committees and unions throughout the world have discussed this important issue in their ethical codes. For instance, the International Council of Nurses published nurses' ethical codes in 2012, taking into consideration the environmental issues and observations ([@B48]). Unfortunately, there are no such ethical codes in Iran, and aside from the ethical code of research on animals ([@B49]) and medical research codes (only in one paragraph) ([@B50]), there is no clause among the thirteen written codes which specifically address environmental issues.
Another item related to this role is the support of research projects. The subthemes mentioned in the last topic as physicians' professional duties depend on the support of healthcare system administrators in order to be realized. Thus, writing papers, presenting statistics, preparing scientific and valid reports, and conducting relevant research on the subject require the support of healthcare system authorities. Moreover, in order to establish green hospitals and make proper use of telemedicine, the essential substructures should be provided by healthcare system administrators, and research ethical committees should be bound to study and assess the rate of pollution from an ethical viewpoint. Supervising this vital item is one of the duties of the heads of faculties.
*The third role*
Healthcare system administrators should ask the authorities for explanations regarding environmental crises, because although problems such as air pollution are environmental in nature, the healthcare system will be responsible for the resulting harms and damages. Therefore, ethical sensitivity and concern about such problems are essential for healthcare system authorities.
To summarize, environmental issues should be taken into consideration in the light of medical ethics to determine the ethical duties of the medical society.
Limitations of the study
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Since the topic was rather new, some of the experts did not have an adequate mental background in the subject and were therefore unable to cooperate in their full potential. In order to remove this obstacle, we provided them with the question guide ([@B42]). Furthermore, in spite of the attempt to reach data saturation in the studied specialties, some of them may not have been prioritized in the interviews; however, almost all of the relevant specialties were considered according to the literature review.
Conclusion
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Ideas on medical professionalism at all three levels (interpersonal, intrapersonal and public) and physician's social responsibilities are frequently overlapping. Both require the physician to be accountable to the communities and the society in which they serve. Therefore, the physician should be increasingly prepared to respond to the growing environmental challenges and their social consequences.
The present research shed a light on some aspects of this mutual relationship, suggesting that policymakers consider these aspects when establishing laws, supervisory mechanisms, and educational and research policies to create a positive attitude and control physicians' conduct regarding environmental issues. Since there is not much positive attitude in the field of environment, one of the priorities of the healthcare system can be to establish an appropriate attitude through different means, for instance by way of education.
This study was extracted from the researcher's Ph.D. dissertation supported by Tehran University of Medical Sciences.
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